光応答制御に基づく生命現象の解明とがん・老化研究への応用

平成２５年度〜平成２７年度私立大学戦略的研究基盤形成支援事業

研

究

成

果

報

告

書

平成２８年５月
学校法人名：

学校法人学習院

大 学 名：

学習院大学

研究組織名：

理学部

研究代表者名： 花岡 文雄（学習院大学理学部生命科学科）

目

次

I.

はじめに・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・

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II.

研究組織・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・

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III.

研究課題 1

放射線が生命に及ぼす影響と耐性機能の解明・・・

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花岡文雄・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・

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菱田 卓・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・

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安達 卓・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・

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IV.

V.

研究課題 2

光の情報を受け取る分子メカニズムの解明・・・・・・

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岡田哲二・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・

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岡本治正・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・

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清末知宏・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・

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研究課題 3光を利用した生命機能の解明と発がん制御技術の開発

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中村浩之・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・

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小島修一・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・

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馬渕一誠・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・

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西坂崇之・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・

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I.

はじめに
太陽は、地球における生命の誕生から今日に至るその繁栄に極めて重要な役割を果たしてきた。太陽光には様々な波長の光（電磁波）が含まれており、人間の目に見える可視光は、生物に様々な情報を与え、環境への適応能力の獲得に貢献している。一方、可視光の範囲外にある光は、環境情報としての役割の他に、遺伝情報物質である DNA に損傷を与え発がんの原因になるなど、生体システムの維持にとって脅威となる一面を持っている。
本研究では、微生物から高等動植物に至る様々な生物について、分子レベルから個体レベ
ルに至るあらゆる階層を研究対象として、様々な波長の光が生物に与える影響や情報としての光が関わる生命機能を明らかにし、さらに、それらの情報を基に生物の光情報による環境
適応システムの解明と、光を利用した生命機能の解析技術や発がん制御技術の創出を目的とした。具体的には以下の三つの研究課題、すなわち「①放射線が生命に及ぼす影響と耐性機能の解明（花岡／菱田／安達）」、「②光の情報を受け取る分子メカニズムの解明（岡田／岡本／清末）」、「③光を利用した生命機能の解明と発がん制御技術の開発（中村／小島／
馬渕／西坂）」に取り組んだ。
各研究課題には、それぞれ３ないし４名の研究者を配置し、菱田教授、清末教授、馬渕教
授にそれぞれの課題の取りまとめ役を依頼し、全体を研究代表者の花岡が総括するという体制をとった。３つの研究課題は互いに密接に関係しており、すべて学習院大学生命科学科・
大学院自然科学研究科の生命科学専攻に所属しているので、月に２回、定期的に開かれる専攻会議の場で議論した。本プロジェクトの途中から中村教授が東工大に異動したが、その後も本学の非常勤講師を務めており、頻繁に議論を行った。さらに中村教授以外のメンバー
は地理的にも近接した二つの建物で研究を行っており、必要なときにはいつでも行き来して情報交換を行った。
各研究室には基本的に１名の助教がおり、教授とともに本プロジェクトに関与した。また、本プロジェクトで雇用したポスドクは、PD 研究員という身分でプロジェクトを推進した。さらに全体４〜５名のリサーチアシスタントと各研究室数名から 10 名近くの大学院生も積極的に本プロジェクトに参加し、主にマンパワーとして貢献した。主要な装置、設備の利用・管理について、メーカーによる説明会を開催したり、研究参加者からの個別相談に対応したりといった技術的サポートをプロジェクトメンバーの研究室に所属する各助教が、また各種申請書・報告書等の作成支援および経理業務といった事務的サポートを理学部事務室が中心となってそれぞれ行ない、円滑な研究遂行のための支援体制を構築した。
共同研究機関としては、プロジェクトの途中から中村教授の異動先である東工大の資源化学研究所が主たるものであるが、それ以外にも国内外の複数の研究機関と積極的に共同研

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究を進めた。これら３つの研究テーマのメンバーがグループ内及びグループ間で共同研究やセミナーなどを行い、互いに連携しながら研究を推進した。中村は、平成 25 年 9 月 1 日から
東京工業大学の資源化学研究所に教授として転出したが、その後も共同研究機関の立場でプロジェクトに参加した。西坂は平成 25 年度まで「最先端次世代研究開発支援プログラム」に参画中のため、初年度は連携研究者として参加した。
建物としては、本プロジェクトの前身である「生体システムの環境応答に関する分子細胞生物学的研究 —老化・がん化の制御に向けてー」の予算から補助を受けた南 7 号館（自然科学研究棟：整備年度 H21）に 1,718 m2 と、旧くから存在・使用している南 6 号館（生命分子科学
研究所：整備年度 H4）に 448 m2 が存在し、それぞれ 7 研究室（当初は 8 研究室）と 2 研究室がプロジェクトを推進した。本プロジェクトの途中から、中村教授は東工大の資源化学研究所に教授として転出し、そちらの建物でプロジェクトを推進した。
本事業で助成され購入した「アコースティックソルビライザー」および「デジタルマイクロスコープ」は、それぞれ稼働時間約 450 時間／年と 500 時間／年、また別予算で購入した「オールインワン蛍光顕微鏡システム」、「共焦点スキャナユニット」、「倒立顕微鏡 Ti-E パーフェクト
フォーカスシステム」等の顕微分光関連設備については、それぞれ約 1,200 時間／年、100 時
間／年、1,000 時間／年の稼働時間が実績として残された。これらの設備は、上記二つの建物に設置され、プロジェクト参加研究員 10 名（現在 9 名）、助教 10 名（現在 9 名）、PD 研究員
1 名に加え、大学院生約 15 名、学部生約 45 名が有効に利用した。
本プロジェクトの構成員の多方面の卓越した研究を基盤に、多様な生物が様々な波長の光にどのように応答し、適応しているのかを分子レベルで解析し、そのメカニズムを明らかにすることを目標とした。さらにそのメカニズムに欠陥が生じたとき、老化やがん化といったプロ
セスがどのように促進されるのかを明らかにすることによって、これら高次生命現象を制御する方策を探ることも目指した。

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II.

研究組織

研究課題１．放射線が生命に及ぼす影響と耐性機能の解明
研究者名
花岡 文雄

菱田

卓

（取りまとめ役）

安達卓

プロジェクトでの研究課題

プロジェクトでの役割

遺伝子損傷の細胞応答とがん化・ 紫外線等による DNA 損傷が個体のがん化・老化に及ぼす影響とその防御
老化機構の研究
DNA 損傷ストレスに対する耐性機 電離放射線や紫外線などの微量かつ慢性的な DNA 損傷ストレスに対する構の解明
生物の適応機構の解明
放射線に対する細胞死耐性の機 放射線が導く細胞死応答に対して耐性をもつ細胞における分子機構の解構
明

研究課題２．光の情報を受け取る分子メカニズムの解明
研究者名
岡田 哲二
岡本 治正

清末 知宏

プロジェクトでの研究課題

プロジェクトでの役割

視覚関連タンパク質の動作原理の解
明眼の発生・機能関連新規遺伝子の 光情報の処理にかかわる新規因子の同定
同定
受容体分子構造基盤の解明

高等植物の光環境応答と花成（取りまとめ役）

光周性による成長相転換制御機構の解明

研究課題３．光を利用した生命機能の解明と発がん制御技術の開発
研究者名
中村 浩之

小島 修一
馬渕 一誠
（取りまとめ役）

西坂 崇之

プロジェクトでの研究課題

プロジェクトでの役割

光を利用した生命機能解明とがん 光触媒による生命機能解明のための治療
新しいケミカルプローブの開発を行うとともに、ホウ素と中性子の核反応による新しい放射線療法のためのホウ素薬剤開発を行う
生体防御関連タンパク質の構造お タンパク質の活性制御領域の構造・機よび生化学的解析
能解析と新規阻害剤の開発
細胞分裂構造の制御メカニズムの 細胞のがん化と、がん細胞の浸潤運
解明
動のメカニズムの理解
光学顕微鏡技術を駆使した生体分 光学顕微鏡技術の開発によって生体子の動態の可視化
分子やその超構造の動態を明らかにし、生命機能のより詳細な理解に貢献する

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III. 研究課題１ 「放射線が生命に及ぼす影響と耐性機能の解明」
本課題では、遺伝子 DNA に損傷を与える主要な環境要因である紫外線や電離放射線に着目し、これらの要因による DNA 損傷が生物にどのような影響を及ぼすか、そして生物はこれらの DNA 損傷にどのようにして耐性を獲得するのかを中心に解析した。特に紫外線への曝露によって高頻度に皮膚がんを発症するヒト遺伝病、色素性乾皮症（XP）、中でもバリアント
群 XP の原因遺伝子産物である XPV タンパク質（Polη ）の構造と機能について、タンパク質レ
ベル、細胞レベル、個体レベルなど様々な観点から研究を行った。また放射線や紫外線による細胞の耐性に DNA の相同組換え機構が働いていること、そして出芽酵母においては慢性
的な紫外線ストレスが染色体の倍数化を引き起こすという興味深い結果が得られており、それらのメカニズムについて明らかにする試みがなされた。一方、ショウジョウバエのオス成虫がもつ内部生殖器官である附属腺を用いて、放射線ストレスへの細胞応答の代表である細胞死に対して耐性になる場合とそうでない場合との比較を行い、興味深い結果が得られた。
以下に、これらの研究について、それぞれの研究担当者からの解説と業績リスト、そして代表的な論文 2 編ずつを掲載する。

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遺伝子損傷の細胞応答とがん化・老化
教授 花岡 文雄
助教 横井 雅幸
助教 川元 洋子（2013 年度のみ）[目的]
遺伝子 DNA は種々の内的あるいは外的な要因によって、その複製を阻害されたり、損傷を受けたりしている。そのような状態を放置すると個体に老化やがん化をもたらす。特に日光中の紫外線には地球上の全ての生物が例外なく曝されており、DNA にシクロブタン型
ピリミジン二量体（cyclobutane pyrimidine dimer: CPD）や 6-4 光産物といった損傷が生じる。生物は、こうした紫外線損傷を修復したり、DNA 合成期に乗り越えたり（translesion
synthesis: TLS）する能力を備えているが、ヒトにはそうした能力に欠損を持つ遺伝的疾患が複数存在する。中でも色素性乾皮症（xeroderma pigmentosum: XP）の患者は皮膚の早期老化および高い頻度での皮膚がんの発症を特徴とする。本研究では我々のグループが発見したバリアント群 XP の責任遺伝子産物で、CPD を効率良く正確に乗り越える DNA ポ
リメラーゼであるポル・イータ（Polη）1) を中心に、ヒトおよびマウスの TLS ポリメラーゼの生理的な機能やその X 線結晶構造 2) に基づいた変異体を解析し、TLS ポリメラーゼの欠損や変異による個体のがん化・老化の分子的な基盤を明らかにすることを目的とする。
[結果と考察]1. TLS ポリメラーゼの多彩な機能について(1) Polηが体細胞超突然変異に働く分子メカニズムの解明
B 細胞において、V(D)J 組換え後に免疫グロブリン遺伝子に起きる体細胞超突然変異（somatic hypermutation: SHM）は、免疫グロブリンに多様性をもたらすために必須のメカニズムである。これまでの研究から、RGYW と WA（R は purine; Y:は pyrimidine; W
は A または T）という二つの SHM のホットスポットのうち WA に関しては Polηがメインな役割を果たしていると考えられている。今回、我々は米国 NIH のグループなどとの共同研究により、ヒト Polηの触媒活性を担う部分タンパク質（1-432）とプライマー末端の鋳型側の WA モチーフが TA または AA、あるいは非 WA の配列（CA または GA）との共結晶を作成し、
それらの X 線結晶構造を解析した。
その結果、WA モチーフの T に対して dGTP
が wobble 塩基対を形成する際に Gln-38 と Arg-61 とが安定化に働いていること、プライマー末端の W（T:A または A:T）の弱い塩基対と Polηとの相互作用が WA モチーフへの Gの誤塩基取り込みに導くことが明らかになった。二つの WA モチーフの中でも AA よりも
TA 配列が A から G への変異をより起こし易いこと、また Polηは T:G 誤塩基対を効率良く伸長し、突然変異を完成させることが判明した 3)。
(2) DNA の再複製における TLS ポリメラーゼの役割
DNA の再複製は、遺伝子重複などのゲノム不安定性に寄与する異常複製の主要なプロセスである。しかしどの DNA ポリメラーゼがこのプロセスに働いているかは明らかになっていない。本研究では、DNA 複製に必須の因子である Cdt1 を阻害することによって、1 細
胞周期に一度しか DNA 複製が起きないようにしている重要な因子である geminin を5

siRNA 処理によって DNA 再複製を誘導する系 4)を用い、Polη、ポル・カッパ（Polκ）、
ポル・イオタ（Polι）、REV1 などの忠実度の低い Y ファミリー・ポリメラーゼやポル・
デルタ（Polδ）やポル・イプシロン（Polε）などの複製型ポリメラーゼの DNA 再複製への関与を調べた。その結果、これらの TLS 型や複製型の DNA ポリメラーゼはヒト U2OS細胞の再複製部位へとリクルートされること、またこれらのポリメラーゼを RNAi によってノックダウンすると geminin レベルの低下によって誘導される「DNA の再複製」が抑制されることが分かった 5)。興味深いことに、Y ファミリー・ポリメラーゼは複製型ポリメラーゼとは異なる経路で再複製に働いていることが示唆された。さらに cyclin E の過剰発現による DNA の再複製にも Polηと Polκが関与していること、また Polηの触媒活性が必要なことも明らかとなった。
2. シトシンを含む CPD とそれに対する Polηの働き(1) CPD 中で安定なシトシンの誘導体
CPD は紫外線による DNA の損傷の中で主要なものであるが、通常、実験に使用されるのはほとんどチミン-チミン二量体である。それは DNA 中のシトシンが生理的な条件下でも不可逆的に脱アミノ化を受けてウラシルになりやすく、CPD を形成するとさらに脱アミノ化が促進されるので、実用的でないのである。先行研究で、5-メチルシトシン（mC）はシトシンよりも安定なこと、mC を含む CPD もシトシンを含む CPD よりも安定なことが示されていたので、DNA 合成機でオリゴ DNA を合成する際、さらに安定と思われる N4-mC（mCm）をチミンと共に CPD 形成させた T[]mCm を有機合成し使用した。その結果、
T[]mCmを含むオリゴ DNA の寿命は生理的条件下で約 1 週間と比較的長く、ヒト Polηを用いた invitro の TLS 反応において T[]mCm の mCm の反対側に dGMP の取り込みだけでなく、TMPも相当な頻度（約 6/16 の割合）で取り込まれること、また TMP が取り込まれた場合、そこで DNA 合成が停止しやすいことが分かった 6)。

(2) 野生型および Polη欠損マウス皮膚における紫外線誘発突然変異の塩基配列特異性

λファージをベースに、lacZ 遺伝子をレポーターにしたプラスミドを持つマウスを野生型および Polη欠損マウスと掛け合わせ、背中の皮膚に UVB を照射して、表皮と真皮における紫外線誘発突然変異の頻度（mutant frequency: MF）とスペクトルを解析した 7)。表皮および真皮のいずれにおいても、Polη-/-マウスは、野生型（Polη+/+）およびヘテロ（Polη+/-）マウスに比べ高い UVB 誘発 MF を示した。野生型とヘテロとでは有意な差は見られなかった。野生型と Polη-/-マウスで UVB 誘発および未処理の条件下での変異スペクトルを比較したところ、いずれにおいても UVB 照射に特異的で dipyrimidine 部位での C からT への transition がメインであった。しかしながら変異スペクトルを詳細に検討したところ、野生型の上皮では 5’-TCG-3’配列での変異がドミナントであるのに対し、Polη-/-マウス
ではそのような配列特異性が見られなかった。このことは、Polη-/-マウスでは CPD の乗り越えに変異を起こしやすい TLS ポリメラーゼが働いており、特に 5’-TCG-3’配列がホットスポットになっていることを示唆している。
3. 複数の TLS ポリメラーゼ間での機能の連携
以前、我々は Polηと Polιの単独あるいは二重欠損マウスの紫外線照射皮膚がん誘発実験により、Polη／Polι二重欠損マウスでは、Polη単独欠損マウスと比べ UVB 誘発皮膚6

がんの頻度はあまり変わらないが、Polη単独欠損マウスではもっぱら上皮性のがんを生じるのに対し、Polι単独欠損あるいは Polη/Polι二重欠損マウスにおいては、Polη単独欠損マウスでは見られない肉腫の形成を認めた 8)。これらのことは、Polηも Polιもマウスの UVB による皮膚がん誘発を抑制していること、またその抑制の部位、組織等が異なることを示唆している。そこで大腸菌 rpsL 遺伝子を約 100 コピー、レポーターとしてゲノム中に有するマウスを上記のポリメラーゼ欠損マウスと掛け合わせキメラマウスを作成し、これらのマウス表皮を用いて、UVB 誘発 MF と変異スペクトルを解析した。予想通り、Polη単独欠損マウスは野生型に比べ非常に高い UVB 誘発 MF を示し、Polι単独欠損では野生型とほとんど変わらなかった。興味深いことに、Polη/Polι二重欠損マウスにおいてはPolη単独欠損マウスに比べ、UVB 誘発 MF が低かった。このことは、Polηが欠損している状況で、Polιは Polηの代わりに働くが、その際、誤塩基を対合しやすいことを示している。変異スペクトルの解析から、Polη単独欠損でも Polη/Polι二重欠損でも、UVB 誘
発変異のほとんどはジピリミジン部位に起きていること、Polη単独欠損で変異は G:C と

A:T の両方の塩基対で起きていることから in vivo で Polηはシトシンとチミンのいずれのピリミジンを含む二量体も正しく乗り越えることが分かった。さらに Polη/Polι二重欠損
では、G:C から A:T へのトランジションが Polη単独欠損に比べ有意に低下することから、
Polιはシトシンを含む二量体の乗り越え時に誤対合を起こしやすいことが示唆された 9)。

[まとめ]Polηが体細胞超突然変異に働いていることは、様々な免疫学的な解析から分かっていたが、今回、ヒト Polηの立体構造が安定な部分タンパク質と様々な鋳型・プライマー末端の共結晶の構造解析から、実体としてヒト Polηが変異の誘導に機能しているメカニズムを明らかにしたことは大きな意義がある。また発がんとの関係で重要な事象である「DNA の再複製」に複製型ポリメラーゼだけでなく、TLS 活性を持つ Y ファミリーポリメラーゼも機能していることは大変興味深い。特に Polηの触媒活性が必要である点は、がん化のメカニズムを考える上でも重要である。
これまでシトシンを含む CPD の研究は、その不安定さゆえにチミン-チミン二量体に比べて遥かに立ち後れていた。今回、N4-メチル-5-メチルシトシンとチミンの二量体が比較的安定に存在することが分かり。これまで実験的にアプローチ出来なかった点に大きく寄与することが期待される。
今回の研究で、
UVB 誘発皮膚がんの突然変異頻度と変異スペクトルが色々と解析出来た。
こうした研究は、皮膚がんだけでなく、一般的にがんの発生にどのような変異が重要であるかを知るための大事なステップとなるであろう。
[参考文献]
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7. Ikehata, H., Chang, Y., Yokoi, M., Yamamoto, M., and Hanaoka, F. (2014) Remarkableinduction of UV-signature mutations at the 3’-cytosine of dipyrimidine sites except at 5’-TCG-3’
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10. Matsumoto, S., Fisher, E. S., Yasuda, T., Dohmae, N., Iwai, S., Mori, T., Nishi, R., Yoshino, K.,
Sakai, W., Hanaoka, F., Thomä, N.H., and Sugasawa, K. (2015) Functional regulation of thedamage-recognition factor DDB2 by ubiquitination and interaction with xeroderma
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and Hanaoka, F. (2015) UV-induced mutations in epidermal cells of mice defective in DNA
polymerase η and/or ι. DNA Repair 29, 139-146. 査読有
15. Akita, M., Tak, Y. S., Shimura, T., Matsumoto, S., Okuda-Shimizu, Y., Shimizu, Y., Nishi, R.,
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Hanaoka, F., Structure and function of mammalian DNA polymerase eta with special referenceon translesion synthesis and somatic hypermutation. 京都大学大学院医学研究科・免疫ゲノ
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2. 川元－尾崎洋子、水島恒裕 出芽酵母 COP9 シグナロソームの発現系構築. 第 13 回日本
3.

蛋白質科学会年会、ポスター発表（2013 年 6 月 12-14 日、鳥取）

花岡文雄

がんとは何か？

―その発生のメカニズムを探る―

ーラム ―学習院大学理学部の研究力―

読売サイエンスフォ

ここまで来た！がん研究最前線

招待講演（2013 年 7 月 27 日、東京）

4. 越坂部晃永、堀越直樹、立和名博昭、香川亘、安田武嗣、花岡文雄、菅澤薫、岩井成憲、

胡桃坂仁志 紫外線損傷 DNA を含むヌクレオソームの生化学的および構造生物学的解

5.
6.

析、第 86 回日本生化学会大会、口頭発表・ポスター発表（2013 年 9 月 11-13 日、横浜）

川元－尾崎洋子、水島恒裕 出芽酵母 COP9 シグナロソームの発現系構築、第 86 回日本生化学会大会、ポスター発表（2013 年 9 月 11-13 日、横浜）

横井雅幸 ゲノム情報維持における DNA ポリメラーゼ・イオタの役割、国立遺伝学研
究所研究集会「染色体 DNA の安定維持の分子メカニズム」、
口頭発表（2013 年 9 月 27-28
日、三島）

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8.

Sekimoto, T., Oda, T., Masutani, C., Hanaoka, F., and Yamashita, T., Y-family polymerases

participate in oncogene-induced DNA re-replication. 第 72 回日本癌学会学術総会、ポスタ
ー発表（2013 年 10 月 3-5 日、横浜）

Yokoi, M., Sakurai, Y., and Hanaoka, F., DNA polymerse ι, as a member of genome guardians.

第 72 回日本癌学会学術総会、ポスター発表（2013 年 10 月 3-5 日、横浜）

9. 越坂部晃永、立和名博昭、堀越直樹、香川亘、安田武嗣、花岡文雄、菅澤薫、岩井成憲、
胡桃坂仁志 紫外線損傷 DNA を含むヌクレオソームの構造生物学・生化学的解析、第22 回 DNA 複製・組換え・修復ワークショップ、ポスター発表（2013 年 11 月 20-22 日、
仙台）

10. 倉島公憲、小田司、関本隆志、小林広美、尤礼佳、花岡文雄、山下孝之 c-Myc 発現亢

進による複製ストレスにおけるポリメラーゼ η と PML の関与、第 22 回 DNA 複製・組換え・修復ワークショップ、ポスター発表（2013 年 11 月 20-22 日、仙台）

11. 小林百合香、宮澤沙絵、水野武、立澤桜子、花岡文雄、鳥越秀峰 マウステロメア結合

タンパク質 Pot1a/b と DNA ポリメラーゼ α の相互作用解析、第 22 回 DNA 複製・組換
え・修復ワークショップ、ポスター発表（2013 年 11 月 20-22 日、仙台）

12. 泉雅子、水野武、今本尚子、阿部知子、花岡文雄 ヒト Mcm10 と Mcm2-7 複合体の相互作用とその制御機構の解析、第 22 回 DNA 複製・組換え・修復ワークショップ、ポスター発表（2013 年 11 月 20-22 日、仙台）

13. 水野武、小林百合香、宮澤沙絵、泉雅子、花岡文雄、鳥越秀峰 DNA ポリメラーゼ αを複製フォークに呼び込む因子群 iPAOBs の提唱、第 22 回 DNA 複製・組換え・修復ワ
ークショップ、ポスター発表（2013 年 11 月 20-22 日、仙台）

14. 櫻井靖高、横井雅幸、花岡文雄 皮膚細胞における TLS ポリメラーゼの役割分担、第22 回 DNA 複製・組換え・修復ワークショップ、口頭発表（2013 年 11 月 20-22 日、仙台）
15. 金尾梨絵、増田雄司、花岡文雄、益谷央豪 ヒト細胞における PCNA の翻訳後修飾による複製阻害回避機構の解析、第 22 回 DNA 複製・組換え・修復ワークショップ、口頭発表（2013 年 11 月 20-22 日、仙台）

16. 安田武嗣、香川亘、斉藤健吾、荻朋男、鈴木健裕、堂前直、日野拓也、中沢由華、早乙女（中邑）愛、下等宝光、Genet, M., 花岡文雄、菅澤薫、田嶋克史、胡桃坂仁志 ヒ

ト Rad52 タンパク質のアセチル化による DNA 二重鎖切断修復制御、
第 22 回 DNA 複製・組換え・修復ワークショップ、口頭発表（2013 年 11 月 20-22 日、仙台）

17. Hanaoka, F., Role of DNA polymerase eta in somatic hypermutation. The 3rd Frence-JapaneseCancer Meeting、招待講演（2013 年 11 月 20-23 日, Toulouse, France）

18. 水野武、渋谷麻美、花岡文雄、今本尚子 哺乳類細胞核内の異常タンパク質を排除する

タンパク質品質管理機構に働くタンパク質分解系の解析、第 31 回染色体ワークショッ
プ・第 12 回核ダイナミクス研究会、口頭発表（2013 年 11 月 25-27 日、箱根）

19. 内山雅司、小田嶋嶺太、花岡文雄 分裂酵母 Rev1 の過剰発現による chk1 欠損株のシス
プラチン感受性の相補は損傷乗り越え機構と細胞周期チェックポイント機構が相互に協調することを示す、第 36 回日本分子生物学会年会、ポスター発表（2013 年 12 月 3-6
日、神戸）
20. 水野武、渋谷麻美、花岡文雄、今本尚子

11

哺乳類細胞核内の異常タンパク質を排除す

るタンパク質品質管理機構に働くタンパク質分解系の解析、第 36 回日本分子生物学会
年会、ポスター発表（2013 年 12 月 3-6 日、神戸）

21. 阿曽沙織、照沼淳子、金谷秀人、内山雅司、花岡文雄 分裂酵母 Rev3 の APMI 型 PCNA
結合領域は Rev3/Rev7 と Cdc1/Cdc2 から成る DNA Polymerase zeta;複合体形成に必須である、第 36 回日本分子生物学会年会、ポスター発表（2013 年 12 月 3-6 日、神戸）

22. 船岳朋弘、内山雅司、花岡文雄 分裂酵母 Rev1 BRCT 領域の機能解析、第 36 回日本
分子生物学会年会、ポスター発表（2013 年 12 月 3-6 日、神戸）

23. 渋谷卓未、横井雅幸、Yang, W., 花岡文雄 ヒト DNA ポリメラーゼ η の W297 を中心とした疎水性ポケットとヌクレオチドの相互作用に関する研究、第 36 回日本分子生物学会年会、ポスター発表（2013 年 12 月 3-6 日、神戸）

24. 倉島公憲、小田司、関本隆志、小林広美、尤礼佳、花岡文雄、山下孝之 c-Myc による
DNA 複製ストレスに DNA ポリメラーゼ η が関与する、
第 36 回日本分子生物学会年会、ポスター発表（2013 年 12 月 3-6 日、神戸）

25. 栗山磯子、水野武、赤木純一、横井雅幸、花岡文雄、吉田弘美、水品善之 DNA ポリ

メラーゼ η を阻害するビタミン K3 誘導体と紫外線照射の併用効果の影響、第 36 回日
本分子生物学会年会、ポスター発表（2013 年 12 月 3-6 日、神戸）

26. 渡部周二、堀岡敬太、吉政隆、小田島千尋、花岡文雄、浴俊彦

染色体動態制御に必

須な線虫 RNA 干渉タンパク質 DRH-3 の分子機能解析、第 36 回日本分子生物学会年会、ポスター発表（2013 年 12 月 3-6 日、神戸）

27. 関本隆志、小田司、倉島公憲、花岡文雄、山下孝之 ポリメラーゼ η は、発がんシグ
ナルが誘導する DNA 再複製に関与する、第 36 回日本分子生物学会年会、ポスター発表（2013 年 12 月 3-6 日、神戸）

28. 金尾梨絵、増田雄司、花岡文雄、益谷央豪 ヒト細胞における PCNA ホモ 3 量体の翻訳後修飾による DNA 損傷トレランス制御、第 36 回日本分子生物学会年会、ポスター発表、（2013 年 12 月 3-6 日、神戸）

29. 浴俊彦、村上康文、花岡文雄 2 種類のオリジントラップ法によるヒト複製オリジン断片の単離と配列解析、第 36 回日本分子生物学会年会、ポスター発表（2013 年 12 月 3-6
日、神戸）
30. Yokoi, M., Sakurai, Y., Hando, N., Morita, D., and Hanaoka, F., Physiological role of TLS

polymerases in mouse skin upon UV irradiation. International Conference, Kyoto, 2014:
Replication, Repair and Transcription; coupling mechanisms and chromatin dynamics forgenome integrity. 招待講演（2014 年 2 月 4-5 日、京都）

31. Kanao, R., Masuda, Y., Hanaoka, F., and Masutani, C., Regulation of DNA damage tolerance

distinct from Polη-mediated translesion synthesis. International Conference, Kyoto, 2014:
Replication, Repair and Recombination; coupling mechanisms and chromatin dynamics forgenome integrity. 招待講演（2014 年 2 月 4-5 日、京都）

32. Yang, W., Lee, Young-Sam, Zhao, Y., Nakamura, T., Biertümpfel, C., Yamagata, Y., Hua, Y.,
and Hanaoka, F., All road leads to DNA-lesion bypass: mechanisms of translesion synthesis.

International Conference, Kyoto, 2014: Replication, Repair and Recombination; couplingmechanisms and chromatin dynamics for genome integrity. 招待講演（2014 年 2 月 4-5 日、
京都）

1233. Osakabe, A., Tachiwana, H., Horikoshi, N., Kagawa, W., Yasuda, T., Hanaoka, F., Sugasawa, K.,
Iwai, S., and Kurumizaka, H., Structural and biochemical analyses of the nucleosome

containing UV-damaged DNA. International Conference, Kyoto, 2014: Replication, Repair andRecombination; coupling mechanisms and chromatin dynamics for genome integrity. 招待講演（2014 年 2 月 4-5 日、京都）

34. Hanaoka, F., Defective translesion DNA synthesis and links to human disease. International
Symposium on Xeroderma Pigmentosum and Related Diseases: Disorders of DNA Damage
Response –Bench to Bedside- 招待講演（2014 年 3 月 5-7 日、神戸）

35. Akagi, J., Hashimoto, K., Yokoi, M., Ohmori, H., Iwai, S., Moriya, M., and Hanaoka, F.,
Site-specific replicative analysis of 6-4 photoproduct using a series of TLS polymerases

deficient mouse cells. International Symposium on Xeroderma Pigmentosum and RelatedDiseases: Disorders of DNA Damage Response –Bench to Bedside- ポスター発表（2014 年 3

月 5-7 日、神戸）

36. Sakurai, Y., Yokoi, M., Morita, D., and Hanaoka, F., Specific role of translesion synthesis

polymerases eta and iota in UV-induced mutagenesis of skin cells and tissues. International

Symposium on Xeroderma Pigmentosum and Related Diseases: Disorders of DNA Damage

Response –Bench to Bedside- ポスター発表（2014 年 3 月 5-7 日、神戸）

37. Kanao, R., Masuda, Y., Hanaoka, F., and Masutani, C., Regulation of DNA damage torelancedistinct from Polη-mediated translesion synthesis in human cells. International Symposium on

Xeroderma Pigmentosum and Related Diseases: Disorders of DNA Damage Response –Benchto Bedside- 口頭発表（2014 年 3 月 5-7 日、神戸）

38. Ikehata, H., Yokoi, M., Yamamoto, M., and Hanaoka, F., In vivo UVB-induced mutation
spectrum in the skin epidermis of XP variant model mice. International Symposium on

Xeroderma Pigmentosum and Related Diseases: Disorders of DNA Damage Response –Benchto Bedside- 口頭発表（2014 年 3 月 5-7 日、神戸）

39. Yokoi, M., Sakurai, Y., Sugimoto, T., and Hanaoka, F., Association between global genome
NER and translesion synthesis in UV sensitivity of MEFs and UV-induced tumorigenesis in
mous skin. International Symposium on Xeroderma Pigmentosum and Related Diseases:

Disorders of DNA Damage Response –Bench to Bedside- 口頭発表（2014 年 3 月 5-7 日、
神戸）
40. Masuda, Y., Kanao, R., Hanaoka, F., and Masutani, C., Two different types of interactions

between PCNA and Y-family DNA polymerases have distinct roles for translesion DNA
synthesis. International Symposium on Xeroderma Pigmentosum and Related Diseases:

Disorders of DNA Damage Response –Bench to Bedside- 口頭発表（2014 年 3 月 5-7 日、神戸）
41. Sekimoto, T., Oda, T., Kurashima, K., Hanaoka, F., and Yamashita, T., DNA polymerase η
promotes DNA rereplication. International Symposium on Xeroderma Pigmentosum andRelated Diseases: Disorders of DNA Damage Response –Bench to Bedside- 口頭発表（2014
年 3 月 5-7 日、神戸）

42. 花岡文雄 損傷トレランスと突然変異生成に働く乗り越え複製ポリメラーゼ、日本薬学会第 134 年会、特別講演（2014 年 3 月 28-30 日、熊本）

1343. 金尾梨絵、増田雄司、花岡文雄、益谷央豪 ヒト細胞における PCNA ホモ三量体の翻訳後修飾による複製阻害回避機構、ポスター発表（2014 年 3 月 28-30 日、熊本）

44. Takaoka, S., Yokoi, M., Nakanishi, A., Yamamoto, M., and Hanaoka, F., Mammalian GyrI-likegene has an essential role in early mouse embryonic development. 第 47 回日本発生生物学会
年会、ポスター発表（2014 年 5 月 28-30 日、名古屋）

45. 花岡文雄 損傷を乗り越える DNA ポリメラーゼについて、平成 26 年度日本生化学会関東支部例会、特別講演（2014 年 6 月 14 日、水戸）

46. Akagi, J., Hashimoto, K., Yokoi, M., Ohmori, H., Iwai, S., Moriya, M., Ogawa, K., and Hanaoka,
F., Effects of sequence context on error-prone translesion synthesis past 6-4 photoproducts.

Gordon Research Conferences on Mutagenesis ポスター発表（2014 年 6 月 15 日-20 日、Girona, Spain）

47. 花岡文雄 損傷トレランス研究の 40 年：SOS 応答から損傷乗り越えポリメラーゼへ、
第 36 回光医学・光生物学会、特別講演（2014 年 7 月 25 日-26 日、大阪）

48. Sakurai, Y., Yokoi, M., Murakumo, Y., and Hanaoka, F., Roles of translesion synthesis

polymerases eta and iota in UV-induced mutagenesis in mice. DNA Polymerases: Biology,

Diseases and Biomedical Applications. ZING Conference 2014、招待講演 （2014 年 8 月 31
日-9 月 4 日、Cambridge, UK）

49. Masuda, Y., Kanao, R., Ohmori, H., Hanaoka, F., and Masutani, C. Different types ofinteractions between PCNA and Y-family DNA polymerases for translesion DNA synthesis.

DNA Polymerases: Biology, Diseases and Biomedical Applications. ZING Conference 2014、口頭発表（2014 年 8 月 31 日-9 月 4 日、Cambridge, UK）

50. 花岡文雄 損傷乗り越え合成ポリメラーゼと発がん、第 73 回日本癌学会学術総会、シンポジウム「クロマチン制御と発がん」（2014 年 9 月 25-27 日、横浜）

51. 倉島公憲、小田司、関本隆志、花岡文雄、山下孝之 DNA ポリメラーゼ η （Polη ）は
c-myc による複製ストレスを抑制する、第 73 回日本癌学会学術総会、ポスター発表（2014 年 9 月 25-27 日、横浜）

52. 櫻井靖高、横井雅幸、塚本徹哉、小田司、魏民、山下孝之、鰐淵英機、立松正衛、村雲

芳樹、花岡文雄 紫外線照射 DNA の損傷乗り越え複製において DNA ポリメラーゼイ

ータとイオタが果たす生理的役割、第 73 回日本癌学会学術総会、ポスター発表（2014年 9 月 25-27 日、横浜）

53. 益谷央豪、金尾梨絵、増田雄司、花岡文雄 PCNA の翻訳後修飾による損傷トレランス制御機構の解析、日本放射線影響学会 第 57 回大会 ワークショップ「紫外線誘発 DNA損傷に対する生物の防御戦略とその分子基盤、口頭発表（2014 年 10 月 1-3 日、鹿児島）

54. 池畑広伸、横井雅幸、山本雅之、花岡文雄 色素性乾皮症バリアント群モデルマウス皮膚における UVB 誘発突然変異スペクトルの解析、日本放射線影響学会 第 57 回大会、
口頭発表（2014 年 10 月 1-3 日、鹿児島）

55. Sakurai, Y., Yokoi, M., and Hanaoka, F. Roles of DNA polymerases eta and iota in UV-induced

mutagenesis. The 5th US-Japan DNA Repair Meeting、口頭発表（2014 年 10 月 28-31 日、
鳴門）

56. Osakabe, A., Tchiwana, H., Horikoshi, N., Kagawa, W., Yamamoto, J., Yasuda, T., Hanaoka, F.,
Sugasawa, K., Iwai, S., and Kurumizaka, H. Structures of nucleosomes containing UV-damaged

14

DNA bases and the damaged base recognition mechanism by DNA repair proteins. 5th

US-Japan DNA Repair Meeting、口頭発表（2014 年 10 月 28-31 日、鳴門）57. Yang W., Lee, Y-S., Gregory, M., Nakamura, T., Biertuempfel, C., and Hanaoka, F. Mechanisms

for translesion DNA synthesis. 5th US-Japan DNA Repair Meeting、口頭発表（2014 年 10 月
28-31 日、鳴門）

58. Masutani, C., Kanao, R., Masuda, Y., and Hanaoka, F. Relevance of simultaneousmono-ubiquitination of multiple units of PCNA homo-trimers in DNA damage tolerance. 5th
US-Japan DNA Repair Meeting、口頭発表（2014 年 10 月 28-31 日、鳴門）59. Sekimoto, T., Oda, T., Kurashima, K., Hanaoka, F., and Yamashita, T. Y-family polymerases are

involved in oncogene-induced aberant replication. 5th US-Japan DNA Repair Meeting、口頭発表（2014 年 10 月 28-31 日、鳴門）60. 横井雅幸 アミノ酸置換変異体を用いたヒト Polη の機能解析、国立遺伝学研究所研究

集会「染色体 DNA の安定維持の分子メカニズム」、口頭発表（2014 年 11 月 6-7 日、三島）

61. Hanaoka, F., Sakurai, Y., and Yokoi, M. Roles of mammalian DNA polymerases eta and iota in

UV-induced mutagenesis. The 9th 3R Symposium、招待講演 （2014 年 11 月 17-21 日、御殿場）

62. Masutani, C., Kanao, R., Masuda, Y., and Hanaoka, F. Relevance of simultaneousmono-ubiquitinations of multiple units of PCNA homo-trimers in DNA damage tolerance. The
9th 3R Symposium、ポスター発表（2014 年 11 月 17-21 日、御殿場）

63. Masuda, Y., Kanao, R., Ohmori, H., Hanaoka, F., and Masutani, C. Interactions between PCNA
and Y-family DNA polymerases. The 9th 3R Symposium、ポスター発表（2014 年 11 月 17-21
日、御殿場）
64. Hanaoka, F. Differential roles of translesion synthesis polymerases eta and iota in UV-induced

mutagenesis. The 4th Japan-France Cancer Workshop、招待講演（2014 年 11 月 19-21 日、
京都）

65. 大熊芳明、秋本勇亮、山本誠士、飯田智、久武幸司、花岡文雄、田中亜紀 転写初期における転写開始複合体のダイナミックな変化、第 37 回日本分子生物学会年会、口頭発
表・ポスター発表 （2014 年 11 月 25-27 日、横浜）

66. 泉雅子、水野武、今本尚子、阿部知子、花岡文雄 ヒト Mcm10 と Mcm2-7 複合体の相互作用の解析、第 37 回日本分子生物学会年会、ポスター発表（2014 年 11 月 25-27 日、
横浜）

67. 安田武嗣、香川亘、斎藤健吾、荻朋男、花岡文雄、菅澤薫、胡桃坂仁志、田嶋克史 ヒ

ト RAD52 タンパク質のアセチル化制御、第 37 回日本分子生物学会年会、ポスター発
表（2014 年 11 月 25-27 日、横浜）

68. 金尾梨絵、増田雄司、花岡文雄、益谷央豪 変異体 PCNA 発現ヒト細胞を用いた DNA

損傷トレランス制御の解析、第 37 回日本分子生物学会年会、ポスター発表（2014 年 11
月 25-27 日、横浜）

69. 秋元勇亮、田中亜紀、飯田智、花岡文雄、大熊芳明 転写コファクターPC4 は基本転写

因子 TFIIE と結合して転写開始から伸長への移行段階に機能する、第 37 回日本分子生物学会年会、ポスター発表（2014 年 11 月 25-27 日、横浜）

15

70. 内山雅司、花岡文雄 分裂酵母 TLS ポリメラーゼ Rev1 は CDK inhibitor Rum1 を通じて
G1/S における細胞周期遅延を引き起こす、第 37 回日本分子生物学会年会、ポスター発
表（2014 年 11 月 25-27 日、横浜）

71. 阿曽沙織、照沼淳子、内山雅司、花岡文雄、大森治夫、分裂酵母 DNA ポリメラーゼ ζ
における Rev3 と Rev3 の相互作用について、第 37 回日本分子生物学会年会、ポスター

発表 （2014 年 11 月 25-27 日、横浜）

72. 越阪部晃永、立和名博昭、堀越直樹、香川亘、山元淳平、安田武嗣、花岡文雄、菅澤薫、岩井成憲、胡桃坂仁志

ヌクレオソームにおける紫外線損傷塩基の収納およびその認識機構の解析、第 37 回日本分子生物学会年会、ポスター発表（2014 年 11 月 25-27 日、
横浜）
73. 渋谷卓未、横井雅幸、Wei Yang、花岡文雄 プリンアナログはヒト DNA ポリメラーゼ

η の W297 を介してその活性を阻害する、第 37 回日本分子生物学会年会、ポスター発

表（2014 年 11 月 25-27 日、横浜）

74. 森田大輝、櫻井靖高、伊藤若菜、横井雅幸、花岡文雄 マウス皮膚由来細胞の紫外線感受性における不活性型 Polη の発現の影響、第 37 回日本分子生物学会年会、ポスター発

表 （2014 年 11 月 25-27 日、横浜）

75. 赤木純一、豊田武士、Young-Man Cho、横井雅幸、大森治夫、花岡文雄、小川久美子 損

傷乗り越え型 DNA ポリメラーゼ η・ι・κ 三重欠損細胞の変異原に対する高感受性を用いた新規遺伝子毒性検出法の検討、第 37 回日本分子生物学会年会、ポスター発表（2014年 11 月 25-27 日、横浜）

76. 小林百合香、荘司健太、水野武、伊藤岳、花岡文雄、今本尚子、鳥越秀峰 マウステロ

メア結合タンパク質 Tpp1、Tin2 に依存した Pot1a/b の核局在と複合体形成解析、第 37

回日本分子生物学会年会 ポスター発表（2014 年 11 月 25-27 日、横浜）

77. 金尾梨絵、増田雄司、花岡文雄、益谷央豪 ヒト細胞における PCNA の翻訳後修飾による DNA 損傷トレランス制御機構の解析、がん支援活動シンポジウム、ポスター発表（2015 年 1 月 27-28 日、東京）

78. Hanaoka, F., Functional roles of TLS polymerases in mouse skin upon UV irradiation. Gordon

Research Conferences on Mammalian DNA Repair、招待講演（2015 年 2 月 8-13 日、Ventura,USA）

79. 櫻井靖高、横井雅幸、塚本徹哉、魏 民、鰐淵英機、村雲芳樹、花岡文雄 紫外線に誘発される突然変異において DNA ポリメラーゼ・イータとイオタが皮膚細胞と組織で果たす役割、第 104 回日本病理学会総会 ポスター発表（2015 年 4 月 30-5 月 2 日、名古屋）

80. Hanaoka, F., Forty years of DNA damage tolerance. 15th International Congress of Radiation
Research (ICRR 2015), 招待講演（2015 年 5 月 25-29 日、京都）

81. 水野武、石田竜次、下仲基之、花岡文雄、今本尚子 哺乳類細胞核内のタンパク質品質管理機構の解明、第 67 回日本細胞生物学会大会、口頭発表（2015 年 6 月 30 日-7 月 2日、東京）
82. 荘司健太、小林百合香、水野武、花岡文雄、今本尚子、鳥越秀峰 マウステロメア結合

タンパク質シェルタリンの複合体形成機構 —Tin2 の機能ドメインを通じて―、第 67回日本細胞生物学会大会、ポスター発表（2015 年 6 月 30 日-7 月 2 日、東京）

16

83. Uchiyama, M., Terunuma, J., and Hanaoka, F., Regulation of the protein level of Rev1 in fission

yeast. Genomic Integrity, ZING Conference 2015, 招待講演（2015 年 8 月 1-5 日、Cairns,
Australia）

84. Osakabe, A., Tachiwana, H., Kagawa, W., Horikoshi, N., Matsumoto, S., Yamamoto, J.,
Hanaoka, F., Sugasawa, K., Iwai, S., and Kurumizaka, H., Structural and biochemical analyses

for accommodation and recognition of UV-damaged bases within nucleosome. 「クロマチン動構造」国際シンポジウム、口頭発表（2015 年 8 月 23〜26 日、淡路）

85. 花岡文雄 私の DNA 修復研究：XPC から XPV へ、新学術領域研究「ゲノム普遍的制御」終了シンポジウム：ゲノム安定性の機構と生命の維持－進化、癌化、老化の理解のために 口頭発表（2015 年 8 月 29-30 日、京都）

86. 赤木純一、横井雅幸、Cho Young-Man、豊田武士、花岡文雄、小川久美子 Hypersensitivityof Polη, ι, and κ triple knockout cells to mutagens is a valuable indicator of genotoxicity. 第 74回日本癌学会学術総会、ポスター発表（2015 年 10 月 8-10 日、名古屋）

87. 金尾梨絵、増田雄司、花岡文雄、益谷央豪

Regulation of DNA damage tolerance bysimultaneous mono-ubiquitinations of multiple-units of PCNA in human cells. 第 74 回 日本
癌学会学術総会、ポスター発表（2015 年 10 月 8-10 日、名古屋）

88. 倉島公憲、関本隆志、小田 司、花岡文雄、山下孝之 Polη, a member of Y-family DNA
polymerases, prevents generation of DNA double strand breaks induced by c-myc expression.
第 74 回日本癌学会学術総会、ポスター発表（2015 年 10 月 8-10 日、名古屋）

89. 横井雅幸、花岡文雄 High concentration of dATP attenuated the catalytic rate and efficiencyof translesion synthesis Polη . 第 74 回日本癌学会学術総会、ポスター発表 （2015 年 10
月 8-10 日、名古屋）

90. 金尾梨絵、柏葉脩一郎、増田雄司、松尾-楠本理加、花岡文雄、益谷央豪

酸化損傷によって誘導される PCNA のモノユビキチン化を制御する新規メカニズムの解析、第 23

回 DNA 複製・組換え・修復ワークショップ、口頭発表（2015 年 10 月 19-21 日、焼津）

91. 益谷央豪、柏葉脩一郎、金尾梨絵、松尾-楠本理加、花岡文雄、増田雄司 PCNA のユビ
キチン化を制御して過酸化水素誘発突然変異を抑制するヒト細胞のメカニズム、
BMB2015 ワークショップ「放射線生物影響の課題に挑む分子生物学研究の力」、口頭発表（2015 年 12 月 1-4 日、神戸ポートアイランド）

92. 花岡文雄 総括、BMB2015 ワークショップ「放射線生物影響の課題に挑む分子生物学研究の力」、口頭発表（2015 年 12 月 1-4 日、神戸ポートアイランド）

93. 安田武嗣、香川 亘、荻 朋男、齊藤健吾、加藤宝光、鈴木健祐、藤堂 直、滝澤和也、

早乙女（中邑）愛、中沢由華、Matthew D. Genet、宇井彩子、花岡文雄、菅澤 薫、岡安隆一、Penny A. Jeggo、胡桃坂仁志、田嶋克史 ヒト RAD52 のアセチル化を介した相同組換えにおけるアセチル化および脱アセチル化酵素の新規機能の解明、BMB2015 ワ

ークショップ「多様な DNA 損傷応答の統合制御機構 2015〜ゲノム不安定性の病態解明
研究、口頭発表（2015 年 12 月 1-4 日、神戸ポートアイランド）

94. 道津貫太郎、大雲剛志、横井雅幸、花岡文雄 酸化損傷剤、臭素酸カリウムへの細胞内感受性における DNA ポリメラーゼ η、ι の働き、BMB2015 ポスター発表（2015 年 12

月 1-4 日、神戸ポートアイランド）

95. 横井雅幸、尾仲紗也加、美島一太、花岡文雄 ヒト Polη のフィンガーおよびリトルフ

17ィンガードメインの変異が CPD の乗り越え合成に及ぼす影響、BMB2015 ポスター発表（2015 年 12 月 1-4 日、神戸ポートアイランド）

96. 小林百合香、荘司健太、水野 武、花岡文雄、今本尚子、鳥越秀峰 マウステロメア結合タンパク質 Tpp1、Tin2 に依存した Pot1a/b の核局在と複合体形成解析、BMB2015 ポ

スター発表（2015 年 12 月 1-4 日、神戸ポートアイランド）

97. 赤木純一、横井雅幸、Young-Man Cho、豊田武士、大森治夫、花岡文雄、小川久美子 損

傷乗り越え型 DNA ポリメラーゼ η・ι・κ 三重欠損細胞は様々な機序の遺伝性毒性物質に感受性を示し、遺伝毒性物質のスクリーニングに有効である、BMB2015 ポスター発表（2015 年 12 月 1-4 日、神戸ポートアイランド）

98. 倉島公憲、関本隆志、小田 司、川端 剛、花岡文雄、山下孝之 Y-family 損傷乗り越え DNA ポリメラーゼ（Y-Pol）の一員 Polη は MYC がん遺伝子の誘導する複製ストレスを軽減する、BMB2015 ポスター発表（2015 年 12 月 1-4 日、神戸ポートアイランド）

99. 越阪部晃永、立和名博昭、堀越直樹、香川亘、松本翔太、山元淳平、花岡文雄、菅澤薫、岩井成憲、胡桃坂仁志 クロマチンにおける紫外線損傷 DNA 認識機構の解析、第 33
回染色体ワークショップ・第 14 回核ダイナミクス研究会、口頭発表（2016 年 1 月 12-14日、松島）100. 金尾梨絵、柏葉脩一郎、増田雄司、松尾（楠本）理加、花岡文雄、益谷央豪 PCNA のモノユビキチン化の制御によるヒト細胞での酸化的 DNA 損傷誘発突然変異抑制機構、
日本薬学会第 136 年会、ポスター発表（2016 年 3 月 27-29 日、横浜）

18

Mechanism of somatic hypermutation at the
WA motif by human DNA polymerase η
Ye Zhaoa,b, Mark T. Gregoryb, Christian Biertümpfelb,1, Yue-Jin Huaa,2, Fumio Hanaokac,2, and Wei Yangb,2aInstitute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou 310029, China; bLaboratory of Molecular Biology, National Institute of Diabetes andDigestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892; and cFaculty of Science, Gakushuin University, Tokyo 171-8588, Japan

Edited by Dinshaw J. Patel, Memorial Sloan–Kettering Cancer Center, New York, NY, and approved March 29, 2013 (received for review February 18, 2013)

Somatic hypermutation is programmed base substitutions in thevariable regions of Ig genes for high-afﬁnity antibody generation.
Two motifs, RGYW and WA (R, purine; Y, pyrimidine; W, A or T),
have been found to be somatic hypermutation hotspots. Overwhelming evidence suggests that DNA polymerase η (Pol η) is responsible for converting the WA motif to WG by misincorporatingdGTP opposite the templating T. To elucidate the molecular mechanism, crystal structures and kinetics of human Pol η substituting
dGTP for dATP in four sequence contexts, TA, AA, GA, and CA,
have been determined and compared. The T:dGTP wobble basepair is stabilized by Gln-38 and Arg-61, two uniquely conserved
residues among Pol η. Weak base paring of the W (T:A or A:T) at
the primer end and their distinct interactions with Pol η lead tomisincorporation of G in the WA motif. Between two WA motifs,
our kinetic and structural data indicate that A-to-G mutation
occurs more readily in the TA context than AA. Finally, Pol η canextend the T:G mispair efﬁciently to complete the mutagenesis.
π–cation stacking| A-to-G transition | immunoglobulinA

fter V(D)J recombination, nascent antibodies producedin B cells usually have low afﬁnity for antigens. Somatic
hypermutation (SHM), which generates mutations in the variable
region at a frequency far beyond the rate of spontaneous mutations, potentially changes the conformation of the antigen-bindingsite and can increase antigen recognition by up to 1,000-fold (1, 2).
Mutations have been observed at all four bases, but two sequencemotifs, RGYW and WA (R, purine; Y, pyrimidine; W, A or T),
have been shown to be the mutation hotspots (3). Although themechanism of SHM and its target selection is incompletely understood, activation-induced cytidine deaminase (AID) (4, 5),which converts cytosine into uracil, for example, in the RGYW
motif, initiates SHM by converting a C:G base pair to a U:G
mismatch. Removal of the U by uracil–DNA glycosylase (UNG)
generates an abasic site in the DNA, which may lead to a variety ofbase substitutions (6–8). For the WA motif, it is suggested thatafter AID-dependent deamination of a cytosine, recognition of the
U:G mismatch by mismatch repair protein MutSα (MSH2–MSH6
heterodimer, in which MSH stands for MutS Homolog) leads to therecruitment of DNA polymerase η (Pol η), and with an incision
made by UNG the ensuing short-patch repair synthesis results inmutations of A:T pairs to G:C (Fig. 1A) (9–11).
Pol η is one of the highly conserved translesion synthesis (TLS)
DNA polymerases found in all eukaryotes and is specialized in
bypassing UV-induced cyclobutane pyrimidine dimers (CPDs) inan error-free manner (12, 13). Deﬁciency of Pol η in humanscauses a variant form of the cancer predisposition syndrome
xeroderma pigmentosum (XP-V) (14). Patients are thousandsof times more likely to develop skin cancer from exposure to
sunlight. Like all Y-family DNA polymerases, human Pol η has
no proofreading 3′–5′ exonuclease activity (15). Puriﬁed Pol η ishighly mutagenic on normal DNA and prefers to misincorporatedGTP opposite dT, thereby generating A-to-G transition on a
newly synthesized strand (13, 16). The base-substitution spectrumof SHM from XP-V patients is altered with severely decreased
mutations at A:T pairs (11, 17, 18). Similar suppression of A:T to
G:C mutations was observed in the POLH-deﬁcient mice (19–21). Interestingly, the MSH2–MSH6 heterodimer interacts and8146–8151 | PNAS | May 14, 2013 | vol. 110 | no. 20

stimulates Pol η activity in vitro (22), and mutations of A:T basepairs were abolished in SHM when both POLH and MSH2 genes
were knocked out (23).
Crystal structures of the polymerase domain of human Pol η(1–432 aa) complexed with different lesion DNA substrates werereported recently (24–27). These structures revealed a uniquelyenlarged active site that can readily accommodate two normal
template bases, a cis–syn thymine dimer (CPD), or to a certain
extent intrastrand cisplatin cross-linked guanines (Pt-GG). In
addition, the “molecular splint” of human Pol η stabilizes the
upstream DNA duplex in a normal B-form conformation, evenin the presence of cross-linked bases by forming numerous salt
bridges and hydrogen bonds with the phosphate backbones,
thus facilitating primer extension after CPD lesions (24–27).
Misincorporation by Pol η, however, has not been investigatedin a sequence-dependent manner or at atomic resolution.
To elucidate the molecular mechanism of Pol η in SHM, we
set out to determine crystal structures of the polymerase domainof human Pol η (1–432 aa) (24–27) in the process of misincorporating dGTP opposite T in the WA motif (TA or AA) andnon-WA sequences (CA or GA) as well as when extending theprimer after a T:G mispair. In addition, steady-state kinetic
parameters are measured to complement structural observations.
Results
Pol η Prefers to Mutate WA to WG. We ﬁrst compare the efﬁciencyof human Pol η incorporating dATP vs. dGTP opposite a template T following a perfectly paired T, A, G, or C at the primer 3′
end (Table S1). The four sequence contexts are labeled as TA,
AA, GA, and CA, respectively, where the second nucleotide, an
A, represents the correct nucleotide to be incorporated, but it
may become G due to misincorporation, for example, in the TA
and AA cases (WA motif). The measured KM and kcat indicatethat human Pol η inserts the correct base (dATP) with a similar
efﬁciency (less than twofold difference), regardless of the sequence context. For misincorporation, the KM for dGTP increases by ∼10-fold compared with dATP in all four sequencevariations, but the catalytic rates (kcat) differ with sequence
contexts (Table S1). The kcat is reduced by 3.6- and 5.6-fold in
the TA and AA case, respectively, and is reduced by 8.0-fold in
the GA case. As for CA, dGTP misinsertion is severely inhibited,
and the kcat is reduced 31-fold. Thus, the relative efﬁcienciesof misincorporation at TA, AA, GA, and CA are 1/40, 1/52, 1/
106, and 1/321, respectively, of the correct incorporation, making the WA motif twofold to eightfold more susceptible to A-toG mutation.

Author contributions: Y.Z. and W.Y. designed research; Y.Z., M.T.G., and C.B. performed
research; Y.Z., Y.-J.H., F.H., and W.Y. analyzed data; and Y.Z. and W.Y. wrote the paper.
The authors declare no conﬂict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates, structure factors, and diffraction data havebeen deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4J9K–4J9S).
1

Present address: Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.

2

To whom correspondence should be addressed. E-mail: wei.yang@nih.gov, 20070117@
gakushuin.ac.jp, or yjhua@zju.edu.cn.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1303126110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1303126110

19

Fig. 1. SHM at the WA motif. (A) Model of SHM targeting
the WA motif through short-patch DNA synthesis. The initial incision targeting the variable region of Ig genes
depends on AID and UNG. Recruitment of Pol η is enhanced
by MutSα, which recognized the U:G mismatch. The template strand is in orange, and the primer strand (subject tomutation) is shown in yellow. The WA motif is highlighted
with A in red. (B) Structural determination of the fourstages of dGTP misincorporation in this study. The W of the
WA motif is highlighted in yellow and the A subject to replacement by G in red. The protein domains of Pol η incontact with DNA and dNTP are outlined and labeled. The
two Mg2+ ions in the active site are shown as purple spheres.

Structures of dGTP Misincorporation Opposite T. Crystal structuresof human Pol η incorporating dATP or its nonreactive analog
2′-deoxyadenosine-5′-[(α,β)-imido]triphosophate (dAMPNPP)
opposite T template have been reported (27, 28). Here we focus
on the structures of dGTP misincorporation. Human Pol η (1–432 aa) complexed with DNA and nonhydrolyzable 2′-deoxyguanosine-5′-[(α,β)-imido]triphosophate (dGMPNPP) opposite
T after A, T, G, or C at the 3′ primer end were crystallized in
the P61 space group. These crystals contain one complex per
asymmetric unit and are isomorphous to the Pol η ternarycomplexes with perfectly base-paired DNA and incoming nucleotides (Materials and Methods and Fig. 1B). The structures
were reﬁned to resolutions between 1.85 and 2.25 Å (Table 1).
The overall protein structures in these misincorporation complexes (AA/G, TA/G, CA/G, and GA/G) are similar to each otherand to that of the correct incorporation (T:dATP) complexes,

except for a slight closing of the ﬁnger and thumb domain as if tosqueeze the primer strand and dGMPNPP toward each other (Fig.
2A and Movie S1). Among the misincorporation complexes, there
are small but perceptible deviations of a loop (Gln-373–Ser-379) inthe little ﬁnger (LF) domain (25). The catalytic triad Asp-13, Asp115, and Glu-116 in the palm domain that chelate the two Mg2+
ions essential for catalysis overlay well with those in the ternarycomplex of dATP incorporation (25). The 7-bp upstream duplex is
kept in the straight B form between the thumb and LF domain as
observed (27, 28) (Fig. 2A).
A clear deviation in the four Pol η misincorporation complexes
is observed at the T:dGMPNPP mispair surrounded by the ﬁnger
and palm domains (Fig. 2A). As typically observed for a T:G
wobble pair with two hydrogen bonds, the templating base
T shifts toward the major groove, and the dGMPNPP shifts toward the minor groove (Fig. 2 B–D). Despite the shift of theData collection
Space group
Lattice constanta, b, c; ÅWavelength, ÅResolution, ÅRsym, %I/σI
Completeness, %Wilson B factor, Å2
Redundancy
Reﬁnement
Resolution, ÅNo. of reﬂections
Rwork/Rfree
No. of atoms
Protein/DNA
Ligand/ion
Water
B factors, Å2
Protein/DNA
Ligand/ion
Water
rms deviations
Bond length, ÅBond angle, °

TA/G

CA/G

AA/G

GA/G

Extension complex

P 61

P 61

P 61

P 61

P 61

98.58
98.58
81.67
1.000030.0–2.03
10.2 (61.5)
12.2 (2.0)
96.3 (97.9)
23.1
4.5 (3.9)

98.43
98.43
81.96
1.000030.0–1.85
8.7 (66.4)
14.6 (2.0)
98.6 (96.8)
18.2
3.8 (3.1)

98.43
98.43
82.01
1.000030.0–2.25
9.8 (59.5)
14.5 (3.0)
99.7 (99.7)
25.8
4.4 (4.4)

98.66
98.66
81.96
1.000030.0–1.95
9.3 (42.6)
19.2 (4.8)
98.6 (100.0)
18.2
6.4 (6.3)

99.51
99.51
81.57
1.541830.0–2.60
13.0 (68.8)
12.4 (2.4)
99.5 (95.7)
34.1
4.8 (4.7)

30.0–2.03
28,41916.4/18.7

30–1.85
38,03017.2/20.8

30.0–2.25
21,43418.0/22.3

30.0–1.95
32,381
16.5/19.9

30–2.60
14,14520.8/23.4

3,366/386
81
261

3,366/366
57
374

3,359/368
46
188

3,366/363
45
326

3,279/383
4298

28.0/30.2
30.9
34.3

18.7/23.6
18.6
25.1

24.1/28.6
16.4
24.0

21.4/23.9
12.7
26.2

37.4/48.5
35.6
36.6

0.006
0.981

0.011
1.169

0.005
0.917

0.010
1.229

0.007
1.102

Data in the highest resolution shell are shown in parentheses.

PNAS | May 14, 2013 | vol. 110 | no. 20 | 8147

Zhao et al.

20

IMMUNOLOGY

Table 1. Data collection and reﬁnement statistics of Pol η ternary complex

Fig. 2. Structures of dGTP misincorporation by Pol η. (A)Structure superposition of Pol η complexed with DNA andnonhydrolyzable dGMPNPP opposite dT in all four sequence
contexts. The protein is shown in ribbon diagrams with palm
domain in pink, thumb in green, ﬁnger in blue, and little
ﬁnger (LF) in magenta. DNA is shown as stick-and-ladder inyellow (primer) and orange (template). Mg2+ ions (purple
spheres) and incoming dGMPNPP (red sticks) are also shown.
(B) Correct (T:dATP) and incorrect (T:dGMPNPP) nascent basepair in complex with Pol η. The conserved residues Gln-38
and Arg-61 are shown in blue sticks. (C) Superposition of
the Pol η active site in dGTP misincorporation (colored) andnormal ternary complexes [gray; PDB ID code 3MR2]. Alterations of primer end and Lys-224 are indicated by red
arrows. The water molecule that participates in Mg2+
coordination is shown as a red sphere. A gray double arrow
indicates the cation–π stacking. (D) Superposition of thenascent (0) and (−1) base pairs in normal productive (gray)and mispaired nonproductive ternary complexes (colored).
The movement of each nucleotide is indicated by red
arrows.

bases, the triphosphate of dGMPNPP remains coordinated by
the two active site Mg2+ ions and situated nearly the same as thatof a correctly paired dAMPNPP (Fig. 2 A and C). Two residues,
Gln-38 and Arg-61, are uniquely conserved among all Pol η
homologs, and both appear to contribute to the misincorporation.
Gln-38 interacts with both thymine and guanine bases in the minor
groove (Fig. 2B); Arg-61 adopts a rotamer conformation thathas not been observed in Pol η–DNA complexes before andmakes hydrogen bonds with O4 of dT and O6 and N7 ofdGMPNPP in the major groove (Fig. 2B).
The largest structural change in the T:dGMPNPP complexes
occurs at the 3′ primer end. Perhaps due to the molecular splint
effect of Pol η on the template strand, the 0.7-Å shift of thetemplating base T toward the major groove leads to a shift of its
immediate neighbor upstream (−1 position) in the same direction, which via base pairing induces a displacement of the 3′
primer end (Fig. 2 C and D). The last base of the primer
strand is no longer stacked with the incoming dGMPNPP and
instead is stacked with the guanidino group of Arg-61. As a result,the 3′-OH shifts >4 Å compared with all Pol η ternary structures
solved to date and is hydrogen bonded with a nonbridging oxygenof the α-phosphate of dGMPNPP (Fig. 2C). Concomitantly, awater molecule replaces the 3′-OH as a ligand for the A-site Mg2+,
and Lys-224 also switches from interacting with the last phosphate group of the primer strand to interacting with the activesite carboxylate Glu-116 and a water molecule (Fig. 2C). In all
four Pol η ternary complexes with the T:dGMPNPP mispair, thedominant conformation is the misaligned primer and dGMPNPP
that are incompatible with the nucleophilic attack.
Unique Structural Features of the WA Motif. The four structures ofPol η misincorporating dGTP do have differences. After close inspection of the electron density maps, we found that in the TA/G
and AA/G structures, which are of the WA motif, there is distinct
positive electron density in the Fo − Fc difference map thatindicates a second conformation of the 3′ primer end with ∼30%
occupancy (Fig. 3A). Although a minor species in the population,
the second conformation of the 3′-OH is highly similar to theprimer end in the normal ternary complex and is aligned with the
α-phosphate of dGMPNPP for the phosphoryltransfer reaction(Fig. 3B). However, in the CA/G or GA/G complexes, the misaligned 3′ end is the only conformational species observed (Fig.
3C). By serendipity, two slightly different DNA sequences wereused, one in the TA/G and GA/G and the other in the AA/G and

Fig. 3. The alternative DNA conformations observed in
TA/G and AA/G that is compatible with dGTP incorporation.
(A) The primer 3′ nucleotide (Thy) in the TA/G structure. The
major (yellow) and the minor conformation (green) of thelast nucleotide is superimposed with the 2Fo − Fc (silver;
contoured at 1.0σ) and Fo − Fc omit map (green; contoured
at 2.5σ), which were calculated without the minor conformation species. (B) The minor conformation of TA/G iscompatible with the phosphoryltransfer reaction. Superposition of the minor conformation of the TA/G structure(colored) with the normal ternary complex (gray; PDB ID
code 3MR2). (C ) Interactions between Arg-61 (blue) andthe −1 base pair (yellow, green primer, and orange template) in TA/G, AA/G, CA/G, and GA/G complexes. The incoming dGMPNPP is shown as semitransparent pink sticks.
Hydrogen bonds and van der Waals contacts are indicatedby color-coded dashes and distances.

8148 | www.pnas.org/cgi/doi/10.1073/pnas.1303126110

Zhao et al.

21

CA/G structures (Table S2). The structural differences observed
between the WA and non-WA motif complexes are therefore
independent of DNA sequence.
Several factors appear to contribute to the minor but productive conformational species found with the WA motif. The
ﬁrst is likely the base-pair strength at the 3′ primer end. Basedon the electron density, when the 3′ primer end is aligned with
the incoming nucleotide in the productive conformation, its
base-pairing partner in the template strand does not appear tohave a second conformation to maintain the proper Watson–
Crick hydrogen bonds. Therefore, the primer end has to break thehydrogen bonds with the template to assume the minor conformational species. In the CA/G and GA/G cases, the G:C or C:G
pair at the 3′ primer end shares three hydrogen bonds despite thebase pair being severely buckled (Figs. 3C and 4A). The reduced
number of hydrogen bonds in A:T pairs and the buckle between
the base pairs (Fig. 4B) may facilitate formation of the second
conformational species in the WA motif. Particularly in the TA/G
case, the T:A base pair retains only one hydrogen bond due to basepair opening (Fig. 3C).
The second factor is the interaction of the base at the 3′ end(−1) with the guanidino group of Arg-61 and with the surrounding bases. It has often been observed that Arg interacts
well with the major groove side O6 and N7 of guanine (Fig. S1),and such polar interactions between Arg-61 and the guaninebase at the 3′ end are apparent (Fig. 3C). As a result, the 3′
guanine in the GA/G structure maintains its base stacking withthe upstream neighbors and has the least buckle (Fig. 4A). Whenthe 3′ primer end is a cytosine (CA/G), the base tilts by ∼25° to
avoid electrostatic repulsion between Arg-61 and N4 of the base,thus resulting in the cytosine stacking with the guanidinium of
Arg-61 instead of its neighboring bases (Fig. 3C and 4A). In bothcases, the 3′ base is structurally stable and does not have an alternative conformation. When a thymine is at the primer 3′ end(TA/G), Arg-61 forms favorable polar interactions with O4 ofthe base, particularly when the T assumes the second conformation that is stacked with its neighboring bases (Fig. 3C). In
contrast, the electrostatic repulsion between Arg-61 and N4 most
likely prevents the cytosine adopting the second conformation.
When an adenine is at the 3′ primer end, the electrostatic repulsion between its N6 and Arg-61 probably leads to the baseoffset in both conformations compared with guanine (Fig. 3C).
The strong propensity of adenine to form base stacking (29)likely leads to the productive conformation in the AA/G structure, where the 3′ primer end stacks with both dGMPNPP and
the upstream base (Fig. 4 A and B).
The third factor is likely the global position of the DNA substrate relative to the polymerase. Among the four misincorporation complexes, the upstream DNA in the CA/G and GA/G
structures are shifted along the DNA helical axis toward theincoming nucleotide and the ﬁnger domain (Fig. 4A). However,in the TA/G complex, it is more similar to the normal ternarycomplex, and the DNA in the AA/G complex is in between.
Favorable van der Waals interactions between the template strand
bases and residues in the LF domain are observed in the WA cases(Fig. 4B), which likely stabilize the DNA in a more native-like
conformation. The shift of the DNA substrate also affects theinteraction between the ﬁnger domain and the downstream
single-stranded DNA. In the TA case, the downstream DNA
conformation is most similar to the undamaged ternary complexes (Fig. 4 A and B). In the other three cases, despite thesame template length as in the undamaged ternary complexes(25, 27), the +1 nucleotide is ﬂipped out and occupies the usual
binding site of the +2 nucleotide, and the hydroxyl group of S62
turns to occupy the void (Fig. 4C).

Fig. 4. Unique interactions found in TA/G and AA/G complexes. (A) Stereoview of the superposition of the four Pol η misincorporation and thenormal ternary complexes. Each complex is color-coded as indicated. The
upward shift of the DNA duplex in GA/G, CA/G, and AA/G complexes is obvious after the entire protein is superimposed. (B) Distinct interactions between the LF domain and the DNA found in TA/G and GA/G. The van der
Waals contacts are indicated by lines formed by open circles. (C) Side-by-side
views of TA/G and GA/G with Pol η shown as gray molecular surface andSer-62 side chain in blue carbon and red oxygen. The shift of the DNA
substrate in GA/G is accommodated by the rotamer change of Ser-62.

rotamer conformation that forms extensive hydrogen bonds
with T:G mispair to favor the dGMPNPP binding. However,Arg-61 also stabilizes the displaced 3′ primer end by cation–πstacking interactions that prevent the polymerization reaction.
Only when A or T is at the primer end does the 3′ primer end
occasionally revert to the reactive conformation for polymerization, as is evident in the alternative conformations. The arrangement of Arg-61 stabilizing the T:G mismatch and stacking
with the base preceding the G is also observed in the postreaction Pol η–product DNA binary complex when a T:G mismatchis at the DNA primer end, but not when it is T:A (Fig. 5A and
Table S3). To delineate the hydrogen bonding vs. the basestacking roles of Arg-61 in SHM of the WA motif, we replaced
Arg-61 by Lys, which has an amino group to mimic electrostaticinteraction and hydrogen-bonding ability of Arg but has greatly
reduced potential to stack with DNA bases (30).
KM and kcat of the R61K mutant Pol η in dATP and dGTP
incorporation in the TA, AA, and CA sequence contexts were
measured (Table S1). When incorporating the correct dATP, KM
of R61K is increased by twofold to threefold, and the overall
efﬁciency is reduced by twofold to fourfold compared with wildtype (WT) Pol η in the three sequence contexts tested. This reduction is not surprising because the guanidino group of Arg-61
forms bifurcated hydrogen bonds with the α and β phosphates of
the nucleotide (28), and Lys, being shorter than the Arg side
chain, is a poor substitute for such interactions. Interestingly,
the trend that the WA motifs are more susceptible to dGTP
misincorporation is the same with R61K as WT Pol η, indicatingthat the positive charge of Arg-61 may be the determinant ininﬂuencing the mutability of the WA motif. Surprisingly, theR61K mutant compared with WT Pol η has a threefold to
PNAS | May 14, 2013 | vol. 110 | no. 20 | 8149

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22

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Arg-61 Plays a Key Role in Misincorporation. Arg-61 adopts astep are examined (Fig. 1B). Human Pol η was cocrystallizedwith a T:A pair or mismatched T:G at the DNA duplex end as
the posttranslocation binary complexes (Fig. 1B and Table S3).
These crystals diffracted X-rays to 1.95 Å (T:A) and 2.35 Å (T:G),
respectively. In these binary complexes, however, a subpopulation of DNA duplex is observed to remain in the productstate rather than fully translocated (Fig. 5B). We suspect thatstacking of Arg-61 with the purines at the primer 3′ end may
cause the sluggish translocation because with a pyrimidine atthe 3′ end there is no sign of untranslocated species (26). The
incomplete translocation is more severe with an T:G mismatch
than T:A base pair (Fig. S2), which may contribute to the 12-foldincrease in KM when extending a T:G mismatch compared withnormal extension (Table S1).
We have also obtained ternary-complex crystals of Pol η incorporating dAMPNPP after a T:G mismatch, and the structure
was determined at 2.6 Å (Table 1). In the presence of an incoming dAMPNPP, the 3′ primer end resumes the near-normal
position despite the wobble T:G pair. The reactants are moreor less superimposable with the perfectly matched substrates(Fig. 4C). To validate the reaction-ready nature of this structure, we measured the extension efﬁciency of the T:G mispair
in solution by Pol η and showed that the catalytic efﬁciency(kcat/KM) is reduced by 32-fold compared with normal DNA
synthesis (Table S1). Pol η is thus more efﬁcient in primer extension after a mismatched base pair than replicative and Bfamily TLS polymerases, whose catalytic efﬁciency is reducedby 10,000 and 100 folds, respectively (32).

Fig. 5. Translocation and extension of T:G mispair. (A) Structures of postinsertion Pol η–DNA binary complexes. The wobble nascent base pair of themisincorporation is shown side by side with the normal incorporation
looking down the DNA helical axis. The template base is shown in orange
and the newly incorporated base in yellow (correct) or red (incorrect). Arg-61
interacts with the mispaired guanine base. (B) Structures of posttranslocation binary complexes. In the T:G mismatch complex (Left), the DNA exhibits
two equal populations of translocated (multicolored) and untranslocated(gray) conformations. The Fo − Fc omit map, which was calculated with 100%translocated population and contoured at 2.5σ in green, superimposes wellwith the untranslocated conformation. (C) Structure of the T:G mismatch
extension complex. The 2Fo − Fc map (gray; contoured at 1.0σ) corresponding to the primer end and incoming nucleotide is superimposed with thereﬁned structure. (D) Superposition of the T:G mismatch extension (colored)
and normal ternary complex structure (gray; PDB ID code 3MR2). The 3′–OHat the primer end and the α-phosphate of dAMPNPP are aligned in themismatch extension complex, albeit slightly shifted relative to the active siteof Pol η.

sixfold higher propensity to make dGTP incorporation, suggesting that stacking of Arg-61 with the primer end actually reduces
the misincoporation efﬁciency (Table 1). Previously, it was
shown that R61A mutation reduces dGTP misinsertion opposite
T (27) and increases nucleotide insertion ﬁdelity in general at thecost of reduced catalytic efﬁciency (31). We deduce that thepositive charge of Arg-61 (or R61K) must be required to stabilize
the T:dGTP mispair (Fig. 2B), thus promoting misincoporation.
Extension of a T:G Mispair by Pol η. For the A-to-G mutation topersist in somatic cells, it is necessary that the DNA primer be
extended after the mismatch during the short-patch DNA synthesis to prevent the misinserted G from being removed by theediting function of a replicative polymerase. Most polymerases
are inefﬁcient in mismatch extension (32). To extend a T:G
mismatch, both the translocation step and the primer extension
8150 | www.pnas.org/cgi/doi/10.1073/pnas.1303126110

Discussion
Pol η binds dGTP tightly during misincorporation with a KM of∼10 μM regardless of sequence context (Table S1), indicating
there is no sequence context preference in the dGTP binding
step. The crystal structures of dGTP misincorporation in all four
sequence contexts show the common feature that the T:G
wobble pair ﬁts well in the active site and interacts snugly withthe conserved Gln-38 and Arg-61 in the major and minor groove(Fig. 2B). These interactions are rather different from other Yfamily DNA polymerases, for example, Pol ι or Dpo4. The incoming dGTP is unable to stack with the primer 3′ end in the Dpo4
misincorporation ternary structure (33) (Fig. S3A). In the Pol ι
case, the mismatched T and G maintains anti–anti conformation,
but the templating base is displaced from its normal position and is
not paired with the incoming dGTP (34) (Fig. S3B). Gln-59, whichis conserved among Pol ι homologs and equivalent of Gln-38 inhuman Pol η, forms a hydrogen bond with only the N2 atom of theguanine base but not with the template T. Steady-state kinetic
measurement indicates that replacing either Gln-38 or Arg-61
by Ala in Pol η dramatically inhibits the misincorporation as well
as bypassing of CPDs (27). We ﬁnd that replacing Arg-61 with
Lys also greatly increases the misincorporation frequency andreduces the catalytic efﬁciency. The equivalent of Arg-61 in Pol ι
is a Lys (35). Nature through evolution probably has selectedArg-61 and Gln-38 in Pol η to maximize the efﬁciency and accuracy for UV–lesion bypass. Pol η-dependent dGTP misincorporation at the WA motif in SHM is likely a byproduct thattakes advantage of the conserved Arg-61 and Gln-38 late in theevolutionary history.
The kcat of dGTP misincorporation is signiﬁcantly reduced
compared with the correct incorporation and differs according to
the base-pair sequence at the primer end. The catalytic efﬁciencies of dGTP misincorporation in the TA and AA contexts arehigher than the GA and CA, and the relative efﬁciency is TA >AA > GA >> CA (Table S1). These results correlate well withthe published SHM spectrum, which shows that the TA mutations
are strongly favored over AA mutations by Pol η (36). The reduced kcat correlates with the displacement of the primer end due
to cation–π interaction mediated by Arg-61. For efﬁcient catalysis, it is essential that the 3′-OH group of the primer end and the
α-phosphate of the incoming dNTP be perfectly aligned. Misalignment between primer end and incoming nucleotide, even
Zhao et al.

23

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Materials and Methods
Crystallization and Structure Determination. Site-directed mutagenesis, protein expression, and puriﬁcation of WT or R61K human Pol η (1–432 aa) were
performed as described (27). Puriﬁed Pol η was stored in 20 mM Tris·HCl,(pH 7.5), 450 mM KCl, and 3 mM DTT. After mixing Pol η and DNA at a
1:1.05 molar ratio, the complex was transferred into 20 mM Tris·HCl (pH7.5), 150 mM KCl, 3 mM DTT, and 5 mM MgCl2 and concentrated to 3 mg/mL
Pol η. To make ternary complexes, an appropriate dNTP analog (purchasedfrom Jena Bioscience) was added. Crystals were grown by the hanging-drop
vapor-diffusion method with optimized reservoir buffer containing 0.1 M
Mes (pH 6.0), 5 mM MgCl2, 19–21% (wt/vol) MPEG2000 (polyethylene glycol
monomethyl ether 2000) (27). The oligos and incoming nucleotides used in
crystallization are summarized in Table S2. Crystals grew to maximal
dimensions with diffraction quality in 3–7 d. Diffraction data were collected
at beamline 22-BM at Advanced Photon Source, Argonne National Laboratory, processed with HKL2000 (37) or XDS (38), and converted to structure
factors by using TRUNCATE (39). Reﬁnement and structure analyses(Table 1 and Table S3) were performed by using COOT (40), PHENIX (41),and PyMOL (www.pymol.org).
Kinetic Measurements. The primer extension assay and steady-state KM andkcat measurements were carried out by using a 5′-ﬂuorescein labeled primer
and normal template oligonucleotides as described (25). The DNA sequences
and nucleotides used in these assays are shown in Table S1. Quantiﬁcation and curve ﬁtting was also performed as described (32).
ACKNOWLEDGMENTS. We thank Drs. R. Craigie and D. Leahy for criticalreading of the manuscript. This work was supported by the Intramural
Research Program of National Institute of Diabetes and Digestive andKidney Diseases, National Institutes of Health (Y.Z., M.T.G., C.B., and W.Y.);a Chinese Ministry of Education scholarship (Y.Z.); National Natural Science
Foundation of China Grant 31210103904 (to Y.-J.H.); and Grants-in-Aid for
Scientiﬁc Research from the Ministry of Education (KAKENHI) (to F.H.).

21. Rogozin IB, Pavlov YI, Bebenek K, Matsuda T, Kunkel TA (2001) Somatic mutation
hotspots correlate with DNA polymerase eta error spectrum. Nat Immunol 2(6):530–536.
22. Wilson TM, et al. (2005) MSH2-MSH6 stimulates DNA polymerase eta, suggestinga role for A:T mutations in antibody genes. J Exp Med 201(4):637–645.
23. Delbos F, Aoufouchi S, Faili A, Weill JC, Reynaud CA (2007) DNA polymerase eta is thesole contributor of A/T modiﬁcations during immunoglobulin gene hypermutation in
the mouse. J Exp Med 204(1):17–23.
24. Ummat A, et al. (2012) Structural basis for cisplatin DNA damage tolerance byhuman polymerase η during cancer chemotherapy. Nat Struct Mol Biol 19(6):628–632.
25. Zhao Y, et al. (2012) Structural basis of human DNA polymerase η-mediated chemoresistance to cisplatin. Proc Natl Acad Sci USA 109(19):7269–7274.
26. Ummat A, et al. (2012) Human DNA polymerase η is pre-aligned for dNTP binding andcatalysis. J Mol Biol 415(4):627–634.
27. Biertümpfel C, et al. (2010) Structure and mechanism of human DNA polymerase eta.
Nature 465(7301):1044–1048.
28. Nakamura T, Zhao Y, Yamagata Y, Hua YJ, Yang W (2012) Watching DNA polymerase
η make a phosphodiester bond. Nature 487(7406):196–201.
29. Kool ET (2001) Hydrogen bonding, base stacking, and steric effects in DNA replication. Annu Rev Biophys Biomol Struct 30:1–22.
30. Sathyapriya R, Vishveshwara S (2004) Interaction of DNA with clusters of amino acids
in proteins. Nucleic Acids Res 32(14):4109–4118.
31. Kondo Y (2005) Studies of in vivo and in vitro function of DNA polymerase eta. PhD
dissertation (Osaka Univ, Osaka).
32. Wang F, Yang W (2009) Structural insight into translesion synthesis by DNA Pol II. Cell
139(7):1279–1289.
33. Vaisman A, Ling H, Woodgate R, Yang W (2005) Fidelity of Dpo4: Effect of metal ions,
nucleotide selection and pyrophosphorolysis. EMBO J 24(17):2957–2967.
34. Kirouac KN, Ling H (2009) Structural basis of error-prone replication and stalling ata thymine base by human DNA polymerase iota. EMBO J 28(11):1644–1654.
35. Alt A, et al. (2007) Bypass of DNA lesions generated during anticancer treatment withcisplatin by DNA polymerase eta. Science 318(5852):967–970.
36. Yavuz S, Yavuz AS, Kraemer KH, Lipsky PE (2002) The role of polymerase eta in somatic hypermutation determined by analysis of mutations in a patient with xeroderma pigmentosum variant. J Immunol 169(7):3825–3830.
37. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276:307–326.
38. Kabsch W (2010) XDS. Acta Crystallogr D Biol Crystallogr 66(Pt 2):125–132.
39. Collaborative Computational Project, Number 4 (1994) The CCP4 suite: Programs forprotein crystallography. Acta Crystallogr D Biol Crystallogr 50(Pt 5):760–763.
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213–221.

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slightly, will inhibit the nucleotidyl-transfer reaction (25). Arg-61
also provides another barrier in misincorporation by impeding
the translocation step as observed in our binary complexes after
misincorporation and before the next round of incorporation.
Misincorporation is much enhanced by the R61K mutant Pol η.
Lys, which has a shorter side chain than Arg and reduced capability to form cation–π stacking with the primer end (Fig. 2C), ismore prone to misincorporate dGTP than WT Pol η (Table 1).
Besides the dominant misaligned conformation, the electron
density in our structures revealed that there is a second population of primer end in the TA/G and AA/G, but not CA/G or
GA/G, complexes that superimposes well with the normal ternarycomplex and supports the chemistry.
Because the R61K mutant polymerase still favors dGTP misincorporation in the WA motif, we suspect that the cation–πstacking between Arg-61 and the primer end is not the main
reason for the WA motif to be an SHM hotspot. The different
stability of A:T and T:A vs. G:C and C:G base pairs most likely
underlies the high efﬁciency of dGTP misincorporation in the
WA motif. In addition, the stacking propensity of the 3′ basewith its neighbors and its electrostatic interactions with Arg-61
may inﬂuence whether the 3′-OH can revert to the reactive conformation and also the probability of such reversion. T and A atthe primer 3′ end are thus found to be more able than G and C
to align with the incoming dGTP and form a productive complex for misincorporation to take place. Together, the strong
dGTP binding, even when it is a mismatch for the template T,
weaker hydrogen bonding between A and T at the 3′ primer
strand end, and efﬁcient mispair extension by Pol η provide themolecular rationale for the conversion of WA motifs to WG
during SHM.

DNA Repair 29 (2015) 139–146

Contents lists available at ScienceDirect

DNA Repair
journal homepage: www.elsevier.com/locate/dnarepair

UV-induced mutations in epidermal cells of mice defective in DNA
polymerase ␩ and/or ␫
Rie Kanao a,b,c,d , Masayuki Yokoi a,c,e , Tsuyoshi Ohkumo a , Yasutaka Sakurai a,c ,Kantaro Dotsu a,c , Shinobu Kura f , Yoshimichi Nakatsu f , Teruhisa Tsuzuki f ,Chikahide Masutani a,d,e , Fumio Hanaoka a,b,c,e,∗a

Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japanc
Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japand
Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japane
Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, Tokyo, Japanf
Faculty of Medical Sciences, Kyushu University, 3-1-1 Higashi-ku, Maidashi, Fukuoka 812-8582, Japanba r t i c l e

i n f oArticle history:
Received 17 November 2014
Received in revised form 2 February 2015
Accepted 3 February 2015
Available online 16 February 2015
Keywords:
DNA polymerase ␩
DNA polymerase ␫Translesion synthesis
UV-induced mutagenesis
Xeroderma pigmentosum variant (XP-V)a b s t r a c t
Xeroderma pigmentosum variant (XP-V) is a human rare inherited recessive disease, predisposed to
sunlight-induced skin cancer, which is caused by deﬁciency in DNA polymerase ␩ (Pol␩). Pol␩ catalyzes
accurate translesion synthesis (TLS) past pyrimidine dimers, the most prominent UV-induced lesions.
DNA polymerase ␫ (Pol␫) is a paralog of Pol␩ that has been suggested to participate in TLS past UV-induced
lesions, but its function in vivo remains uncertain. We have previously reported that Pol␩-deﬁcient andPol␩/Pol␫ double-deﬁcient mice showed increased susceptibility to UV-induced carcinogenesis. Here,
we investigated UV-induced mutation frequencies and spectra in the epidermal cells of Pol␩- and/orPol␫-deﬁcient mice. While Pol␩-deﬁcient mice showed signiﬁcantly higher UV-induced mutation frequencies than wild-type mice, Pol␫ deﬁciency did not inﬂuence the frequencies in the presence of Pol␩.
Interestingly, the frequencies in Pol␩/Pol␫ double-deﬁcient mice were statistically lower than those inPol␩-deﬁcient mice, although they were still higher than those of wild-type mice. Sequence analysis
revealed that most of the UV-induced mutations in Pol␩-deﬁcient and Pol␩/Pol␫ double-deﬁcient mice
were base substitutions at dipyrimidine sites. An increase in UV-induced mutations at both G:C and A:T
pairs associated with Pol␩ deﬁciency suggests that Pol␩ contributes to accurate TLS past both thymineand cytosine-containing dimers in vivo. A signiﬁcant decrease in G:C to A:T transition in Pol␩/Pol␫ doubledeﬁcient mice when compared with Pol␩-deﬁcient mice suggests that Pol␫ is involved in error-prone TLS
past cytosine-containing dimers when Pol␩ is inactivated.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction
Xeroderma pigmentosum (XP) is a genetic disorder characterized by ultraviolet light (UV) sensitivity and increased incidenceof skin cancers. XP has been classiﬁed into eight genetic complementation groups, XP-A–G and XP-V [1]. Cells derived from XP-A
through XP-G patients are deﬁcient in nucleotide excision repair(NER), which repairs a variety of DNA lesions including UV-induced∗ Corresponding author at: Faculty of Science, Gakushuin University, 1-5-1 Mejiro,
Toshima-ku, Tokyo 171-8588, Japan. Tel.: +81 3 3986 0221x6457;
fax: +81 3 5992 1029.
E-mail address: fumio.hanaoka@gakushuin.ac.jp (F. Hanaoka).
http://dx.doi.org/10.1016/j.dnarep.2015.02.006
1568-7864/© 2015 Elsevier B.V. All rights reserved.

25

cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts.
The variant form (XP-V) is characterized by proﬁciency in NER but
deﬁciency in translesion synthesis (TLS). TLS is a mechanism thatprevents replication blockage at a DNA lesion by using specialized
DNA polymerases that incorporate nucleotides opposite the lesion
and continue DNA synthesis past the site of damage [1]. We haveidentiﬁed human DNA polymerase ␩ (Pol␩) as the product of thegene associated with XP-V, POLH. Pol␩ catalyzes accurate translesion synthesis (TLS) past cis–syn thymine–thymine dimers [2–5],
an important mechanism to prevent UV-induced skin cancers inhuman cells.
Mammalian cells are now known to have ﬁfteen DNA templatedependent DNA polymerases, and these are classiﬁed into ﬁve families, A, B, X, Y, and AEP (archaeo-eukaryotic primase superfamily)

140

R. Kanao et al. / DNA Repair 29 (2015) 139–146

according to similarities in their primary structures [6–9]. The
Y-family, which was shown to carry out TLS past some lesions,consists of Pol␩, Pol␫, Pol␬, and REV1 [10]. Pol␫, encoded by the
POLI gene, incorporates one or two nucleotides opposite (6-4)
photoproducts in vitro but cannot bypass these lesions by itself[11,12], although limited Pol␫ dependent bypass of CPD lesions
has been observed depending upon the local sequence context
and metal ion used as a cofactor [11,13–15]. Whereas Pol␫ is aparalog of Pol␩, no human disease related to Pol␫-deﬁciency has
been identiﬁed so far, and its physiological relevance has not beenclariﬁed. Pol␬ is the eukaryotic homologue of the Escherichia coli
DinB protein (DNA polymerase IV) and has been shown to be
involved in accurate TLS past N2 -adducts of dG and also in bothspontaneous and induced mutagenesis [16,17]. Although Pol␬ wassuggested to be involved in NER of UV-induced lesions [18,19], it
seems unlikely that Pol␬ plays an important role in TLS past UV
lesions since the enzyme showed no activity to bypass past CPD
and (6-4) photoproduct in vitro [20]. REV1 has a dCMP transferase
activity, but the protein appears to exert its role in UV-induced
responses, as a scaffold protein interacting with Pol␨, Pol␩, Pol␫,and Pol␬ [10]. Pol␨ is a B-family polymerase containing REV3and REV7 subunits and is believed to function as an “extender”
enzyme after a TLS DNA polymerase (for example, Pol␫) insertinga nucleotide opposite a given DNA lesion [10].
To investigate the physiological roles of Pol␩ and Pol␫, we havepreviously generated mice mutated in Polh and/or Poli genes [21].
Pol␩-deﬁcient mice were generated by inserting a G418 resistance gene cassette into exon 8 of the Polh gene, resulting in
production of a truncated, nonfunctional Pol␩ protein. Using the
129 mouse derived embryonic stem (ES) cell line, which carries a
spontaneous Pol␫ nonsense mutation [22], we obtained Pol␩/Pol␫
double-deﬁcient mice as well as Pol␫-deﬁcient mice. We foundthat the incidence of skin tumors was greatly increased in thePol␩- and Pol␩/Pol␫ double-deﬁcient mice after UV irradiation, that
Pol␩/Pol␫ double-deﬁcient mice started to develop skin tumors
earlier than Pol␩-deﬁcient mice, and that the average number ofskin tumors was higher in the double-deﬁcient mice than in thePol␩-deﬁcient mice. In addition, we found that epithelial and mesenchymal tumors were formed in Pol␩- and Pol␫-deﬁcient mice,
respectively [21]. These results suggest that, in addition to Pol␩,
Pol␫ also participates in suppressing skin carcinogenesis. Other
groups had also shown the importance of Pol␩ and Pol␫ to suppress UV-induced skin tumors. Pol␩-deﬁcient mice, generated bydisruption of exon 4 of the Polh gene, are highly susceptible to UVinduced skin tumors [23]. Pol␩/Pol␫ double-deﬁcient mice, in turn,develop UV-induced skin tumors earlier than Pol␩-deﬁcient mice[24].
To investigate the functions of Pol␩ and Pol␫ in suppressing UVinduced carcinogenesis, we examined the mutation frequencies
and spectra in UV-irradiated and unirradiated epidermis in Pol␩deﬁcient, Pol␫-deﬁcient, and Pol␩/Pol␫ double-deﬁcient mice, using
the rpsL transgene as a mutation reporter sequence. Our results
show that Pol␩ plays crucial roles in suppressing any types of UVinduced base substitution at dipyrimidine sites in vivo and that Pol␫participates in error-prone TLS past UV lesions in the absence ofPol␩.

2. Materials and methods
2.1. Generation of transgenic mice
The rpsL transgenic mouse line (ssw2-14p) used in this study
was derived from C57BL/6J mice, but carries approximately 100
hemizygous copies of the pSSW plasmid; it has been described previously [25]. The pSSW plasmid carries a reporter gene, rpsL from

E. coli, with a dominant streptomycin-sensitive (Sms ) phenotype inbacterial cells, and a kanamycin-resistant (Kmr ) gene. Pol␩- and/orPol␫-deﬁcient mice were established as previously described [21].
Polh+/− , Poli+/− mice were mated with the rpsL transgenic miceto generate rpsLTg/+ Polh+/− Poli+/− mice. By mating the rpsLTg/+
Polh+/− Poli+/− mice with Polh+/− Poli+/− mice, rpsLTg/+ Polh−/− Poli+/+ ,rpsLTg/+ Polh+/+ Poli−/− , and rpsLTg/+ Polh−/− Poli−/− mice were generated. The handling and sacriﬁce of all animals were carried out
in accordance with nationally prescribed guidelines, and ethicalapproval for the studies was granted by the Committee for Animal Experiments, Graduate School of Frontier Biosciences of Osaka
University.

2.2. Preparation of UVB irradiated and unirradiated epidermal
genomes
At 8 weeks of age, after shaving of dorsal hair, mice from each
respective genotype were irradiated with UVB (FL20SE-E; Toshiba,
Tokyo, Japan) at 400 J/m2 . One week after irradiation, these mice
were sacriﬁced, and the irradiated dorsal and unirradiated ventral
skins were collected. These skin samples were treated with 20 mM
EDTA in PBS at 37 ◦ C, and the epidermis was peeled from the dermis using a spatula. The isolated epidermal samples were frozen
in liquid nitrogen and stored at −80 ◦ C. To prepare genomic DNA,the epidermal samples were ﬁrst incubated in lysis buffer (10 mM
Tris–HCl (pH 8.0), 100 mM EDTA, 200 mM NaCl, 0.5% SDS, 0.1 mg/ml
RNaseA) at 37 ◦ C for 1 h, followed by 1 mg/ml Proteinase K for 3 h.
After phenol/chloroform extraction, genomic DNA was obtained byethanol precipitation.

2.3. rpsL mutagenesis assay
Mutation analyses were performed as described [25] with minor
modiﬁcations. Brieﬂy, 10 ␮g of isolated genomic DNA was digested
with 35 U of BanII (TaKaRa, Shiga, Japan) at 37 ◦ C for 3 h, which
incises once the pSSW plasmid, in order to excise the integrated
shuttle vector at unit size. The BanII digested DNA was thentreated with 350 U of T4 DNA ligase (TaKaRa, Shiga, Japan) for 2 h
at 16 ◦ C for self-circularization. One microgram of the resulting
DNA was introduced into E. coli DH10B cells by electroporation(1.8 kV/mm). Then, a portion of the cells were plated onto LB
plates containing kanamycin (50 ␮g/ml), and the remainders wereplated onto plates containing both kanamycin and streptomycin(200 ␮g/ml). These plates were incubated at 28 ◦ C for 48 h. Total
numbers of kanamycin-resistant colonies were calculated fromthe number of colonies formed and the amount of DNA used.
Mutation frequency was calculated as the ratio of total colony
numbers on the plate containing kanamycin and streptomycin
to those on the plate containing kanamycin only. For mutation
spectrum analysis, DNA fragments containing the rpsL gene were
ampliﬁed by PCR from the kanamycin- and streptomycin-resistantcolonies. The primers F (5 -CACCTGATTGCCCGACATTA-3 ) and R(5 -CAGGGTCGGAACAGGAGAGC-3 ) were used for the PCR. The
ampliﬁed PCR products were directly sequenced with the forward
primer (5 -GACGAATTCCCGGTTTGACTGGTC-3 ) and the reverse
primer (5 -GGATTGTCCAAAACTCTACGAG-3 ). Mutations found in
the rpsL coding region and surrounding sequences containing thepromoter and ribosomal recognition regions (from position −120
to position 375) were exhibited in the mutation spectrum.

2.4. Statistical analyses
Statistical analyses for mutation frequency included the Student’s t-test and Fisher’s exact probability test. The CLUSTERM

26

R. Kanao et al. / DNA Repair 29 (2015) 139–146

141

Table 1
Mutation frequencies in UVB irradiated and unirradiated epidermal genomes fromPolh+/+ Poli+/+ , Polh−/− Poli+/+ , Polh+/+ Poli−/− , and Polh−/− Polh−/− mice.

Fig. 1. Mutation frequencies of the rpsL transgene in UVB-irradiated and unirradiated epidermis from Polh+/+ Poli+/+ , Polh−/− Poli+/+ , Polh+/+ Poli−/− , and Polh−/− Poli−/−mice. Mean mutation frequencies of six mice are plotted with standard deviations.
Open and ﬁlled bars indicate data for unirradiated and irradiated epidermis, respectively. All differences in mean mutation frequencies between UV-irradiated andunirradiated mice are statistically signiﬁcant (P = 0.0006 for Polh+/+ Poli+/+ mice,and P < 0.0001 for Polh−/− Poli+/+ , Polh+/+ Poli−/− and Polh−/− Poli−/− mice). Differences between irradiated Polh−/− Poli+/+ mice and irradiated Polh+/+ Poli+/+ mice, andbetween irradiated Polh−/− Poli−/− mice and irradiated Polh+/+ Poli+/+ mice are both
statistically signiﬁcant (P < 0.0001). The difference between irradiated Polh−/− Poli−/−mice and irradiated Polh−/− Poli+/+ mice is statistically signiﬁcant (P = 0.021).

program (http://www.itb.cnr.it/webmutation/) was employed to
predict mutation hotspots [26].

Genotype

UV
irradiation

Aa (×105 )Polh+/+ Poli+/+

−

3.44
4.81
5.25
9.22
7.73
6.56

5
1
3
3
1
2

1.45
0.21
0.57
0.33
0.13
0.30

+

5.46
3.99
3.80
7.81
7.25
3.37

13
15
5
24
23
16

2.38
3.76
1.32
3.07
3.17
4.75

−

3.94
4.67
6.45
2.76
3.10
5.83

9
4
3
4
6
2

2.28
0.86
0.47
1.45
1.94
0.34

+

3.55
2.73
2.96
3.02
3.16
2.55

158
102
124
140
127
87

44.5
37.4
41.9
46.4
40.2
34.1

−

6.75
3.43
3.06
3.15
4.62
3.36

6
1
1
1
1
4

0.89
0.29
0.33
0.32
0.22
1.19

+

3.23
4.95
3.11
3.36
3.62
3.57

10
20
8
7
11
16

3.10
4.04
2.57
2.08
3.04
4.48

−

3.65
5.36
3.51
4.59
3.91
4.24

2
4
2
4
10
10

0.55
0.75
0.57
0.87
2.56
2.36

+

2.16
3.17
2.85
3.42
2.20
2.69

90
109
92
101
75
79

41.7
34.4
32.3
29.5
34.1
29.4

Polh−/− Poli+/+

Polh+/+ Poli−/−

3. Results
3.1. Pol but not Pol suppresses UV-induced mutations inepidermal cells
Polh+/+ Poli+/+ , Polh−/− Poli+/+ , Polh+/+ Poli−/− and Polh−/− Poli−/−mice carrying the rpsL transgene hemizygously were obtained bycrossing of Polh+/− Poli+/− mice with rpsL transgenic mice. Those
mice had its back shaved and irradiated with 400 J/m2 UVB once.
One week after irradiation, the mice were sacriﬁced, and irradiateddorsal skin samples and unirradiated ventral skin samples werecollected and separated into epidermis and dermis. Genomic DNAs
were extracted from the epidermis and subjected to mutation analysis as described in Section 2. Six mice from each genotype were
examined. The number of screened and mutated colonies, together
with mutation frequencies of each individual mouse, is shown in
Table 1. The mean mutation frequencies for six mice are depicted inFig. 1. There was no signiﬁcant difference in the mutation frequencyof the unirradiated epidermal cells among the four genotypes. The
increases in mutation frequency following UV-irradiation were
statistically signiﬁcant in all genotypes. UV-induced mutation frequencies for Polh−/− Poli+/+ (4.07 × 10−4 ) and Polh−/− Poli−/− mice
(3.36 × 10−4 ) were approximately 13- and 11-fold higher, respectively, than that of Polh+/+ Poli+/+ mice (3.07 × 10−5 ), indicatingthat Pol␩ exerts a crucial role for suppressing mutations by UVirradiation. On the other hand, the UV-induced mutation frequency
in Polh+/+ Poli−/− mice (3.22 × 10−5 ) were not signiﬁcantly different from that in wild-type mice (P = 0.82), suggesting that Pol␫
does not inﬂuence UV-induced mutagenesis in the epidermis in thepresence of Pol␩. Interestingly, however, the UV-induced mutation
frequency in the genome from Polh−/− Poli−/− mice was signiﬁcantly lower than that of Polh−/− Poli+/+ mice (P = 0.020), suggestingthat Pol␫ contributes to increasing mutations in the absence of Pol␩.

27

Polh−/− Poli−/−Bb

Mutationfrequency(×10−5 )a
Total number of Kmr colonies, as calculated from the number of Kmr colonies
counted and the amount of DNA sample used for A and B.
b
Total number of Kmr and Smr colonies counted.

3.2. Sequence analysis of mutations
To identify the types and locations of mutations, we sequenced
the rpsL coding region and its upstream region containing the promoter and the ribosomal recognition sequences (from position−120 to position 375) in the mutants. The identiﬁed mutations are shown in Fig. 2A–D. As summarized in Table 2, basesubstitutions were among the most common mutations induced by
UV-irradiation in all mouse genotypes. Statistical analysis revealed
that Polh−/− Poli+/+ and Polh−/− Poli−/− mice had signiﬁcantly
higher base substitutions, namely, 4.03 × 10−4 and 3.22 × 10−4 ,142

R. Kanao et al. / DNA Repair 29 (2015) 139–146

Fig. 2. Site-distribution of UV-induced mutations observed in (A) Polh+/+ Poli+/+ , (B) Polh−/− Poli+/+ , (C) Polh+/+ Poli−/− and (D) Polh−/− Poli−/− mice. Base substitutions areindicated above the wild-type rpsL sequence by the following symbols. A, T, C and G are indicated by circle, square, triangle and diamond, respectively. Deletions andadditions are indicated by an asterisk (*) and plus (+), respectively. Tandem mutations are indicated below the sequence in parentheses. The nucleotide positions, startingfrom the ﬁrst position of the initiation codon, for translation are shown at the right side of the sequence. The start codon and the termination codon are surrounded by boxes.
The promoter sequence and the ribosomal recognition sequence (Shine–Dalgarno sequence; SD) are underlined.

respectively, than Polh+/+ Poli+/+ (2.51 × 10−5 ) and Polh+/+ Poli−/−(2.78 × 10−5 ) mice (P < 0.0001) (Table 3). On the other hand, there
were no statistically signiﬁcant differences in the frequencies of
UV-induced frameshift mutations between the four genotypes. An

important point to note is that most of the UV-induced mutations were found at dipyrimidine sites (Fig. 2A–D and Table 3). The
frequencies of base substitutions at dipyrimidine sites in Polh−/−Poli+/+ and Polh−/− Poli−/− mice were signiﬁcantly higher than those

Table 2
Class-distribution of rpsL mutations.
Polh−/− Poli+/+

Polh+/+ Poli+/+
UV

−

−

+
a

c

Polh+/+ Poli−/−
−

+
c

c

Polh−/− Poli−/−+−+

Base substitutions

0.16 (6)2.62 (83)0.37 (10)43.2 (777)0.12 (3)3.11 (68)0.24 (6)33.2 (547)Transitions
G:C→A:T
A:T→G:C

0.08 (3)0.08 (3)1.58 (50)c0.28 (9)0.22 (6)0.04 (1)c23.5 (422)c
3.95 (71)c0.12 (3)<0.04 (0)2.11 (46)c0.32 (7)c0.16 (4)<0.04 (0)16.4 (271)c
4.00 (66)cTransversions
G:C→T:A
G:C→C:G
A:T→T:A
A:T→C:G<0.03 (0)<0.03 (0)<0.03 (0)<0.03 (0)<0.03 (0)0.03 (1)0.41 (13)0.13 (4)0.04 (1)0.04 (1)0.04 (1)<0.04 (0)5.45 (98)c0.50 (9)c5.95 (107)c
0.56 (10)c<0.04 (0)<0.04 (0)<0.04 (0)<0.04 (0)0.14 (3)<0.05 (0)0.32 (7)0.09 (2)0.04 (1)<0.04 (0)0.04 (1)<0.04 (0)3.94 (65)c0.43 (7)4.73 (78)c0.73 (12)cTandems<0.03 (0)0.19 (6)<0.04 (0)3.34 (60)c<0.04 (0)0.14 (3)<0.04 (0)2.91 (48)c

c

c

c

Others
1 bp deletions
1 bp insertions
Large deletionsb
Large insertionsb

0.24 (9)0.14 (5)<0.03 (0)0.08 (3)0.03 (1)0.60 (19)0.32 (10)c0.10 (3)c0.19 (6)<0.03 (0)0.75 (20)0.49 (13)c<0.04 (0)0.26 (7)<0.04 (0)0.61 (11)0.11 (2)0.28 (5)c0.22 (4)c<0.11 (0)0.45 (11)0.25 (6)0.04 (1)0.16 (4)<0.04 (0)0.41 (9)0.18 (4)0.05 (1)0.14 (3)0.05 (1)1.03 (26)0.44 (11)<0.04 (0)0.55 (14)0.04 (1)1.33 (22)0.30 (5)<0.06 (0)0.97 (16)0.06 (1)Total

0.41 (15)

3.22 (102)1.22 (30)43.9 (788)0.58 (14)3.53 (77)1.27 (32)34.5 (569)

Mutation frequencies (×10−5 ) were determined by total number of mutations identiﬁed from six mice for each of genotypes.
aNumbers in parentheses are the number of independent mutations.
b
Large deletion and large insertion include deletions and insertions of two or more base pairs.
cMutants contained two or three mutations were found.

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143

Table 3
Mutation frequencies (×10−5 ) of UV-induced base substitutions at dipyrimidine sites and other sites, and other mutations, including deletions and insertions.

Base substitutions
At dipyrimidine sites
At other sites
Other mutations

Polh+/+ Poli+/+Polh−/− Poli+/+

Polh+/+ Poli−/−Polh−/− Poli−/−

2.51 ± 1.06
2.31 ± 0.95
0.20 ± 0.21
0.69 ± 0.33

40.3 ± 4.01
40.1 ± 4.19b
0.39 ± 0.28
0.68 ± 0.47

2.78 ± 1.03
2.62 ± 0.81
0.21 ± 0.23
0.44 ± 0.53

32.2 ± 5.85
31.7 ± 5.73
0.67 ± 0.72
1.34 ± 2.57

aMean mutation frequencies of six mice are shown, along with standard deviations.
aValues are signiﬁcantly different from those of non-irradiated Polh+/+ Poli+/+ and Polh−/− Poli−/− mice (P < 0.0001 and P = 0.029, respectively).
b
Values are signiﬁcantly different from those of non-irradiated Polh+/+ Poli+/+ and Polh−/− Poli−/− mice (P < 0.0001 and P = 0.025, respectively).

in Polh+/+ Poli+/+ and Polh+/+ Poli−/− mice (P < 0.0001). These results
indicate that Pol␩ is predominantly responsible for suppressing
mutations upon UV-irradiation, by correctly bypassing UV-induced
pyrimidine dimers.
Interestingly, in Polh−/− Poli−/− mice, the frequency of UVinduced total base substitutions was signiﬁcantly lower than in
Polh−/− Poli+/+ mice (P = 0.029, see Table 3). Similarly, the frequency
of base substitutions at dipyrimidine sites in Polh−/− Poli−/− mice
was also signiﬁcantly lower than in Polh−/− Poli+/+ mice (P = 0.025,
see Table 3). In addition to this, several mutational hotspots
were observed at dipyrimidine sites in the UV-irradiated epidermal genomes of Polh−/− Poli+/+ mice, including −104C, −103T,−104/−103CT, −101G, −79A, −11G, −10G, −11/−10GG, 3G, 16C,
28A, 40C, 73G, 83C, 82/83CC, 88A, 94G, 106C, 127–130A, 170T,
184G, 194C, 208G, 226G, 233C, 262A, 263A, and 272C (Fig. 2B).
Among these, −104C, 40C, 82/83CC, 94G, 106C, 170T, 184G, 194C,
226G, and 262A were no longer mutational hotspots in the UVirradiated Polh−/− Poli−/− mice (Fig. 2D). These results are consistent
with our interpretation that Pol␫ participates in error-prone TLS
past UV-induced lesions in an error-prone manner, especially whenbypassing C within pyrimidine dimers under the Pol␩-deﬁcient
background.
The frequencies for each type of base substitution are depicted
in Fig. 3. The G:C to A:T transition was the most prominent UVinduced mutation in all genotypes. In the genomes of Polh−/−Poli+/+ and Polh−/− Poli−/− mice, the UV-induced mutational frequencies of all types of base substitutions were higher than in
Polh+/+ Poli+/+ and Polh+/+ Poli−/− mice, although G:C to C:G and
A:T to C:G transversions in Polh−/− Poli+/+ mice were not statistically signiﬁcant compared with those in Polh+/+ Poli+/+ mice. Theseobservations strongly suggest that Pol␩ suppresses UV-induced
mutations at both C and T residues in the epidermis in vivo. Interestingly, in Polh−/− Poli−/− mice, the frequency of G:C to A:T transitions
was statistically lower than that in Polh−/− Poli+/+ mice (P = 0.0013),
suggesting that Pol␫ contributes to UV-induced G:C to A:T transitions in the absence of Pol␩, probably by inserting A opposite
cytosine within pyrimidine dimers.

4. Discussion
4.1. Roles of Pol in suppressing UV-induced mutagenesis
In the present study, we analyzed the UVB-induced mutations
occurring in the rpsL transgene in epidermal cells of wild type,Pol␩-deﬁcient, Pol␫-deﬁcient, and Pol␩/Pol␫ double-deﬁcient mice.
Although several studies have investigated the effects of Pol␩
and/or Pol␫ deﬁciencies on UV-induced mutagenesis [24,27–33],
these studies were done using cultured cells in vitro. To the best of
our knowledge, this is the ﬁrst report addressing the roles of Pol␩and Pol␫ in UV-induced mutagenesis in vivo. When irradiated with
UVB, Pol␩-deﬁcient mice showed signiﬁcantly higher mutation frequencies than control mice. Most of the UV-induced mutationsin the genomes of Pol␩-deﬁcient mice were base substitutions at
dipyrimidine sites (both C and T residues). These ﬁndings clearly29

indicate that Pol␩ is crucial for accurate TLS past UV-induced
pyrimidine dimers in the epidermal cells and in the absence ofPol␩, other TLS polymerases carries out mutagenic bypass past
the lesions. These results account for previous ﬁndings that Pol␩deﬁcient mice suffer from high incidences of UV-induced skintumors [21,23,24].
The most prominent UV-induced mutations were G:C to A:Ttransitions in all genotypes. Hydrolytic deamination of the cytosine
in CPDs, which produces uracil-containing CPDs, is thought to playa key role in the UV-induced mutations at G:C base pairs [34–36].
Pol␩ preferentially incorporates dAMP opposite uracil of T-U CPDs[37], resulting in a G:C to A:T transition, if the CPD is repaired
after TLS. This may account for the frequent G:C to A:T transitions
observed in Pol␩-proﬁcient mice. However, the large increase in thefrequency of UV-induced G:C to A:T transitions in Pol␩-deﬁcient
mice indicates that Pol␩ rather functions to suppress such transitions. Similar results showing that the most prominent UV-induced
mutations caused by Pol␩ deﬁciency are G:C to A:T transitions wereobserved in two previous experiments. In the ﬁrst of these, the supF
gene was introduced into XP-V cells and human kidney 293T cells
in which Pol␩ expression was suppressed by siRNA. In the second,the LacZ’ gene was introduced into Burkitt’s lymphoma BL2 cells inwhich the POLH gene was inactivated by gene targeting [28,29,33].
Abundant G:C to A:T transitions were also observed following UVinduced mutation analyses of the HPRT locus in XP-V cells andprimary ﬁbroblasts obtained from Pol␩-deﬁcient mice, although
equivalent levels of G:C to T:A transversions were also observedin these studies [24,27,31]. These observations suggest that Pol␩ iscritical for suppressing at C (some of them might be deaminated)within UV-induced pyrimidine dimers. Although we do not know
which kind of nucleotides Pol␩ incorporates opposite the cytosine
in CPDs, its preference for incorporating dAMP opposite uracil suggests that Pol␩ needs to cooperate with other TLS polymerases or
unknown factors to promote accurate bypass of the lesion. Pol␫is a possible candidate for this role. It is capable of incorporatingdGMP opposite uracil in CPDs with half the efﬁciency of dAMP [14].
Pol␩ also interacts with Pol␫ and UV-induced nuclear foci formation by Pol␫ is largely dependent on Pol␩ [38]. Pol␩ interacts with
REV1 [39]. Supporting this, we have shown that the accumulationof endogenous REV1 in UV-damaged sites in nuclei depends on
Pol␩, and that Pol␩-REV1 interaction is required to suppress spontaneous mutations in human cells [40]. These observations suggestthat coordinated TLS by multiple DNA polymerases may take placein the presence of Pol␩, contributing to greater suppression of basesubstitutions at dipyrimidine sites than by Pol␩ alone.
UVB exposure in mice may produce reactive oxygen species thatgenerate oxidative base damage, such as 8-oxoG [41]. MammalianPol␩ is efﬁcient at incorporating dC opposite 8-oxoG to suppress
G:C to T:A transversion in vitro [42,43]. Furthermore, Lee and Pfeifer
reported that G:C to T:A transversions were predominant in XP-Vand Pol␩-knockdown human cells [44]. Hence, it is reasonable tosuggest that some, if not all, G:C to T:A transversions in UV-exposedPol␩-deﬁcient mice are caused by UV-induced oxidative base damage. On the other hand, there were no signiﬁcant differences inthe spontaneous mutation frequencies in several tissues between

144

R. Kanao et al. / DNA Repair 29 (2015) 139–146

Fig. 3. UV-induced base substitutions observed in Polh+/+ Poli+/+ , Polh−/− Poli+/+ , Polh+/+ Poli−/− and Polh−/− Poli−/− mice. White, dotted, striped, and black bars indicate Polh+/+Poli+/+ , Polh−/− Poli+/+ , Polh+/+ Poli−/− and Polh−/− Poli−/− mice, respectively. The mean mutation frequencies for the six mice are plotted along with their standard deviations.
Note that these mean values differ from the total mutation frequencies listed in Table 3. Values for G:C to A:T, A:T to G:C, G:C to T:A, and A:T to T:A mutations of Polh−/−Poli+/+ mice are signiﬁcantly different from those of Polh+/+ Poli+/+ mice (P < 0.0001, P = 0.0006, P < 0.0001, and P < 0.0001, respectively). Values for G:C to A:T, A:T to G:C, G:C
to T:A, A:T to T:A, and A:T to C:G mutations of Polh−/− Poli−/− mice are signiﬁcantly different from those of Polh+/+ Poli+/+ mice (P < 0.0001, P = 0.0068, 0.0005, P < 0.0001, and
P = 0.042, respectively). The value for the G:C to A:T transition of Polh−/− Poli−/− mice is signiﬁcantly different from that of Polh−/− Poli+/+ mice (P = 0.0013).

one-year-old Pol␩-deﬁcient and -proﬁcient mice [32]. It remains
uncertain whether or not oxidative base damage is involved in theenhancement of mutagenesis in UV-exposed Pol␩-deﬁcient mice.

4.2. Roles of Pol in UV-induced mutagenesis
UV-induced mutation frequencies in epidermal genomes fromPol␫-deﬁcient mice were not signiﬁcantly different from those ofwild-type mice, but the mutation frequency in Pol␩/Pol␫ doubledeﬁcient mice was statistically lower than in Pol␩-deﬁcient mice.
These results imply that Pol␫ would not cause mutations in the
UV-irradiated epidermal genome when Pol␩ is present, but would
cause UV-induced mutations in its absence. This is especially true
for Pol␫ and G:C to A:T transitions, in which the difference is statistically signiﬁcant in Pol␩-deﬁcient mice. This is consistent witha previous report that Pol␫ preferentially incorporates dAMP opposite T-U CPDs in vitro [14], which would give rise to G:C to A:Ttransitions. Pol␫ also preferentially incorporates dTMP opposite TTCPD in vitro [11], which would generate A:T to T:A transversion. In
the experiments described in this report, however, we have not yet
observed any statistical difference in the frequency of transversion
between Pol␩-deﬁcient and Pol␩/Pol␫ double-deﬁcient mice.
Gueranger et al. [33] reported that Pol␫ deﬁciency did notaffect UV-induced mutations in a shuttle vector in the presence ofPol␩ but did reduce some mutational hotspots observed in Pol␩targeted BL2 cells. Wang et al. [31] also reported that suppressionof Pol␫ expression decreased UV-induced mutation frequencies inPol␩-deﬁcient XP-V cells. These observations are consistent with
our ﬁndings that Pol␫ acts as a mutator in the absence of Pol␩. However, Dumstorf et al. [24], investigating primary ﬁbroblasts isolated
from Pol␩- and/or Pol␫-deﬁcient mice, have reported that Pol␫ deﬁciency causes a decrease in UV-induced base substitutions not onlyin the absence of Pol␩ but also in its presence. We cannot explain
the discrepancies between these observations, but it may be possible that the difference depends on the type of cells employed, i.e.

that there is an inherent difference between dermal ﬁbroblasts andepithelial cells. Dumstorf et al. also reported biases for UV-induced
base substitutions between nontranscribed and transcribed strandsof the Hprt locus in wild-type and Pol␩- deﬁcient cells which werelower in Pol␩/Pol␫ double-deﬁcient cells [24]. We did not, however,observe this strand bias. This is probably due to the different transcription status of the Hprt gene and the rpsL reporter gene, i.e. the
Hprt gene is an actively transcribed housekeeping gene but the rpsL
reporter gene has no eukaryotic promotor. So, UV-induced photoproducts could be selectively removed from the transcribed strandof the Hprt gene [45] but not of the rpsL gene.
The lower UV-induced mutation frequency in Pol␩/Pol␫ doubledeﬁcient mice than in Pol␩-deﬁcient mice was unexpected, as it
is inconsistent with previous observations that Pol␩/Pol␫ doubledeﬁcient mice start to develop skin tumors earlier, and go on todevelop higher numbers of skin tumors than Pol␩-deﬁcient mice
[21,24]. It should be noted that UV-irradiation procedures used fortumor formation are different from those used for mutagenesis
analyses, i.e. mice were irradiated every day, with 2 kJ/m2 of UVB,
for 20 weeks, in tumor formation assays, but were only irradiatedonce, with 0.4 kJ/m2 of UVB, in mutagenesis assays. In addition,
we have reported that Pol␩ and Pol␫ deﬁciencies induce epithelial
and mesenchymal tumors, respectively, suggesting that the contributions of Pol␩ and Pol␫ to mutation avoidance in the epidermis
are different from those in the dermis. Petta et al. demonstrated that
Pol␫ is involved in oxidative base damage tolerance independentlyof Pol␩ [46]. Our present study, however, did not show a greaternumber of mutations in UV-irradiated epidermal cells from Pol␫deﬁcient mice. These are issues that we hope to solve in the near
future.

4.3. UV-induced mutagenesis in the absence of Pol
Small numbers of UV-induced G:C to C:G and A:T to C:Gtransversions may suggest that cells do not have DNA polymerases

30

R. Kanao et al. / DNA Repair 29 (2015) 139–146

that efﬁciently incorporate dCMP opposite cytosine, uracil, or
thymine dimers. However, a clear increase in UV-induced transitions in the Pol␩-deﬁcient mice indicates that an error-prone
TLS mechanism exists in the epidermis in vivo. We showed that
Pol␫ partially causes mutations in the UV-irradiated genome in theabsence of Pol␩; however, the majority of these mutations could be
produced by other TLS polymerases. Pol␨ and REV1 are likely candidates for error-prone enzymes. Previous studies using both mouseand human cells have shown that reduced expression or disruption of REV3 or REV7 genes, which encode Pol␨, results in a decrease
in UV-induced mutation frequencies [33,39,47,48]. In human cells,
reduced REV1 gene expression has been reported to decrease thefrequency of UV-induced mutation [39,47,49]. Moreover, a targeted
deletion of the BRCT domain in mouse cells decreased UV-induced
mutation frequency [50]. In addition, Pol␬-deﬁcient and Polkknockdown cells exhibit UV sensitivity [51–53], indicating that thispolymerase is also involved in UV-damage tolerance mechanisms.
Pol␬ participates in nucleotide excision repair (NER) [18,19]. However, it also appears to play a role in TLS past UV-induced lesions,as shown in Xenopus egg extracts and NER-deﬁcient XP-A cells[53–55]. Finally, it should be pointed out that replicative DNA polymerase(s) may add one or more nucleotides (mostly one dAMP) tothe primer terminus before dissociating from it, Pol␨ extending further [56]. If A is inserted opposite to C in pyrimidine dimers in sucha way by a replicative DNA polymerase and extended by Pol␨, itshould result in a G:C-to-A:T transition. Although disruption of the
Rev3 gene results in embryonic lethality in mice [47], Rev1, Rev7 andPol knockout and Rev3 conditional knockout animals have beensuccessfully generated [57–61]. To further understand the mechanisms that protect against and produce mutations and tumors
after UV-irradiation, it will be necessary to perform in vivo UVinduced mutagenesis experiments with mice defective in multiple
TLS polymerases.
4.4. Conclusions
In this study, we show that Pol␩ is important for suppressing
any types of UV-induced base substitution at dipyrimidine sites,
and that Pol␫ acts as a mutator at UV lesions in the absence of Pol␩in epidermal cells.
Conﬂict of interest statement
None declared.
Acknowledgements
We thank all of the members of the Hanaoka Laboratory at Osaka
University for critical discussions and helpful comments. We are
grateful to Dr. Haruo Ohmori (Gakushuin University) for his criticalreading of the manuscript and valuable suggestions. This study was
supported by KAKENHI (Grant-in Aid for Scientiﬁc Research) from
the Ministry of Education, Culture, Sports, Science, and Technologyof Japan (17013053 to F.H.) and by Solution Oriented Research for
Science and Technology from the Japan Science and Technology
Agency.
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32

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ᶵ⬟ゎᯒࠋ㸰㸧៏ᛶⓗ࡞⣸እ⥺ᦆയࢫࢺࣞࢫ࡟ᑐࡍࡿ⪏ᛶ⋓ᚓࡢศᏊᶵᵓࠋ$$%⤖ᯝ࡜⪃ᐹ&$㸯㸧!56 ಟ᚟࡟㛵୚ࡍࡿ኱⭠⳦ 7,8" ࢱࣥࣃࢡ㉁ࡢᶵ⬟ゎᯒ$!"# ┦ྠ⤌᥮࠼ᶵᵓࡣࠊ!56 ࢆṇ☜࡟ಟ᚟ࡍࡿࡇ࡜ࡀ࡛ࡁࡿ၏୍ࡢ࣓࢝ࢽࢬ࣒࡜ࡋ࡚⏕≀㛫࡛㧗ᗘ࡟ಖᏑࡉࢀ࡚࠸ࡿࠋ኱⭠⳦ࡢ 7,8# ࣜࢥࣥࣅࢼ࣮ࢮࡣࠊ!"# ษ᩿ᮎ➃ࡢ๐ࡾ㎸ࡳ࡟ࡼࡾ⏕ࡌࡓ୍ᮏ㙐 !"# ୖ࡛ࢾࢡࣞ࢜ࣇ࢕࣓ࣛࣥࢺࢆᙧᡂࡋࠊ┦ྠ㙐᳨⣴࠾ࡼࡧ !"#㙐஺᥮཯ᛂࢆ⾜࠺ࠋࡉࡽ࡟ࠊ୍ᮏ㙐 !"# ࡟⤖ྜࡍࡿࡇ࡜࡛άᛶ໬ࡉࢀࡓ 7,8# ࡣࠊ595
ㄏᑟ࡜࿧ࡤࢀࡿ !"# ಟ᚟ࡸ⣽⬊ศ⿣ࡢ೵Ṇ࡟㛵ࢃࡿ㑇ఏᏊ⩌ࡢⓎ⌧ࢆㄏᑟࡍࡿࠋ
5/0)8/)01+$:1;2/,2128,$(<$8=0(:(.(:,$>5?@A$ ࣇ࢓࣑࣮ࣜࢱࣥࣃࢡ㉁ࡣࠊཎ᰾ཬࡧ┿᰾⏕≀࡟࠾࠸࡚ᗈࡃᏑᅾࡋࠊᰁⰍయᵓ㐀ࡢືែไᚚ࡜⥔ᣢ࡟ᚲ㡲ࡢᙺ๭ࢆᯝࡓࡋ࡚࠾ࡾࠊ!"#」〇ࡸ㌿෗ࠊᰁⰍయศ㞳࡞࡝ࡢᵝࠎ࡞ᰁⰍయᶵ⬟ࡢไᚚ࡟㛵୚ࡋ࡚࠸ࡿࠋ኱⭠⳦ 7,8" ࢱࣥࣃࢡ㉁ࡣ 5?@ ࣇ࢓࣑࣮ࣜ࡟ᒓࡋ࡚࠾ࡾࠊ!"# ᦆയ౫Ꮡⓗ࡟Ⓨ⌧ࡋࠊ࣐࢞ࣥ⥺ࡸࢡࣟࢫࣜࣥࢡ๣࡛࠶ࡿ࣐࢖ࢺ࣐࢖ࢩࣥ @ ࡞࡝࡟ࡼࡿ !56 ࡢಟ᚟࡟㛵୚ࡋ࡚࠸ࡿࠋࡇࢀࡲ࡛ࡢ
BCD-7,8" ࢆ⏝࠸ࡓ⺯ග㢧ᚤ㙾ゎᯒ࠿ࡽࠊ7,8" ࡣ࣐࢖ࢺ࣐࢖ࢩࣥ @ ౫Ꮡⓗ࡟᰾ᵝయୖ࡛BCD ࣇ࢛࣮࢝ࢫࢆᙧᡂࡍࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓ EAࠋ௒ᅇᡃࠎࡣࠊࡇࡢ !"# ᦆയ౫Ꮡⓗ࡞
BCD-7,8" ࣇ࢛࣮࢝ࢫᙧᡂࡀ !"# ┦ྠ⤌᥮࠼ಟ᚟ࡢ⤌᥮࠼㓝⣲࡛࠶ࡿ 7,8# ࢆḞᦆࡋࡓ⣽⬊࡟࠾࠸࡚ほᐹࡉࢀ࡞࠸ࡇ࡜ࢆぢ࠸ࡔࡋࡓࠋࡉࡽ࡟ࠊ0,8# ࡢኚ␗యࣛ࢖ࣈ࣮ࣛࣜࢆ⏝࠸ࡓࢫࢡ࣮ࣜࢽࣥࢢ࠿ࡽࠊBCD-7,8" ࣇ࢛࣮࢝ࢫᙧᡂ⬟ࢆኻࡗࡓ᪂つࡢ 0,8# ኚ␗㸦0,8#FGHH7㸧
ࢆྠᐃࡋࡓࠋ⯆࿡῝࠸ࡇ࡜࡟ࠊ0,8#FGHH7 ኚ␗ᰴࡣࠊ࣐࢖ࢺ࣐࢖ࢩࣥ @ ࡟ᑐࡋ࡚㧗࠸ឤཷᛶࢆ♧ࡍ୍᪉࡛ࠊ⣸እ⥺࡟ᑐࡍࡿឤཷᛶཬࡧ 7,8# ౫Ꮡⓗ࡞㑇ఏᏊⓎ⌧ㄏᑟ㸦595 ㄏᑟ㸧ࡣ㔝⏕ᆺᰴ࡜ྠ⛬ᗘ࡛࠶ࡗࡓࠋࡇࡢ⤖ᯝࡣࠊ7,8# ࡜ 7,8" ࡜ࡢ㛫࡟ᶵ⬟ⓗ࡞┦஫స⏝ࡀᏑᅾ

33ࡍࡿࡇ࡜ࢆ♧၀ࡋ࡚࠸ࡿࠋࡇࡢ⤖ᯝ࡟୍⮴ࡋ࡚ࠊᢠ C+1I ᢠయࢆ⏝࠸ࡓච␿ỿ㝆ᐇ㦂࠿ࡽࠊ⣽⬊ෆ࡟࠾࠸࡚ C+1I-7,8" ࡜ 7,8# ࡀ」ྜయࢆᙧᡂࡋ࡚࠸ࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋࡉࡽ࡟ࠊ⢭〇ࡋࡓ J;.-7,8" ࡜ 7,8# ࢆ⏝࠸ࡓ⏕໬Ꮫⓗゎᯒ࠿ࡽࠊࡇࢀࡽࡢࢱࣥࣃࢡ㉁ࡣ┤᥋⤖ྜࡋ࡚࠸ࡿࡇ࡜ࡀࢃ࠿ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽࠊ!56 ᮎ➃࡟⏕ࡌࡿ୍ᮏ㙐 !"# 㡿ᇦ࡟ 7,8# ࡀ⤖ྜᚋࠊ7,8" ࡣ 7,8# ࡜ࡢ≀⌮ⓗ┦஫స⏝࡟ࡼࡗ࡚ᦆയ㒊఩࡬ࣜࢡ࣮ࣝࢺࡉࢀࡿࡇ࡜ࡀ᫂
ࡽ࠿࡟࡞ࡗࡓࠋḟ࡟ࠊ0,8" ཬࡧ 0,8#FGHH7 ኚ␗ᰴࡢ᰾ᵝయᵓ㐀࡟ࡘ࠸࡚⺯ග㢧ᚤ㙾ࢆ⏝࠸࡚ほᐹࡋࡓ⤖ᯝࠊ࠸ࡎࢀࡢኚ␗ᰴ࡟࠾࠸࡚ࡶࠊ࣐࢖ࢺ࣐࢖ࢩࣥ @ ࡢฎ⌮ᚋ࡟ᑠࡉ࡞᰾ᵝయࡀከᩘほᐹࡉࢀࡓࠋࡇࡢࡼ࠺࡞ᑠࡉ࡞᰾ᵝయࡢฟ⌧ࡣࠊ㔝⏕ᆺ⣽⬊࡟࠾࠸࡚ࡣほᐹࡉࢀ࡞࠸ࡇ࡜ࡸࠊ኱⭠⳦ࢤࣀ࣒ୖ࡟୍⟠ᡤᏑᅾࡍࡿ」〇㛤ጞⅬ௜㏆ࢆ⺯ගᶆ㆑ࡋࡓሙྜ࡟ࠊᑠࡉ
࡞᰾ᵝయ࡟ࡣᶆ㆑ࡉࢀ࡞࠸ࡶࡢࡀከᩘᏑᅾࡋࡓࡇ࡜࠿ࡽࠊࡇࢀࡽࡣ !56 ࡟ࡼࡗ࡚ษ᩿ࡉࢀ
ࡓ᰾ᵝయ᩿∦࡛࠶ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ௨ୖࡢࡇ࡜࠿ࡽࠊ7,8" ࡣ 7,8# ࡜ࡢ≀⌮ⓗ࡞┦஫స⏝ࢆ௓ࡋ࡚᰾ᵝయ࡬ᒁᅾࡋࠊ!56 ࡀ⏕ࡌࡓ᰾ᵝయᵓ㐀ࡢ⥔ᣢ࡟࡜ࡗ࡚㔜せ࡞ᙺ๭ࢆᯝࡓ
ࡋ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗࡓࠋ௨ୖࡢ⤖ᯝ࠿ࡽࠊ7,8" ࢱࣥࣃࢡ㉁ࡣ᰾ᵝయୖ࡛ !56 ࡀ⏕ࡌࡓ௜㏆࡟ᒁᅾࡋࠊ!56 ࡢ୧ᮎ➃࡟⤖ྜࡋࡓ 7,8# ࡜」ྜయࢆᙧᡂࡍࡿࡇ࡜࡟ࡼࡗ࡚᰾
ᵝయᵓ㐀ࡢᏳᐃᛶࢆಖ㞀ࡍࡿ࡜ඹ࡟ࠊ7,8# ࡟ࡼࡿ┦ྠ⤌᥮࠼཯ᛂࡢಁ㐍࡟㛵୚ࡋ࡚࠸ࡿ࡜
⪃࠼ࡽࢀࡿࠋ$$㸰㸧៏ᛶⓗ࡞⣸እ⥺ᦆയࢫࢺࣞࢫ࡟ᑐࡍࡿ⪏ᛶ⋓ᚓࡢศᏊᶵᵓ$⣸እ⥺࡟ࡼࡗ࡚⏕ࡌࡿࣆ࣑ࣜࢪࣥ஧㔞యࡣ !"# 」〇ࡸ㌿෗ࢆ㜼ᐖࡍࡿࡓࡵࠊ⏕≀ࡣࣆ࣑ࣜࢪࣥ஧㔞యࢆಟ᚟࡛ࡁࡿࢾࢡࣞ࢜ࢳࢻ㝖ཤಟ᚟㸦"K7㸧ᶵᵓࡢ࡯࠿ࠊ!"# 」〇㜼ᐖࢆゎᾘࡍࡿ !"#$ ┦ྠ⤌᥮࠼ཬࡧᦆയࢺࣞࣛࣥࢫᶵᵓࢆᣢࡗ࡚࠸ࡿࠋࡇࢀࡲ࡛ࡢ◊✲࠿ࡽࠊᡃࠎ
ࡣ⮬↛⎔ቃ࡛ၥ㢟࡜࡞ࡿ៏ᛶⓗప⥺㔞⋡ࡢ⣸እ⥺↷ᑕ$ >8=0(2;8$+(L-'(.,$MN4$@OMNA$ ୗ࡛㓝ẕ⣽⬊ࢆᇵ㣴ྍ⬟࡞⿦⨨ࢆస〇ࡋࠊ៏ᛶⓗ࡞ !"# ᦆയࢫࢺࣞࢫ࡟ᑐࡍࡿ⪏ᛶᶵᵓࡢゎ᫂ࢆ⾜ࡗ࡚ࡁࡓࠋᮏᐇ㦂⣔ࢆ⏝࠸ࡓ◊✲࠿ࡽࠊᚑ᮶ࡢ▷᫬㛫ࡢᛴᛶ⣸እ⥺↷ᑕ࡛ࡣ᭱ࡶ㧗࠸⮴Ṛᛶࢆ♧ࡋࡓ "K7 ⤒㊰ࡢḞᦆᰴࡀ @OMN ⎔ቃ࡛ࡣቑṪ㜼ᐖࢆᘬࡁ㉳ࡇࡉ࡞࠸୍᪉࡛ࠊ
!"# ᦆയࢺࣞࣛࣥࢫ⤒㊰ࡢḞᦆᰴࡀቑṪ㜼ᐖࢆᘬࡁ㉳ࡇࡍࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓ PAࠋࡉࡽ࡟ࠊ
"K7 ࡢḞᦆᰴ࡛ࡣ @OMN ⎔ቃୗ㸦ࠥPQ ᫬㛫㸧ࡢ⣽⬊ቑṪ࡟కࡗ࡚ &ń7 ᆺⅬ✺↛ኚ␗ࡢ㢖ᗘࡀ㌿෗ࡢ㗪ᆺ㙐≉␗ⓗ࡟㢧ⴭ࡟ቑ኱ࡍࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓ GAࠋ௒ᅇࠊ"K7 Ḟᦆᰴ$
>UDG¨Aࢆ⏝࠸࡚ࠊ@OMN ⎔ቃୗ࡛⥅௦ᇵ㣴࡟ࡼࡿ㛗᫬㛫㸦ࠥ㸴᪥㛫㸧࡟Ώࡿ✺↛ኚ␗㢖ᗘࡢᐃࢆ⾜ࡗࡓ࡜ࡇࢁࠊᇵ㣴㛤ጞᚋᩘ᪥ࡣ✺↛ኚ␗㢖ᗘࡢ㢧ⴭ࡞ୖ᪼ࡀほᐹࡉࢀࡓ୍᪉

࡛ࠊᇵ㣴㸱᪥┠௨㝆࡟✺↛ኚ␗㢖ᗘࡀᛴ⃭࡟పୗࡍࡿ࡜࠸࠺⯆࿡῝࠸⤖ᯝࡀᚓࡽࢀࡓࠋࡑ
ࡇ࡛ࠊ@OMN ⎔ቃ࡛⥅௦ᇵ㣴ࡋࡓ 01'EQ¨⣽⬊ࡢᰁⰍయ !"# ࢆࣃࣝࢫࣇ࢕࣮ࣝࢻࢤࣝ㟁ẼὋື࡟ࡼࡗ࡚ศ㞳ࡋࡓࠋࡑࡢ⤖ᯝࠊྠࡌ⣽⬊ᩘࡢࢤࣀ࣒ !"# ࢆࢧࣥࣉࣝ࡜ࡋ࡚౑⏝ࡋ࡚࠸ࡿ࡟ࡶ࠿࠿ࢃࡽࡎࠊᇵ㣴㛤ጞ᫬࡟ẚ࡭ࠊ㸱᪥┠௨㝆ࡢᰁⰍయࡢࣂࣥࢻᙉᗘࡀ᫂ࡽ࠿࡟ᙉ࠸
ࡇ࡜ࡀࢃ࠿ࡾࠊ⣽⬊࠶ࡓࡾࡢ !"# ྵ㔞ࡢቑຍࡀ㉳ࡇࡗ࡚࠸ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋࡑࡇ࡛⥅௦ᇵ㣴᫬ࡢ⣽⬊ࡢ !"# ྵ㔞ࢆ C#@5 ゎᯒ࡟ࡼࡾㄪ࡭ࡓ࡜ࡇࢁࠊ㸱᪥┠௨㝆࡟஧ಸయ໬ࡋ
ࡓ⣽⬊ࡢ๭ྜࡀ㢧ⴭ࡟ቑ኱ࡍࡿࡇ࡜ࢆぢ࠸ࡔࡋࡓࠋࡋࡓࡀࡗ࡚ࠊ✺↛ኚ␗㢖ᗘࡢᛴ⃭࡞పୗࡣࠊ⣽⬊ࡢ஧ಸయ໬㸦㑇ఏᏊࢆ㸰ࢥࣆ࣮ᣢࡘࡇ࡜㸧ࢆ཯ᫎࡋࡓࡶࡢ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡓࠋ
ࡉࡽ࡟ࠊ୍ಸయཬࡧ஧ಸయ UDGƦ ⣽⬊ࡢ @OMN ឤཷᛶࢆẚ㍑ࡋࡓ࡜ࡇࢁࠊ஧ಸయ⣽⬊ࡀ
᭷ព࡟ @OMN ࡟ᑐࡋ࡚⪏ᛶࢆ♧ࡍࡇ࡜ࡀࢃ࠿ࡗࡓࠋࡑࡇ࡛஧ಸయ⣽⬊ࡢฟ⌧࡜ @OMN ⪏ᛶ⋓ᚓࡢ㛵㐃ᛶࢆㄪ࡭ࡿࡓࡵ࡟ࠊ!"# ᦆയᛂ⟅࡟㛵ࢃࡿ㑇ఏᏊࢆḞᦆࡋࡓ UDGƦ ⣽⬊ࢆస34

〇ࡋࡓࠋࡑࡢ⤖ᯝࠊ┦ྠ⤌᥮࠼ಟ᚟࡟㛵ࢃࡿ 7#!RE ࢆḞᦆࡋࡓ 01'EQ¨$01'RE¨⣽⬊ࡣࠊ஧ಸయ≉␗ⓗ࡞ @OMN ⪏ᛶ⬟ࡀኻࢃࢀ࡚࠾ࡾࠊ@OMN ࢫࢺࣞࢫ࡟ࡼࡿ஧ಸయ⣽⬊ࡢฟ⌧ࡶほᐹࡉࢀ࡞࠿ࡗࡓࠋ௨ୖࡢ⤖ᯝ࠿ࡽࠊ୍ಸయ⣽⬊࡟ẚ࡭࡚஧ಸయ⣽⬊ࡀ @OMN ࢫࢺࣞࢫ⎔ቃ࡟ᑐࡋ࡚⪏ᛶ࡛࠶ࡾࠊ஧ಸయ≉␗ⓗ࡞ @OMN ⪏ᛶࡢ⋓ᚓ࡟ࡣ┦ྠ⤌᥮࠼ᶵᵓࡀ㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗࡓࠋᡃࠎࡢ௨๓ࡢ◊✲࠿ࡽࠊ@OMN ⎔ቃ࡛ࡣ」〇㜼ᐖࡀ㢖⦾࡟⏕ࡌ࡚࠸ࡿࡇ࡜ࡀ♧ࡉࢀ࡚࠾ࡾࠊ௒ᅇࠊ஧ಸయ⣽⬊ࡀ @OMN ࡟ᑐࡋ࡚⪏ᛶࢆ♧ࡍཎᅉ࡜ࡋ࡚ࠊ
」〇㜼ᐖࡢゎᾘ࡟┦ྠᰁⰍయ㛫ࡢ⤌᥮࠼཯ᛂࡀ㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀ⪃࠼ࡽࢀࡿࠋ$$%ࡲ࡜ࡵ&$!"# ┦ྠ⤌᥮࠼ࡣࠊ⮴Ṛⓗ࡞ᙳ㡪ࢆཬࡰࡍ !56 ࡢಟ᚟ࡸ !"# 」〇㜼ᐖࡢゎᾘ࡟࠾࠸࡚㔜せ࡞ᙺ๭ࢆᯝࡓࡋ࡚࠾ࡾࠊࡑࡢไᚚᶵᵓࡢ◚⥢ࡣ㌿ᗙࡸ㔜」ࠊᰁⰍయ෌⦅ࠊᰁⰍయ႙ኻ
࡞࡝ࡢᰁⰍయ୙Ᏻᐃᛶࡢཎᅉ࡜࡞ࡗ࡚࠸ࡿࠋ
௒ᅇᡃࠎࡣࠊ
኱⭠⳦ཬࡧฟⱆ㓝ẕࢆ⏝࠸࡚ !"#┦ྠ⤌᥮࠼ࡢไᚚ࣓࢝ࢽࢬ࣒࡟㛵ࡋ࡚ヲ⣽࡞ゎᯒࢆ⾜࠸ࠊࡑࢀࡒࢀ !56 ಟ᚟ཬࡧ」〇㜼ᐖࡢゎᾘ࡟࠾ࡅࡿ !"# ┦ྠ⤌᥮࠼ࡢ᪂ࡓ࡞ᙺ๭ࢆ᫂ࡽ࠿࡟ࡋࡓࠋ኱⭠⳦ࡢ◊✲࡛ࡣࠊ5?@ࣇ࢓࣑࣮ࣜ࡟ᒓࡍࡿ 7,8" ࢱࣥࣃࢡ㉁ࡀ 7,8# ࡟౫Ꮡࡋ࡚ !56 㒊఩࡟ࣜࢡ࣮ࣝࢺࡉࢀࠊ
!56
࿘㎶ࡢ᰾ᵝయ㸦ᰁⰍయ㸧ᵓ㐀ࢆᏳᐃ໬ࡍࡿࡇ࡜࡛┦ྠ⤌᥮࠼ಟ᚟ࡢಁ㐍࡟㛵୚ࡋ࡚࠸ࡿࡇ
࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋཎ᰾⏕≀࡟ࡣ┿᰾⏕≀ࡢࢥࣄ࣮ࢩࣥࡢࡼ࠺࡞」〇ᚋࡢጜጒᰁⰍయ㛫ࢆ
᮰ࡡࡿ 5?@ ࢱࣥࣃࢡ㉁ࡀᏑᅾࡋ࡞࠸ࡇ࡜࠿ࡽࠊ!56 ࡀ⏕ࡌࡓሙྜࡢ !"# ᮎ➃㒊ࡢ✵㛫ⓗ࡞ไᚚᶵᵓ࡟ࡘ࠸࡚ࡣ୙࡛᫂࠶ࡗࡓࠋ௒ᅇࡢ⤖ᯝࡣࠊ7,8" ࡀ !56 ಟ᚟࡟࠾࠸࡚≉␗ⓗ࡟ᶵ⬟ࡍࡿ 5?@ ࢱࣥࣃࢡ㉁࡛࠶ࡾࠊ!56 ࿘㎶ࡢᰁⰍయືែไᚚᶵᵓ࡟㔜せ࡞ᙺ๭ࢆᯝࡓ
ࡋ࡚࠸ࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓⅬࡣᴟࡵ࡚㔜せ࡞Ⓨぢ࡛࠶ࡿࠋࡲࡓࠊฟⱆ㓝ẕࢆ⏝࠸ࡓ◊✲࠿ࡽࡣࠊ៏ᛶⓗ࡞⣸እ⥺ࢫࢺࣞࢫ⎔ቃ࡟࠾࠸࡚ࠊ⣽⬊ࡣᰁⰍయࡢಸᩘᛶࢆኚ໬ࡉࡏࡿࡇ࡜
࡛ࢫࢺࣞࢫ⪏ᛶࢆ⋓ᚓࡋ࡚࠸ࡿ࡜࠸࠺⯆࿡῝࠸⌧㇟ࢆ᫂ࡽ࠿࡟ࡋࡓࠋࡉࡽ࡟ࠊࡇࡢ⪏ᛶ⋓
ᚓ࡟ࡣ !"# ┦ྠ⤌᥮࠼ᶵ⬟ࡀᚲ㡲࡛࠶ࡿࡇ࡜ࡀࢃ࠿ࡗࡓࠋಸᩘయ໬ࡣࠊࣄࢺࢆྵࡵࡓከࡃ
ࡢ⏕≀࡟࠾࠸࡚ほᐹࡉࢀࡿᬑ㐢ⓗ࡞⌧㇟࡛࠶ࡾࠊ⏕≀✀ࡲࡓࡣ⤌⧊ࡢ✀㢮ẖ࡟ࡑࡢ⏕⌮ⓗ࡞ព⩏ࡣ␗࡞ࡗ࡚࠸ࡿࡶࡢࡢࠊඹ㏻ࡍࡿᴫᛕ࡜ࡋ࡚ࠊಸᩘయ໬ࡣከᵝᛶࢆ⏕ࡴཎືຊ࡜࡞
ࡗ࡚࠸ࡿࡇ࡜ࡀ⪃࠼ࡽࢀࡿࠋࡋࡓࡀࡗ࡚ࠊ៏ᛶⓗ࡞⣸እ⥺ࢫࢺࣞࢫ࡟ᑐࡍࡿಸᩘయ໬ࡢ⌧㇟ࡣࠊࢫࢺࣞࢫ⪏ᛶࡲࡓࡣ⎔ቃ㐺ᛂᛂ⟅ࡢ୍ࡘ࡜⪃࠼ࡿࡇ࡜ࡀ࡛ࡁࡿࠋࡉࡽ࡟ࠊಸᩘయ໬
ࢆྵࡵࡓᰁⰍయ୙Ᏻᐃᛶࡢቑ኱ࡣࣄࢺࡢࡀࢇ⣽⬊࡟࠾࠸࡚㢧ⴭ࡟ほᐹࡉࢀࡿ≉ᚩࡢ୍ࡘ࡜
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8+1:U-:,';1/,'$;218/;]1/;(2$(<$!21#S$
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7,8#$U0(/,;2$0,80);/.$./0)8/)01+$:1;2/,2128,$(<$8=0(:(.(:,.$>5?@A-+;3,$7,8"$
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21.8,2/$!"#$./012'.$12'$,a);U10/;/;(2;2I$(<$8=0(:(.(:,.$;2$KS$8(+;S$@,++$7,U(0/.$QZ$YXR-YYRS$$%⥲ㄝ➼&$ES ⳻⏣ ༟㸦PHER㸧ࢤࣀ࣒ࢆྖࡿ࢖ࣥࢱ࣮࣓࢔㸸㠀ࢥ࣮ࢻ !"# 㡿ᇦࡢ !"# ᦆയᛂ⟅㸦໬
ᏛྠேࠊUU-EbQ㸧$PS ⳻⏣ ༟㸦PHER㸧ࣀ࣮࣋ࣝ໬Ꮫ㈹㸸!"# ಟ᚟ࡢศᏊ࣓࢝ࢽࢬ࣒ࡢゎ᫂㸦ࣃࣜࢸ࢕ࠊ
N(+SGH$"(SEPZ$UUQG-QR㸧$$$%ཱྀ㢌Ⓨ⾲&$ES ẟ㇂ᮧ㈼ྖࠊஂಖ⏣ె஀ࠊோᮌᏹ඾ࠊ⳻⏣ ༟ !"# ஧㔜㙐ษ᩿ಟ᚟࡟㛵୚ࡍࡿ 5?@ࣇ࢓࣑࣮ࣜࢱࣥࣃࢡ㉁ 7,8" ࡢᶵ⬟ゎᯒࠋ➨ EH ᅇ PE ୡ⣖኱⭠⳦◊✲఍㸦PHEG ᖺ ᭶ PH-PE ᪥ࠊ㟼ᒸ㸧$PS ẟ㇂ᮧ㈼ྖࠊ᪂஭ᗣኴࠊ⳻⏣ ༟ ┦ྠᰁⰍయ㛫ࡢ !"# ┦ྠ⤌᥮࠼ไᚚ࣓࢝ࢽࢬ࣒
ࡢゎᯒࠋ➨ XR ᅇ᪥ᮏ㑇ఏᏛ఍㸦PHEG ᖺ Y ᭶ EY-PE ᪥ࠊ៞ᛂ⩏ሿ኱Ꮫ᪥ྜྷ࢟ࣕࣥࣃࢫ㸧$GS ẟ㇂ᮧ㈼ྖࠊ᪂஭ᗣኴࠊ⳻⏣ ༟ ┦ྠᰁⰍయ㛫ࡢ⤌᥮࠼࡟࠾ࡅࡿฟⱆ㓝ẕ 50.P ࣮࣊ࣜ࢝ࢮࡢᙺ๭ࠋ➨ PP ᅇ !"# 」〇࣭⤌᥮࠼࣭ಟ᚟࣮࣡ࢡࢩࣙࢵࣉ㸦PHEG ᖺ EE ᭶ PH-PP᪥ࠊ௝ྎ㸧$
QS ẟ㇂ᮧ㈼ྖࠊ㔠Ꮚెᶞࠊோᮌᏹ඾ࠊ⳻⏣ ༟ !"# ஧ᮏ㙐ษ᩿ಟ᚟࡟㛵୚ࡍࡿ 7,8"
ࡢ !"# ┦஫స⏝࣓࢝ࢽࢬ࣒ࡢゎᯒࠋ➨ EE ᅇ PE ୡ⣖኱⭠⳦◊✲఍㸦PHEQ ᖺ  ᭶ R-᪥ࠊ┒ᒸ㸧$RS ྜྷ⏣㯞⨾ࠊẟ㇂ᮧ㈼ྖࠊ⳻⏣ ༟ !"# ᦆയࢫࢺࣞࢫᛂ⟅࡟㛵୚ࡍࡿࣄࢫࢺࣥኚ␗య

36ࡢ⥙⨶ⓗゎᯒࠋ➨  ᅇ᪥ᮏ㑇ఏᏛ఍㸦PHEQ ᖺ Y ᭶ Eb-EY ᪥ࠊ⁠㈡㸧$⳻⏣ ༟ ៏ᛶⓗ࡞⣸እ⥺ᦆയࢫࢺࣞࢫ࡟ᑐࡍࡿ⪏ᛶ⋓ᚓ࡟㛵୚ࡍࡿࢾࢡࣞ࢜ࢯ
࣮࣒ᵓ㐀ࠋ➨ Rb ᅇ᪥ᮏᨺᑕ⥺ᙳ㡪Ꮫ఍㸦PHEQ ᖺ EH ᭶ E-G ᪥ࠊ㮵ඣᓥ㸧$$bS ẟ㇂ᮧ㈼ྖࠊ⳻⏣ ༟ ┦ྠᰁⰍయ㛫ࡢ⤌᥮࠼ไᚚᶵᵓ࡟࠾ࡅࡿฟⱆ㓝ẕ 50.P ࡢᙺ๭ࠋ➨ GP ᅇᰁⰍయ࣮࣡ࢡࢩࣙࢵࣉ࣭➨ EG ᅇ᰾ࢲ࢖ࢼ࣑ࢡࢫ◊✲఍㸦PHEQ ᖺ EP ᭶ ER-Eb
᪥ࠊᗈᓥ㸧$XS ẟ㇂ᮧ㈼ྖࠊᡞ⩚࠶ࡍ⨾ࠊோᮌᏹ඾ࠊ⳻⏣ ༟ ஧ᮏ㙐 !"# ࡢษ᩿ಟ᚟࡟㛵୚ࡍࡿ
7,8" ࡢᶵ⬟ゎᯒࠋ➨ EE ᅇ PE ୡ⣖኱⭠⳦◊✲఍㸦PHER ᖺ  ᭶ Q-R ᪥ࠊ⁠㈡㸧$YS ▼ẟ኱㍜ࠊẟ㇂ᮧ㈼ྖࠊ㛗㇂ᕝࡺࡁࠊᒾ㷂༤ྐࠊ⳻⏣ ༟ ฟⱆ㓝ẕ࡟࠾ࡅࡿ」〇ࢫࢺࣞࢫᛂ⟅࡟㛵ࢃࡿ᪂ࡓ࡞ᅉᏊࡢ᥈⣴ࠋ➨ Xb ᅇ᪥ᮏ㑇ఏᏛ఍
㸦PHER ᖺ Y ᭶ PQ- ᪥ࠊᮾ໭኱Ꮫᕝෆ໭࢟ࣕࣥࣃࢫ㸧$
EHS ሷධᣅ㤿ࠊẟ㇂ᮧ㈼ྖࠊ⏣୰ಟᖹࠊ⳻⏣ ༟ ៏ᛶⓗ࡞ప⥺㔞⋡⣸እ⥺↷ᑕୗ࡟࠾ࡅ
ࡿฟⱆ㓝ẕ "K7 Ḟᦆᰴࡢືែゎᯒࠋ➨ Xb ᅇ᪥ᮏ㑇ఏᏛ఍㸦PHER ᖺ Y ᭶ PQ- ᪥ࠊᮾ໭኱Ꮫᕝෆ໭࢟ࣕࣥࣃࢫ㸧$
EES ẟ㇂ᮧ㈼ྖࠊ⳻⏣ ༟ ฟⱆ㓝ẕ 50.P ࣮࣊ࣜ࢝ࢮ࡜ ?).XE-?:.Q ࢾࢡࣞ࢔࣮ࢮ࡟ࡼ
ࡿ┦ྠᰁⰍయ㛫ࡢ⤌᥮࠼ไᚚᶵᵓࠋ➨ PG ᅇ !"# 」〇࣭⤌᥮࠼࣭ಟ᚟࣮࣡ࢡࢩࣙࢵࣉ
㸦PHER ᖺ EH ᭶ EY-PE ᪥ࠊ㟼ᒸ㸧$$%࣏ࢫࢱ࣮Ⓨ⾲&$
EPS Ḉᮏ೺㑻ࠊẟ㇂ᮧ㈼ྖࠊ⳻⏣ ༟ ฟⱆ㓝ẕࢆ⏝࠸ࡓࢪ࣮ࣥࢱ࣮ࢤࢸ࢕ࣥࢢ࡟㛵୚ࡍࡿ᪂つ㑇ఏᏊࡢ᥈⣴ࠋ➨ PP ᅇ !"# 」〇࣭⤌᥮࠼࣭ಟ᚟࣮࣡ࢡࢩࣙࢵࣉ㸦PHEG ᖺ EE
᭶ PH-PP ᪥ࠊ௝ྎ㸧$
EGS Ḉᮏ೺㑻ࠊẟ㇂ᮧ㈼ྖࠊ⳻⏣ ༟ ฟⱆ㓝ẕࢆ⏝࠸ࡓࢪ࣮ࣥࢱ࣮ࢤࢸ࢕ࣥࢢ࡟㛵୚ࡍࡿ᪂つᅉᏊࡢྠᐃࠋ➨  ᅇ᪥ᮏศᏊ⏕≀Ꮫ఍ᖺ఍㸦PHEG ᖺ EP ᭶ G- ᪥ࠊ⚄ᡞ㸧$
EQS ẟ㇂ᮧ㈼ྖࠊஂಖ⏣ె஀ࠊᑠᯘ୓ᕼᏊࠊ㔠Ꮚెᶞࠊோᮌᏹ඾ࠊ⳻⏣ ༟ 5?@ ࣇ࢓࣑࣮ࣜࢱࣥࣃࢡ㉁ 7,8" ࡢ !"# ஧㔜㙐ษ᩿ಟ᚟࡟࠾ࡅࡿᙺ๭ࠋ➨  ᅇ᪥ᮏศᏊ⏕≀Ꮫ
఍ᖺ఍㸦PHEG ᖺ EP ᭶ G- ᪥ࠊ⚄ᡞ㸧$
ERS ẟ㇂ᮧ㈼ྖࠊ᪂஭ᗣኴࠊ⳻⏣ ༟ 7(+,.$ (<$ /=,$ 5188=10(:T8,.$ 8,0,];.;1,$ 50.P$=,+;81.,$;2$;2/,0-=(:(+(I),$0,8(:*;21/;(2$0,U1;0ࠋ➨ Y ᅇ G7 ࢩ࣏ࣥࢪ࣒࢘㸦PHEQ
ᖺ EE ᭶ Eb-PE ᪥ࠊ㟼ᒸ㸧$ 㔜᳃ ᝆࠊẟ㇂ᮧ㈼ྖࠊ⳻⏣ ༟ ฟⱆ㓝ẕ ?I.E ࢱࣥࣃࢡ㉁ࡢᶵ⬟ࢻ࣓࢖ࣥࡢゎᯒࠋ➨ Gb ᅇ᪥ᮏศᏊ⏕≀Ꮫ఍ᖺ఍㸦PHEQ ᖺ EE ᭶ PR-Pb ᪥ࠊᶓ὾㸧$
EbS ྜྷ⏣㯞⨾ࠊẟ㇂ᮧ㈼ྖࠊ⳻⏣ ༟ !"# ᦆയࢫࢺࣞࢫᛂ⟅࡟ᙳ㡪ࢆཬࡰࡍࣄࢫࢺࣥኚ
␗యࡢゎᯒࠋ➨ Gb ᅇ᪥ᮏศᏊ⏕≀Ꮫ఍ᖺ఍㸦PHEQ ᖺ EE ᭶ PR-Pb ᪥ࠊᶓ὾㸧$
EXS [,T1:)01Z$[SZ$12'$+LVKLGD7 7,I)+1/;(2$(<$;2/,0-=(:(+(I),$0,8(:*;21/;(2$*T$/=,$50.P$=,+;81.,$;2$ 5188=10(:T8,.$8,0,];.;1,S$ ࢤࣀ࣒ࢆᨭ࠼ࡿ㠀ࢥ࣮ࢻ !"# 㡿ᇦࡢᶵ⬟㸦PHER ᖺ X ᭶ b-X ᪥ࠊῐ㊰㸧$
EYS ▼ẟ኱㍜ࠊẟ㇂ᮧ㈼ྖࠊ㛗㇂ᕝࡺࡁࠊᒾ㷂༤ྐࠊ⳻⏣ ༟ ฟⱆ㓝ẕ࡟࠾ࡅࡿ」〇ࢫࢺࣞࢫᛂ⟅࡟㛵ࢃࡿ᪂ࡓ࡞ᅉᏊࡢ᥈⣴ࠋ➨ PG ᅇ !"# 」〇࣭⤌᥮࠼࣭ಟ᚟࣮࣡ࢡࢩࣙ
ࢵࣉ㸦PHER ᖺ EH ᭶ EY-PE ᪥ࠊ㟼ᒸ㸧$37

PHS 㛗㇂ᕝࡺࡁࠊẟ㇂ᮧ㈼ྖࠊ㔜᳃ᝆࠊ⳻⏣༟ 」〇ࢫࢺࣞࢫᛂ⟅࡟࠾ࡅࡿฟⱆ㓝ẕ

?I.E ࡢᙺ๭ࠋ➨ PG ᅇ !"# 」〇࣭⤌᥮࠼࣭ಟ᚟࣮࣡ࢡࢩࣙࢵࣉ㸦PHER ᖺ EH ᭶ EY-PE᪥ࠊ㟼ᒸ㸧$PES ẟ㇂ᮧ㈼ྖࠊ⳻⏣ ༟ ┦ྠᰁⰍయ㛫ࡢ⤌᥮࠼ไᚚ࣓࢝ࢽࢬ࣒ࡢゎᯒࠋ➨ GX ᅇ᪥ᮏศᏊ⏕≀Ꮫ఍ᖺ఍࣭➨ XX ᅇ᪥ᮏ⏕໬Ꮫ఍኱఍ྜྠ኱఍㸦PHER ᖺ EP ᭶ E-Q ᪥ࠊ⚄ᡞ㸧$
PPS ,VKLJH 'SZ$ [,T1:)01Z$ [S +DVHJDZD < ,ZDVDNL + DQG +LVKLGD 7$ #$ I,2,/;8$.80,,2$<(0$I,2,.$/=1/$.)UU0,..$/=,$/,:U,01/)0,$.,2.;/;];/T$(<$:I.E-UDG¨F,++.$;2$5188=10(:T8,.$8,0,];.;1,S ➨ GX ᅇ᪥ᮏศᏊ⏕≀Ꮫ఍ᖺ఍࣭➨ XX ᅇ᪥ᮏ⏕໬Ꮫ఍኱఍ྜྠ኱఍㸦PHER ᖺ EP ᭶ E-Q ᪥ࠊ⚄ᡞ㸧$$38

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 41, pp. 29229 –29237, October 11, 2013
© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

RecA Protein Recruits Structural Maintenance of
Chromosomes (SMC)-like RecN Protein to DNA Double-strand
Breaks*□S

Received for publication, May 14, 2013, and in revised form, August 21, 2013 Published, JBC Papers in Press, August 25, 2013, DOI 10.1074/jbc.M113.485474

Kenji Keyamura‡1, Chikako Sakaguchi§1, Yoshino Kubota§, Hironori Niki¶, and Takashi Hishida‡§2
From the ‡Department of Life Science, Graduate School of Science, Gakushuin University, Tokyo 171-8588, the §Research Institutefor Microbial Diseases, Osaka University, Osaka 565-0871, and the ¶Microbial Genetics Laboratory, Genetic Strains ResearchCenter, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan

Escherichia coli RecN is an SMC (structural maintenance ofchromosomes) family protein that is required for DNA doublestrand break (DSB) repair. Previous studies show that GFPRecN forms nucleoid-associated foci in response to DNA damage, but the mechanism by which RecN is recruited to the
nucleoid is unknown. Here, we show that the assembly of GFPRecN foci on the nucleoid in response to DNA damage involves
a functional interaction between RecN and RecA. A novel RecA
allele identified in this work, recAQ300R, is proficient in SOS
induction and repair of UV-induced DNA damage, but is deficient in repair of mitomycin C (MMC)-induced DNA damage.
Cells carrying recAQ300R fail to recruit RecN to DSBs and accumulate fragmented chromosomes after exposure to MMC. The
ATPase-deficient RecNK35A binds and forms foci at MMC-induced DSBs, but is not released from the MMC-induced DNA
lesions, resulting in a defect in homologous recombination-dependent DSB repair. These data suggest that RecN plays a crucial role in homologous recombination-dependent DSB repair
and that it is required upstream of RecA-mediated strand
exchange.

Escherichia coli, and E. coli HR is a paradigm for understanding
HR-related processes in all organisms (3–5).
In E. coli, the repair of DSBs is initiated by RecBCD, which
generates 3! single-stranded DNA (ssDNA) tails at DSB sites via
its helicase and nuclease activities; the ssDNA tails are thensubstrates for homologous pairing by RecA protein (4, 6). The
RecF pathway is involved in the daughter strand gap repair inwild-type cells (3). RecF also provides an alternative pathway
for HR-dependent DSB repair in recBC mutants when two
additional nucleases, ExoI and SbcCD, are inactivated (7, 8).
However, recent studies suggest that the RecFOR pathway may
play a crucial role in DSB repair in bacterial species other thanE. coli (9 –13). In both pathways, RecA is loaded onto thessDNA tail to form a nucleoprotein filament at the DSB sites.
The RecA strand exchange activity generates recombinationintermediates via its strand exchange activity (4, 14), which arethen processed either by the Holliday junction resolvase,
RuvABC, or by the RecG helicase to produce mature products(15–17). In addition, RecA is essential for the induction of the
SOS response (18). RecA assembled on ssDNA stimulates selfcleavage of the LexA repressor, which in turn induces downstream SOS genes (19 –21).
Structural maintenance of chromosomes (SMC) proteins are
ubiquitous proteins that maintain and modulate chromosome
structure in prokaryotic and eukaryotic cells (22, 23). E. coli
RecN is a highly conserved, DNA damage-inducible SMC-likeprotein in bacteria that has two SOS boxes in its promoter
region. Therefore, the expression of recN is tightly regulated by
the LexA repressor (24 –26). GFP-RecN forms nucleoid-associated foci in response to DNA damage and forms aggregates in
the cytoplasm (27). RecN aggregates are then degraded by the
ClpXP protease (27, 28), which is required for efficient recovery
from DNA damage. E. coli recN mutants are highly sensitive toionizing radiation, bleomycin, and the DNA cross-linking agent
mitomycin C (MMC), but not to UV irradiation (29, 30).
Mutants of recN are defective in conjugational recombinationin recBC sbcBC strains (24), suggesting that RecN plays a role in
the RecF pathway. However, RecN is also required for RecBCD-

DNA double-strand breaks (DSBs)3 are serious genomic
lesions that are potentially lethal at the cellular level. DSBs arecaused by exogenous agents such as ionizing radiation, chemical mutagens, reactive oxygen species, and replicative stress (i.e.
collapsed replication forks) (1, 2). In bacteria, homologous
recombination (HR) plays a major role in repairing DSBs. HR
enzymes and pathways have been extensively characterized in* This work was supported by grants-in-aid for scientific research on priority

areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T. H.).
□S
This article contains supplemental Figs. S1–S3.
1
Both authors contributed equally to this work.
2
To whom correspondence should be addressed: Dept. of Life Science, Graduate School of Science, Gakushuin University, Tokyo 171-8588, Japan. Tel.:
81-3-3986-0221; E-mail: takashi.hishida@gakushuin.ac.jp.
3
The abbreviations used are: DSB, DNA double-strand break; HR, homologous recombination, MMC; mitomycin C, SMC, structural maintenance ofchromosomes; ECFP, enhanced cyan fluorescent protein.

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Background: RecN is an SMC (structural maintenance of chromosomes) family protein that is required for DNA doublestrand breaks (DSBs) repair.
Results: We identified a RecA mutant that is deficient in interacting with RecN.
Conclusion: A functional interaction between RecN and RecA is required for assembly of RecN at the sites of DSBs.
Significance: RecN is critical for protecting the structural integrity of chromosomes during DSBs repair.

Localization of RecN Protein to DNA Breaks
dependent repair of DSBs (31–33). Thus, RecN plays a specificrole in the repair of DNA DSBs, and its role is not limited to asingle branch or subpathway of HR.
In this study, we examine the mechanism by which RecN is
recruited to the nucleoid in response to DNA damage. We showthat a functional interaction between RecN and RecA is
required for assembly of RecN foci at MMC-induced DSBs;
conversely, conditions that abrogate or disrupt a stable RecNRecA interaction lead to chromosome fragmentation and lossof cell viability in cells exposed to MMC. The RecN ATPase is
not required for formation of RecN-DSB foci, but is required forrelease of RecN from DSBs and completion of RecA/HR-dependent DSB repair. These data demonstrate that the SMC-likeprotein RecN plays a crucial role in promoting RecA-dependent DSB repair.

EXPERIMENTAL PROCEDURESMedia and General Methods—Standard methods for E. coli
genetics and recombinant DNA techniques were as describedby Miller (34) and Sambrook et al. (35). Ampicillin (50 !g/ml),
tetracycline (10 !g/ml), chloramphenicol (100 !g/ml), andkanamycin (30 !g/ml) were used where indicated. Mitomycin
C (2.5 mg/ml) was dissolved in 10 mM Tris-HCl (pH 8.5) buffer.
Sensitivity to UV damage was measured as described previously(36). To measure sensitivity to MMC, cultures were grown in
LB broth to an A650 of "0.4, serially diluted, spotted onto LB
medium containing the indicated concentration of MMC, andincubated at 37 °C.
Bacterial Strains and Plasmids—Strains used in this study
were isogenic with BW25141 (37) except for strains with PBADI-SceI. Wild-type strains and deletion mutants were provided
by the National BioResource Project (NBRP) (38). The strains
carrying the PBAD-I-SceI were a gift from S. M. Rosenberg (39).
The strains carrying the inducible fluorescent repressor gene(araC PBAD-lacI-ecfp) and the lacO array were described previously (40). A fragment containing the SOS promoter andopen reading frame of recN was cloned into the low copy plasmid pSCH19, generating pRecN (27). RecN was tagged with anenhanced GFP cassette at its NH2 terminus to generate
pSG101. Arabinose-inducible pBAD GFP-recN (pTF271) wasconstructed as described previously (27). recNK35A was generated from pUC19-recN by site-directed PCR mutagenesis, andit was substituted for wild-type recN in pSG101 to generate
pSG105. The structures of recombinant plasmids were confirmed by DNA sequencing.
Isolation of recA Mutant—A library of recA mutants was generated by carrying out PCR-mediated random mutagenesis, as
described previously (36). The resultant recA mutant clones
were transformed into a recA strain. The transformants were
resuspended in M9 salts, plated on LB plates containing ampicillin, and then irradiated with UV (20 J/m2). After overnightincubation, UV-resistant colonies were replica-plated on LB
plates containing MMC (1 !g/ml). Clones that grew very poorly
or did not grow at all on the MMC plates were selected.
SOS Induction—SOS induction was assessed by measuring
the degradation of LexA as described previously (36). Log phasecultures were treated with MMC (0.5 !g/ml), and aliquots weretaken at multiple time points for immunoblot analysis with

29230 JOURNAL OF BIOLOGICAL CHEMISTRYRESULTS
RecA Is Required for the Formation of Nucleoid-associated
GFP-RecN Foci in MMC-treated Cells—When DNA is damaged or replication is inhibited, ssDNA-bound RecA becomes
conformationally active and promotes cleavage of the LexA
repressor, which results in the induction of SOS genes including recN (21, 44). Previously, we showed that GFP-RecN formedfoci on nucleoids after DNA damage (27). This implied that
RecN could be recruited to the nucleoid at a step after RecA is
loaded onto damaged DNA. To specifically examine thissequence of events, we measured GFP-RecN foci in a #recA
strain. Wild-type recN is a part of the SOS regulon, and its
expression is completely dependent on activated RecA. Therefore, this experiment was performed in cells that expressed
GFP-recN under control of the inducible PBAD promoter. Fig.
1A shows that arabinose-inducible GFP-recN fully complements the MMC sensitivity of #recN, when cells are grown in
the presence of arabinose, but not when cells are grown in thepresence of glucose. Furthermore, GFP-RecN foci form in thecytoplasm of both MMC-treated and untreated wild-type cells
and #recA cells, whereas nucleoid-associated GFP-RecN fociform in wild-type MMC-treated cells but are absent in #recA

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anti-LexA (BioAcademia), anti-RecA (BioAcademia), or antiRecN (26). LexA resynthesis was inhibited by adding chloramphenicol (100 !g/ml) to the cultures 10 min before adding
MMC.
Fluorescence Microscopy and Localization Analysis of
GFP-RecN—Exponentially growing cultures were treated with
0.5 !g/ml MMC at 37 °C. Cells were fixed with methanol,
stained with 1 !g/ml DAPI (4!, 6!-diamidino-2-phenylindole),
and spread (1–2 !l) on a cover glass. GFP fluorescence was notaffected by prior fixation of the sample. Fluorescence microscopy was performed on a Zeiss Axioplan2 (41). Scale bars of 5 or
10 !m are shown in the figures. Images of both nucleoids (blueas a color signal) and GFP-RecN foci (green as a color signal)
were merged on identical cells. In the merged image, GFPRecN foci localized on the nucleoid appeared as a light blue
color, whereas GFP-RecN foci in the cytoplasm appeared as agreen color. The localization of GFP-RecN foci was determinedbased on these visual criteria, and more than 150 individual
cells were scored for each strain.
To observe the ori1 loci (15 kbp distance from oriC) on chromosomes, we constructed wild-type and #recN strains carrying
both a lacO array inserted into the ori1 locus and an inducible
fluorescent repressor gene (araC PBAD-lacI-ecfp) (40, 42, 43).
Cells were grown at 37 °C for 90 min in LB medium containing
0.2% arabinose and 1 !g/ml MMC and then analyzed by fluorescence microscopy.
Effect of I-SceI-induced DSBs on the Localization of RecN—To
detect DSB-induced RecN foci, #recA strains carrying the
PBADI-SceI cassette in the chromosome and a single I-SceI recognition site at the codA21 locus in the F! episome (39) weretransformed with pSG101 (gfp-recN) and with pRecA or
pRecAQ300R. When cultures had reached early log phase, I-SceI
was induced by the addition of 0.2% arabinose (w/v). One hour
after the addition of arabinose, aliquots were taken and examined under the microscope.

Localization of RecN Protein to DNA Breaks

MMC-treated cells (Fig. 1, B and C). These results suggest that
RecA is required to recruit GFP-RecN to DNA damage sites in
MMC-treated cells.
Isolation of a recA Mutant That Mimics the Phenotype of#recN—Although RecA plays the central role in recombinational repair and is the master inducer of the SOS pathway, it isunclear what functions of RecA are required to recruit RecN to
the nucleoid in MMC-treated cells. The phenotype of #recA or
SOS-deficient lexA3 cells (45) differs from the phenotype of#recN cells; the former are hypersensitive to MMC and UV,
whereas the latter are hypersensitive to MMC but insensitive to
UV (Fig. 2A). Therefore, a library of recA mutants was generated and screened for mutants that provide resistance to UV
but confer sensitivity to MMC. Three candidate mutants wereisolated from "2,000 clones. All three mutants carry an arginine substitution at the highly conserved C-terminal Gln-300 of
RecA (Fig. 2B). Fig. 2C shows that this allele, recAQ300R, fullycomplements the UV sensitivity of #recA cells, but does notcomplement their MMC sensitivity. In the wild-type strain,
MMC-induced DNA damage leads to proteolytic cleavage of
LexA, the repressor of the SOS regulon, and induces the SOS
response (Fig. 2D). Similarly, recAQ300R is capable of inducing
expression of the SOS regulon and specific proteolytic cleavageof LexA in MMC-treated cells (Fig. 2D). Thus, recAQ300R is
proficient in the DNA damage-induced SOS response, and its
phenotype is similar to the phenotype of #recN.
Nucleoid Fragmentation in MMC-treated #recN andrecAQ300R Cells—To explore these results further, #recA,
recAQ300R, #recN, and #ruvB cells were stained with DAPI andexamined by fluorescence and phase-contrast microscopy forgenome integrity and cell morphology. Under conditions of
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FIGURE 2. Effect of recAQ300R on DNA repair and SOS response. A, sensitivity of cells to MMC and UV irradiation. 10-fold serial dilutions of the indicatedstrains were spotted on LB plates. DNA damage was induced by either MMC
or UV irradiation. B, Q300 is conserved in bacterial RecA orthologs. A map of
the E. coli RecA region between amino acids 290 and 305 is shown. Ec, E. coli;
Hi, H. influenzae; Bs, B.subtilis; Tt, Thermus thermophilus; Dr, D. radiodurans. C,
sensitivity of recAQ300R cells to MMC and UV irradiation was examined as in A.
D, RecAQ300R is proficient in SOS induction. Protein extracts from cells treatedwith MMC for the indicated times were prepared and analyzed by Western
blot using anti-RecA, anti-RecN, or anti-LexA antibodies. For the LexA degradation assay, chloramphenicol (100 !g/ml) was added at time 0 to inhibit
resynthesis of LexA protein.

exponential growth in the absence of MMC, all cells had a normal morphology, with two centrally located nucleoids per cell(Fig. 3A). Wild-type cells treated with MMC for 90 min became
highly filamented with elongated, evenly spaced nucleoids (Fig.
3A). This morphology is typical of SOS-activated cells (46). By
contrast, a large fraction of MMC-treated SOS-defective #recA
were anucleate, and filamentous cells were hardly detected (Fig.
3A). RuvABC resolvasome branch-migrates and resolves Holliday junctions, and inactivation of any of the three Ruv functions blocks resolution of recombinational repair intermediates(3). Therefore, RuvB plays a role in the later steps of HR. MMCtreated ruvB mutants formed both filamentous and anucleate
cells. As reported previously (47), the nucleoids of filamentous
ruvB cells were centrally located and little to no DNA migrated
to cell poles, which is in contrast to the morphology of filamentous wild-type cells (i.e. well partitioned nucleoids). This indicates that the accumulation of intermediates of HR-mediated
DSB repair results in chromosome nondisjunction and the production of anucleate cells. MMC-treated #recN and recAQ300R

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FIGURE 1. RecN foci in wild-type and !recA cells with or without DNA
damage. A, #recN cells carrying either an arabinose-inducible GFP-recN gene(pTF271) or a pBAD vector (pTF200) were diluted and spotted onto LB plateswith or without MMC (0.5 !g/ml) in the presence of either glucose or arabinose. B, the subcellular localization of GFP-RecN foci in response to MMCinduced damage. Wild-type or #recA cells carrying pTF271 were exposed to
MMC (0.5 !g/ml) followed by the addition of arabinose (0.05%, w/v) to induce
GFP-RecN. The panels show GFP/DAPI-merged images of cells 30 min after
the addition of arabinose. Scale bar indicates 2.5 !m. C, quantitative analysis
of GFP-RecN foci. For cells incubated with or without MMC, "150 cells were
examined. The results represent the average of at least three independentmeasurements. Error bars indicate S.D.

Localization of RecN Protein to DNA Breaks

fragments. For this purpose, oriC was labeled indirectly, via
LacI-ECFP (enhanced cyan fluorescent protein) bound to an
ectopic tandem array of Lac repressor-binding sites (240 $lacO) at the ori1 locus (15 kb counterclockwise of oriC) (40).
LacI-ECFP was expressed from the chromosomally integrated
gene under the control of the PBAD promoter. Wild-type and#recN cells were treated or not with MMC and visualized using
fluorescence microscopy. The results revealed 2– 4 ori1 foci percell in the majority of wild-type and #recN cells in the absenceof MMC. In these cells, all nucleoids contained at least one ori1
focus (Fig. 3C). When treated with MMC, the number of ori1
foci per nucleoid increased significantly in wild-type and #recN
cells, and the foci were distributed throughout the elongatednucleoid (Fig. 3C). Notably, %15% of #recN cells carried nucleoids lacking ori1 foci, whereas such nucleoids were infrequentcells were as filamentous as wild-type cells, but had an abnormal morphology characterized by multiple, short, diffuse
nucleoids (Fig. 3A). In wild-type cells, the number of nucleoids
per cell was largely unaffected by exposure to MMC, whereas
the number of nucleoids per cell increased when #recN andrecAQ300R cells were exposed to MMC (Fig. 3B). Furthermore,abnormal nucleoid morphology was not generally observed in
UV-irradiated #recN and recAQ300R cells (supplemental Fig.
S1). These results support the conclusion that recAQ300R is aphenocopy of #recN. Our interpretation of this result is that
RecN is dysfunctional in the recAQ300R mutant.
We hypothesized that the abnormal nucleoid morphology of
MMC-treated #recN cells might reflect the presence of unrepaired DSBs and chromosome fragmentation. Therefore, afluorescence-based method was used to visualize chromosome

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FIGURE 3. Morphological changes in MMC-treated wild-type, !recN, and recA cells. Exponentially growing cells were fixed and stained with DAPI andanalyzed by fluorescence microscopy. A, the panels show DAPI images of cells incubated for 90 min in the presence or absence of MMC (1 !g/ml). Nucleoids
are visualized as a light blue color. B, quantitative analysis of nucleoids. For cells with or without MMC-induced DNA damage, %200 cells were examined. The
results represent the average of at least three independent measurements. Error bars indicate S.D. C, localization of a LacI-ECFP to the nucleoid. The wild-type
and #recN strains carry an ectopic tandem array of lacO at ori1 (15 kb counterclockwise of oriC). The panels show merged images of LacI-ECFP (light blue) andnucleoids (dark blue). White arrows indicate nucleoids that fail to bind LacI-ECFP, and by implication, oriC-lacking nucleoids. D, quantification of cells lacking ori1
foci. Wild-type and #recN cells were treated with MMC for 90 min and examined by fluorescence microscopy. For cells with or without MMC-induced damage,%200 cells were examined.

Localization of RecN Protein to DNA Breaks

FIGURE 4. Nucleoid-associated RecN foci in response to MMC-induced
DNA damage. A, MMC damage-induced RecN foci in recAQ300R cells. Cells
carrying the SOS-inducible GFP-recN (pSG101) were exposed to MMC for 60
min. The panels show GFP/DAPI-merged images of cells. Scale bar indicates
2.5 !m. B, quantitative analysis of GFP-RecN foci. For cells with MMC damage,%150 cells were examined. The results represent the average of at least three
independent measurements. Error bars indicate S.D.

in wild-type cells (&1.4%) (Fig. 3D). These results demonstrate
the presence of aberrant nucleoids lacking oriC in MMCtreated #recN cells, which likely represent subchromosomal
fragments.
RecAQ300R Is Defective in Recruiting RecN to Nucleoids in
MMC-treated Cells—The results described above suggest that
RecAQ300R does not recruit RecN to the nucleoid, under conditions where wild-type RecA does so (i.e. in MMC-treatedwild-type cells). To explore this further, GFP-RecN foci were
quantified in MMC-treated #recA #recN cells expressing SOSinducible GFP-recN and either wild-type recA or recAQ300R.
After exposure to MMC for 60 min, %90% of wild-type cells
contained nucleoid-associated GFP-RecN foci (Fig. 4, A and B).
By contrast, &5% of cells expressing recAQ300R had nucleoidassociated GFP-RecN foci, whereas the number and fraction ofcells with cytoplasmic GFP-RecN foci was higher in cells
expressing recAQ300R than that in cells expressing wild-type
recA (Fig. 4, A and B). These results indicate that RecA is
required for the formation of MMC-induced, nucleoid-associated RecN foci and that RecAQ300R has a specific defect in thisfunction/role.
RecA Is Required to Recruit RecN to sites of DSBs—To examine the recruitment of RecN to a unique DSB site in RecAproficient cells, I-SceI was used to introduce a site-specific DSB
into a strain that carries the PBADI-SceI cassette on the chromosome and a single I-SceI recognition site on the F! episome (39).
Appropriately engineered cells were transformed with a plasmid expressing GFP-recN from its native SOS-inducible promoter, grown to early log phase and exposed to arabinose to
induce I-SceI. Control cells were grown in medium lacking
arabinose. VspI endonuclease digestion resulted in a 1.8-kb
fragment containing the I-SceI cleavage site. I-SceI digestion
produced two fragments, one of which with a size of 1.2 kb
hybridized to the site 1 probe (Fig. 5A). The kinetics of DSB
formation was monitored by Southern blot analysis of VspIdigested DNA isolated from samples taken at different times
after the addition of 0.2% arabinose or glucose to the culture. A
1.2-kb fragment was not detected when cells were maintained
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in glucose-containing medium, whereas it was detected within
30 min in wild-type cells proficient for RecBCD after the addition of arabinose (Fig. 5, A and B). The intensity of the 1.2-kb
fragments increased with time, reaching a maximum intensity"1 h after the addition of arabinose. Similar results wereobtained when the site 2 probe was used to detect the I-SceI
cleavage site (Fig. 5A and supplemental Fig. S2). One possible
explanation for the kinetics of DSB formation is that I-SceI
digestion is not synchronous in the entire population, and thebreaks may be repaired very efficiently. Thus, the only breaks
generated just before samples were taken might be detected bySouthern blotting. This is consistent with previous studies
using chromosomally integrated I-SceI site, where DSB products are readily detected in wild-type cells even after 1 h of
I-SceI induction (48).
Fig. 5C shows that nucleoid-associated GFP-RecN foci weredetected in cells that expressed I-SceI and carried an F! episomewith an I-SceI cleavage site. By contrast, GFP-RecN foci were
not observed when the same cells were grown in glucose-containing medium (to repress I-SceI) or if the cells did not carry an
I-SceI-sensitive F! episome (Fig. 5C). Furthermore, the numberof nucleoid-associated GFP-RecN foci was much lower inrecAQ300R mutant cells (&1%) than in cells expressing wild-type
recA (18%) (Fig. 5D). These results indicate that, in wild-type
cells, a single I-SceI-induced DSB induces an SOS response andpromotes the formation of nucleoid-associated GFP-RecN fociin a RecA-dependent manner.
RecN ATPase Activity Is Required for Release from Growth
Arrest in Cells with DNA Damage—RecN has a typical SMC
family protein domain structure, including an extensive, centrally located coiled-coil domain and globular N- and C-terminal domains with Walker A and Walker B nucleotide-binding
motifs, respectively (49). A previous study showed that substitution of Lys-35 with alanine in the Walker A motif resulted in
a complete loss of RecN DNA repair activity in vivo (50). Biochemical characterization of Deinococcus radiodurans RecN
showed that RecNK67A (an lysine-to-alanine substitution at
position 67, which corresponds to E. coli RecN Lys-35) abolished ATPase activity, but did not impair ATP binding in vitro(49). Fig. 6A shows that expression of recNK35A in a #recN background conferred sensitivity to MMC that was equivalent tothat of #recN. The overproduction of RecNK35A rendered wildtype cells sensitive to MMC (Fig. 6A), demonstrating that
recNK35A is a dominant-negative allele of recN. GFP-RecNK35A
formed nucleoid-associated foci in %80% of MMC-treatedcells, and these foci failed to form in cells expressing recAQ300R(Fig. 6B). This result indicates that the ATPase activity of RecN
is not required for formation of nucleoid-associated RecN foci.
However, #recN cells expressing wild-type GFP-recN resumed
normal cell growth, and nucleoid-associated GFP-RecN focidissociated after exposure to MMC was terminated (Fig. 6C).
By contrast, #recN cells expressing GFP-recNK35A became
highly filamented, acquired fragmented nucleoid structures,and retained GFP-RecNK35A foci for 2 h after exposure to MMC
was terminated (Fig. 6C). These results demonstrate that
ATPase-defective RecNK35A is recruited to sites of DNA damage, but may not be properly released because of the defects in
HR-mediated repair of MMC-induced DSBs.

Localization of RecN Protein to DNA Breaks

MMC-induced cell-cycle arrest. This results in highly filamented cells with nucleoid-associated GFP-RecNK35A foci andfragmented nucleoid structures. One possible explanation forthe presence of persistent foci associated with damaged DNA in
recNK35A cells is that RecNK35A is recruited to DSBs, but is notreleased from DSB sites because it lacks ATPase activity; undersuch conditions, mutant RecNK35A DNA damage foci persist
and accumulate, which interferes with RecA-mediated synaptic
steps in the HR pathway.
This study also reveals that #recN and recAQ300R cells are
hypersensitive to MMC but not to UV. A previous study
showed that cells expressing recA#C17 (a deletion mutant lacking residues 336 –352) are hypersensitive to MMC but not to
UV (51). Here, we confirm that recA#C17 cells are sensitive to
MMC, although they are less sensitive than #recN andDISCUSSION
Previous studies demonstrate that RecN protein forms both
nucleoid-associated and cytoplasmic foci in cells exposed to
DSB-inducing agents and that cytoplasmic RecN aggregates aredegraded by the ClpXP protease (27). Here, we demonstratethat RecN is recruited to nucleoids in a RecA-dependent manner. We characterize a novel recA allele, recAQ300R, which promotes expression of SOS-inducible genes but does not promoteformation of nucleoid-associated RecN foci. RecN accumulates
at a unique I-SceI-induced DSB in wild-type recA cells but not
in recAQ300R cells. Thus, we conclude that RecA plays an essential role in DNA damage-induced expression of recN and theassembly of RecN foci at the sites of DSBs. ATPase-deficient
recNK35A mutants are proficient in forming nucleoid-associated foci at DSBs, but fail to resume growth after release from29234 JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 5. RecA is required for the assembly of RecN at the sites of DSBs. A, Southern blot of the flanking region of the I-SceI site before and after inductionof I-SceI. Exponentially growing cells were cultured, and either arabinose (Ara) was added to induce I-SceI or glucose (Glu) was added as a control. Cells weretaken at the indicated times. The DNA digested with VspI was analyzed on a 1% agarose gel and detected by Southern analysis using site 1 probe. The top panel
illustrates the DNA sequences flanking the I-SceI cleavage site in the codA21::miniTn7Kan locus on the F! episome. The location of VspI cut sites and the sizes of
the DNA fragments after VspI digestion are shown. Site 1 and site 2 regions were used for Southern blot analysis. B, quantitation of Southern blot analysis. I-SceI
break refers to the levels of the 1.2-kb fragment resulting from DSB formation. C, GFP-RecN foci at a unique I-SceI-induced nascent DSB. The panels show
GFP/DAPI-merged images of cells with or without a single I-SceI recognition site. recA# cells expressing SOS-inducible GFP-recN and either wild-type recA or
recAQ300R were incubated for 1 h in the presence of arabinose to induce I-SceI. Scale bar indicates 2.5 !m. D, quantitative analysis of GFP-RecN foci. The results
represent the average of at least three independent measurements. Error bars indicate S.D.

Localization of RecN Protein to DNA Breaks

recAQ300R cells (supplemental Fig. S3). We found that nucleoidassociated GFP-RecN foci form normally in MMC-treated
recA#C17 cells (supplemental Fig. S3), indicating that thedefects in the response to MMC in recAQ300R cells are not a
result of a dysfunctional RecA C-terminal domain. However, it
still remains possible that the C-terminal region of RecA plays arole in modulating RecN function at a later step in the repair/
response to MMC-induced DSBs.
Previous studies suggest that the SOS response plays a critical role in DSB repair in E. coli but not in Bacillus subtilis (52).
Indeed, unlike in E. coli, the expression of B. subtilis RecN
appears to be SOS-independent (53), and GFP-B. subtilis RecN
foci associate with DSBs before RecA is recruited to the DNA
lesion (10). By contrast, E. coli recN is typical of SOS-regulated
genes in that its expression is tightly repressed in unstressed
cells. This suggests that E. coli RecN participates in HR repair of
DSBs after RecA senses DNA damage. Consistent with this, the
OCTOBER 11, 2013 • VOLUME 288 • NUMBER 41

present study indicates that RecA actively recruits RecN to
DSBs. These results may reflect species-specific attributes ofE. coli and B. subtilis HR pathways. The purified B. subtilis
RecN (and also D. radiodurans RecN) binds to DNA and has
DNA-stimulated ATPase activity in vitro (54, 55). Unfortunately, it has been difficult to purify E. coli RecN because it isrelatively insoluble and highly susceptible to degradation (datanot shown). A recent study showed that Haemophilus influenzae RecN can be purified to near homogeneity and is fully functional in E. coli (50). Purified Haemophilus influenzae RecN
does not bind DNA, and DNA had no significant effect on Haemophilus influenzae RecN ATPase activity in vitro, which contrasts with the activity of B. subtilis RecN. This observation
supports our conclusion that E. coli RecN is recruited to DSBs
through its interaction with RecA. Thus, the difference in the
DNA binding specificities of RecN orthologs may explain theirdifferent affinities for their respective bacterial nucleoids. How-45

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FIGURE 6. RecNK35A is deficient in HR-mediated recovery after exposure to MMC. A, sensitivity of cells to MMC. The indicated strains were grown in LB. Cells
were diluted and spotted onto LB with or without MMC (0.5 !g/ml). B, subcellular localization of GFP-RecNK35A. The panels show GFP/DAPI images of #recN
cells containing SOS-inducible GFP-recNK35A after 30 min of incubation in the presence of MMC. Quantitative analysis of GFP-RecNK35A foci is shown to the right.
The results represent the average of at least three independent measurements. C, wild-type and recNK35A cells were treated with MMC for 10 min and thentransferred to MMC-free medium (t ' 0). At the indicated time points, cells were analyzed for the presence of RecN foci. The right panels show the GFP/DAPI
images of cells at the indicated time after transfer to MMC-free media. Scale bar indicates 2.5 !m. Error bars indicate S.D.

Localization of RecN Protein to DNA Breaks
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K12. Mol. Gen. Genet. 190, 162–167
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Acknowledgments—We are grateful to S. M. Rosenberg for providing
the E. coli strains. We thank T. Kado for technical assistance. We
thank the members of the Hishida laboratory for stimulating
discussions.
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ever, our data do not exclude the possibility that E. coli RecN
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SMC proteins are highly conserved ATPases whose role inhigher order chromosome organization and dynamics is conserved from bacteria to humans. DSBs are one of the most cytotoxic forms of DNA damage, and therefore, the repair of DSBs is
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reaction is formation of a RecA-ssDNA nucleoprotein filament.
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In future studies, it will be interesting to investigate how
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approaches to examine this and other questions concerning theroles of RecN and RecA will be required. The results of suchstudies should advance our understanding of the mechanism of
DSB repair in prokaryotic and eukaryotic cells.

Localization of RecN Protein to DNA Breaks

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Supplemental Data:

WT

!recN

!recA /
pRecA (WT)

!recA

!recA /
pRecAQ300R

+ UV

5 !m

Figure S1. Morphological changes in UV-treated cells. Exponentially growing cells were
irradiated with UV (20 J/m2) and samples were taken at 90 min after UV irradiation. Cells
were fixed and stained with DAPI, and analyzed by fluorescence microscopy.

48

A

B
Glu

Time (min) 0

1.8 kb
0.6 kb

30

60

90

90

: Arabinose: Glucose

30
I-SceI break (%)

Ara

20
10
0

0

30

60

90 (min)Figure S2. Southern blot analysis of DNA extracted at different times after DSB induction.
(A) Exponentially growing cells were cultured with addition of arabinose (for induction of I-SceI)or glucose (as a control). Cells were taken at the indicated times. The DNA was digested with VspI
and probed with a site2 fragment. (B) Quantitation of Southern blot analysis. “I-SceI break” refers
to the levels of the 0.6-kb fragment resulting from DSB formation.

49

A

MMC (0.5 !g/ml)

Control

UV (20 J/m2)!recA /vector!recA /pRecA(WT)!recA /pRecAQ300R!recA /pRecA"C17

B

C
100

% of focirecA!C17

Nucleoid
Cytoplasmic

50

5 !m

MMC (1 !g/ml, 60 min)0

recA!C17

Figure S3. Assembly of RecN at the sites of DSBs in recA!C17 mutants. (A) Sensitivity of cells to MMC
and UV irradiation. Ten-fold serial dilutions of the indicated strains were spotted onto LB plates containing
the 0.5 ! g/ml MMC. For UV irradiation, the indicated strains were spotted onto LB plates, DNA damage was
induced by UV. (B) MMC damage-induced RecN foci in recA!C17 cells. Cells carrying the SOS inducible
GFP-recN were exposed to MMC for 60 min. The panels show GFP/DAPI-merged images of cells. (C)Quantitative analysis of GFP-RecN foci. For cells with MMC damage, >150 cells were examined. The results
represent the average of at least three independent measurements.

50

RecA Protein Recruits Structural Maintenance of Chromosomes (SMC)-like RecN
Protein to DNA Double-strand Breaks
Kenji Keyamura, Chikako Sakaguchi, Yoshino Kubota, Hironori Niki and Takashi
HishidaJ. Biol. Chem. 2013, 288:29229-29237.
doi: 10.1074/jbc.M113.485474 originally published online August 25, 2013

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細胞死耐性の分子メカニズムの解析
教授 安達 卓助教 谷口喜一郎[目的]
一般に、アポトーシスという細胞死現象の発見は、1972 年の Kerr 夫妻（Scotland）によるものとされている。しかしながら、放射線によって導かれる細胞死は、典型的なアポトーシスでありながら、その発見は 100 年以上も前のことであり、様々な動物組織の間でその応答に差があることが研究されていた。例えば、分裂頻度が高く、分化レベルが浅く、再生能力の高い組織（即ち、現在で言う組織幹細胞をもつ再生組織）では、この細胞死は容易に誘導されるが、逆に非再生系組織の倍数化細胞やがん組織にある増殖細胞などでは誘導されなくなるという性質は、広く多細胞動物一般に認められ、Bergonie-Tribondeauの法則として 1906 年に報告されたものである。しかし、これを説明する分子メカニズムについては、現在でも十分に解明できてはいない（参考文献１、２）。この仕組みの解明は、
単に放射線に対する防御能を知るのみならず、原子力発電等の放射線を利用した諸技術の発展に伴って必要となるもので、また近年展開されるガンの放射線治療を改良する際にも、重要な情報を与えると考えられ、社会的にも大きな意義をもつ研究であると推察される。
本研究では、遺伝的解析に優れたモデル生物であるショウジョウバエを用い、前立腺相同の非再生系生殖器官である附属腺に焦点をあてて、そこに自然に生じている、分化細胞の細胞死耐性の分子メカニズムを明らかにすることを目的とした。
[結果と考察]ショウジョウバエのオス成虫がもつ内部生殖器官の一つ、附属腺は、幹細胞をもたない
非再生組織であり、一旦生じた分化細胞は、生涯にわたって長く維持される必要がある。
そのために、これらの細胞では、各種の細胞死誘導性刺激に対し抵抗性が生じていることが期待された。そこで、エックス線照射（DNA 損傷）・パラコート投与（活性酸素上昇）・
reaper 遺伝子強制発現（Initiator Caspase 活性化）・Dcp-1 遺伝子強制発現（effector
Caspase 活性化）など、様々な細胞死誘導性刺激を与えて細胞死応答を調べ、再生系組織における応答との比較を行った。その結果、これらいずれの刺激を与えても、再生系組織
では細胞死が誘導されたが、附属腺細胞においては、エックス線照射・パラコート投与・
reaper 遺伝子強制発現では、予想どおり顕著な抵抗性を見せて細胞死が誘導されなかった
ものの、Dcp-1 遺伝子強制発現のみが細胞死を誘導できることが分かった。従って、細胞死
抵抗性は細胞死誘導経路の中では、effector Caspase 上流で生じることが推察された（図１）。
そこで、ショウジョウバエにおける２種の effector Caspase パラログである、Dcp-1 とDrice について、内在性の遺伝子発現量を、翅原基（再生組織）におけるそれと比較した。
その結果、Dcp-1 では顕著に、Drice においても一定程度の発現量の低下を観察することができ、これらの発現抑制が、この組織における細胞死耐性の一因であることが予測された。

70

図１．

一般に知られているアポトーシスの誘導経路（左）、および各因子の各組織における発現を RT-PCR で検出した実験結果（右）ショウジョウバエにおいて細胞死耐性を見せる組織は、成虫にある附属腺だけではなく、幼虫における唾液腺・脂肪体・消化管や、成虫における後腸などが、その性質を見せる。
これら様々な組織における細胞死耐性が共通したメカニズムによってもたらされているかどうか調べるために、まず reaper 遺伝子強制発現による細胞死誘導性を確認したところ、附属腺細胞と同様に、細胞死を誘導することはできなかった。よってその耐性の制御点も、
附属腺細胞と同様に、initiator Caspase の下流である。そこで次に、Dcp-1 の強制発現による効果を検証したところ、幼虫脂肪体では附属腺細胞と同様に細胞死が誘導されたが、成虫後腸では附属腺細胞とは異なり、依然として細胞死耐性を見せ続けた。従って、この組織における細胞死耐性制御点は effector Caspase よりさらに下流にあるか、また独立の経路において存在していると予測された。実際、遺伝子発現量を調べると、幼虫脂肪体や成虫後腸においても、Dcp-1 の明瞭な発現低下が認められるのに加えて、成虫後腸ではもう一つの effector Caspase である Drice や Initiator Caspase である Dronc、また effector
Caspase 下流の CAD (Caspase-Activated DNase)などの発現も低く、また幼虫脂肪体では、
Apf-1（アポプトソーム構成因子）の発現量が特異的に低いなどの特徴が認められた。
これらの知見を総合すると、個体の内部で様々な組織が見せる細胞死耐性は、effector
Caspase の発現量低下という共通した性質が基盤にある一方で、各組織で独自の遺伝子の発現が抑えられるという多様化した仕組みと組み合わされて成り立っていることが明らかとなった。なお、これらの遺伝子発現量調節が、ヒストンのメチル化状況や microRNA に
よって影響を受けている訳ではないことを、これらの各現象に関わる遺伝子である dUTX
（ヒストン脱メチル化酵素）や Drosha（miRNA プロセシング RNase）のノックダウンによって検証済みである。

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一方、Dcp-1 遺伝子のシス調節領域にあるエンハンサー／サイレンサーを介したシンプルな転写調節が、これらの細胞死耐性組織における Dcp-1 発現低下を招いているのかどうかを知るために、Dcp-1 遺伝子の 5’非翻訳領域にリポーター遺伝子 lacZ が挿入されたショウ
ジョウバエ系統 P{PZ}dcp-1[02131]を用い、その lacZ 発現を調べた。その結果、オス成虫
附属腺・幼虫脂肪体・成虫後腸のいずれにおいても翅原基より低い発現量が観察された。
さらに、もう一つの effector Caspase である Drice のエンハンサー／プロモーター制御下で、成虫附属腺や幼虫脂肪体において低レベルで Dcp-1 遺伝子を発現する形質転換系統、
Drice-Dcp-1 を作成し、その個体において細胞死誘導が起き得るかどうかについて調べている。現在までのところ、エックス線照射による細胞死誘導は、成虫附属腺・幼虫脂肪体・
成虫後腸のいずれにおいても認められなかった。しかし、これは細胞死抑制の原因が Dcp-1にはないことを直ちに示すものではないと考えられる。なぜなら、これらの組織においては DNA 損傷と reaper 遺伝子発現誘導の間を介在する p53 遺伝子の発現が抑制されていることが知られ、このために、下流の Caspase 経路を活性化することが出来なくなっていると考えられる。そこで今後さらに、この Drice-Dcp-1 バックグラウンドにおいて、reaper
遺伝子を強制発現させて細胞死が誘導されるかどうかを確認することで、Dcp-1 発現量低下
が細胞死耐性獲得の主要な原因であることを明確にしたいと考えている（谷口ら未発表）。
以上に加えて、この研究と密接に関連する３つの研究を、並行して進めている。一つは、附属腺細胞の老化細胞死に関するものである。上述のように、附属腺細胞はストレス性の刺激に対する細胞死応答を起こし難いが、老化が極度に進んだ時には、機能破綻した一部の細胞が細胞死に向かう（谷口ら未発表）。この際にどのような分子メカニズムが働くのかについて調べているが、その過程で、附属腺としてのマスターコントロール遺伝子である
Paired（ホメオドメイン含有タンパク質）を初めとした様々な因子の発現量が減少する上、３種類の細胞内シグナルが同時に働くことで、細胞死耐性を解除していることが判明した（谷口ら未発表）。この研究過程では、老化した附属腺と若齢の附属腺との間に認められる遺伝子発現量の差の網羅的な解析を、次世代シーケンサーを用いた RNA-seq より行った。
これにより判明した発現量が低下する数十個の遺伝子については、細胞死耐性をもたらす
責任候補遺伝子として位置づけて、各遺伝子の RNAi に基づく老化細胞死検定を進めて、幾つかの重要な遺伝子が絞り込まれるに至っている。ここまでの経過においては、本助成金により購入されたキーエンス社デジタルマイクロスコープ VHX-2000 が効力を発揮し、細胞死検定を迅速に進めることができた。
他の関連する研究として、倍数化によって附属腺に類似した細胞死抑制を見せる消化管細胞において、マスターコントロール遺伝子 GATAe の発現抑制が示す表現型を調べている（参考文献３）。眼の発生におけるマスターコントロール遺伝子 Pax6 に関する初期の研究で良く知られるように、マスターコントロール遺伝子の発現消失は一般に細胞死を起こし、
ホメオティック遺伝子の発現消失が、細胞分化タイプの転換を起こすのとは対照的である（参考文献４）。附属腺と消化管のどちらにおいても、マスターコントロール遺伝子の発現が失われ、器官のアイデンティティを失った際の応答には、共通の仕組みが予想される。

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ショウジョウバエのオス成虫附属腺は、それを構成している上皮組織の細胞のすべてが、常に核を２つずつもつという珍しい特徴をもっている。我々は、その２核化のメカニズムについても研究を進めてきたが（参考文献５）、通常の細胞分裂時には、分裂細胞は上皮層から頂端側へ逸脱して分裂するが、附属腺において細胞質分裂を伴わない核分裂が起きる
際には、核分裂細胞は上皮層から逸脱することなく、隣接した上皮細胞と細胞側面を接着させたまま、核が頂端基底方向に分かれることが分かった。この事実が意味していることは、通常の増殖組織では比較的自由に ON/OFF 制御されている細胞間接着が、附属腺ではOFF することが困難な状態にあると考えられる。すると、アポトーシスの際に、周辺細胞やマクロファージが貪食するために生じるアポトーシス小体などへの細胞形態変化も、困難になっている状態が考えられる。実際、附属腺細胞が極度に老化した時に起きる老化細胞死においても、細胞はアポトーシス小体を作ることなく、そのままの形で内部の消化が進む。一方、アポトーシスの過程は、周囲の細胞が貪食しないと進行しないことが知られていて（参考文献６）、これら一連の事実により、附属腺細胞はアポトーシスを起こせないようになっているという仕組みも考えられる。
[まとめ]
以上のように、附属腺のもつ細胞死耐性がどのような仕組によるのか明らかにしてきた。
その基本として、effector Caspase の発現抑制のように共通した仕組みがあるのに加えて、各耐性組織に固有の遺伝子発現抑制が組み合わされている。また、老化が進んだ状況では、マスターコントロール遺伝子ほかの遺伝子発現低下に加え、平常時には働かない３種類の細胞内シグナルが働くことにより細胞死耐性が解除され、細胞死へ向かうことが分かった。
[参考文献]

1. Mehrotra, S., Maqbool, S. B., Kolpakas, A., Murnen, K., and Calvi, B.
R. .Endocycling cells do not apoptose in response to DNA rereplication genotoxic
stress. Genes Dev., 22: 3158-3171 (2008).
2. Hassel, C., Zhang, B., Dixon, M., and Calvi, B. R. Induction of endocycles
represses apoptosis independently of differentiation and predisposes cells to genome
instability. Development 141: 112-123 (2014).
3. Okumura, T., Takeda, K., Kuchiki, M., Akaishi, M., Taniguchi, K., andAdachi-Yamada, T. GATAe regulates intestinal stem cell maintenance anddifferentiation in Drosophila adult midgut. Dev. Biol., 410: 24-35 (2016).
4. Takemura, M. and Adachi-Yamada, T. Repair responses to abnormalities inmorphogen activity gradient. Dev. Growth Differ., 53: 161-167 (2011).
5. Taniguchi, K., Kokuryo, A., Imano, T., Minami, R., Nakagoshi H., andAdachi-Yamada, T. Isoform-specific functions of Mud/NuMA mediate binucleationof Drosophila male accessory gland cells. BMC Dev. Biol., 14: 46 (2014).
6. Li, W. and Baker, N. E. Engulfment is required for cell competition. Cell, 129:
1215-1225 (2007).

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[原著論文]1.

Okumura, T., Takeda, K., Taniguchi, K. and Adachi-Yamada, T. βν integrin inhibits
chronic and high level activation of JNK to repress senescence phenotypes in
Drosophila adult midgut. PLoS One, 9, e89387 (2014).

2.

Taniguchi, K., Kokuryo, A., Imano, T., Minami, R., Nakagoshi H., andAdachi-Yamada, T. Isoform-specific functions of Mud/NuMA mediatebinucleation of Drosophila male accessory gland cells. BMC Dev. Biol., 14: 46(2014).

3.

Okumura, T., Takeda, K., Kuchiki, M., Akaishi, M., Taniguchi, K., andAdachi-Yamada, T. GATAe regulates intestinal stem cell maintenance anddifferentiation in Drosophila adult midgut. Dev. Biol., 410: 24-35 (2016).

[総説等]

1.

Takemura, M. and Adachi-Yamada, T. “Apoptosis during cellular pattern formation”
in Apoptosis, InTech (2013).

[口頭発表]【国外学会】
1.

Analysis of βν integrin mutant showing a premature aging phenotype in Drosophila
adult midgut
奥村高志, 武田晃司, 谷口喜一郎, 安達卓
55rd Annual Drosophila Research Conference (2014.3.26-30, アメリカ, サンディエゴ)

2.

Isoform-specific functions of Mud/NuMA mediate binucleation of Drosophila maleaccessory gland cells
谷口喜一郎, 國領顯彦, 今野貴夫, 南竜之介, 中越英樹, 安達卓
55rd Annual Drosophila Research Conference (2014.3.26-30, アメリカ, サンディエゴ)

3.

Cytokinesis-deficient binucleation in Drosophila accessory gland for providing
plasticity of organ size
谷口喜一郎, 國領顯彦, 今野貴夫, 坂田瑠美, 南竜之介, 中越英樹, 安達卓
BSCB/BSDB/JSDB Joint Spring Meeting 2012 (2012.4.15-18, イギリス, コヴェントリ)

4.

The tissue-specific integrin, βνintegrin, is required for proper proliferation ofintestinal stem cells in the Drosophila adult midgut
奥村高志, 安達卓

74

BSCB/BSDB/JSDB Joint Spring Meeting 2012 (2012.4.15-18, イギリス, コヴェントリ)
5.

Cytokinesis-deficient binucleation in Drosophila accessory gland for providing
plasticity of organ size
谷口喜一郎, 國領顯彦, 今野貴夫, 坂田瑠美, 南竜之介, 中越英樹, 安達卓
53rd Annual Drosophila Research Conference (2012.3.7-11, アメリカ, シカゴ)

【国内学会】
1.

ショウジョウバエ中腸から分泌されるホルモンによる寿命制御
武田晃司, 奥村高志, 山口未音, 朽木めぐみ, 谷口喜一郎, 安達卓
第38回日本分子生物学会年会・第88回日本生化学会大会合同大会 (2015.12.1-4, 神戸)

2.

ショウジョウバエ雄蛹期附属腺における二核化とcell cycle arrestの関係
越田惠子, 谷口喜一郎, 岡田朋也, 中越英樹, 安達卓
第38回日本分子生物学会年会・第88回日本生化学会大会合同大会 (2015.12.1-4, 神戸)

3.

ショウジョウバエ雄附属腺の細胞二核化におけるDppシグナルの関与
若松憲之, 谷口喜一郎, 岡田朋也, 中越英樹, 安達卓
第38回日本分子生物学会年会・第88回日本生化学会大会合同大会 (2015.12.1-4, 神戸)

4.

ショウジョウバエ組織における生理的アポトーシス耐性の解析
谷口喜一郎, 中越英樹, 安達卓
第38回日本分子生物学会年会・第88回日本生化学会大会合同大会 (2015.12.1-4, 神戸)

5.

ショウジョウバエ各種器官の近遠軸成長におけるDachsousの役割について
岡田朋也, 坂田瑠美, 安部翔太, 谷口喜一郎, 安達卓
第37回日本分子生物学会年会 (2014.11.25-27, 横浜)

6.

Isoform-specific functions of Mud/NuMA mediate binucleation of Drosophila maleaccessory gland cells
谷口喜一郎, 國領顯彦, 今野貴夫, 南竜之介, 中越英樹, 安達卓
第11回日本ショウジョウバエ研究会 (2014.6.4-6, 金沢)

7.

Acquisition of apoptosis-resistances in Drosophila accessory gland exocrine cells
谷口喜一郎, 安達卓
新学術領域研究「上皮管腔組織形成」第１回国際シンポジウム (2013.6.22-23, 札幌)

8.

Mud/NuMAはショウジョウバエ雄生殖器附属腺において細胞二核化を制御する
谷口喜一郎, 國領顯彦, 今野貴夫, 南竜之介, 中越英樹, 安達卓
第65回日本細胞生物学会大会 (2013.6.19-21, 名古屋)

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Taniguchi et al. BMC Developmental Biology (2014) 14:46
DOI 10.1186/s12861-014-0046-5

RESEARCH ARTICLE

Open Access

Isoform-specific functions of Mud/NuMA mediatebinucleation of Drosophila male accessory glandcells
Kiichiro Taniguchi1, Akihiko Kokuryo2,3, Takao Imano2,3, Ryunosuke Minami4, Hideki Nakagoshi4and Takashi Adachi-Yamada1,2,3*

Abstract
Background: In standard cell division, the cells undergo karyokinesis and then cytokinesis. Some cells, however,such as cardiomyocytes and hepatocytes, can produce binucleate cells by going through mitosis without
cytokinesis. This cytokinesis skipping is thought to be due to the inhibition of cytokinesis machinery such as thecentral spindle or the contractile ring, but the mechanisms regulating it are unclear. We investigated them bycharacterizing the binucleation event during development of the Drosophila male accessory gland, in which all cells
are binucleate.
Results: The accessory gland cells arrested the cell cycle at 50 hours after puparium formation (APF) and in the middleof the pupal stage stopped proliferating for 5 hours. They then restarted the cell cycle and at 55 hours APF entered theM-phase synchronously. At this stage, accessory gland cells binucleated by mitosis without cytokinesis. Binucleating
cells displayed the standard karyokinesis progression but also showed unusual features such as a non-round shape,
spindle orientation along the apico-basal axis, and poor assembly of the central spindle. Mud, a Drosophila homolog of
NuMA, regulated the processes responsible for these three features, the classical isoform MudPBD and the two newly
characterized isoforms MudL and MudS regulated them differently: MudL repressed cell rounding, MudPBD and MudS
oriented the spindle along the apico-basal axis, and MudS and MudL repressed central spindle assembly. Importantly,
overexpression of MudS induced binucleation even in standard proliferating cells such as those in imaginal discs.
Conclusions: We characterized the binucleation in the Drosophila male accessory gland and examined mechanismsthat regulated unusual morphologies of binucleating cells. We demonstrated that Mud, a microtubule binding protein
regulating spindle orientation, was involved in this binucleation. We suggest that atypical functions exerted by threestructurally different isoforms of Mud regulate cell rounding, spindle orientation and central spindle assembly inbinucleation. We also propose that MudS is a key regulator triggering cytokinesis skipping in binucleation processes.
Keywords: Drosophila, Male accessory gland, Binucleation, Cytokinesis, Central spindle, Cell rounding, Spindleorientation, Mud* Correspondence: Takashi.Adachi-Yamada@gakushuin.ac.jp
1
Department of Life Science, Faculty of Science, Gakushuin University, Tokyo
171-8588, Japan
2
Institute for Biomolecular Science, Gakushuin University, Tokyo 171-8588,
Japan
Full list of author information is available at the end of the article© 2014 Taniguchi et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.

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Page 2 of 18

Background
Most eukaryotic cells contain only a single nucleus because
the karyokinesis in the M phase of the cell cycle is followedby cytokinesis. In certain cells, however, such as cardiomyocytes and hepatocytes, cytokinesis does not always
occur, which results in cells containing two nuclei [1,2].
Sarcomere assembly is a possible factor repressing cytokinesis in cardiomyocytes [1,3], and insulin signaling plays apart in the generation of binucleate hepatocytes [2].
The production of binucleate cells is thought to result
from the repression of certain phases of cytokinesis,
such as formation of the contractile ring and ingressionof the cleavage furrow. Cytokinesis occurs only when
there is sufficient activation of Rho GTPase at the division plane. After chromosome segregation, a prominent
bundle of microtubules, called the central spindle, forms
between the spindle poles [4-6]. The centralspindlin
complex, consisting of kinesin-6 and RhoGAP, moves toward the plus ends of the microtubules, corresponding
to the cell equator, and associates with RhoGEF [7]. The
RhoGEF thus specifically activates Rho GTPase at thedivision plane. Rho signaling activates effector proteins,
such as diaphanous and Rho kinase, that in turn activate
the formation of the actin contractile ring that completes
cell division by pinching the daughter cells apart [8].
Loss of this cytokinesis machinery results in incomplete
cytokinesis and produces binucleate cells [7,9,10]. Thereis, however, no solid evidence that normal binucleation
events are regulated by inhibiting the functions ofcytokinesis components, and little is known about the
key regulators repressing the formation of cytokinesis
machinery during binucleation. On the other hand,recent studies have shown a link between binucleationand inhibition of the cytokinesis machinery in cancer
cells [11,12].
To investigate the mechanism by which cytokinesis isskipped during binucleation, we used as a model system
the Drosophila male accessory gland, which produces
seminal fluid proteins promoting reproductive success,such as the sex peptide Acp70A [13,14]. The exocrine
epithelial cells in the male accessory gland, both themain cells and the secondary cells, are obviously binucleate (Figure 1A) [15]. We previously showed thatbinucleation increases the plasticity of the cell shape,
thereby enabling the volume of the accessory gland cavity to change [16], but the mechanisms of binucleation
have remained unclear.
In the work reported here, we investigated the binucleation event in the accessory gland primordia, which wascharacterized by synchronous entry into the M phase aftera cell-cycle-arrested interval during the mid-pupal developmental stage following standard cell proliferation in theearly stage. We found that the binucleation results notfrom cell fusion but from mitosis without cytokinesis. We

examined the mechanisms of binucleation by focusing on
various morphological features different from those ofstandard dividing cells. We propose that isoform-specificfunctions of the microtubule binding protein Mud, a Drosophila homolog of NuMA, are the key regulators in binucleation of the Drosophila male accessory gland cells.

Results
Accessory gland epithelial cells are binucleatedsynchronously in the mid-pupal stage by mitosis without
cytokinesis

We first determined whether binucleation of the accessory gland epithelial cells is a result of skipping cytokinesis (as in cardiomyocytes). We observed thedevelopmental stages and M-phase entry by using anantibody against phospho-histone H3 (P-H3), a marker
for M-phase chromatin. Until 50 hours after puparium
formation (APF), the accessory gland epithelial cells randomly entered the M phase but did not produce binucleate cells (Additional file 1: Figure S1A–E, A’–E’ andJ) (Figure 1D). That is, standard cell division occurred.
Subsequently, the cells arrested their cell cycle and delayed their M-phase entry for about 5 hours (50-55APF)(Additional file 1: Figure S1F and F’) (Figure 1D). The
secondary cells then entered the M phase at 55 hours
APF (Figure 1B and D) (Additional file 1: Figure S1G
and G’), and the main cells entered the M phase at
60 hours APF (Figure 1C and D) (Additional file 1: Figure S1H and H’). We also found that the mitotic wave
for binucleation in the main cell population initiated at
the middle zone of the accessory gland lobe and propagated to the proximal and distal parts (Additional file 1:
Figure S2). These results indicate a unique cell cycle
regulation in this organ development. Importantly, thesynchronous entries into the M phase accompanied
the production of binucleate cells (Additional file 1:
Figure S1K and Figure S2). No cytokinesis was evident
in this M phase (Figure 2F–J and F’–J’). After binucleation, the accessory gland epithelial cells did not enter asubsequent M phase (Additional file 1: Figure S1I and I’,
Figure S3) but showed a single round of the S phase,indicated by PCNA-GFP labeling (Additional file 1:
Figure S3), indicating that endoreplication occurred(Figure 1D). Thus the accessory gland epithelial cells,
both secondary and main cells, became octaploid cellswith two tetraploid nuclei. In the following section, we
describe our examination of binucleation in the main
cells. The secondary cells probably binucleated in thesame way the main cells did.
Central spindle assembly and actin-contractile ringformation are inhibited during binucleation

We next identified the cytological differences between
standard cell division and binucleation in order to obtain

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Page 3 of 18

Figure 1 Synchronous binucleation of Drosophila maleaccessory gland cells occurs in the pupal stage. (A) Adult
accessory gland epithelium labeled as indicated at the bottom left.
Main cells (nuclei stained both green and magenta) and secondary
cells (nuclei stained only magenta) are shown. The inset at thebottom right depicts an adult male abdomen (gray) and the
reproductive systems around the hindgut. Posterior is to the right.
Scale bar, 10 μm. (B and C) Synchronous entry into M phase insecondary (B) and main (C) cells in the accessory glands during
mid-pupal binucleation stages. Labels as indicated at the bottom
right. Scale bars, 50 μm. (D) Schematic diagram showing cell cycletransition of epithelial cells in accessory gland during thepupal stage.

clues to the mechanisms of cytokinesis skipping. For
this purpose, we compared cell division in the early
pupal accessory gland primordium and binucleation in
the mid-pupal one (Figure 1D). We focused on the following three differences. First, during the standard cell
division in the early stage of accessory gland development, the M-phase cells were apically extruded androunded (Figure 2A–C and A’–C’), as is widely found in
the standard epithelia [17]. During binucleation, however, cells were retained in the epithelial monolayer anddid not show rounding (Figure 2F–H and F’–H’ compared with A–C and A’–C’). Second, the spindle orientation in standard mitosis was parallel to the epithelial
plate (Figure 2A–C and A’–C’) [17]. The spindle formed
during binucleation, in contrast, was always oriented perpendicular to the epithelial plate (Figure 2F–H and F’–H’).
Third, in standard cell division, the central spindles between segregated chromatids arose from anaphase to telophase, and then an actin contractile ring formed at thedivision plane (Figure 2D,E,D’ and E’) [4-6,8]. During
binucleation, in contrast, the central spindle was not properly assembled (Figure 2I,J,I’ and J’). Consequently, thesubsequent formation of the contractile ring was also incomplete at telophase (Figure 2J and J’), although thecleavage furrow was slightly formed at late anaphase(Figure 2I and I’). On the other hand, other components of the mitotic spindle of binucleating cells looked
normal, including the metaphase spindle (Figure 2G
and G’ compared with B and B’) [6] and kinetochore
microtubules (Figure 3C compared with A) [6,18]. Also
apparently normal in the binucleating cells was the Mphase progression indicated by the relationship between
chromatid segregation (Figure 2G,H,G’ and H’ compared with B,C,B’ and C’) [18] and the decay of cyclin B(Figure 3D and D’ compared with B and B’) [19]).
Forced activation of Rho GTPase in binucleation stage
produces actin contractile ring

Looking at the above three differences for clues to themechanism of binucleation in the experiments intended
to produce artificially rounded cells, we saw that cell78

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Figure 2 Central spindle and contractile ring are not formed during binucleation. Photomicrographs showing cross-sectional views of cells (A–O)
and their schematic diagrams (A’–O’) are arrayed from left to right according to the M phase progression. (A–E) Main cells during cell division stage inearly pupa (30–35 hours APF). (F–J) Main cells during binucleation stage in mid-pupa (60–65 hours APF). (K–O) Binucleation-stage main cells in which
pebble was overexpressed just before binucleation. Arrowheads in (D, E, I, J, N and O) indicate equatorial planes in late anaphase and telophase during
cell division and binucleation. Cells are labeled as indicated at the bottom of (E). Scale bar in (A), 5 μm, is applicable to (A–O).

cytokinesis progression (Additional file 1: Figure S4E–H)(Table 1) [23-25]. On the other hand, in centrosomin andSas-4 mutants, weak assembly of the central spindle andan abnormal contractile ring could be observed at low frequencies (6% in centrosominHK21 and 9% in Sas-4S2214).
However, we found that these induction-of-cytokinesisfeatures were observed even in the cells showing normal(vertical) spindle orientation. Thus these results mean that
contribution of the centrosome to the cytokinesis skipping
during binucleation may be nonessential or even trivial.
Mis-assembly of the central spindle and incomplete formation of the contractile ring strongly induced skipping of

extrusion and rounding did not seem to affect binucleation. In the binucleation stage neither overexpression of
Sterile20-like kinase, which is related to cortical rigidity
and cell rounding in standard cell division [20,21], nor
knockdown of the adherens junction protein DE-cadherin encoded by shotgun [22], which maintains epithelial stability, led to cytokinesis (Additional file 1: Figure
S4A–D) (Table 1). Binucleation also seemed to not beaffected by the orientation of the mitotic spindle. This isbecause disruption of vertical spindle orientation in the
binucleation stage by using a mutant for the centrosome
protein Centrosomin or Sas-4 did not cause ectopic

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Figure 3 Metaphase spindle formation and metaphase-anaphase transition are normal during binucleation. (A and C) Localization of polo tokinetochores in main cells in metaphase during cell division in early pupa (A) and during binucleation in mid-pupa (C). Cells are labeled as indicated at
the bottom of (A). En face views (A) and cross-sectional views (C). Scale bar, 5 μm, is applicable to (A and C). (B, B’, D and D’) Levels of cyclin B. En
face images of the wing disc epithelium in third-instar larva as an example of proliferating tissue (B and B’) and the accessory gland epithelium in
mid-pupa as an example of binucleating tissue (D and D’). Cells in (B and D) are labeled as indicated at the bottom right of (B). Intensities of cyclin B::
GFP in (B) and (D) are represented by a rainbow-color scale, with red meaning high intensity and blue meaning low intensity. Magenta dashed lines
indicate outlines of mitotic cells in various M-phase subphases. Scale bar in (B), 10 μm, is applicable to (B, B’, D and D’).

cells in telophase was too low to sufficiently activate
Rho1 signaling for cytokinesis.

cytokinesis during binucleation. The formation of theactin-contractile ring requires sufficient activation of Rho
GTPase at the division plane [8]. To activate Rho GTPase
encoded by Rho1, we temporarily elevated the level of the
RhoGEF pebble around the binucleation stage. Pebble activates a Rho1 signaling cascade that phosphorylates
MRLC (a regulatory light chain of non-muscle myosin II)
encoded by spaghetti squash (sqh) to form the contractile
ring [5,7,8]. Overexpression of pebble resulted in F-actin
accumulation at the cleavage furrow, a sign of contractile
ring formation, and in furrow ingression during the binucleation stage (Figure 2K–O and K’–O’) (Table 1). We also
found that overexpression of the activated form of sqh(sqhD20.D21) induced the formation of the contractile
ring (Additional file 1: Figure S4I) (Table 1). Furthermore, we tested a moderate overexpression of wild-type
sqh or Septin-2 to see whether cells in the binucleation
stage have a latent ability to create a contractile ring.
Since the septin proteins are contractile ring components that act together with actomyosin and microtubules [26], we could easily see contractile ringformation in the telophase cells (Additional file 1:
Figure S4J–M) (Table 1). These results indicate that thelevel of central spindle assembly in accessory gland

Mud regulates central spindle assembly, spindleorientation and cell rounding during binucleation

The insufficient activity of Rho1 signaling in binucleationis thought to be due to the insufficient assembly of thecentral spindle from anaphase to telophase, so we hypothesized that factors that repressed the central spindle assembly would be key regulators in skipping cytokinesis.
Moreover, as stated above, the reduced central spindle assembly during binucleation should be accompanied by anon-round cell shape and orientation of the mitotic spindle along the apico-basal axis. Thus we examined candidate factors – such as mitotic kinesins (kinesin-like protein
at 10A, kinesin-like protein at 61 F, Pavarotti and no distributive disjunction) [4], microtubule-associated proteins(chromosome bows and Eb1) [4], microtubule-severing
proteins (katanin 60, spastin) [27], Par proteins (bazooka,
par-1) [28] and spindle orientation proteins (rapsynoid, G
protein αi subunit and mushroom body defect (mud))[29-31], with regard to their effects on cellular phenotypesof accessory gland cell binucleation. We found that loss of
mud disrupted normal binucleation phenotypes, including

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Table 1 Effect of various genetic manipulations on central spindle assembly and contractile ring formation during
binucleation
Manipulation

Genotype

Frequencies of phenotypes in mitotic behavior
Central spindle assemblyContractile ring formation

None (wild-type)

Canton-S

0% (N = 42, 7)

0% (N = 42, 7)(5%, partial*)(5%, partial**)Induction of cell rounding

fng-Gal4 (Tub-Gal80TS) > sterile 20-like kinase

0% (N = 34, 7)

0% (N = 34, 7)(12%, partial*)(18%, partial**)0% (N = 37, 6)

0% (N = 37, 6)(8%, partial*)(14%, partial**)6% (N = 36, 6)

6% (N = 36, 6)

fng-Gal4 (Tub-Gal80TS) > shotgun.IR

Disruption of centrosome***

Activation of contractile ring formation

centrosominHK21 homozygote(11%, partial*)(8%, partial**)Sas-4S2214 homozygote

9% (N = 34, 7)

9% (N = 34, 7)(18%, partial*)(29%, partial**)fng-Gal4 (Tub-Gal80TS) > pebble

0% (N = 34, 6)

44% (N = 34, 6)(56%, partial*)(41%, partial**)6% (N = 33, 6)

39% (N = 33, 6)(64%, partial*)(48%, partial**)11% (N = 36, 6)

14% (N = 36, 6)(47%, partial*)(53%, partial**)3% (N = 36, 7)

14% (N = 36, 7)(22%, partial*)(42%, partial**)fng-Gal4 (Tub-Gal80TS) > sqhD20.D21

sqh-GFP

Septin-2-GFP

Numbers of cells and tissues (pairs of lobes) observed are shown as (N = cells, tissues).
*A phenotype class with partial assembly of central spindle-like structure.
**A phenotype class with partial accumulation of F-actin accompanied by furrow progression.
***Very low frequency of cells shows obvious misorientation of spindle axis.

reduced central spindle assembly and other features of cellmorphology during binucleation (Figure 4) (Tables 2 and
3). mud encodes the Drosophila homolog of NuMA,
which is associated with microtubules and plays a role inmicrotubule polymerization and determination of thespindle orientation [29-31]. In mud4 hemizygotes andmud-knockdown cells, abnormally clear central spindle
assembly and contractile ring formation were seen even in
the binucleation stage (Figure 4B–E and B’–E’ comparedwith Figure 2I, J, I’ and J’) (Table 2 and Table 3). Loss of
mud was also associated with other morphological defects,
such as abnormal spindle orientation and cell rounding(Figure 4H–J and H’–J’ compared with Figure 2H and H’)(Table 2 and Table 3). We thus confirmed that one copyof a chromosomal duplication encompassing the mudgene region (Figure 4A) rescued these defects (Figure 4F,
G, F’ and G’) (Table 2). These results suggest that Mudcontributes to various cell morphologies during binucleation. We considered its repression of the central spindle
assembly to be a major cause of binucleation because in
mud4 hemizygotes, we frequently observed cytokinesis
progression even when neither horizontal spindle orientation nor cell rounding was observed (Figure 4B–E and B’–E’) (Additional file 2: Table S1).

Mud represses development of central spindle during
binucleation

We showed that Mud repressed central spindle assemblyand led to cytokinesis skipping. We made further observations of the central spindle assembly in wild-type andmud-knockdown cells in the binucleation stage. In particular, we used Pav::GFP (GFP-fusion of Pavarotti, acentralspindlin component) as a marker for the plus
ends of the microtubules in the mitotic spindle [32,33].
Regarding the control results, during the standard cell
division at the early pupal stage, Pav::GFP was localized
to the midzone of the central spindle at telophase, as reported previously (Figure 4K). During binucleation, in
contrast, we never observed Pav::GFP-accumulated microtubules around the cell equator region (Figure 4L).
Detailed observation of Pav::GFP accumulation revealed
that the impairment of central spindle assembly during
binucleation was not due to impaired initiation of
microtubule assembly. That is because the Pav::GFP accumulation around cell equator at anaphase was similarly observed in the case of dividing cells, implyingthat the central spindle precursor could develop at early
anaphase (Additional file 1: Figure S5F and F’ comparedwith A and A’). But during binucleation, impaired

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Figure 4 Loss-of-function for mud erases various characteristics of binucleation. (A) Schematic diagram of the mud transcriptional unit and three
representative splicing variants of mud (coding regions are in magenta). Regions corresponding to mudS.IR (green) and chromosomal duplication in Dp(1;3)DC281 (yellow) are also shown. (B–G) Cross-sectional views of main cells in late anaphase (B, D and F) and telophase (C, E and G) during the
binucleation stage in mutants hemizygous for mud4 (B and C), in knockdown for mud (D and E) and in mutants hemizygous for mud4
rescued by one copy of Dp(1;3)DC281 (F and G). Cells are labeled with phalloidin (magenta), anti-α-Tub antibody (green), and anti-P-H3
antibody (blue). Arrowheads in (C, E and G) indicate equatorial planes. Scale bar in (B), 5 μm, is applicable to (B–G). (B’–G’) Schematic diagramsof (B and G). (H–J) Three representative types of spindle orientation and cell shapes (bottom). Cross-sectional views of main cells in late anaphaseduring the binucleation stage in mutants hemizygous for mud4 are shown. Cells are labeled with phalloidin (magenta) and anti-P-H3 antibody (blue).
Scale bar in (H), 5 μm, is applicable to (H–J). (H’–J’) Schematic diagrams of (H–J). (K–N) Main cells expressing Pav::GFP plus ends marker in telophasein wild-type (K,L) and mud-knockdown (M,N) cells. Cell division stage in early pupa (K, en face view) and binucleation stage in mid-pupa (L–N, crosssectional views) are shown. Cells are labeled with anti-α-Tub antibody (magenta), Pav::GFP fluorescence with anti-GFP antibody (green) and anti-P-H3
antibody (blue). Arrowheads in (K and N) and curly brackets in (L and M) indicate the localization of Pav::GFP on microtubules. Scale bar in (K),
5 μm, is applicable to (K–N).

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Table 2 Ability of Mud variants to rescue binucleation defects in mud mutants
Genotype

Frequencies of phenotypes in mitotic behavior during binucleation

Wild-type (control)
mud4 hemizygote (mud4/Y) +rescue construct (RC)No RC

RC = DC281
RC = mudPBD
RC = mudL
RC = mudS

Central spindle
assembly

Contractile ringformation

Cell rounding

Abnormal spindleorientation***0% (N = 42, 7)

0% (N = 42, 7)

0% (N = 49, 5)

6% (N = 49, 5)(5%, partial*)(5%, partial**)82% (N = 40, 9)

87% (N = 40, 9)

30% (N = 40, 8)

40% (N = 40, 8)(15%, partial*)(13%, partial**)0% (N = 30, 4)

0% (N = 30, 4)

8% (N = 38, 4)

5% (N = 38, 4)(40%, partial*)(57%, partial**)63% (N = 30, 4)

67% (N = 30, 4)

37% (N = 30, 4)

17% (N = 30, 4)(33%, partial*)(33%, partial*)

30% (N = 43, 9)

30% (N = 43, 9)

12% (N = 41, 7)

44% (N = 41, 7)(51%, partial*)(56%, partial**)13% (N = 40, 8)

18% (N = 40, 8)

34% (N = 44, 7)

20% (N = 44, 7)(45%, partial*)(33%, partial**)Numbers of cells and tissues (pairs of lobes) observed are shown as (N = cells, tissues).
*A phenotype class with partial assembly of central spindle-like structure.
**A phenotype class with partial accumulation of F-actin accompanied by furrow progression.
***Spindle orientated horizontally (0° ± 45°) rather than vertically in metaphase.
DC281: Chromosomal duplication encompassing the mud gene region.

to the midzone of the central spindle (Figure 4N). We also
found that this localization was correlated with the central
spindle formation. Cells with partially assembled central
spindles showed a partial localization of Pav::GFP at the
midzone (Figure 4M), whereas cells with strongly assembled central spindles showed a clear localization (Figure 4N).
These results suggest that Mud represses microtubule
polymerization so much that microtubule filaments are nottargeted around the cell equator.

microtubule bundling or destabilization of the microtubules was apparent at late anaphase (Additional file 1:
Figure S5G–J and G’–J’ compared with C–E and C’–E’).
As a result, during binucleation the development of microtubules was insufficient for their interdigitation at thespindle midzone. We also observed that the contractile
ring component Peanut (Pnut), a Septin family protein,
was localized around the cleavage furrow at anaphase but
diffused during telophase (Additional file 1: Figure S6D–F
and D’–F’ compared with A–C and A’–C’). These results
imply that the contractile ring begins to form during anaphase and is degraded during telophase.
We also checked whether the loss of mud affected the
localization of Pav::GFP during binucleation. In mudknockdown cells, the localization of Pav::GFP was restricted

Isoform-specific functions of Mud regulate various traits
in binucleation

Although Mud is known to promote polymerization of
microtubules [34], our results showed that Mud may be
a negative regulator of spindle formation during theTable 3 Effects of knockdown for mud on binucleation and asymmetric cell division (asym. cell div.)
RNAi target

Frequencies of phenotypes in mitotic behaviors during
binucleation

None (fng-Gal4)

mud.IR
mudS.IR + Dcr2

asym. cell div.

Central spindle assemblyContractile ring formation

Cell rounding

Abnormal spindle orientation***Multi-bristle

0% (N = 35, 6)

0% (N = 35, 6)

5% (N = 40, 6)

8% (N = 40, 6)

0% (NW = 60)(0%, partial*)(3%, partial**)

80% (N = 35, 7)

86% (N = 35, 7)

28% (N = 40, 5)

35% (N = 40, 5)

85% (NW = 40)(20%, partial*)(14%, partial**)27% (N = 37, 5)

29% (N = 37, 5)

5% (N = 42, 5)

31% (N = 42, 5)

0% (NW = 40)(51%, partial*)(41%, partial**)Numbers of cells and tissues (pairs of lobes) observed are shown as (N = cells, tissues).
Numbers of adult wings observed are shown as (NW = wings).
*A phenotype class with partial assembly of central spindle-like structure.
**A phenotype class with partial accumulation of F-actin accompanied by furrow progression.
***Spindle orientated horizontally (0° ± 45°) rather than vertically in metaphase.

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functions that regulate central spindle assembly andspindle orientation during binucleation.
To identify isoform-dependent functions of Mud, we
tested the ability of MudPBD, MudL and MudS to rescue
the various defects in binucleation in mud hemizygotes.
Overexpression of mudPBD (Additional file 1: Figure S7
and Figure S8) rescued only their abnormal spindle orientation (Figure 6A–C and A’–C’), reducing the frequency ofabnormal spindle orientation from 40% in mud4/Y to 17%
in mud4/Y + mudPBD (Table 2). Overexpression of mudS
(Additional file 1: Figure S7 and Figure S8) rescued
their abnormally enhanced central spindle assembly(Figure 6H–J and H’–J’), reducing the frequency of central spindle assembly from 82% in mud4/Y to 13% in
mud4/Y + mudS (Table 2). mudS also partially rescued theabnormal spindle orientation (Figure 6G and G’), reducing
the frequency of abnormal spindle orientation from 40%
in mud4/Y to 20% in mud4/Y + mudS (Table 2). In contrast, the overexpression of mudL (Additional file 1: Figure
S7 and Figure S8) effectively rescued the cell rounding

binucleation process. The above results also indicate that
unknown functions of Mud regulate central spindle assembly, spindle orientation, and cell rounding. Interestingly, mud generates various splicing variants that havedifferent C-termini (Additional file 1: Figure S3A andreferred to in http://flybase.org). To understand therelationship between these unknown functions of Mudand its structural isoforms that had not fully been analyzed, we compared the functions of three different splicing variants (Figure 4A) (Additional file 1: Figure S7): a
Pins binding domain (PBD)-containing isoform(MudPBD), a PBD-lacking longer isoform (MudL) and a
PBD-lacking shorter isoform (MudS). MudPBD is wellknown major variant that regulates spindle polarity in a
Pins/LGN-dependent manner and promotes microtubule polymerization [29-31,34]. MudL and MudS,
both of which lack the Pins/LGN-binding domain(Figure 4A) (Additional file 1: Figure S7), have not yet
been examined and their functions are unclear. We
thus expected that mudL or mudS would exhibit novel

Figure 5 mudS is not required for the spindle orientation during asymmetric cell division. (A) Schematic diagram of molecular structures ofhuman NuMA1 and Drosophila Mud. Shared domains are as indicated at the bottom. (B) Amino acid sequence alignment of the C-terminal regions of
NuMA1-s and MudS. The blue box indicates short isoform- specific regions in NuMA1-s (amino acids 1701–1763) and MudS (amino acids 1880–1933).
Red, orange and yellow overlays indicate similarities as indicated at the bottom. (C) Schematic diagram of SOP lineage in Drosophila wing margin. Cells
indicated at the bottom right are produced in this lineage. Mud forms a complex with Gα and Pins that is localized asymmetrically (red crescent). (D–F)
Bristles on adult anterior wing margin in wild-type (D), in knockdown of all mud isoforms (E), and in knockdown specific to mudS (F). Arrowheads in (E)indicate multi-bristle phenotypes.

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Figure 6 Three types of mud splicing isoforms differently regulate morphologies of binucleating cells. (A–J) Rescue of mud mutant phenotypes
by each mud isoform. Cross-sectional views of main cells in metaphase (A, D and G), late anaphase (B, E and H) and telophase (C, F, I and J) during
the binucleation stage in mutants hemizygous for mud4 with overexpression of FLAG::mudPBD (A–C), FLAG::mudL (D–F) or FLAG::mudS (G–J).
Cells are labeled with phalloidin (magenta), anti-α-Tub antibody (green) and anti-P-H3 antibody (blue). The cell in (I) shows neither a central
spindle assembly nor furrow progression. The cell in (J) shows furrow progression but no central spindle assembly. Arrowheads in (C, F, I andJ) indicate equatorial planes. Scale bar in (A), 5 μm, is applicable to (A–J). (A’–J’) Schematic diagrams of (A–J). Cross-sectional views of
mudS- knockdown main cells in metaphase (K), late anaphase (L) and telophase (M) during the binucleation stage. Cells are labeled withphalloidin (magenta), anti-α-Tub antibody (green) and anti-P-H3 antibody (blue). Arrowhead in (M) indicates an equatorial plane. Scale bar in(A), 5 μm, is applicable to (K–M). (K’–M’) Schematic diagrams of (K–M). Effects on cell morphologies in each of the three rescued genotypes((A–C): mud4 hemizygotes rescued by mudPBD, (D–F): mud4 hemizygotes rescued by mudL, (G–J): mud4 hemizygote rescued by mudS) and in
mudS-knockdown cells (K–M) are listed under each set of diagrams.

central spindle assembly, MudPBD and MudS each play arole in orienting the spindle axis along the apico-basal
axis, and MudS also strongly represses central spindle
assembly.

phenotype found in mud hemizygotes (Figure 6D and D’),
reducing the frequency of cell rounding from 30% in
mud4/Y to 12% in mud4/Y + mudL (Table 2). mudL also
partially rescued the abnormally enhanced assembly ofcentral spindle (Figure 6E, E’, F and F’), reducing the frequency of central spindle assembly from 82% in mud4/Y
to 30% in mud4/Y + mudL (Table 2), but its ability was obviously less than that of mudS (compare the reduction to
30% by mud4/Y + mudL with the reduction to 13% by
mud4/Y + mudS). These results imply that Mud has thefollowing isoform-dependent functions during binucleation: MudL represses cell rounding and weakly represses

MudS orients mitotic spindle along the apico-basal
polarity and inhibits cytokinesis

The above overexpression results suggest that MudS
contributes to cytokinesis skipping during binucleation
more than MudL does, but the functions of MudS during
Drosophila development have not yet been reported. We
therefore tried to determine whether endogenously

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Figure 7 Model for binucleation and isoform-specific functions of Mud. Schematic diagrams of cell division and binucleation are shown.
In binucleation, MudS changes mitosis from cell division to binucleation by (1) reorienting the mitotic spindle from horizontal to vertical along
the apico-basal axis and (2) repressing assembly of the central spindle. MudPBD is also required at this time for orienting the spindle along theapico-basal axis. MudL, in contrast, (3) represses mitotic cell rounding and may assist in the process of cytokinesis skipping by (4) partially repressing
the assembly of the central spindle.

expressed mudS actually regulates binucleation in theaccessory gland. To do so, we performed a mudS -specificknockdown (Figure 4A and Figure 6H–K and I’–K’)(Additional file 1: Figure S7 and Figure S8) and foundthat the mudS -knockdown cells showed abnormally enhanced assembly of the central spindle (Figure 6L, L’, M
and M’) and abnormal orientation of the mitotic spindle(Figure 6K and K’) (Table 3). These results strongly suggest that MudS represses central spindle assembly andorients the spindle axis vertically but does not regulate
cell rounding.
MudPBD is known to regulate spindle orientation during asymmetric cell division, so we tried to determine
whether the spindle orientation-regulating function of
MudS during binucleation is independent of that of
MudPBD. We tried to do that by determining whether aknockdown of MudS affected asymmetric cell division in
the sensory organ precursor cells (SOPs) on the adultwing margins (Figure 5C). The spindle orientation ofthis asymmetric cell division is known to be regulated by
MudPBD in a Pins- and Gαi-dependent manner, and lossof this function results in a multi-bristle phenotype on
the adult wing (Figure 5C). In fact, the knockdown of all

Mud isoforms caused abnormal spindle orientation andthe multi-bristle phenotype (arrowheads in Figure 5E
compared with D) (Table 3) [29,35,36]. The knockdown of
mudS alone, in contrast, did not induce the multi-bristle
phenotype (Figure 5F) (Table 3), although it effectively
caused spindle orientation changes during binucleation(Figure 6I and I’) (Table 3). These results suggest that eventhough MudS regulates spindle orientation during binucleation, it is not involved in the spindle orientation during asymmetric cell division of SOPs.
As shown above, MudS seems to convert the mitotic
morphologies from the cell-division type (horizontal
spindle orientation and cytokinesis progression) to the
binucleation type (vertical spindle orientation and cytokinesis skipping) (Figure 6) (Table 2 and Table 3). To
examine this hypothesis, we tested the ability of MudS
to convert the mitotic morphologies of cells in the early
pupal accessory gland primordia, in which standard cell
division occurs. The results showed that the spindle orientation changed from horizontal to vertical (Figure 8E compared with Figure 2D). Furthermore, binucleate cells
appeared with some frequency (arrowhead in Figure 8F).
This effect of overexpression of mudS on cell morphologies

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Figure 8 Overexpression of mudS is sufficient for converting dividing cells into binucleating ones. (A–F) Cross-sectional views of main cells inearly pupal proliferating stage in which mudPBD (A and B), mudL (C and D) or mudS (E and F) are overexpressed. Cells in telophase (A, C andE) or interphase (B, D and F) are shown. Cells are labeled with phalloidin (magenta), anti-α-Tub antibody (green in A, C and E), anti-LamDm0
antibody (green in B, D and F) and anti-P-H3 antibody (blue). Arrowheads in (A, C and E) indicate equatorial planes. The arrowhead in (F) indicates abinucleate cell. Scale bar in (A), 5 μm, is applicable to (A, C and E). Scale bar in (B), 5 μm, is applicable to (B, D and F). (G, G’, I, I’, K and K’)
Wing imaginal discs in which FLAG::mudPBD (G), FLAG::mudL (I) or FLAG::mudS (K) is induced in their dorsal compartment (labeled with GFP).
Cells are labeled with phalloidin (magenta), GFP (green) and anti-FLAG antibody (blue). Gray-scale images in (G’, I’ and K’) are of the blue
channels in (G, I and K). Scale bar in (G), 100 μm, is applicable to (G, G’, I, I’, K and K’). (H, J and L) Magnified views of (G, I and K) aroundthe dorso-ventral boundaries. Magenta and green channels are shown. Cells with expression of mudS were enlarged in volume (L), possibly asa result of cytokinesis defects. (H’, J’ and L’) Cross-sectional images reconstructed by using a stack of confocal sections at cyan lines in (H, I
and L). The epithelium with expression of mudS has an abnormally folded or layered structure (arrowhead in L’). Scale bar in (H), 10 μm, isapplicable to (H, H’, J, J’, L and L’).

S1). This may indicate that standard mitosis and binucleation have very different regulations of the cell cycle
and cytokinesis. In fact, although in most tissues in
Drosophila standard mitosis can occur normally without
the spindle checkpoint [37], we previously demonstrated
that the knockdown of mad2, a spindle checkpoint component, frequently causes a defect in chromosomal segregation in accessory gland cells [16]. This is consistent
with our proposal that binucleation is regulated by a system different from the one regulating standard mitosis.

can be seen not only in the accessory gland but also in thewing imaginal disc (Figure 8K, K’, L and L’). Overexpression of mudPBD or mudL, in contrast, affected neither spindle orientation nor cytokinesis (Figure 8A–D, G–J and G’–J’). These results suggest that MudS can convert mitotic
morphologies from the cell-division type to the binucleation type.

Discussion
Regulation of M-phase entry in binucleation of Drosophilamale accessory gland

We characterized the final M-phase entry that contributed to binucleation of cells in the Drosophila maleaccessory gland (Figure 1D) (Additional file 1: FigureS1). We showed that the entry into the binucleation
stage took place with a two-step cell cycle transition: cell
cycle arrest for 5 hours and subsequent synchronousentry into the M phase (Figure 1D) (Additional file 1:
Figure S1). Thus, standard cell division and binucleation
are separated by a 5-hour interval of cell cycle arrest(Additional file 1: Figure 1D) (Additional file 1: Figure

Morphological features of mitotic cells during
binucleation

We identified three morphological features characteristicof binucleation: the non-round shape of mitotic cells,
the apico-basal orientation of the mitotic spindle andthe poor assembly of the central spindle (Figure 2F–J
and F’–J’ compared with A–E and A’–E’). The reductionof central spindle assembly could directly repress cytokinesis, but we did not find the significance of the vertical orientation of the spindle or the non-round shape of87

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the cell. We can explain it as follows. If most cells of theepithelium synchronously enter the M phase with thecell rounding phenotype, the stability of the monolayer
epithelium may be severely disrupted. In fact, we observed a severe defect in epithelial stability after the
binucleation stage in a mud mutant in which the cells
were rounded during the binucleation stage (data notshown). In addition, if the spindles in columnar cells arehorizontally oriented, they will be less stable than theywould be if they were vertically oriented because a spindle is aligned more stably along a longer axis than ashorter one [38]. Vertical orientation of the spindle andlack of cell rounding may thus be appropriate for synchronous binucleation of columnar cells, but these morphological features do not directly regulate cytokinesis
skipping.

from one another (Figure 6, Figure 5, Figure 8 and
Figure 7). It is known that the mud gene produces four isoforms (RH, RI, RJ and RL) that contain the Pins/LGN binding domain (PBD) and three isoforms (RF, RG and RK)
that do not contain it (Figure 5A) (Additional file 1: FigureS7) (http://flybase.org). Moreover, the three PBD-lacking
isoforms are structurally classified into the following two
types: a longer isoform (RF) that simply skips the PBDencoding exons by alternative splicing, and two shorter
isoforms (RG and RK) that contain a shorter isoformspecific exon instead of the PBD-encoding exons(Figure 5A) (Additional file 1: Figure S7).
At least one of the PBD-containing isoforms (RL) is
functional and known to regulate the spindle orientation
in a Pins/LGN-dependent manner in asymmetric cell
division [29-31]. In contrast, although mudL and mudS
are actually transcribed [34] there is no evidence that
MudL and MudS are functional and have Pins/LGN-independent functions. As in the Drosophila gene mud,splicing variants also occur in human NuMA1, and thesevariants encode a longer isoform (NuMA1-l), a shorter
isoform (NuMA1-s) and a medium isoform (NuMA1-m)[39]. Although the functions of NuMA1-m and NuMA1-s
are unclear, NuMA1-l has an LGN-binding domain in the
C-terminal region and determines the spindle polarity inan LGN-dependent manner, the same as in the case of
Drosophila MudPBD (Figure 5A) [40]. NuMA1-m and
NuMA1-s, in contrast, like Drosophila MudL and MudS
do not have an LGN-binding domain in the C-terminal
region (Figure 5A). Interestingly, we found sequencesimilarities between human NuMA1-s and Drosophila
MudS in their C-terminal domains, including the shorter
isoform-specific regions (Figure 5B). These similarities
suggest that MudS in Drosophila functions similarly to
NuMA1-s in humans.

Mechanisms by which Mud regulates central spindle
assembly, spindle orientation, and cell rounding during
binucleation

We propose that Mud is a key factor in regulating
binucleation. We demonstrated that Mud functions in a
way that represses central spindle assembly, orients the
mitotic spindle along the apico-basal axis and inhibits
mitotic cell rounding during mitosis (Figure 7). We
found a clue as to how Mud represses the central spindle assembly. In standard cell division, during late anaphase the microtubules of the central spindle arepolymerized in order to target their plus ends at thecell equator [32,33], and we confirmed this in our experiments in which the plus ends were labeled with
Pav::GFP (Figure 4K). During binucleation, in contrast,
the Pav::GFP marker did not sufficiently target the cell
equator (Figure 4L). This implies that the polymerizationof the microtubules of the central spindle is insufficient totarget them at the cell equator, and the central spindle
therefore does not develop completely. We also showedthat the Mud is needed in order to repress the growthof microtubules. In fact, the knockdown of mud promoted the growth of microtubules that targeted theirplus ends at the cell equator even in the binucleation
stage (Figure 4M and N). However, the underlying molecular mechanisms of Mud in repressing polymerizationof microtubules remain unclear. Moreover, the questionof how Mud regulates the mitotic spindle orientation
along the apico-basal axis and how it inhibits mitotic cellrounding are also unclear. The logical next step will be torelate the isoform of each Mud to various effector molecules regulating the orientation and the rounding.

Isoform-dependent functions of Mud mediate various
morphological changes of binucleating cells

We showed that the functions of mudPBD, mudL and
mudS are independent during binucleation. The repression of mitotic cell rounding was a MudL-specific function. In contrast, changing the orientation of the mitotic
spindle along the apico-basal axis was controlled by both
MudPBD and MudS (Figure 6 and Figure 7) (Table 2 andTable 3). MudPBD was previously reported to be required
for the spindle orientation during asymmetric cell division [29,35,36]. We showed, however, that MudS is not
associated with the spindle orientation during asymmetric cell division (Figure 5F). On the other hand, theoverexpression of mudS but not mudPBD reoriented thespindle along the apico-basal axis in dividing cells(Figure 8A and E). These results suggest that MudPBD and
MudS regulate the spindle orientation independently.

Alternative splicing of mud/NuMA produces three typesof structurally different proteins

We showed that the three types of alternative splicing
products MudPBD, MudL and MudS have distinct functions

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The function repressing central spindle assembly during binucleation was also shared by MudL and MudS,
but we showed that MudS contributed the most to
repressing spindle assembly. In fact, the overexpressionof mudS effectively rescued the mud4 mutant phenotype,the abnormally enhanced assembly of the central spindle(Figure 6I and J) (Table 2). In addition, like the mud4
mutant, a mudS -specific knockdown abnormally enhanced central spindle assembly in the binucleation
stage (Figure 6L and M) (Table 3). MudL, in contrast,
only partially repressed the central spindle assembly during binucleation (Figure 6F) (Table 2). Moreover, overexpression of mudS, but not mudL, inhibited cytokinesis to
produce binucleate cells in dividing cells such as theearly pupal accessory gland cells (Figure 8D and F) andthe larval wing disc cells (Figure 8J and L). This also suggests that mudS mainly contributes to the repression ofcentral spindle assembly (Figure 7).

expression of NuMA is known to be correlated with the
production of cancer cells in mammals [43]. Our findingof atypical functions of Mud may contribute to the understanding of the relationship between NuMA and tumor
progression.

Methods
Drosophila strains

Canton-S and w1118 were used as wild-type strains andthe following mutant alleles were used: a functional nullallele centrosominHK21 [25], a strong loss-of-function allele Sas-4S2214 [23], and a strong loss-of-function allele
mud4 [34]. Dp(1:3)DC281 is a chromosome with a duplication of the mud gene region [44]. fngNP5399 (fng-Gal4,
Gal4 Enhancer Trap Insertion Database, http://kyotofly.
kit.jp/stocks/GETDB/getdb.html) expresses Gal4 in the
pupal accessory gland epithelial cells and larval-pupal
wing disc (data not shown). AyGal4, apterousMD544 (apGal4), and Act5C-Gal4 have been described previously[45,46]. hs-FLP was used as the source of the FLP recombinase [47]. Tub-Gal80TS was used for the TARGET
system [48]. UAS-Sterile20-like kinase, UAS-pebble andUAS-sqhD20.D21 express the wild type of Sterile20-likekinase [49], the wild type of pebble [50] and a constitutively active forms of sqh [51]. The strains 103962 (UASshotgun.IR, VDRC), mudJF02911 (UAS-mud.IR, Transgenic
RNAi project) [52] and VALIUM20-mCherry (UASmCherry.IR, Transgenic RNAi project at Harvard Medical School) [52] express inverted repeat RNAs (which
form hairpin loop double-stranded RNAs) for shotgun,
mud, and mCherry. The strains mus209.ΔNhe::GFP(PCNA-GFP) [53], Ubi-Cyclin B::GFP [54], sqh::GFP[55] and Septin2::GFP [56] have been described previously. poloCC01326 (polo::GFP) is a protein trap line ofpolo (FlyTrap, http://cooley.medicine.yale.edu/flytrap/
index.aspx#page2) [57].

Conclusions
We described the binucleation event of the Drosophilamale accessory gland during pupal development andanalyzed the cellular mechanisms regulating this binucleation. We characterized a unique cell cycle regulation in the developing accessory gland: the M-phaseentry for binucleation occurred synchronously at 55
APF after a cell cycle arrest for 5 hours. We also foundthat Mud, the Drosophila homolog of mammalian
NuMA, regulated various features of the binucleating
cells, such as a non-round shape, spindle orientation
along the apico-basal axis, poor assembly of the central
spindle and cytokinesis skipping. It is known that Mudbinds Pins to determine the mitotic spindle orientation
during the standard cell division or asymmetric cell division [29-31,41,42]. Interestingly, we found atypicalfunctions of Mud that depended on three types of splicing isoforms, each differently regulating the above
various features of binucleating cells. We concluded
that MudPBD, which is a well-known isoform having a
PBD (Figure 5A) (Additional file 1: Figure S7), oriented
the spindle along the apico-basal axis. MudL, one of the
newly characterized isoforms and simply lacking a PBD,
inhibited the mitotic cell rounding and weakly impaired
the central spindle assembly (Figure 7). MudS, another
newly characterized isoform, containing a shorter
isoform-specific domain instead of a PBD-containing domain (Figure 5A) (Additional file 1: Figure S7), oriented
the spindle along the apico-basal axis and strongly impaired the central spindle assembly (Figure 7). Importantly, overexpression of MudS induced an ectopic
binucleation even in the cell division stage, whereas overexpression of MudPBD or MudL did not (Figure 8). These
results suggest that MudS is an important regulator triggering cytokinesis skipping in binucleation. Abnormal

Immunostaining and microscopic analysis

The dissected accessory glands were fixed with 4% formaldehyde (Wako) and stained using standard immunostaining protocols. For the DNA staining, the fixedsamples were pretreated with RNase (Wako, 0.025 mg/ml) for 15 minutes at 37°C and then stained with propidium iodide (Invitrogen, 1:500). Rhodamine-phalloidin(Invitrogen, 1:40) was used to stain the filamentous actin(F-actin). The following primary antibodies were used:
rabbit anti-phospho-histone H3 (P-H3) polyclonal antibody (Millipore, 1:200), mouse anti-α-tubulin (α-Tub)
monoclonal antibody (Sigma, 1:50), rat anti-α-tubulin(α-Tub) monoclonal antibody (Millipore, 1:25), rabbit
anti-PKCζ (C-20) polyclonal antibody cross-reactingwith Drosophila aPKC (Santa Cruz, 1:200), mouse anticoracle (Cora) monoclonal antibody (Developmental
Studies Hybridoma Bank, 1:20), mouse anti-lamin Dm0

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Gal80TS/UAS-pebble; fng-Gal4/+, w/Y; Tub-Gal80TS/
UAS-sqhD20.D21; fng-Gal4/+). To knockdown shotgun by
expressing shorgun.IR in the accessory gland primordia
just before binucleation (Additional file 1: Figure S4A
and B) (Table 1), pupae reared at 19°C for 80 hours APF
were incubated at 29°C for 20 hours (genotypes: w/Y;
Tub-Gal80TS/UAS-shotgun.IR; fng-Gal4/+). To moderately express mudS in the accessory gland primordia justbefore binucleation (Figure 6D–G), pupae reared at 19°C
for 110 hours APF were incubated at 26°C for 5 hours(genotype: mud4/Y; Tub-Gal80TS/+; fng-Gal4/UASFLAG::mudS). To express mudPBD, mudL and mudS in
the accessory gland primordia in the cell-division stage(Figure 5G–J), pupae reared at 19°C for 40 hours APF
were incubated at 29°C for 5 hours (genotypes: w/Y;
Tub-Gal80TS/+; fng-Gal4/UAS-FLAG::mudPBD, w/Y;
Tub-Gal80TS/+; fng-Gal4/UAS-FLAG::mudL , w/Y; TubGal80TS/+; fng-Gal4/UAS-FLAG::mudS). Pupae weredissected immediately after the target gene inductions
described above.

(LamDm0) monoclonal antibody (Developmental Studies
Hybridoma Bank, 1:40), mouse anti-peanut (Pnut)
monoclonal antibody (Developmental Studies Hybridoma Bank, 1:5) and mouse anti-FLAG M2 (FLAG)
monoclonal antibody (Sigma, 1:200). The following secondary antibodies were used: Cy3-conjugated donkey
anti-mouse IgG (Jackson ImmunoResearch, 1:200), Cy2conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, 1:200), Cy5-conjugated donkey anti-mouse IgG(Jackson ImmunoResearch, 1:200), Alexa Fluor 488conjugated donkey anti-rat IgG (Millipore, 1:200) andCy5-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, 1:200). Stained samples were mounted in
50% glycerol/PBS containing 0.25% n-propyl gallate(Wako) and observed with an ECLIPSE TE2000-U witha Digital ECLIPSE C1 and C1Si confocal system (Nikon).
Images were processed using EZ-C1 Gold Version 3.70
(Nikon), Adobe Photoshop CS3 Extended (Adobe Systems), and Adobe Illustrator CS3 (Adobe Systems).
Detection of formation of central spindle and contractile
ring

Construction of plasmids

We used microtubule bundles and actin filaments asmarkers for the central spindle and contractile ring, respectively. In central spindle assembly, microtubule filaments bundle together, forming a large structure thatcrosses the cell equator. We regarded microtubulerelated structures having these features (i.e., bundlingand equator-crossing) with fewer microtubule filaments
as partially assembled central spindles. However, if thefilaments neither associated with each other nor crossed
the cell equator, we did not regard the structures as central spindles.
The accumulation of actin filaments in the equatorial
region of the cell membrane is usually associated withformation of a cleavage furrow, and we considered acontractile ring to have partially formed if either of these
two features was observed.

pP-Acp70A-Stinger:
The upstream enhancer of Acp70A (-477 to -34) was
amplified from the genome DNA of Canton-S with a
PCR (primer set #1 in Additional file 2: TableS2) andsubcloned into T vector pMD20 (TaKaRa) by using TA
cloning. The fragment for the Acp70A enhancer wasdigested with BglII and NotI and subcloned into pHStinger (Drosophila Genomics Resource Center (DGRC))
to construct pP-Acp70A-Stinger.
pUAS-FLAG::mudPBD-attB (see Additional file 1: Figure S7,
Figure S8 and Additional file 2: Table S1):
The genomic fragment encompassing three subisoforms of mudPBD (mud-RH, mud-RI, and mud-RL in
Additional file 1: Figure S7) (from just after the startcodon to the stop codon) was amplified with a PCR (primer set #2 in Additional file 2: Table S2) from the BAC
clone CH322-147E14 (P[acman] Resources) [58] andsubcloned into T vector pMD20 (TaKaRa) by using TA
cloning. The forward primer also included the Kozak sequence, start codon and FLAG tag sequence. The seventh intron (eighth intron in the case of mud-RJ), which
included the mudS -specific exon, was removed fromthe fragment with an inverse PCR (primer set #3 inAdditional file 2: Table S2) and ligated to generate a
FLAG-tagged protein-coding region including mud-RH,
mud-RI and mud-RL. This fragment was digested with
NotI and KpnI and subcloned into pUASattB (FlyC31,
http://www.flyc31.org/) [59] to generate pUAS-FLAG::
mudPBD-attB.
pUAS-FLAG::mudL-attB (see Additional file 1: Figure S7,
Figure S8 and Additional file 2: Table S1):

Temporal expression of genes using the TARGET system

The TARGET system [48] was used for the temporal expression of genes in the pupal accessory gland epithelium. To restrict the expression of the target genes byactivation of Gal80TS, flies were reared at a permissive
temperature (19°C). To permit moderate expression of
the target genes by weak activation of Gal80TS, flies werereared at a semi-permissive temperature (26°C).
To fully express Sterile20-like kinase, pebble andsqhD20.D21 in the accessory gland primordia just before
binucleation (Figure 2K–O) (Table 1), pupae reared at
19°C for 110 hours after puparium formation (APF) wereincubated at 29°C for 5 hours (genotypes: w/Y;
Tub-Gal80TS/UAS-Sterile20-like kinase; fng-Gal4/+, w/Y;
Tub-Gal80TS/UAS-shotgun.IR; fng-Gal4/+, w/Y; Tub-

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2A, w*; M{3xP3-RFP.attP'}ZH-51C with ΦC31-mediatedsite-specific integration to generate transgenic lines.

The genomic fragment encompassing a mudL (mudRF in Additional file 1: Figure S7) (from just after thestart codon to the stop codon) was amplified with PCR(primer set #2 in Additional file 2: Table S2) from the
BAC clone CH322-147E14 (P[acman] Resources) [58]and subcloned into T vector pMD20 (TaKaRa) by using
TA cloning. The forward primer also included the Kozak
sequence, start codon and FLAG tag sequence. The seventh
intron, which included the PBD-encoding exons and mudSspecific exon, was removed from the fragment with an inverse PCR (primer set #4 in Additional file 2: Table S2) andligated to generate a FLAG-tagged Mud-RF-coding fragment. This fragment was digested with NotI and KpnI and
subcloned into pUASattB (FlyC31, http://www.flyc31.org/)[59] to generate pUAS-FLAG::mudL-attB.
pUAS-FLAG::mudS-attB (see Additional file 1: Figure S7,
Figure S8 and Additional file 2: Table S2):
The genomic fragment encompassing two subisoforms of
mudS (mud-RG and mud-RK in Additional file 1: Figure S7)(from just after the start codon to the stop codon) was
amplified with a PCR (primer set #5 in Additional files 2:
Table S2) from the BAC clone CH322-147E14 (P[acman]
Resources) [58] and subcloned into T vector pMD20(TaKaRa) by using TA cloning. The forward primer also
included the Kozak sequence, start codon and FLAG tag
sequence. The FLAG-tagged fragment, which included
both mud-RG and mud-RK, was digested with NotI and
KpnI and subcloned into pUASattB (FlyC31, http://www.
flyc31.org/) [59] to generate pUAS-FLAG::mudS-attB.
pUAS-mudS.IR-attB (see Additional file 1: Figure S7,
Figure S8 and Additional file 2: Table S2):
The genomic fragment including the mudS -specificexon and upstream intron with a splice donor and acceptor (Additional file 1: Figure S7) was amplified with a
PCR (primer set #6 in Additional file 2: Table S2) fromthe genome DNA of Canton-S and subcloned into T
vector pMD20 (TaKaRa) by using TA cloning (#1). The
mudS -specific exon without the upstream intron was
also amplified with a PCR (primer set #7 in Additional
file 2: Table S2) from the genome DNA of Canton-S and
subcloned into T vector pMD20 (TaKaRa) by using TA
cloning (#2). The plasmid #1 was digested with NotI and
XhoI, and the plasmid #2 was digested with EcoRI and
NotI. Both of the digested fragments were combined in asingle plasmid pUASattB (FlyC31, http://www.flyc31.
org/) [59] to generate pUAS-mudS.IR.

RT-PCR to verify the expression of UAS-mud isoforms

Total RNA was extracted from third-instar larvae ofthe following four genotypes: w/Y;; hs-Gal4/UASFLAG::mudPBD, w/Y;; hs-Gal4/UAS-FLAG::mudL, w/Y;;
hs-Gal4/UAS-FLAG::mudS, and w/Y;; hs-Gal4/+ (negative control). Before the extraction of RNA, larvae were
heat-shocked twice at 37°C for 45 minutes and subsequently incubated at 25°C for 2 hours to reach the highlevel expression of UAS targeted genes. The cDNA ofeach genotype was synthesized using the oligo-dT primer with PrimeScript RT-PCR kit (TaKaRa). Two primer sets (#8 and #9 listed in Additional file 2: Table S2of the supplementary material) that specifically amplify
FLAG::mud transgenes but not endogenous mud genes(Additional file 1: Figure S8) were used for the PCR.
Semi-quantitative RT-PCR to verify isoform-specificknockdown by UAS-mudS.IR

Total RNA was extracted from third-instar larvae of thefollowing two genotypes: w/Y; Act5C-Gal4/UAS-mCherry.
IR (control) and w/Y; Act5C-Gal4/+; +/UAS-mudS.IR(knockdown for mudS). The cDNA of each genotype wassynthesized using the oligo-dT primer with PrimeScript
RT-PCR kit (TaKaRa). Expressions of genes were normalized by using Rpl32 as a reference gene. The three sets ofprimers #10, #11 and #12 listed in Additional file 2:
Table S2 of the supplementary material (Additional file
1: Figure S7) were used for the PCR.
Animal ethics

All animals used in above genetic experiments wereanesthetized with carbon dioxide before each mating. Allprocedures complied with guidelines of the Animal Ethics
Committee of Gakushuin University.

Additional files
Additional file 1: Figures S1, S2, S3, S4, S5, S6, S7 and S8 are
included.
Additional file 2: Tables S1 and S2 are included.
Abbreviations
APF: After puparium formation; mud: mushroom body defect; PBD: Pins
binding domain; NuMA: Nuclear mitotic apparatus; RhoGAP: GTPase
activating protein for Rho; RhoGEF: Guanine nucleotide exchange factor for
Rho GTPase; M phase: Mitotic phase; Acp70A: Accessory gland protein 70A;
P-H3: Phospho-histone H3; PCNA: Proliferating cell nuclear antigen;
GFP: Green fluorescence protein; DE-cadherin: Drosophila epithelial cadherin;
MRLC: Myosin regulatory light chain; sqh: spaghetti squash; Par: Partitioningdefective protein; Pav: Pavarotti; Pnut: Peanut; SOP: Sensory organ precursor
cell; fng: fringe; ap: apterous; Act5C: Actin 5C; UAS: Upstream activation
sequence; Tub: Tubulin; TARGET: Temporal and regional gene expression
targeting; IR: Inverted repeat; aPKC: atypical protein kinase C; Cora: Coracle;
LamDm0: lamin Dm0.

Generation of transgenic fly lines

The pP-Acp70A-Stinger vector was injected into y, w flylines with standard protocols to generate transgeniclines. The pUAS-FLAG::mudPBD-attB, pUAS-FLAG::
mudL-attB, pUAS-FLAG::mudS-attB and pUAS-mudS.IRattB vectors were injected into y1, M{vas-int.Dm}ZH-2A,
w*;; M{3xP3-RFP.attP}ZH-86Fb or y1, M{vas-int.Dm}ZH-

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Competing interests
The authors declare that they have no competing interests.

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the way. Nat Rev Mol Cell Biol 2008, 9:355–66.
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Authors’ contributions
KT obtained and analyzed all of the cytological data and helped in all
experiments performed by other authors. AK discovered the synchronous andwavelike M-phase entry at binucleation. TI discovered the particular cell polarity
during binucleation and examined PCNA::GFP expression. RM and HN analyzed
the reproductive data and contributed to manuscript preparation. TAY directed
the project. The manuscript was written by KT and TAY. All authors read andapproved the final manuscript.

Acknowledgments
We thank S. Cohen, C. Gonzalez, K. Matsuno, J. Raff, R. Saint, A. Carpenter, theBloomington Stock Center, Kyoto Stock Center, the National Institute of
Genetics, Mitsubishi Kagaku Institute of Life Sciences and the VDRC Stock
Center for supplying us with fly stocks, and we thank the Developmental
Studies Hybridoma Bank at the University of Iowa for supplying us with
antibodies. We also thank BestGene Inc. for generating new transgenic flylines. We are grateful to M. Kamakura for technical advice.
Author details
1
Department of Life Science, Faculty of Science, Gakushuin University, Tokyo
171-8588, Japan. 2Institute for Biomolecular Science, Gakushuin University,
Tokyo 171-8588, Japan. 3Department of Biology, Graduate School of Science,
Kobe University, Kobe 657-8501, Japan. 4Graduate School of Natural Scienceand Technology, Okayama University, Okayama 700-8530, Japan.
Received: 13 May 2014 Accepted: 11 December 2014

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Contents lists available at ScienceDirect

Developmental Biology
journal homepage: www.elsevier.com/locate/developmentalbiology

GATAe regulates intestinal stem cell maintenance and differentiation
in Drosophila adult midgut
Takashi Okumura a, Koji Takeda a,c, Megumi Kuchiki a, Marie Akaishi a, Kiichiro Taniguchi a,
Takashi Adachi-Yamada a,b,naDepartment of Life Science, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan
Institute for Biomolecular Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japanc
Graduate Course in Life science, Graduate school of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japanbar t ic l e i nf o

a b s t r a c t

Article history:
Received 16 July 2015
Received in revised form
30 November 2015
Accepted 19 December 2015

Adult intestinal tissues, exposed to the external environment, play important roles including barrier andnutrient-absorption functions. These functions are ensured by adequately controlled rapid-cell metabolism. GATA transcription factors play essential roles in the development and maintenance of adultintestinal tissues both in vertebrates and invertebrates. We investigated the roles of GATAe, the Drosophila intestinal GATA factor, in adult midgut homeostasis with its ﬁrst-generated knock-out mutant aswell as cell type-speciﬁc RNAi and overexpression experiments. Our results indicate that GATAe is essential for proliferation and maintenance of intestinal stem cells (ISCs). Also, GATAe is involved in the
differentiation of enterocyte (EC) and enteroendocrine (ee) cells in both Notch (N)-dependent and -independent manner. The results also indicate that GATAe has pivotal roles in maintaining normal epithelial homeostasis of the Drosophila adult midgut through interaction of N signaling. Since recent reports showed that mammalian GATA-6 regulates normal and cancer stem cells in the adult intestinal
tract, our data also provide information on the evolutionally conserved roles of GATA factors in stem-cell
regulation.
& 2015 Published by Elsevier Inc.

Keywords:
Drosophila
Adult midgut
Intestinal stem cell
GATA transcription factor
GATAe

1. Introduction
The adult gastrointestinal tract is continually exposed to exogenous stress and damage. Under this condition, gastrointestinal
tissues have barrier, digestive, and absorptive functions, which aresupported by a high cellular metabolic rate. Therefore, intestinal
stem cell (ISC) maintenance is important for preserving intestinal
physiological functions to constantly provide mature gastrointestinal epithelial cells including enterocytes (ECs) and enteroendocrine cells (ees) throughout an organism's life. Also, thismechanism has been reported to be conserved among mammals
and Drosophila (Crosnier et al., 2006; Micchelli and Perrimon, 2006;
Ohlstein and Spradling, 2006; Simons and Clevers, 2011; Weissman,2000). Between both animals, similar genetic regulation of ISCs
including their proliferation and differentiation has also been reported (Jiang and Edgar, 2012; Takashima and Hartenstein, 2012).
GATA transcription factors are evolutionarily conserved and play
important roles in cell proliferation, differentiation, and survival ofnCorresponding author at: Department of Life Science, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan.
E-mail address: Takashi.Adachi-Yamada@gakushuin.ac.jp (T. Adachi-Yamada).

multiple developing and adult tissues/organs (Ayanbule et al., 2011;
Molkentin, 2000; Murakami et al., 2005). Those in hematopoiesis anddeveloping heart and endodermal tissues have been extensively studied and in various multicellular organisms (Ayanbule et al., 2011). In
vertebrates, GATA-4, GATA-5, and GATA-6 of the six GATA factors show
distinct expression patterns in adult gastrointestinal tissues (Fanget al., 2006). GATA-4 and GATA-5 tend to be highly expressed in
proximal tracts, such as the stomach, jejunum and/or ileum, while
GATA-6 is expressed throughout the gastrointestinal tract in mice andhumans (Fang et al., 2006; Haveri et al., 2008). Consistently, GATA-4 isessential for region-speciﬁc identities (Bosse et al., 2006) and show
lineage-speciﬁc expression. Brieﬂy, GATA-4 shows high- and low-level
expression in EC/proliferating crypt and Paneth cells, respectively(Bosse et al., 2006; Dusing and Wiginton, 2005). With regard to GATA5, it is expressed in secretory lineages such as ee, Paneth, and goblet
cells (Dusing and Wiginton, 2005). On the other hand, GATA-6 ismainly expressed in mature ee and immature proliferating cells in thecrypt (Dusing and Wiginton, 2005). Accordingly, each of these GATA
members are at least in part involved in the proliferation or differentiation of immature cells in the intestinal crypt, and gene expression
in differentiated cells (Beuling et al., 2012; Beuling et al., 2011; Bosseet al., 2006; Divine et al., 2004; Haveri et al., 2008). Furthermore, GATA
factors are involved in gastrointestinal diseases, including cancer, with

http://dx.doi.org/10.1016/j.ydbio.2015.12.017
0012-1606/& 2015 Published by Elsevier Inc.

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interaction of signal transduction such as Wnt and BMP signaling (Linet al., 2012; Tsuji et al., 2014; Whissell et al., 2014; Zheng and Blobel,2010). However, in invertebrates including the Drosophila, few studiesof GATA factors have been reported regarding the maintenance of theadult digestive tract. Therefore, essential evolutionally conserved features in GATA functions in gut maintenance have remained elusive.
In Drosophila, ﬁve GATA transcription factor genes, pannier (pnr,also known as GATAa), serpent (srp, also known as GATAb), grain (grn,also known as GATAc), GATAd, and GATAe, are known (Abel et al.,
1993; Lin et al., 1995; Okumura et al., 2005; Ramain et al., 1993;
Rehorn et al., 1996; Winick et al., 1993). Of them, srp, grn, GATAd, andGATAe are expressed in embryonic, larval, and/or adult midguts, acounterpart of the mammalian small intestine, whose epithelium isderived from the endoderm (Murakami et al., 2005; Okumura et al.,2005; Senger et al., 2006). The srp and GATAe are required for speciﬁcation and differentiation of the endoderm forming larval midgut
epithelium (Okumura et al., 2005; Rehorn et al., 1996; Reuter, 1994).
In addition, srp expression in the endoderm disappears at embryonicstages, while GATAe continues to be expressed from embryonic toadult stages (Okumura et al., 2005). In adult midgut homeostasis,
GATAe has recently been reported to be required for maintenance of
the EC morphological structure, digestive function, and intestinal
gene expression (Buchon et al., 2013). On the other hand, the function of grn and GATAd in the midgut has remained unknown.
In this study, to determine whether GATA factors are involved
in regulation of Drosophila adult midgut homeostasis, we conducted RNAi treatment for the ﬁve Drosophila GATA factor genes in
ISCs and their daughter cells enteroblasts (EBs). We found that
GATAe RNAi only induced some defective phenotypes including adecrease in ISCs/EBs. We also conﬁrmed similar defects with theﬁrst-generated knock-out mutant of GATAe. Furthermore, GATAeknocked out cells indicated the possibility that GATAe is essential
for EC and ee differentiation in an N-dependent and independent
manner. Based on observations of the new GATAe knock-out mutation, we found that GATAe is required for normal adult midgut
development. Combined with previous reports, our results indicate that GATAe contributes to the development and maintenance of Drosophila midgut epithelia throughout life.

(Bardet et al., 2008) and UAS-GFPS65T. The hs-FLP provided FLP recombinase under heat-shocked temperature, tub-GAL80ts ubiquitously overexpressing the temperature-sensitive GAL80 protein(McGuire et al., 2003), and FRT82B, FRT82B,tub-GAL80, FRT19A, andtub-GAL80,hs-FLP,wn,FRT19A used for mosaic analysis with repressible
cell marker (MARCM), were also obtained from BDSC.
Flies were cultured in standard medium at appropriate temperature and females were used in all experiments in this study.
2.2. Generation of GATAe knock-out ﬂy
Knock-out of the GATAe locus was conducted by ends-out genetargeting, as previously reported (Rong and Golic, 2000). To construct
the pW25-GATAe-KO vector, the 5′ and 3′ homologous fragments were
ampliﬁed by polymerase chain reaction (PCR) from w1118 genomic
DNA with the following primers; 5′-CACCGCGGCCGCATGGTCTGCAAAACTATCTCAC-3′, 5′-CACCGGTACCTGCGTTGTCTGCTGTTCCAT-3′,
5′-CACCGGCGCGCCGGCGCTCCAAGTTTAC-3′, and 5′-CACCCGTACGCTGCAAGTGCATTAGTGTG-3′, then the ampliﬁed fragments were subcloned into the NotI-Acc65I and AscI-BsiWI sites of the pW25 vector(Drosophila Genomics Resource Center). The transgenic donor linestransformed with the pW25-GATAe-KO construct were generated by
BestGene Inc. Male donor ﬂies were crossed with w;;hsp70-FLP, hsp70I-SceI females and the progeny were heat-shocked at 37 °C for an hour
three times at day 2 and 3 after egg laying. The F1 females with mosaic
or white eyes were collected and crossed with w;hsp70-FLP males, andF2 males and females with red eyes were screened and ﬁve lines, inwhich the pW25-GATAe-KO construct was replaced with GATAe
genomic locus, were obtained. The replacement was investigated bygenomic PCR with the following primers; 5R 5′-GAATTGAATTGACGCTCCGT-3′, 3R 5′-GTCCGGTTGTTTTCGTGCTC3′, 1 5′-GGTGTGGGTAGCTAATTGG-3′, 2 5′-GATCGTGATCATGATACGAC-3′, 3 5′-TCGCAGGGAGTCAACCTGA-3′, 4 5′-AGCAGCTTCAACTGCTGG-3′, and 5 5′-ATTGACTCTGTGGCGTTGAT-3′. The resulting deletion covered almost all
the coding region of GATAe, as shown in Fig. 2A, and this line was used
as a loss of the function allele of GATAe (termed GATAe1) in this study.
To select the 1st instar larvae of GATAe1 homozygotes, the green
balancer TM3,twi-GAL4,UAS-2  GFP was used.
2.3. Immunostaining and in situ hybridization

2. Materials and methods
2.1. Fly stocks
Canton-S and w1118 were used as wild-type strains. The following
RNAi lines obtained from the Vienna Drosophila RNAi Center (VDRC)
were used for knock-down of GATA family genes and N; pnr(#101522), srp (#112327), grn (#105192), GATAd (#50364), GATAe(#10420), and N (#100002). We also obtained the other GATAe RNAilines (#33748 and #34641) from the Bloomington Drosophila Stock
Center (BDSC). The Df(3R)sbd45 is a deﬁciency line uncovering the
GATAe locus. The reporter lines for the Delta (Dl) transcription,
N-signaling activity, JAK-STAT-signaling activity, and c-Jun N-terminal
kinase (JNK)-signaling activity were Dl05151 (Dl-lacZ), Gbeþ Su(H)m8lacZ, 10  STAT92E-GFP, and pucE69 (puc-lacZ), respectively (Bach et al.,2007; Biteau et al., 2008; Furriols and Bray, 2001; Martin-Blanco
et al., 1998). The GAL4 lines used were NP6267 (esg-GAL4) (Hayashi
et al., 2002) and Ay-GAL4 (Ito et al., 1997). The following upstream
activating sequence (UAS) strains were used: UAS-FLAG::GATAe producing a functional tagged form of the GATAe protein (gift from
Murakami), UAS-GATAe::V5-6  His producing a functional fusion
protein tagged with V5 epitope and 6  His and its effect is weaker
than FLAG::GATAe (personal communication) (gift from Murakami),
UAS-NICD (Go et al., 1998), UAS-p35 (BDSC), UAS-DIAP-myc (gift fromHay) (Umemori et al., 2007), and UAS-rpr (DGRC #108447). The following UAS lines were also obtained from BDSC; UAS-Apoliner

Larval, pupal, and adult midguts dissected at appropriate stages
were ﬁxed with 4% paraformaldehyde. After being washed withphosphate buffered saline (PBS) containing TritonX-100 (PBT), immunostaining was done with the following primary antibodies; rat
anit-GFP (Nacalai Tesque, 1:200), chick anti-β-galactosidase (β-gal)(abcam, 1;200), rabbit anti-phospho histone H3 (pH3) (Upstate Biotech,
1:200), mouse anti-Prospero (Pros) (DSHB, 1:100), mouse anti-Armadillo (Arm) (DSHB, 1:50), mouse anti-Delta (Dl) (DSHB, 1:50), mouseanti-dual-phosphorylated ERK (dpERK) (Sigma-Aldrich, 1:200), andrabbit anti-Pdm1 (provided by Xiaohang, 1:200). Secondary antibodies
used were Cy3- and DyLight649-conjugated anti-mouse IgG (Jackson
ImmunoResearch, 1:200), Alexa Fluors 488-conjugated anti-rat IgG(Jackson Immuno Research, 1:200), Alexa Fluors 555-conjugated and
DyLight649-conjugated anti-rabbit IgG (Jackson ImmunoResearch,
1:200), and DyLight649-conjugated anti-chick IgY (Jackson ImmunoResearch, 1:200). Rhodamine-conjugated and Alexa Fluors 647conjugated phalloidin (Molecular Probe, 1:100) were used to stain ﬁlamentous actin (F-actin). Nuclei were stained with DAPI (SIGMA).
For in situ hybridization, the adult midgut ﬁxed with 4% paraformaldehyde was washed with PBS containing 0.1% Tween 20.
After they were treated with 10 μg/ml proteinase K and reﬁxed
with 4% paraformaldehyde, hybridization with digoxygenin-labeled RNA probes was done at 60 °C overnight. After washing, they
were treated with a 0.1% blocking regent (Roche) and reacted withan anti-Digoxygenin antibody labeled with alkaline-phosphatase

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Fig. 1. GATAe RNAi in ISC/EBs caused a decrease of their cell number and morphological defects. (A–G) Frequency and morphology of the esg-positive cells in the esgGFPts (A, D, andF) and esgGFPts 4GATAeIR (B, E, and G) PMG. Green is esg-positive cells. Magenta in A and B, D and E, and F and G were stained with anti-Arm antibody, anti-Arm and anti-Pros
antibodies, and phalloidin, respectively. Insets in F and G are magniﬁcations. (C) Frequency of esg-positive cells in FOV of esgGFPts (24.0471.23 at 2 days old, 30.3570.77 at 7 days
old, and 26.3370.39 at 14 days old) and esgGFPts 4GATAeIR (23.9371.73 at 2 days old, 22.1771.64 at 7 days old, and 18.1671.06 14 days old) midguts. The esg-positive cells in FOVof 5 or 6 midguts were counted. Error bars indicate standard error of means (S.E.M). P-values were calculated using Student's t test. (H and I) PMG of esgGFPts 4NIR (H) andesgGFPts 4GATAeIR þ NIR (I). Green and magenta are esg-positive and Pros-positive cells. (J–L) GATAe mRNA in the PMG of wild-type (J and K) and esgGFPts 4NIR (L) was detected with
antisense (J and L) but not sense (K) probes (magenta). Green indicates Pros- (J’, J”, K’ and K”) and esg-positive (L’ and L”) cells. Blue and white indicate nuclei stained with DAPI.

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Fig. 2. Novel knock-out mutation of GATAe (GATAe1) inhibited normal epithelial regeneration. (A) Replacement of GATAe locus induced by targeted homologous
recombination. Green arrows indicate primers used in conﬁrming the replacement. The results of genomic PCR with several pairs of primers showed precise replacement. (B–K) MARCM clones of control (B–E), GATAe1 (F–I), and GATAe1 expressing FLAG::GATAe (J and K) in the PMGs at 4 (B, F, and J), 7 (C, G, and K), 11 (D and H),
and 14 (E and I) days after clone induction (ACI). Insets are magniﬁcation of the boxes. The MARCM clones and nuclei were marked with GFP (green) and stained withDAPI (blue). (L) Graph showing average cell number in each genotype of MARCM clones at 4 and 7 days ACI. Average cell numbers are 4.78 7 0.78 (day 4) and5.71 7 0.57 (day 7) in control clones, 1.67 7 0.07 (day 4) and 1.70 7 0.08 (day 7) in GATAe1 clones, 1.86 7 0.20 (day 4) and 2.39 7 0.14 (day 7) in GATAe1 expressing FLAG::
GATAe clones, and 1.92 7 0.09 (day 4) and 2.05 7 0.11 (day 7) in GATAe1 expressing p35 clones. Error bars indicate S.E.M. P-values were calculated using Student's t test.
The number of counted clones is in parentheses.

(Roche, 1:7000). Then, the anti-Digoxygenin antibody was detected using FastRed (Roche). The cDNAs of GATAe and βint-ν wereused as templates for antisense and sense RNA probes preparedusing a DIG RNA labeling kit (Roche).

The stained adult midgut was mounted in 80% glycerol andanalyzed with Nikon Digital Eclipse C1 and C1Si confocal microscopes (Nikon). The images were processed with EZ-C1 3.90 Free
Viewer (Nikon).

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Fig. 3. GATAe1 disrupted ISC proliferation and maintenance. (A–E) MARCM clones of control (A and C), GATAe1 (B and D), and GATAe1 expressing FLAG::GATAe (E) immnostainedwith anti-pH3 (magenta in A and B) and anti-Dl (magenta in C–E) antibodies. A’, B’, C’-C”’, D’-D”’, and E’-E”’ are magniﬁcations of the boxes in A, B, C, D, and E, respectively. The
yellow dotted lines indicate the outline of the MARCM clones. The yellow arrows in A and B indicate mitotic (pH3-positive) cells. The magenta arrows in C–E indicate examplesof Dl-positive cells. The MARCM clones and nuclei in A–E were marked with GFP (green) and stained with DAPI (blue). (F) Frequency of MARCM clones containing Dl-positive
cells in each genotype at 7 days ACI. The number of counted clones is in parentheses. (G) Clone size and ratio of clones containing no, one, and two Dl-positive cells at 7 days ACI.

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2.4. TUNEL assay

3. Results

The dissected adult midguts were ﬁxed with 4% paraformaldehyde in PBS and cell death was detected with the Apoptag kit(Millipore Corporation, Billerica, MA, USA).

3.1. GATAe-RNAi in ISCs/EBs affects midgut epithelial renewal

2.5. Temperature shift assay
In the RNAi and overexpression experiments with the TARGET
system (McGuire et al., 2003), adult ﬂies carrying tub-GAL80ts combined with esg-GAL4 were raised at a permissive temperature (18 °C).
One-day-old ﬂies were placed and cultured at a non-permissive
temperature (29 °C) until dissection at the appropriate day old. In
GATAe knock-down experiments by RNAi during larval and pupa
stages, the given genotypes of the 1st larvae or white pupa raised at
18 °C were placed and cultured at 29 °C to the adult stages. Eachgenotype used in this assay is described in the Figure legends.
2.6. Mosaic analysis
To generate MARCM clones (Lee and Luo, 1999), given genotypes
ﬂies were raised and cultured at 25 °C until 7 days old, heatshocked at 37 °C for 2 h 4 times, and additionally cultured for an
appropriate number of days. The MARCM and twin-spot clones
were also induced at the 3 rd instar larval stages and examined at
the adult stages.
When the GATAe RNAi experiment with Ay-GAL4 (Ito et al., 1997;
Struhl and Basler, 1993) was conducted, adult ﬂies were raised andcultured at 18 °C until 7 days old, heat-shocked at 37 °C for 20 min,and cultured at 25 °C for an appropriate number of days.
2.7. Quantiﬁcation and statistical analysis
To examine the frequency of esg-positive cells in the GATAe RNAi
experiment (Fig. 1C), the images of the posterior midgut of control(w;esg-GAL4,UAS-GFPS65T/þ;tub-GAL80ts/þ) and GATAe RNAi (w;esg-GAL4,UAS-GFPS65T/UAS-GATAe RNAi; tub-GAL80ts/þ ) ﬂies stainedwith anti-GFP and anti-Pros antibodies were obtained using a
confocal microscope. Then the total numbers of cells and esg-positive cells in the ﬁeld of view (FOV) were manually counted in theposterior midgut using EZ-C1 3.90 Free Viewer (Nikon). Examples of
FOV (636.5 μm  636.5 μm) images are shown in Fig.1A and B.
To count the cell number per clone of controls (hs-ﬂp/þ;Act5CGAL4,UAS-GFPS65T/þ;FRT82B,tub-GAL80/FRT82B), GATAe1 (hs-ﬂp/ þ;
Act5C-GAL4,UAS-GFPS65T/þ;FRT82B,tub-GAL80/FRT82B,GATAe1),
GATAe1 þUAS-FLAG::GATAe (hs-ﬂp/ þ;Act5C-GAL4,UAS-GFPS65T/UASand
GATAe1
FLAG::GATAe;FRT82B,tub-GAL80/FRT82B,GATAe1),
S65T
þUAS-p35 (hs-ﬂp/þ ;Act5C-GAL4,UAS-GFP/UAS-p35;FRT82B,tubGAL80/FRT82B,GATAe1) generated in the posterior midgut with
MARCM, we stained the adult midguts with DAPI and anti-GFP
antibody, obtained images with the confocal microscope, andcounted the cells in each MARCM clone (Fig. 2L and Fig. 4D). The
frequencies of Dl-, Pdm1- and Pros-positive cells in each genotype
clone were also counted after staining with anti-Dl, anti-Pdm1,
and anti-Pros antibodies (Fig. 3F, and G, Fig. 6I, and J and Supplemental Fig. 4B).
In FLAG::GATAe and GATAe::V5-6  His overexpression with MARCM,
the frequency of each clone size was counted (Supplemental Fig. 3E).
The genotypes used were hs-ﬂp/þ; Act5C-GAL4,UAS-GFPS65T/UAS-FLAG::
GATAe; FRT82B,tub-GAL80/FRT82B,GATAe1 and tub-GAL80,hs-FLP,w*,
FRT19A/FRT19A; Act5C-GAL4,UAS-GFPS65T/þ; GATAe::V5-6  His/þ .
The P-values were calculated using student's t-test as needed.

To determine whether the Drosophila GATA factors were involvedin ISC regulation, we expressed each UAS-RNAi-construct against theﬁve GATA factor genes, pnr, srp, grn, GATAd, and GATAe. The escargot(esg)-GAL4 driver under the control of GAL80ts, a temperature-sensitive GAL80, combined with UAS-GFP (esgGFP ts) speciﬁcally induced
strong expression of the above constructs in both ISCs and EBs at thepermissive temperature of 29 °C but not at the non-permissive
temperature of 19 °C (Fig. 1A) (Micchelli and Perrimon, 2006). Of theﬁve GATA factor genes, we found that GATAe RNAi (GATAeIR) caused afrequent decrease in esg-positive cells in the posterior midgut (PMG)
when the ﬂies were cultured at 29 °C for 7 days and 14 days but notfor 2 days after eclosion (Fig. 1B and C). We also observed morphological and positional defects of esg-positive cells. The normal ISCs/
EBs showed a pyramidal morphology and was located in an epithelial
niche adjacent to the basement membrane (Fig. 1D and F). On theother hand, GATAeIR-expressed ISCs/EBs showed a slightly larger,
spherical morphology and detachment from the basal region (Fig. 1E
and G compared with D and F). Similar defects were observed withthe other RNAi constructs (#34641 and #33746) that direct against
the different regions of GATAe mRNA (Supplemental Fig. 1A and B).
Also, the morphological defects were not suppressed even by cooverexpression of the anti-apoptotic factors, p35 and Diap1 (Supplemental Fig. 1C and D).
In addition, GATAeIR driven with esgGFPts resulted in a thin midgut
tube (Fig. 1B and G compared with 1 A and F). This atrophy probably
suggests that GATAeIR induces an insufﬁcient epithelial renewal. Indeed, GATAeIR inhibited normal growth of the MARCM clones (Supplemental Fig. 1E–J) when compared with pnrIR, srpIR, grnIR and
GATAdIR. GATAeIR also inhibited ISC- and ee-like tumor formationinduced by N RNAi (NIR) with esgGFP ts, (Fig. 1H and I) (Micchelli andPerrimon, 2006). Thus, GATAe-knock-down in ISCs/EBs affected
continuous epithelial renewal of the Drosophila adult midgut.
3.2. GATAe is expressed in all types of adult midgut epithelial cells
GATAe transcripts were previously detected from the adultmidgut by RT-PCR and microarray analysis (Buchon et al., 2013;
Okumura et al., 2005). To further examine which epithelial cell
types express GATAe in the adult midgut, we conducted in situ
hybridization for GATAe mRNA. With its antisense but not sense
probe, GATAe mRNA was detectable at similar levels in all types of
epithelial cells; ISC/EB (diploid cells), ee (Pros-positive cells) andEC (polyploid cells) (Fig. 1J–K”). Also, GATAe was expressed in ISCand ee-like tumors induced by NIR (Fig. 1L–L”). This was consistent
with the tumor suppression induced by GATAeIR (Fig. 1I). The GATAe expression implied that it plays important roles in all types ofadult midgut epithelial cells.
3.3. Generation of novel GATAe knock-out mutant
Previously, GATAe functions in developmental and adult midguts were studied through RNAi experiments (Buchon et al., 2013;
Okumura et al., 2005). In this study, to more directly evaluate
GATAe roles, we generated a knock-out mutant of GATAe (GATAe1)
by using a homologous recombination–based gene targeting
technique (Fig. 2A) (Rong and Golic, 2000). Most of the coding
region of GATAe was replaced with the w þ gene in GATAe1, which
was veriﬁed by genomic PCR with primers corresponding to the
GATAe locus and the knock-out construct pW25 (Fig. 2A). All individuals of GATAe1 homozygotes were died at the embryonic orthe 1st instar larval stages with morphological and digestive defects in the midgut (Supplemental Fig. 6A–D). The Df(3 R)sbd45, aPlease cite this article as: Okumura, T., et al., GATAe regulates intestinal stem cell maintenance and differentiation in Drosophila adult99
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7

Fig. 4. GATAe depletion in ISC/EB-like small cells did not predominantly undergo cell death. (A) GATAe1 MARCM clone labeled with GFP (green) at 7 days ACI. TUNEL staining(magenta in A and A’) was rarely detected in the GATAe1 clones (arrows in A and A’). A’ is the single channel of TUNEL staining. (B) Adult PMG treated by rpr overexpression
with esgGFPts for 1 day. Almost all esg-positive cells (green) disappeared. (C) Adult PMG expressing Apoliner and GATAeIR that were induced by esgGFPts for 21days. GFP (green
in C and C’) and RFP (C and C”) both were tethered to plasma membrane, indicating that the caspase pathway was not activated. C’ and C” are single channels of GFP (green)
and RFP (magenta) in the box of C, respectively. (D) Graph showing proportion of GATAe1 clone size at 4, 7, 11, and 14 days ACI. (E) Requirement of GATAe in ISC maintenanceincluding its proliferation, Dl expression, diploid state, and survival.

deﬁciency line uncovering the GATAe locus failed to complement
the lethality of GATAe1 (data not shown), suggesting that GATAe is
essential for survival.
3.4. GATAe is required for ISCs proliferation
Next, we generated GATAe1 homozygous clones in the adult
PMG with MARCM (Lee and Luo, 1999). Control clones showednormal growth at days 4, 7, 11, and 14 after clone induction (ACI)(Fig. 2B–E and L). In contrast, GATAe1 clone growth was inhibited
under the same condition (Fig. 2F–I and L) and the clones largely
stayed at the basal region (Fig. 6D and Supplemental Fig. 2I). In
addition, at day 7 but not day 4 ACI, the GATA1 clone growth inhibition was slightly rescued by FLAG::GATAe overexpression(Fig. 2J–L). This slightness was probably due to the dominant effectof GATAe overexpression that affected ISC maintenance, as described in the discussion (Supplemental Fig. 3). Combined with theabove results (Fig. 1 and Supplemental Fig. 1), we concluded that
GATAe is required for epithelial renewal in the midgut.
We next investigated whether GATAe is required for ISC proliferation. To examine this, we conﬁrmed the appearance of mitotic ISCs in GATAe1 clones with anti-phospho-Histone H3 (pH3)antibody. The result was that any mitotic ISCs were not observedin GATAe1 clones at day 7 ACI (Fig. 3A and B). Similarly, the pH3positive cells also disappeared in the GATAe RNAi-treated midgut
with esgGFPts (Supplemental Fig. 1K and L). These results suggestthat GATAe is required for ISC proliferation.
Numerous signal transductions have been reported to be involved in promoting ISC proliferation. We asked whether the two
major signaling pathways to induce ISC proliferation, Janus kinase/

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Fig. 5. GATAe1 suppressed EC and ee differentiation. (A–H) MARCM clone cells of control (A and B), GATAe1 (C and D), GATAe1 expressing FLAG::GATAe (E and F), and GATAe1
expressing p35 in the PMG at day 7 ACI. The Pdm1 (magenta in A, C, E and G) and Pros (yellow in A’, C’, E’ and G’) expressions were detected with their antibodies. The crosssections of each MARCM clone in B, D F, and H were stained with phalloidin (magenta). Insets are magniﬁcations of the boxes. White and yellow arrows indicate Pdm1- andPros-positive cells in the clones. Magenta arrows indicate brush border (bb). MARCM clones were marked with GFP (green) and their outlines in the insets are indicated bydotted lines. Yellow numerals in A”’, C”’, E”’ and G”’ indicate cell numbers in the GFP-positive clone(s) in insets. Nuclei were stained with DAPI (blue and white). (I and J)
Graphs showing frequency of Pdm1- (I) and Pros- (J) positive cells in each genotype clone. The number of counted cells is represented at the top.

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Fig. 6. GATAe1 caused cell growth inhibition without any N-signaling inactivation. (A–E) ISC (A and C) and transient (B, D, and E) MARCM clones of control (A and B), GATAe1(C and D), and GATAe1 expressing Nintra (E) in the adult PMGs. The Gbe-Su(H)m8-lacZ (magenta) and Dl (yellow) expression were detected in A–D. The MARCM clones andnuclei were labeled with GFP (green) and DAPI (blue). Right panels are magniﬁcation of the boxes. The yellow dotted lines indicate the outline of the MARCM clones (A–D). (Fand G) PMG expressing NIR (F) and co-expressing GATAe::V5-6  His and NIR (G) with esgGFPts. F’ and G’ are magniﬁcations of the boxes in F and G, respectively. The esgpositive cells were marked with GFP (green). The Pros-positive cells (magenta) were also marked with anti-Pros antibody. Nuclei (blue) were stained with DAPI. (H) Predictedgene regulatory pathway necessary for EC and ee differentiation.

signal transducer and activator of transcription (JAK-STAT) andepidermal growth factor (EGF), are affected by GATAeIR by using
10  STAT92E-GFP and dual-phosphorylated ERK (dpERK) antibody,respectively (Jiang and Edgar, 2009; Jiang et al., 2009). However,
we did not detect any obvious alteration in them in theesgRFPts 4 GATAeIR midguts (Supplemental Fig. 1M and N). Thissuggested that the decrease in ISC proliferation in GATAe1 was notcaused by a decrease in JAK-STAT or EGF signaling.
3.5. GATAe is essential for maintaining stemness of ISCs
A possible explanation for the disappearance of proliferative cells
is due to ISC loss. To conﬁrm this, we examined whether GATAe1
clones had diploid cells expressing Dl, an ISC molecular marker(Ohlstein and Spradling, 2007). In normal midguts, about half of thecontrol MARCM clones were ISC clones that were multicellular andcontained Dl þ cells, while the other half were transient clones thatwere Dl- single cells at day 7 ACI (Fig. 3C, F, and G). On the other
hand, the frequency of GATAe1 MARCM clones containing Dl þ cells
drastically decreased (Fig. 3D, F, and G). Similarly, a decrease in Dl þ
and Dl-lacZ þ cells was observed in the esgGFPts 4GATAeIR midguts at
14 days old (Supplemental Fig. 2A–D). Under the same condition,
RNAi against the other GATA factors did not abolish Dl expression
both at 7 and 14 days old (Supplemental Fig. 2E–H and data notshown). Furthermore, we frequently observed the appearance ofGATAe1 ISC clones without any diploid cells (Supplemental Fig. 2I and
J). Instead, as observed in the esgGFP ts 4GATAeIR midgut, they haveslightly larger nuclei when compared with the other diploid cells
such as Dl þ ISC, mitotic ISC, and Pros þ ee (Fig. 3D, Supplemental
Fig. 2I and J, and Supplemental Fig. 4). These results suggest that

GATAe is required for ISC's identities and properties. On the other
hand, we also found that the ISC disappearance in the GATAe1 mutant
partially rescued by FLAG::GATAe overexpression (Fig. 3E, F, and G) as
in the case of clone growth (Fig. 2).
Next, to conﬁrm whether GATAe-depleted ISCs underwent apoptosis that triggered their elimination, we conducted TUNEL assay for
the GATAe1 clones. However, TUNEL-positive cells were rarely observed in small GATAe1 clone cells (Fig. 4A). Consistently, GATAe1
clone size did not alter from 4 to 11 days ACI (Fig. 4D) and was not
rescued enough by p35 overexpression (Fig. 2L). Furthermore, a decrease in esg þ cells by GATAeIR (Fig. 1B, G and Supplemental Fig. 1)
was not comparable to that observed in the overexpression of an
apoptotic gene reaper, which immediately caused esg þ cell disappearance (Fig. 4B). Moreover, the residual esg þ cells did not show
up-regulation of c-Jun N-terminal kinase (JNK) signaling monitoredwith puckered (puc-lacZ) (data not shown), TUNEL-positive cells (datanot shown), and activation of the caspase pathway monitored with
Apoliner (Fig. 4C). Apoliner indicated the translocation of EGFP but
not RFP from the plasma membrane into the nuclei when apoptotic
cascade was activated (Bardet et al., 2008). These results suggest thatthe growth inhibition of GATAe1 clones is not due to apoptosis. From
the above observations, we also collectively concluded that GATAe is
required for maintaining the stemness of ISCs (Fig. 4E).
3.6. GATAe regulates EC and ee differentiation in an N-dependentand independent mannerIn this study, we also examined whether GATAe was requiredfor EC and ee differentiation. To conﬁrm this, we ﬁrst observed
their speciﬁc differentiation markers including morphological

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characteristics and gene expression in GATAe1 MARCM clones (Leeet al., 2009; Micchelli and Perrimon, 2006; Ohlstein and Spradling,2006). In control clones at 7 days ACI, differentiated ECs showedPdm1 expression in the polyploid nucleus and an apicallywell-developed brush border (Fig. 5A and B). Differentiated ees
had Pros expression in the diploid nucleus (Fig. 5A’). On the other
hand, GATAe1 clones showed a frequent decrease in Pdm1 þ andPros þ cells at 4 and 7 days ACI (Fig. 5C–C”, I, J, and Supplemental
Fig. 4A and B). They also did not have any well-developed brushborders (Fig. 5D). Moreover, the decrease in ECs and ees in the
GATA1 clones was partially and completely rescued by FLAG::GATAe
overexpression (Fig. 5E–E”, F, I, and J, and Supplemental Fig. 4B).
Furthermore, EC disappearance was not inhibited with p35 forced
expression even in an equivalent size of EC-like cells (Fig. 5G, H
and I, and Supplemental Fig. 4B). Similarly, ee loss was not rescued
with p35 forced expression (Fig. 5G’ and J, and Supplemental
Fig. 4B). These results indicated that GATAe is required for EC andee differentiation.
Previous reports suggested that EC and ee differentiation required
N signaling activation that was stimulated by Dl of ISCs (Kapuria
et al., 2012; Ohlstein and Spradling, 2007). This allow us to speculatethat a GATAe depletion inﬂuenced N signaling activation because wefound that GATAe depletion affected Dl expression (Fig. 3D, F, G, andSupplemental Fig. 2). Therefore we examined N signaling levels withGbeþ Su(H)m8-lacZ, a reporter construct for N signaling activity(Furriols and Bray, 2001). In control ISC clones, some small GbeþSu
(H)m8-lacZ þ cells (EBs) appeared at 7 days ACI (Fig. 6A). Under thiscondition, the control transient clone differentiated into a large EC in
which GbeþSu(H)m8-lacZ expression had already disappeared(Fig. 6B). On the other hand, GATAe1 ISC clones also contained aGbeþ Su(H)m8-lacZ þ cell (Fig. 6C). Also, GATAe1 transient clone cells
were small and their Gbeþ Su(H)m8-lacZ expression still remained(Fig. 6D). These results were opposed to our expectation that GATAe
depletion decreased GbeþSu(H)m8-lacZ expression. However, it waspossible that this appearance of GbeþSu(H)m8-lacZ þ cells in GATAe1
clones was due to transactivation from Dl þ ISCs located inside theirearly-stage clones and/or on the adjacent outside of them. Some Dl þ
cells were observed around GATAe1 clones (Fig. 6A–D) and all ISCs in
GATAeIR driven with esgGFPts did not suppress GbeþSu(H)m8-lacZexpression for 7 and 14 days (data not shown). Accordingly, we did
not experimentally reveal that GATAe was required for N signaling
activation. However, from the above results of Dl disappearance(Fig. 3D, F, G and Supplemental Fig. 2), we do not exclude the possibility that GATAe contributes to the N signaling activation throughmaintaining Dl expression.
Since excess N-signaling activation promotes EC differentiation
including their growth (Kapuria et al., 2012; Micchelli and Perrimon, 2006), we tried to rescue the growth defect of GATA1 cellswith forced expression of NICD, an active form of N. This could not,however, completely induce a large EC-like cell in GATAe1 clones(Fig. 6E). Conversely, the GATAe overexpression could not rescue
EC differentiation defects induced by N-signaling inhibition(esgGFPts 4 NIR) (Fig. 6F and G). Combined with above result of Dl
disappearance, we concluded that GATAe is required for EC and ee
differentiation in N-dependent and independent manner (Fig. 6H).

4. Discussion
It has previously been reported that GATAe transcripts weredetected in the adult midgut (Fig.1) (Buchon et al., 2013; Okumura
et al., 2005), suggesting that GATAe has an important role in adultmidgut homeostasis. A recent report showed that GATAe RNAi in
ECs consistently affected the adult midgut epithelial structure(Buchon et al., 2013). We further examined GATAe roles through
GATAe knock-out, knock-down, and overexpression experiments,

which revealed that GATAe has essential roles in midgut epithelial
renewal where N signaling was involved.
4.1. GATAe maintains stemness of ISCs
We discuss that GATAe depletion abolishes ISC proliferation(Fig. 1–3 and Supplemental Fig. 1). This is the ﬁrst evidence that
GATAe is required for Drosophila ISC proliferation. Also, we foundthat GATAe depletion induces the disappearance of Dl þ ISCs without enough cell death/elimination and instead, slightly larger nuclear cells than diploid cells frequently appeared (Figs. 1 and 3 andSupplemental Figs. 2, and 4). Thus, GATAe depletion probably converted ISCs into non-proliferative cells. These observations suggestthat GATAe function is required for maintaining the stemness of
ISCs. In the Drosophila adult midgut, the epithelial basal region is astem-cell niche where ISCs are anchored to the basement membrane (BM) with integrin. An inhibition of myospheroid (mys) encoding one of the two Drosophila integrin β subunits in ISCs/EBs
induced ISCs differentiation (Goulas et al., 2012). In some cases,
GATAe depletion showed the ISC's detachment (Fig. 1G), but any
alteration of Mys protein levels was not observed (data not shown).
Meanwhile, GATAe depletion decreased an expression of βint-ν,
encoding the other integrin β subunit (Supplemental Fig. 5). However, its null homozygotes did not show ISC loss and detachment(Okumura et al., 2014). It is thought that Perlecan (Pcan), an extracellular matrix component, is the other candidate for targeting
GATAe responsible for this phenotype. The lack of trol encoding Pcan
autonomously caused ISC detachment from BM, proliferative cell
disappearance, Dl þ cell loss, and inhibition of EC and ee differentiation, but not apoptosis and JAK-STAT and EGF signaling alteration (You et al., 2014). These phenotypes were very similar to
those when GATAe was depleted. It is a possible that GATAe may
regulate an anchor of ISCs to the BM through trol and maintain ISCs.
Drospohila adult midgut epithelial cells including ISCs are generated from their progenitors that express esg in the embryonic,
larval, and pupa stages (Micchelli, 2012). Also, recent reports haveindicated that esg regulates the stemness of ISCs in the adult midgut(Korzelius et al., 2014; Loza-Coll et al., 2014). Thus, esg is involved instemness maintenance in the Drosophila midgut throughout life.
Similarly, GATAe continues to be expressed from embryonic to adultmidgut (Okumura et al., 2005). In addition, GATAe depletion caused
defects in larval esg þ cells and affected adult midgut development
but not the other internal and external organs during the pupa
stage (Supplemental Fig. 6E-N and data not shown). Furthermore,
GATAe is required for development of the most posterior region of
the adult midgut epithelium derived from the hindgut-proliferating-zone cells (Supplemental Fig. 6O, (Takashima et al., 2013). Although we did not strictly conﬁrm if GATAe depletion affected esg
expression levels in ISC, GATAe is required for maintaining stemness
probably via the esg functions in Drosophila midgut development
and maintenance throughout life.
GATAe overexpression did not largely rescue GATAe1 clone
growth inhibition (Fig. 2J–L) and Dl expression disappearance(Fig. 3D, F, and G). We observed that the FLAG::GATAe and GATAe::
V5-6  His overexpression with esgGFPts both resulted in a decrease in small esg þ cells (Supplemental Fig. 3A, B compared with
Fig. 1A). However, they did not affect survival, mitosis, and Dl andGbeþSu(H)m8-lacZ expression of esg þ cells (Supplemental Fig. 3C,
D and data not shown). In addition to the decrease in small
esgGFP þ cells, we found that some EC-like polyploid cells showed
a weak esgGFP þ signal when FLAG::GATAe and GATAe::V5-6  His
were overexpressed with esgGFPts (Supplemental Fig. 3A and B).
This suggests that GATAe overexpression promotes EC and/or ee
differentiation that inhibits ISC self-renewal. Indeed, 90%  of their
MARCM clones were composed of single EC-like polyploid orPros þ cells (Supplemental Fig. 3F-J). Also, the EC-like polyploid

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cells expressed Pdm1 and a well-developed brush border (Supplemental Fig. 3G-I). This was consistent with the above resultsthat GATAe overexpression could rescue EC and ee differentiation
defects (Fig. 6E, F, I, and J). Thus, GATAe overexpression inhibited
ISC self-renewal but not its differentiation.
Mouse GATA-4 and GATA-6 are expressed in the crypt-proliferative cells of the small intestine (Beuling et al., 2011; Bosse et al.,2006). Similarly, human GATA-6 is expressed in the small intestine(Haveri et al., 2008). GATA-6 conditional knock-out reduces cryptproliferative cells that lead to a decrease in villus height and epithelial cell number in the distal ileum where GATA-6 but not GATA-4
is expressed (Beuling et al., 2011). Both GATA-4 and GATA-6 knockout also induce similar defects in the proximal small intestinewhere both are expressed (Beuling et al., 2011; Walker et al., 2014).
In the distal ileum, GATA-6 knock-out also affects components andtargets of Wnt- and N-signaling pathways as well as causes a decrease in crypt-cell proliferation (Beuling et al., 2011). This casemight be similar to GATAe depletion, which decreases Dl expression
and ISC proliferation (Fig. 3 and Supplemental Fig. 2). In addition,
GATA-6 directly enhances expression of LGR5 that activates Wnt
signaling and represses BMP4 expression in the stem cells of colorectal cancer (Tsuji et al., 2014; Whissell et al., 2014). Thus, GATA
factors regulate signal transductions in the adult ISCs of both the
Drosophila midgut and mammal small intestine.

11

into a detailed common function of GATA factors in EC differentiation
among multicellular animals.
In GATAe1 MARCM clone experiments, although the average sizeof GATA1 clones without any dividing cells was not altered between
days 4 (1.67 cell) and 7 (1.70 cell) (Fig. 2L and Fig. 4D), the frequencyof Pdm1 þ and Pros þ cells decreased during days 4–7 (EC: 20.6%12.63%, ee: 5.67%-0.42%) (Fig. 5I and J). Some GATA1 cells consistently showed weak Pdm1 expression (Supplemental Fig. 4A).
These facts imply that, in addition to their differentiation process,
GATAe is also required for maintenance of EC's and ee's differentiatedstates. In addition, to conﬁrm the fate of undifferentiated GATAe1
clone cells (Dl-, Pdm1-, and Pros- cells), we tried to observe theirolder clones at 21 day ACI. But, in this condition, we did not ﬁnd any
GATAe1 clone cells because they were eliminated probably by cell
death or apical extrusion (data not shown). A recent study suggested
that GATAe may control the physiologically functional genes regulating digestive, metabolism, structure, and defense response in theadult midgut (Buchon et al., 2013). Also, GATAe regulates similar intestinal genes in the larval midgut (Okumura et al., 2007; Senger
et al., 2006). In mammal intestine, GATA-4, GATA-5, and GATA-6
directly and indirectly regulate a subset of functional gene expressions (Aronson et al., 2014). Thus, in addition to ISC regulation/
maintenance, GATA factors regulate differentiation and maintenanceof other types of intestinal cells, which are crucial for physiological
function both in mammal and Drosophila adult intestines.

4.2. GATAe regulates EC and ee differentiation in N-dependent andindependent manner
Acknowledgments
The Drosophila adult midgut epithelium mainly consists of two
types of differentiated cells, EC and ee (Marianes and Spradling,2013; Micchelli and Perrimon, 2006; Ohlstein and Spradling,2006). In their speciﬁcation and differentiation, N signaling plays
an important role (Bardin et al., 2010; Beehler-Evans and Micchelli,
2015; Kapuria et al., 2012; Ohlstein and Spradling, 2007). We
showed that EC and ee differentiation were disrupted in GATAe1
clones (Fig. 5), which were not rescued by excess N-signaling activation (Fig. 6). This suggests that GATAe is intrinsically requiredfor EC and ee differentiation independently of N signaling.
A high- or low-level activation of the N-signaling pathway in EBs
was stimulated from Dl expressed in neighboring ISCs (de Navascues et al., 2012). The activation subsequently regulates EC andee differentiation (Kapuria et al., 2012; Perdigoto et al., 2011). We
found that GATAe was required for Dl expression (Fig. 3 and Supplemental Fig. 2). These facts imply that GATAe also has an extrinsicrole in EC and ee differentiation via Dl expression. In mouse distal
ileum, GATA-6 deletion similarly down-regulates expression of the
Delta-like 1 encoding the N ligand (Beuling et al., 2011). Conversely,
N-signaling alteration did not affect GATA-6 transcripts as in thecase of GATAe transcripts in N-depleted cells (Fig. 1) (Beuling et al.,2011). A mechanism by which GATA factors regulate N-signaling
ligand may be evolutionally conserved in EC and ee differentiation.
While GATA-4 and -5 showed regional expression patterns, GATA6 was constantly expressed throughout the mouse small intestine(Buchon et al., 2013; Fang et al., 2006). GATA-6 is also expressed in
the large intestine of mice and humans (Fang et al., 2006; Haveri
et al., 2008). In summary, GATA-6 is the only GATA factor gene expressed in the distal ileum and colon. In these regions, in addition toa decrease in crypt-cell proliferation, GATA-6 conditional deletion
decreases differentiated ee and Paneth cells, which are replaced withgoblet-like cells (Beuling et al., 2011; Dusing and Wiginton, 2005).
This suggests that GATA-6 plays an important role also in mammalian secreted-cell differentiation. On the other hand, the GATA-6 rolein EC differentiation has been unclear since its conditional deletion
results in a dramatic decrease in ECs constructing villi, which isprobably caused by the limitation of crypt-proliferative cells. Spatiotemporal studies with Drosophila techniques may provide insight

We thank Bruce A. Hay, Ryutaro Murakami and Xiaohang Yang forproviding UAS-DIAP-myc, GATAe::V5-6  His and UAS-FLAG::GATAe
strains and anti-Pdm1 antibody. Fly stocks and antibodies were also
obtained from the Bloomington Drosophila Stock Center (Indiana University), Drosophila Genetic Resource Center (Kyoto Institute of Technology), the Vienna Drosophila RNAi Center (VDRC) and Developmental
Studies Hybridoma Bank (University of Iowa). We also thank BestGene
Inc. for generating transgenic ﬂy lines. We thank members of AdachiYamada laboratory for critical comments and discussion. This work was
supported by grants from Naito Foundation and MEXT*-Supported
Program for the Strategic Research Foundation at Private Universities,
2008–2012 (Grant number S0801006) (*Ministry of Education, Culture,
Sports, Science and Technology)

Appendix A. Supplementary material
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.ydbio.2015.12.017.

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IV. 研究課題 2 「光の情報を受け取る分子メカニズムの解明」
本課題では、生物が光刺激をどのように受容し、その情報を細胞内へとどう伝達して最終的に個体レベルでどう応答していくのかについて、種々の実験系を用いて解析した。特に G タ
ンパク質共役受容体（GPCR）の原子レベルでの解析から、光受容が GPCR の構造変化によってどのように活性変化をもたらすのか、またアフリカツメガエル初期胚における眼の発生時に転写因子とシグナル伝達分子の遺伝子発現が時空間的にどのような変化を示すのかなどについて GPCR 型遺伝子の発現と併せて解析した。一方、モデル生物の一つであるシロイヌ
ナズナの光周性花成経路で働く花成促進因子 FKF1 の機能欠損変異体（遅咲き変異体）を用いて、それを早咲きに変える変異体を単離し解析した。この現象に関与する遺伝子を同定することにより、光の受容から花成促進までの経路を明らかにすることが出来るはずである。以下、これらの研究について、それぞれの研究担当者からの解説と業績リスト、そして代表的な論文２編ずつを記載する。

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受容体分子構造基盤の解明
教授 岡田 哲二
助教 高橋 清大[目的]
生物に対する光の作用は生態系の主要な側面を左右し、かつ多岐にわたるものである。
光は、高等動物での視覚のように環境情報を与える物理的なシグナルとなるが、それに対して適切な応答を示すことは生物の基本的かつ重要な機能である。分子レベルでみると、応答の第一段階を担う光受容体をはじめとして、活性調節・異常が直接的・間接的に光応
答機能を左右するタンパク質に関する構造情報は、生命現象の理解に不可欠なものである。
真核生物の光受容体を含む G タンパク質共役受容体や微生物のレチナールタンパク質（ロドプシン）を中心とした 7 回膜貫通タンパク質の構造解析等により活性発現・調節機構を明らかにするとともに、他分子との相互作用を介して機能発現を行う緑内障関連タンパク質の構造・機能解析を行うことを目的とする。
[結果と考察]a. 構造未知の視覚関連タンパク質
光受容膜タンパク質として、活性化に伴う構造変化機能が損なわれるロドプシン変異体のうち、後述の網膜色素変性症との関連が最近明らかになったもの（W126L）や、GPCR と微生物ロドプシンの双方に対して部分的な配列相動性がみられるタンパク質に着目し、大量発現系の構築から結晶化を目的とする研究を継続して行っている。緑内障に関与する水
溶性タンパク質 OPTN に関しては、ヒト由来の全長および N 末端側、C 末端側の約半分に相当するタンパク質を大腸菌により大量発現させ、アフィニティーカラムやゲルろ過による精製を行った。これらの精製試料から最近生成した結晶のうち、全長タンパク質については高エネルギー加速器研究機構 PF-NW12A ビームラインでの放射光共同利用実験で、3.0
Å 分解能を超える X 線回折が確認できた。この結晶生成に至る過程では、OPTN 分子の会合状態を詳細に調べ、溶液環境に依存した 2, 3, 6 量体に相当する成分間の相対量変化の様子が明らかとなった。また、緑内障との関連が強く示唆されている OPTN での変異 1 を導入したものと結合タンパク質 TBK1 との複合体の精製および結晶化を目的とする研究も進めている。
b. 受容体活性化に伴う構造変化の配列依存性（原著論文 1）
既知の高分解能構造に関する詳細かつ系統的な解析は、受容体機能を理解するうえで極めて有効な手段と考えられる。膜貫通へリックス領域の構造共通性が高いことが明らかになってきた GPCR スーパーファミリーは、そのような研究の対象として前例のない適性を備えている。特に興味深い課題は、如何にして多種多様な細胞外刺激（低分子、高分子リガ
ンド、光など）が細胞質側表面における共通性のある構造変化へと変換されるのかという点である。本研究では、近年明らかになってきた GPCR の活性型構造について、受容体サブタイプ間における共通性と特異性に関する知見を得るために、独自の手法による解析を行った。対象としたのは、ロドプシン、b2 アドレナリン受容体、A2A アデノシン受容体で、特に活性化に重要と考えられている TM3 および TM6 の変位等に着目した。TM6 の細胞質

107

側における変位は、いずれの受容体においても剛体運動を基本とするものであることが確認された。一方、細胞外側の変位に着目すると、TM3 および TM6 のいずれにおいても受容体特有のパターンがみられることが明らかになった。具体的には、ロドプシンにおけるTM6 細胞外側での顕著な変位幅、A2A アデノシン受容体の TM3 細胞外部分における非剛体的な構造変化が特筆すべき結果として得られた。
c. 受容体活性調節と視覚疾患との関連
網膜色素変性症は主要な視覚関連疾患であり、光受容体における変異との関連を示唆する報告が数多くなされている。共同研究者により新たに見出された、日本人家族における常染色体劣性網膜色素変性症（adRP）患者でのロドプシン
変異のうち、膜貫通へリックス 3（TM3）のW126L が生じうる影響について、分子動力学法（MD）によるシミュレーションを行った。光吸収の有無による効果の違いについても調べるた
め、11 シスレチナール結合の不活性型（ロドプシン）、およびレチナールを持たない活性型（オプシン）の 2 つに対応する結晶構造をモデルと

図 1：W126L オプシン表面におけるして用いた。シミュレーション前後での変化がTM3 の変化最も顕著であったのはオプシン構造における

緑：MD 前、赤：MD 後

TM3 の細胞質側で、TM6 の方向への有意な変位として観察された（図 1、原著論文 2）。この結果は、光活性化において必要と考えられている TM3-TM6 間の細胞質側表面での空間形成 2 が、W126L 変異によって損なわれる可能性を示すものである。過去の変異タンパク質を用いた報告では、W126L では 11 シスレ
チナール結合活性を保持しているものの、光依存的な G タンパク質活性化機能が低下する
ことが示されている 3 が、本研究結果はそれらの分子メカニズムを説明するものということができる。すなわち、W126 は膜貫通へリックス 4 および 5 の近傍アミノ酸と重要な疎水性相互作用に寄与しており、それらが光活性化プロセスにおいても保持されている必要があるのではないかと考えられる。W から L への置換は、これらの疎水性相互作用を弱める
ため、光活性化されたタンパク質において TM6 の外向き変位が起こるとともに同じ方向への TM3 の変位が起ってしまい、空間形成が阻害されるという説明ができる。この部位における疎水性相互作用の重要性は b2 アドレナリン受容体でも指摘されている 4 ことから、ロドプシンファミリーの GPCR における機能制御の観点からも重要な知見が得られたと考えられる。
d. 受容体不活性状態における構造の分子進化的保存性
膜貫通型のタンパク質分子において、その折りたたまれた状態での空間的な配置の多様性と必須機能との関連は、これまで研究することが困難であった。しかし、7 回膜貫通へリックスという基本モチーフを共有する GPCR スーパーファミリーの高分解能構造の蓄積により、細胞内外をつなぐ情報伝達機能の中でも特に真核生物で高度に進化してきたと思われ
る 3 量体 G タンパク質を介した系について、その作動原理を探るための解析が可能となってきた。本研究では、分子内 Cα間距離情報の系統的な解析により、アミノ酸配列的に極め

108

て多様な受容体スーパーファミリーの機能発現において最も保存された空間配置を明らかにすることを試みた。空間配置の保存性評価には、
単純ながらもこれまで報告例のないスコアリング法を導入し、既知のほぼ全ての不活性型結晶構造モデルを対象とした解析を行った。各へリックスごとの解析結果からは、スーパーファミ
リー全体かサブファミリーのみかによらず、TM3 の細胞質側部分での極めて高い保存性が定
量的に明らかになった。予想外な結果として得られたのは、スーパーファミリー全てにおいて
最も保存性が高かったへリックス間配置は、
TM1-TM3 および TM1-TM6 にみられ、特にTM1 の細胞質側部分がどちらにも寄与していることであった（図 2、原著論文 3）。上述のように、GPCR において共通すると思われる活性化
機構は、
TM3-TM6 間での細胞質側空間形成である。従って本研究で得られた知見は、その分子メカニズムの基盤として重要な活性化前における空間配置条件を示すものともいえる。
e. 微生物レチナールタンパク質をモデルとした
分子内距離情報解析法の提唱
上述のように、GPCR の高分解能構造モデルの蓄積により、これまでに得ることが困難であっ 図 2：GPCR スーパーファミリー構造におた機能発現機構に関する情報抽出が可能となっける分子内へリックス間距離の保存性てきた。一方、その研究において採用したスコ

青：TM1-TM3、紫：TM1-TM6

アリング法については、膨大な情報量に対して比較対象となる報告例が存在しない。従って、同様の手法を他のタンパク質あるいはタン
パク質ファミリーに適用し、その結果を踏まえることによって更に有効な解析へと発展させることが重要である。本研究では、GPCR と同様の 7 回膜貫通へリックス構造をもつ微生物のレチナールタンパク質（ロドプシン）を対象として、DSA （distance scoring analysis）と略称を付与した解析を行った。また、その手法を簡便に適用するためのツール（score-analyzer）作成にも着手し、プロトタイプ（v01）も論文とともに公開した。微生物レチナールタンパク質の中で、最も構造研究が行われてきたのは高度好塩菌由来の光駆動性プロトンポンプとして知られるバクテリオロドプシン（bR）である。様々な変異体や光反応状態の構造も含め、数多くのモデルが PDB（Protein Data Bank）エントリーとして登録されている。これらは互いに極めて類似した構造であるが、DSA の適用によって分子内非可動部位ともいえる部分あるいはへリックス間配置に関する情報が得られた。すな
わち、bR の膜貫通へリックスのうち A-B, B-C, C-D という隣接するペアにおいては、特にB-C 間の保存性が高いことが明らかになった。さらに配列の異なる 13 種の微生物レチナー
ルタンパク質を解析対象とした結果からは、いずれの場合にも機能として必須であるレチ

109

ナール結合に重要と考えられるへリックス C-G間における保存性の高さが明らかになった（図 3、原著論文 4）。特に、レチナール結合部位である
へリックス G の Lys 側鎖の末端は、この保存されたへリックス間部位のほぼ中間に位置しており、進化的な観点からも興味深い知見が得られたといえる。これらの解析結果は、GPCR スーパーファミリーについて行った際に得られたスコア
値やそれらの距離依存性の評価においても貴重な比較対象となるものである。また、今後さらに多様なタンパク質あるいはタンパク質ファミリーへと研究を進めるための方針・計画策定等へも生かすことができる。
[まとめ]
タンパク質発現から結晶化・構造解析に関する研
究は、長期計画の下での継続的な遂行が必要であるが、特に緑内障関連タンパク質については顕著な進展がみられている。既知の構造モデルからの新たな情報抽出研究は、既に立ち上げている７回
膜貫通領域に関するデータのポータルサイト（ HeptahelicalTransmembrane

Structure

Portal, http://www.gses.jp/7tmsp/）での研究対象 図 3：微生物レチナールタンパク質にお公開に基づいて進行してきており、今後さらに広 ける分子内へリックス間距離の保存性
範なタンパク質ファミリーに関する解析コンテン

緑：TM3-TM7

ツ・ツール提供へと発展できる。
[参考文献]
1. Rezaie T, Child A, Hitchings R, Brice G, Miller L, et al. (2002) Adult-onset primary
open-angle glaucoma caused by mutations in optineurin. Science 295, 1077-1079.
2. Farrens DL, Altenbach C, Yang K, Hubbell WL, Khorana HG. (1996) Requirementof rigid-body motion of transmembrane helices for light activation of rhodopsin.
Science 274, 768-770.
3. T. A. Nakayama and H. G. Khorana, “Mapping of the amino acids inmembrane-embedded helices that interact with the retinal chromophore in bovine
rhodopsin,” The Journal of Biological Chemistry, vol. 266, no. 7, pp. 4269–4275,
1991.
4. S. G. F. Rasmussen, H.-J. Choi, J. J. Fung et al., “Structure of a nanobody-stabilized
active state of the 𝛽2 adrenoceptor,” Nature, vol. 469, no. 7329, pp. 175–180, 2011.

110[原著論文]1. Nakamura S, Itabashi T, Ogawa D, Okada T (2013) Common and distinct mechanisms ofactivation of rhodopsin and other G protein-coupled receptors. Sci Rep 3, 1844.

2. Katagiri S, Hayashi T, Akahori M, Itabashi T, Nishino J, Yoshitake K, Furuno M, Ikeo K,

Okada T, Tsuneoka H, Iwata T (2014) RHO mutations (p.W126L and p.A346P) in two Japanesefamilies with autosomal dominant retinitis pigmentosa. J Ophthalmol 2014, 210947.

3. Kinoshita M, Okada T (2015) Structural conservation among the rhodopsin-like and other G
protein-coupled receptors. Sci Rep 5, 9176.

4. Asano M, Ide S, Kamata A, Takahasi K, Okada T (2016) Sequence and intramolecular distancescoring analyses of microbial rhodopsins. F1000Res 5, 165.

[総説等]

1. 岡田哲二、ロドプシンにみるGPCRの活性化機構－光刺激と薬剤刺激、実験医学 31(3),375-381 (2013).
[その他]

1.

2.

3.

4.

5.

6.

Okada, T. (2013) F1000Prime Recommendation

DOI: 10.3410/f.717991028.793473251

http://f1000.com/prime/717991028#eval793473251
Okada, T. (2014) F1000Prime Recommendation

DOI: 10.3410/f.718204478.793489214

http://f1000.com/prime/718204478#eval793489214
Okada, T. (2014) F1000Prime Recommendation

DOI: 10.3410/f.718437268.793498172

http://f1000.com/prime/718437268#eval793498172
Okada, T. (2014) F1000Prime Recommendation

DOI: 10.3410/f.718632616.793500914

http://f1000.com/prime/718632616#eval793500914
Okada, T. (2015) F1000Prime Recommendation

DOI: 10.3410/f.725624587.793508508

http://f1000.com/prime/725624587#eval793508508
Okada, T. (2015) F1000Prime Recommendation

DOI: 10.3410/f.725590722.793509916

http://f1000.com/prime/725590722#eval793509916

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SUBJECT AREAS:
BIOPHYSICSCOMPUTATIONAL BIOLOGY AND
BIOINFORMATICS

Common and distinct mechanisms ofactivation of rhodopsin and other G
protein-coupled receptors
Sumire Nakamura, Takeshi Itabashi, Daisuke Ogawa & Tetsuji Okada

X-RAY CRYSTALLOGRAPHYSTRUCTURAL BIOLOGY

Received
22 March 2013
Accepted
30 April 2013
Published
15 May 2013

Correspondence andrequests for materials
should be addressed toT.O. (tetsuji.okada@gakushuin.ac.jp)

Department of Life Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan.

Detailed and systematic examination of high-resolution structural data is a rational strategy forunderstanding the function of biological macromolecules. G protein-coupled receptors (GPCRs) are an
exceptionally valuable superfamily of proteins for such analysis. The most intriguing question is how avariety of extracellular stimuli evoke structural changes in the intracellular surface of the receptors. The
recent active-like crystal structures of GPCRs provide information for uncovering common and distinct
mechanisms of light-induced and ligand-induced activation. Based on systematic structural alignment, wehave analyzed 3 receptors (rhodopsin, b2 adrenergic receptor, adenosine A2A receptor) and demonstrate thatthe extracellular movement of helix VI is significantly different between rhodopsin and the other 2
receptors, and that the extracellular side of helix III exhibits distinct features in the 3 receptors. Thesefindings not only emphasize the specialization of rhodopsin as a photoreceptor but also provide insights into
the mechanism leading to rearrangement of helix VI.

M

ost of the external stimuli for eukaryotic cells, such as chemical substances, photons, neurotransmitters,
and hormones are captured by G protein-coupled receptors (GPCRs), which are defined by a heptahelicaltransmembrane core domain. A majority of proteins in the GPCR superfamily belong to the so-calledrhodopsin family, which share several key residues in their transmembrane helices. Their activity at the intracellularsurface to catalyze the GDP/GTP exchange on the a-subunit of heterotrimeric G proteins is primarily determinedby the type of ligand bound to the extracellular side of the transmembrane region. Even without ligand binding,
GPCRs exhibit some basal activity, which is then enhanced or attenuated by binding of an agonist or inverseagonist, respectively1. Antagonists are another category of ligands that maintain GPCRs in their low activity state.
Recent crystallographic studies on GPCRs have validated previous spectroscopic studies2, demonstrating thatthe key intramolecular event during activation is the displacement of transmembrane helix VI at the intracellularside3–5. However, the manner in which this change is regulated by various ligands acting on the extracellular side is
not well understood. While there are many crystallographic models of GPCRs that describe a variety of structuralstates of activity in the presence of different bound ligands (agonist, antagonist, and inverse agonist), systematiccomparison of data obtained for different receptors has not been reported quantitatively.
Each of the crystallographic models inevitably suffers from various sources of artifacts and/or errors, as has
been documented for another class of heptahelical membrane proteins, bacteriorhodopsins, whose proton
pumping movement has been extensively studied using crystallographic techniques6. Nonetheless, statisticallysignificant features should become apparent by averaging as many datasets as possible.
Of the more than 15 receptors of known structure, agonist-bound forms are available only for bovine rhodopsin7–9, b110 and b2 adrenergic receptors4,11, the A2A adenosine receptor5,12, the NTSR1 neurotensin receptor13,
the 5-HT1B and 5-HT2B serotonin receptors25,26. Unfortunately, the agonist-bound and inactive-like states arequite similar to each other in the case of the b1 receptor. Furthermore, the structure of the NTSR1, 5-HT1B and5-HT2B receptors has not yet been obtained in the inactive-like state. As a result, detailed analysis on its activation
is currently feasible only for the remaining 3 receptors.
While several previous reviews on the structure of GPCRs provide a number of insights into their mechanismof activation14–16, systematic quantitative analysis is yet to be performed. We have recently reported a rational
procedure for analyzing the experimental transmembrane structures of GPCRs, based on the defined selection
and superimposition of heptahelical bundles consisting of 200 residues17. By extending this approach, we identify
previously undiscovered structural changes accompanying the activation of these receptors. The findings and themethodology will be valuable for understanding the action of ligands on other receptors of unknown structure.

SCIENTIFIC REPORTS | 3 : 1844 | DOI: 10.1038/srep01844

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Table 1 | PDB entries used for this study
PDB ID
receptor

Inactive-like

Active-likerhodopsin

1GZM-A
1U19-Ab2 receptor

2RH1
3D4S
3NY8
3NY9
3EML
3VG9
4EIY

2X72
3PQR
3PXO
4A4M
3P0G
3SN6

A2A receptor

2YDO
2YDV
3QAK

Results
Of all the heptahelical bundles of GPCRs archived on our website(www.gses.jp/7tmsp), we used 18 sets of coordinates in the present
study (Table 1), consisting of 9 inactive (2 of rhodopsin, 4 of b2
receptor, and 3 of A2A receptor) and 9 active-like (4 of rhodopsin,
2 of b2 receptor, and 3 of A2A receptor) states. All of these structurescontain the same sequence ranges of the transmembrane domains
superimposed onto the same reference coordinates—the inactive
state of b2 receptor (2RH1)—by secondary structure matching18.
The aligned structures are shown in Fig. S1. The structural differences between the inactive and active-like states were then analyzed
using 8 pairs for rhodopsin, 8 pairs for b2 receptor, and 9 pairs for
A2A receptor. Accordingly, we anticipated that the degree of averaging would be comparable for these 3 receptors. The details of thecriteria for the selection of the coordinates are described in the
Methods.
Fig. 1 shows the averaged calculated Ca displacement of 200 transmembrane residues between the inactive and active-like states of the
3 receptors. The most prominent feature commonly observed was an
oscillating pattern at the cytoplasmic side of helix VI (residue no. 145, 160). Although the magnitude of displacement in this region
differs significantly among the receptors, which is most likely due
to the current limitation of available crystallization conditions forstabilizing the fully active state for each receptor, the positions of thepeaks match well with each other, indicating that similar rotational

Figure 1 | Averaged displacement (Å) between the inactive and activelike states of the Ca atoms of 200 residues of the 3 receptors. Red:rhodopsin, blue: b2 receptor, green: A2A receptor. The borders between theadjacent helices are shown with gray bars at the top of the panel, while thepositions of seven *.50 residues are shown with short black bars near thezero line of the graph.
SCIENTIFIC REPORTS | 3 : 1844 | DOI: 10.1038/srep01844

movement occurs around the axis of this helix upon activation. It isalso noteworthy that, at the extracellular side of helix VI (residue no.
160 , 176), a significantly larger change occurs in rhodopsin than inb2 receptor and A2A receptor.
The cytoplasmic side of helix III (residue no. 75 , 89) is anotherregion where these receptors exhibit substantial displacement of 1.5, 2 Å, which has been attributed in part to the translational shift
toward the extracellular side5.
By averaging the values for the 3 receptors, structurally conserved
residue positions were obtained, as listed in Table S1. Overall, residues around the highly conserved sequence positions (*.50 inBallesteros-Weinstein [BW] numbering19, where * is the serial number of helix) in helices I, II, IV, and VI appear to be fixed during
activation. In particular, of the most structurally conserved 21 residues, 14 residues are in helix I (1.47, 1.48, and 1.50 , 1.54) and II(2.48 and 2.50 , 2.55). This finding is consistent with the notion thatthe so-called N-D pair (side chain interaction between 1.50 and 2.50)
in these helices plays an important role as an intramolecular scaffold
during activation.
To gain additional insight into the structural changes shown in
Fig. 1, we analyzed the axis of each helix III and VI by helanal20 (Figs
S2 and S3). Examples of the inactive states are shown in Fig. 2. Eachaxis consists of a series of points penetrating about the center of ahelix, with the number of points obtained for each axis equal to thenumber of residues minus 2. Thus, for convenience, we consider thatboth the N- and C-terminal residues lack the corresponding points(Fig. 2 and 3). As previously described, helix III of inactive rhodopsin
is buried more deeply into the core of the heptahelical bundle fromthe center to the extracellular side than not only in the b2 and A2A
receptors shown here, but also in other receptors of a known structure17. It is also noticeable that the extracellular part of this helix in
the A2A receptor is significantly distorted in the inactive-like state.
We first examined the traces of all axes and confirmed that assignment of the inactive and active-like states was reasonable for theProtein DataBank (PDB) entries shown in Table 1. The distancesof the axis points were then calculated for all possible inactive/activelike pairs of a receptor. The average values are shown in Fig. 3. In thecase of helix VI, the pattern of distance plots is consistent with the 3
receptors (Fig. 3, lower). Reflecting the kinked shape in the inactive
state and rotational movement around an axis upon activation, aroughly linear increase of displacement occurs toward the cytoplasmic side of this helix in rhodopsin and the b2 receptor. In contrast,

Figure 2 | Projection view of the axis points of helices III and VI in theinactive state from the cytoplasmic side. Magenta: rhodopsin (1U19-A),
cyan: b2 receptor (2RH1), light green: A2A receptor (3VG9). Ca trace of b2
receptor is also shown in gray. Ballesteros-Weinstein numbers
corresponding to the endpoints of the axes are also shown.

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Figure 4 | Changes in the axis points of helix III during the activation of
the 3 receptors. Top: rhodopsin. Magenta: inactive (1U19-A),
red: active-like (3PXO). Middle: b2 receptor. Cyan: inactive (2RH1),
blue: active-like (3P0G). Bottom: A2A receptor. light green: inactive(3VG9), green: active-like (3QAK). Corresponding axis points in theinactive and active-like states are connected by light green bars.

Figure 3 | Averaged difference distance (Å) plot between inactive andactive-like states of 3 receptors. Upper: helix III, lower: helix VI. The
horizontal axis (BW number) in the upper panel is reversed so that thecytoplasmic side is in the left side of both panels. The borders between the 5sections stacked along the normal of the membrane plane17 are shown withgray bars at the top of the panel.

the magnitude of displacement does not change significantly toward
the extracellular side. The A2A receptor exhibits a similar pattern ofchanges, although the increase in the cytoplasmic side is smaller andnot linear. Remarkably, the change in the extracellular side of thishelix is significantly larger in rhodopsin than in the other 2 receptors,
and the point of minimum displacement appears to be shifted from
6.48 , Pro6.50 in the b2 and A2A receptors to 6.44 , 6.46 in rhodopsin. As shown in Fig. 2, the inactive backbone of helix III in
rhodopsin deviates from the other receptors from , 3.40 to theremainder of the extracellular region. Importantly, 3.40 is the position directly in contact with 6.44. These observations suggest that themore buried inactive orientation of helix III and the larger movementin the extracellular side of helix VI are distinct features of rhodopsin,and that while interpreting these, the large differences of the retinal
ligand shape and position before and after activation must be considered (see Discussion below).
We also found that the inactive state orientation of helix VI of b2
receptor is much closer to the active-like helix VI of rhodopsin in theextracellular region than to that of the inactive rhodopsin. This isconsistent with the finding that the degree of axis movement in theextracellular side of helix VI upon activation is larger in rhodopsin
than in the b2 receptor.
It has been noted that the activation of GPCRs involves a translational shift of helix III toward the extracellular side5,12,14. If the
movement of this helix is only translational, the displacement of axis
SCIENTIFIC REPORTS | 3 : 1844 | DOI: 10.1038/srep01844

points would not exhibit a significant change depending on the position in the helix. Fig. 3 (upper panel) shows that the fairly flat patternobserved in the intracellular side is consistent with such a mechanism. However, the change in the pattern of displacement considerably differed among the 3 receptors in the extracellular side. The
larger movement observed in rhodopsin is consistent with its distinct
arrangement of this helix in the inactive state. On the other hand, anapparently irregular pattern was found in the extracellular side of thishelix in the A2A receptor, suggesting that some localized deformation
occurred in this region.
To clarify the differences observed between these receptors, representative axes of inactive and active-like helix III are shown in
Fig. 4. It is obvious that the translational shift of this helix toward
extracellular side appears to be, at least in part, a result of twistingaround the long axis. The degree of translation in the cytoplasmicside is likely to be determined by the pattern of twisting, and it varies
substantially among the receptors. In the A2A receptor, the pattern oftwisting in the cytoplasmic side is distinct from that in the extracellular side (Fig. 4, bottom). In fact, a simple superimposition calculation fails to fit the inactive and active-like axes with each other,suggesting that helix III of the A2A receptor would not behave as arigid-body during activation.
In order to analyze the rearrangement of helix III in more detail,dihedral angles (Phi, Psi) of each residue were obtained with DSSP21(Fig. S4) and then averaged separately for the inactive and active-likehelices (Fig. 5). In the A2A receptor, significant changes in the dihedral angles occur at 3.30 , 3.31, depending upon the type of the boundligand. For comparison, the results of a similar calculation for helix
VI are shown in Fig. 6. As anticipated from the kinked shape, deviation from the ideal values was observed around 6.48 in the 3 receptors. The angle, however, does not change significantly, regardless ofthe type of the bound ligand, indicating that this helix behaves almost
as a rigid-body in these receptors, as suggested previously8.

Discussion
The present study is based on the comparison of a similar number ofstructures (8 , 9) for each of the 3 receptors. Although the quality ofeach structure varies substantially in terms of the claimed resolutions, from 1.8 (4EIY) to 3.5 Å (3P0G), it is unlikely that the findings
reported here are affected significantly by such differences, given that
we focus only on the backbone rearrangement of the receptors.
The possibility that our procedure of fitting of all the inactive and114

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Figure 5 | Phi/Psi (6) plot of helix III. Top: rhodopsin, middle: b2 receptor, bottom: A2A receptor. Left: averaged Phi/Psi angles; pink: Phi angles ofinactive-like states, red: Phi angles of active-like states, cyan: Psi angles of inactive-like states, blue: Psi angles of active-like states. Right: differences
between averaged Phi (red), Psi (blue) angles of active-like and inactive-like states.

active-like structures to the inactive b2 receptor might be inappropriate is also unlikely because we observed the same degree of movements and/or shifts of helices III and VI as those documented inprevious crystallographic studies on the activation of the 3 receptors.
In addition, the results of fitting independent statistics, such as
Phi/Psi angle changes, further validate our procedure of model
manipulation.
Given the emerging consensus view that a given GPCR can assume
multiple conformational states1,22, averaging available crystal structure models into 2 states, inactive and active-like, might be valid only
for identifying substantial rearrangements of the helices. Nonetheless, our findings suggest that quantitative analyses based on sucha 2 state approximation provide detailed information on not only
SCIENTIFIC REPORTS | 3 : 1844 | DOI: 10.1038/srep01844

common mechanisms but also receptor-specific mechanisms ofactivation.
The activity of rhodopsin changes dramatically upon photon
absorption, and it is accompanied by the isomerization from 11-cis
to all-trans of a covalently bound retinal ligand. 11-cis-retinal is
known to act as a strong inverse-agonist, and the differences between
the 2 forms of retinal with regard to the shape and position in thebinding pocket of opsin are remarkably large compared with ligands
bound to other receptors. Therefore, the magnitude of overall
rearrangement of the helices in rhodopsin may be larger than theother receptors. This finding is in line with the findings that thedisplacement in the extracellular side of the helical bundle is larger(Fig. 1), especially in helices III and VI (Fig. 3). Although the plots in

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Figure 6 | Phi/Psi (6) plot of helix VI. Top: rhodopsin, middle: b2 receptor, bottom: A2A receptor. Left: averaged Phi/Psi angles; pink: Phi angles ofinactive-like states, red: Phi angles of active-like states, cyan: Psi angles of inactive-like states, blue: Psi angles of active-like states. Right: differences
between averaged Phi (red), Psi (blue) angles of active-like and inactive-like states.

Figures 1 and 3 may be influenced by the manner of fitting themultiple structures, the increase in the distance between the 2 helices
of rhodopsin at the extracellular side was confirmed by measuring
the interhelical Ca distances, which is independent of how active-likestructures were fit onto the inactive ones (Fig. S5). We speculate thatan extension of such an analysis would be useful for obtaining further
insights into the relative positional changes among the 7 helices.
The outstanding local twist of helix III observed in the activation of
the A2A receptor is likely to be related to its curved shape in theinactive state, the degree of which is slightly different in the 2 states,
namely, with (3VG9) or without (3EML and 4EIY) an inactivating
antibody bound at the cytoplasmic side. Therefore, it is likely that the
backbone rearrangement upon agonist binding at the extracellular
SCIENTIFIC REPORTS | 3 : 1844 | DOI: 10.1038/srep01844

side of this helix would directly affect the interaction with helix VI
from the middle to the intracellular side of the membrane.
The Phi/Psi angles of 3.30 , 3.31 in the A2A receptor change to aremarkable extent depending on the type of the bound ligand (Fig. 5).
The side chains of these 2 residues in the 3 receptors do not point
toward the interior of the helical bundle and do not appear to be
directly involved in ligand binding. It should be noted that deviations
in Phi/Psi angles at these residues from the canonical values are larger
in the receptor with an inactivating antibody bound to an intracellular surface than that without it. This observation is in agreementwith the finding that the overall twist at the extracellular side of helix
III is significantly enhanced in the antibody-bound A2A receptor(Fig. S2). Upon agonist binding, the overall shape of this helix116

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becomes more like a regular a-helical structure that exhibits little
bending. It is likely that there are many other receptors of unknown
structure that contain a kink at the extracellular side of helix III in theinactive structure. In such cases, a localized twisting motion would bepossible depending on the type of a bound ligand, leading torearrangement of the short segment in the helix.
In summary, the present study highlights the importance ofquantitative analysis of the experimentally available structures to
identify common and distinct rearrangement of transmembrane
helices induced upon the activation of the 3 members of rhodopsin-like GPCRs. In particular, rearrangement of helix III was foundto be a function of the type of receptor. While it has been wellrecognized that the interaction between this helix and helix VI is a
key event in the regulation of the GPCR activity, the mechanism
should be considered taking into account the non-rigid-body-likenature of the polypeptide backbone.

Methods
Selection and structural alignment of heptahelical transmembrane bundles werecarried out as previously described17. Briefly, all the polypeptides from PDB entries
were superimposed onto the inactive-like state of b2 receptor (2RH1) by secondary
structure matching18, and 7 helices consisting of 200 residues were isolated based onunambiguous sequence alignment.
The active-like structures of rhodopsin can be classified into 2 groups, a ligand-free
opsin form and an all-trans-retinal agonist-bound form. Because the backbone
coordinates of these structures are quite similar to each other, we chose only agonistbound structures for this study. Among the alternatives of inactive structures of the b2
and A2A receptors, the coordinates listed in Table 1 were selected by taking higherresolution structures. Of the 3 agonist-bound structures of b2 receptor reported todate, 3PDS was not used because of its negligible backbone deviation from theinactive-like structure.
DSSP analysis showed that all of the helices III and VI assume regular a-helicalsecondary structure throughout the entire lengths, with the exception of the cytoplasmic terminals of 3NY8, 3NY9, and 3.30 , 3.31 of 3VG9. Therefore, all helices III
and VI were analyzed by helanal to determine the axes of the a-helices. Consequently,
an axis point corresponding to 3.30 of 3VG9 appeared to be slightly displaced fromthe expected position within the helix. Although this error does not affect the findings
described in this paper, we replaced the coordinate of the point by the averaged valuesof the preceding and the following points.
Several of the PDB entries contain 2 alternative coordinates for each of a few
residues. Because deviations between the 2 Ca positions were negligibly small, we
chose conformer A for all cases. Distances between the inactive and active-like axis
points were obtained with Chimera23 and CCP4MG24 while that of entire heptahelicalbundles were calculated by inserting the coordinates in a conventional equation. Phi/Psi angles were obtained by DSSP.
For interhelical Ca distance analysis, the coordinates in the inactive and active-likestructures were first averaged separately for each of the 3 receptors, and the intramolecular Ca distances were calculated for all possible pairs in helices III and VI of theaveraged inactive and active-like structures. The differences of the distances at eachpairs of positions were then obtained between the inactive and active-like structures.
All graphs were prepared with IgorPro (WaveMetrics), and the remaining figures
were prepared with CCP4MG24 and Discovery Studio Visualizer (Accelrys).
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receptors. Trends Pharmacol. Sci. 28, 397–406 (2007).
2. Farrens, D. L., Altenbach, C., Yang, K., Hubbell, W. L. & Khorana, H. G.
Requirement of rigid-body motion of transmembrane helices for light activationof rhodopsin. Science 274, 768–770 (1996).
3. Park, J. H., Scheerer, P., Hofmann, K. P., Choe, H. W. & Ernst, O. P. Crystal
structure of the ligand-free G-protein-coupled receptor opsin. Nature 454,
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4. Rasmussen, S. G. et al. Structure of a nanobody-stabilized active state of the b(2)adrenoceptor. Nature 469, 175–180 (2011).
5. Xu, F. et al. Structure of an agonist-bound human A2A adenosine receptor. Science
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6. Hirai, T., Subramaniam, S. & Lanyi, J. K. Structural snapshots of conformationalchanges in a seven-helix membrane protein: lessons from bacteriorhodopsin.
Curr Opin Struct Biol. 19, 433–439 (2009).
7. Choe, H.-W. et al. Crystal structure of metarhodopsin II. Nature 471, 651–655(2011).
8. Standfuss, J. et al. The structural basis of agonist-induced activation inconstitutively active rhodopsin. Nature 471, 656–660 (2011).
9. Deupi, X. et al. Stabilized G protein binding site in the structure of constitutively
active metarhodopsin-II. Proc Natl Acad Sci USA 109, 119–124 (2012).
10. Warne, T. et al. The structural basis for agonist and partial agonist action on ab(1)-adrenergic receptor. Nature 469, 241–244 (2011).
11. Søren, G. F. et al. Crystal structure of the b2 adrenergic receptor–Gs protein
complex. Nature 477, 549–555 (2011).
12. Lebon, G. et al. Agonist-bound adenosine A2A receptor structures reveal common
features of GPCR activation. Nature 474, 521–525 (2011).
13. White, J. F. et al. Structure of the agonist-bound neurotensin receptor. Nature 490,
508–513 (2012).
14. Hulme, E. C. GPCR activation: a mutagenic spotlight on crystal structures. Trends
Pharmacol Sci. 34, 67–84 (2013).
15. Lebon, G., Warne, T. & Tate, C. G. Agonist-bound structures of G protein-coupled
receptors. Curr Opin Struct Biol. 22, 482–490 (2012).
16. Unal, H. & Karnik, S. S. Domain coupling in GPCRs: the engine for induced
conformational changes. Trends Pharmacol Sci. 33, 79–88 (2012).
17. Okada, T. Comparative analysis of the heptahelical transmembrane bundles of G
protein-coupled receptors. PLoS One 7, e35802 (2012).
18. Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool forfast protein structure alignment in three dimensions. Acta Crystallogr D60,
2256–2268 (2004).
19. Ballesteros, J. A. & Weinstein, H. Integrated methods for the construction of
three-dimensional models and computational probing of structure-function
relations in G protein-coupled receptors. Methods Neurosci 25, 366–428 (1995).
20. Bansal, M., Kumar, S. & Velavan, R. HELANAL: a program to characterize helix
geometry in proteins. J Biomol Struct Dyn 17, 811–819 (2000).
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2577–2637 (1983).
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23. Pettersen, E. F. et al. UCSF Chimera--a visualization system for exploratory
research and analysis. J Comput Chem 25, 1605–1612 (2004).
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Acknowledgments
Supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Author contributions
S.N., T.I., D.O. performed analysis, T.O. designed research, performed analysis, and wrote
the manuscript.

Additional information
Supplementary information accompanies this paper at http://www.nature.com/
scientificreports
Competing financial interests: The authors declare no competing financial interests.
License: This work is licensed under a Creative Commons
Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of thislicense, visit http://creativecommons.org/licenses/by-nc-sa/3.0/
How to cite this article: Nakamura, S., Itabashi, T., Ogawa, D. & Okada, T. Common anddistinct mechanisms of activation of rhodopsin and other G protein-coupled receptors. Sci.
Rep. 3, 1844; DOI:10.1038/srep01844 (2013).

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OPEN
SUBJECT AREAS:MEMBRANE BIOPHYSICSSTRUCTURAL BIOLOGY

Structural conservation among therhodopsin-like and other G
protein-coupled receptors
Mikitaka Kinoshita & Tetsuji Okada

Received
27 November 2014
Accepted
24 February 2015
Published
17 March 2015

Correspondence andrequests for materials
should be addressed toT.O. (tetsuji.okada@gakushuin.ac.jp)

Department of Life Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan.

Intramolecular remote coupling within the polypeptide backbones of membrane proteins is difficult toanalyze owing to the limited structural information available at the atomic level. Nonetheless, recent
progress in the crystallographic study of G protein-coupled receptors (GPCRs) has provided an
unprecedented opportunity for understanding the sophisticated architecture of heptahelicaltransmembrane (7TM) bundles. These 7TM bundles can respond to a wide range of extracellular stimuli
while retaining the common function of binding trimeric G proteins. Here we have systematically analyzed
select sets of inactive-like 7TM bundles to highlight the structural conservation of the receptors, in terms of
intramolecular Ca-Ca distances. Distances with the highest scores were found to be dominated by the
intrahelical distances of helix III, regardless of the choice of bundles in the set, indicating that the
intracellular half of this helix is highly conserved. Unexpectedly, the distances between the cytoplasmic sideof helix I and the extracellular region of helix VI provided the largest contribution to the high score
populations among the interhelical pairs in most of the selected sets, including class B, C and frizzled
receptors. These findings are expected to be valuable in further studies of GPCRs with unknown structure
and of other protein families.

A

lmost all eukaryotic organisms utilize G protein-coupled receptors (GPCRs) for sensing both external andinternal signals, such as those from chemical substances, hormones, and photons1,2. Recent advances inthe structural study on the receptors of this superfamily have provided a wealth of information, promoting
a wide range of researches, including for example, the rational design of drugs with improved efficacy andspecificity3,4. However, a substantial number of unexplored targets found from unicellular eukaryotes to humans
remain to be investigated in atomic detail.
A set of coordinates for a given family of proteins can be utilized to develop reliable methods for predicting thestructure of related proteins, and to gain insights into the mechanisms underlying molecular evolution. There arefew membrane protein families suitable for conducting detailed analysis using a wealth of data with preferable
variations in both the structure and the sequence. The transmembrane domains of GPCRs, accommodating boththe functionally common and the variable parts within a bundle of seven helical segments confined in the lipid
bilayer, offer an excellent template model in terms of the biological significance of their structure/activity
relationship and the moderate structural and sequence variations.
Previous studies on the arrangement of the seven transmembrane (7TM) helices of the inactive-like crystal
structures of GPCRs have confirmed that the positions near to the highly conserved residues in the rhodopsin
family, designated as *.50 (asterisk indicates helix number) with Ballesteros & Weinstein (BW) numbering5,
exhibit relatively small positional deviations among receptors6. However, the degree of conservation in terms of
the intramolecular distances has not yet been quantitatively examined. RMSD values, often used for evaluating
similarity between a pair of receptors, are not suitable for determining whether any common features areconserved among a set of receptors. Furthermore, recently determined structures of non-rhodopsin family
GPCRs have demonstrated that the apparently important helix VI of the 7TM bundles deviates significantly
from that of the rhodopsin family receptors7–9. This finding has led to questions regarding the types of structural
conditions that are essential to maintaining a common function.
To address these issues, we performed an extensive distance analysis that included calculation of all the
pairwise intramolecular distances between the Ca atoms within 200 residue 7TM bundles. Conservation wasscored according to the coefficient of variation calculated for the receptors selected in a given set. By testing
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various sets of receptor combinations, we found that this simple
method of analysis provided valuable insights into the architectureof the GPCR superfamily.

Results
Receptor selection. The 7TM bundles, each containing 200 residues,
were defined as previously reported for rhodopsin family receptors6.
Thus, 200 3 199/2 5 19,900 values were examined for each of thebundles. Initially we analyzed 59 inactive bundles that were available
until July 2013 (set 0) that had a crystallographic resolution higher
than 3.6 Å (supporting table S1). The results obtained from this set
led us to perform a further detailed study, and the list of the structures
examined is summarized in supporting table S2. For instance, set 1
was composed of the structures of the rhodopsin family that wereavailable until January 2014, and that had a resolution higher than 3.3
Å. Thus, set 1 contained 6 rhodopsin (4 bovine, 2 squid), 10
adrenergic (5 each for b1 and b2), 5 A2A adenosine, 7 chemokine(5 CXCR4, 2 CCR5), 4 other amine (2 dopamine, 1 histamine, 1
muscarinic), 6 opioid (2 kappa, 2 N/OFQ, 1 delta, 1 mu), 1
sphingosine and 1 thrombin receptor bundles. In this set, the total
number of bundles was decreased to 40, reducing possible bias
toward the adrenergic receptors for which the greatest number ofcrystal structures is available. To evaluate the findings obtained forthe rhodopsin family receptors, set 2 was defined as set 1 with theaddition of three non-rhodopsin family bundles, one class B CRF1
receptor and two chains of smoothened receptor. From set 2, the fivesubfamilies- rhodopsin, adrenergic, adenosine, chemokine, andopioid receptors were removed to form set 3, 4, 5, 6, and 7,
respectively, to see whether any subfamily specific features exist. In
sets 8 and 9, a maximum of three bundles or one bundle, respectively,
for each of the receptors was included, to reduce any possible bias
from larger contribution from rhodopsin family receptors. Thus, set
9 contained 18 unique receptor bundles; however this set was still
biased toward the rhodopsin family, as it contained 16 rhodopsin
family members and 2 non-rhodopsin family members. This was
simply due to the current limited number of available structures
for non-rhodopsin family receptors. Recently reported class C
receptor10 was also taken into account in set 10, which contained
set 9 and mGluR1 receptor.
Since our current analysis focused only on extracting information
on structural features that are relatively invariable among subfamiliesof receptors that vary from each other by at least more than 1.5 Å inthe overall RMSD for the TM region, any possible effects of various
modifications (that mostly result in changes of , 1 Å in overall
RMSD) applied to each receptor (Table S2) did not appear to affectthe results significantly. As shown below, this is confirmed by the factthat we could detect a subtype–specific feature in adrenergic receptors for which both modified and unmodified structures were
included in the analysis.
Ranking procedure. For each of the Ca-Ca pair distances in the sets,
the average, maximum, minimum, and standard deviations were
calculated. As a simple and rational measure (score) of theconservation among the bundles, we chose the inverse ofcoefficient of variation, wherein this coefficient is the average
divided by the standard deviation. The scores obtained in thecurrent study varied from , 1.5 to , 250. With this scoring, thehigher numbers (ranks with a smaller number) were dominated by
Ca pairs of adjacent amino acid positions, and these were omitted
from the ranking because they do not appear to provide muchinformation on structural conservation.
Figure 1 shows the distribution of 19,900 Ca pairs in set 1 over thewhole scoring range with or without the contribution of 193 adjacent
Ca pairs. The cumulative count plot (Fig. 1B) indicates that Ca pairs
found in the top 1,000 ranks have scores greater than approximately
50, which corresponds to a 0.4 Å standard deviation for the average
SCIENTIFIC REPORTS | 5 : 9176 | DOI: 10.1038/srep09176

Figure 1 | Score distribution of 19,900 Ca- Ca pairs. Calculated for set 1
containing forty 7TM bundles of rhodopsin family receptors with (blankbars) or without (filled bars) the contribution of 193 adjacent pairs. A.
Counts in each score range. B. Cumulative counts.

distance of 20 Å, and is fairly higher than the average score of 29.4
obtained for all 19,900 pairs. Since the top 1,000 pairs amounted toonly 5% of the total, detailed analysis of this population is a reasonable approach for examining the intramolecular distance conservation among the receptors in a given set.
Data statistics. We next investigated whether the population ofhigher scoring Ca pairs was biased toward either long or shortdistances. Figure 2a shows the distribution of the average distances
for the 19,900 Ca pairs in set 1. The maximum value of around 21 Å
includes, for example, intrahelical distances separated by four
pitches. Comparison of the average distance distribution for the Capairs ranked within the top 1,000 score with this distribution showedthat both were similar (Fig. 2B), indicating that the current scoring
was not biased toward short or large distance populations.
The relationship between the distance and the score was further
examined using a scatter plot of the 19,900 Ca pairs (Fig. 3) of set 1.
As described earlier, the population with scores ranging from 90 to
200 at the distance of approximately 3.8 Å originates from adjacentpairs of amino acids. The remainder of the distribution appeared
uniform across the whole range of distances. While the lower limitof the scores tended to increase slightly at longer distances, ourdetailed analysis focused only the scores higher than approximately
50. Therefore, these statistics support the assumption that the present simple scoring system would be useful in extracting the conserved arrangement of backbone among the receptors.
Evaluation of high rank pairs. We classified the top 1,000 ranking
Ca pairs into 28 helix pairs (7 intrahelical and 21 interhelical) based1192

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Figure 3 | Correlation between score and the average distance of the
19,900 Ca- Ca pairs in set 1.

Figure 2 | Average distance distribution of Ca- Ca pairs in set 1. A. 19,900
pairs including the contribution of 193 adjacent pairs. B. The
top-ranked 1,000 pairs without the contribution of 193 adjacent pairs. The
point for 1000th rank included in panel B corresponds to rank 1193 inpanel A, and a point corresponding to 193 adjacent pairs at a distance of ,3.8 Å, identical to that shown in panel A, has been omitted from panel B tomake its vertical scale appropriate.

on the helices to which the Ca belonged. Because the total number of
Ca pairs differs among the 28 helix pairs depending on the length(number of amino acids) of each helix, the Ca-Ca counts in the top
1,000 were compared among the 28 helix pairs after normalization.
For instance, the total number of intrahelical Ca-Ca distances of helix
III was 581 since we had selected its TM region from 3.22 to 3.55 (34residues). Therefore, the 220 Ca-Ca distances found in the top 1,000
ranked pairs of set 1 corresponded to 37.9% of the total (see below
and Fig. 4B). In the same way, we obtained the patterns ofcontribution from each of the 28 helix pairs to the top 1,000
ranked pairs for the 11 sets (from set 0 to set 10).
Figure 4 shows the cumulative numbers (ratios to the total) of the
Ca pairs found in the top 1,000 ranks for 28 helix pairs for sets 0 and
1, both of which contain only rhodopsin family bundles. The corresponding data containing non-rhodopsin receptors are shown in figure 5 (sets 2, 8, 9, and 10, the latter of which had a smaller proportionof rhodopsin family members) and in supporting figure S1 (sets 3 to7, for each of which a specific receptor subfamily was omitted fromset 2).
The higher ranked Ca-Ca distances in all the sets were mainly fromthe intrahelical part of helix III, regardless of the inclusion of nonrhodopsin family receptors (Fig. 4, 5, Fig. S1). In the extreme cases of
set 5 (with no adenosine receptors), set 9 (bundles representing 18
unique receptors), and set 10 (set 9 plus class C receptor), nearly 60%(approximately 335 of 561) of the possible Ca pairs in this helix wereranked in the top 1,000. These observations quantitatively demSCIENTIFIC REPORTS | 5 : 9176 | DOI: 10.1038/srep09176

onstrate that the overall arrangement of this helix is much more conserved than that of the other helices among all the GPCRs whose
structure is known. A slight increase in the higher ranked Ca pairs fromhelix III in set 5 is likely due to the uniquely distorted character of thishelix in the adenosine A2A receptor, as has been documented previously11.
Next we examined the conservation pattern in more details,
according to the previously described horizontal sectioning of the
7TM bundle of 200 residues6 (Table 1). As shown in Fig. S2, wedefined five sections to each of the helices. Thus, the possible numberof intersection pairs is 630 (105 intrahelical and 525 interhelical), forthe 7TM bundle. For each of these pairs, the total number of possible
Ca-Ca combination varied from 9 (section 1 of helix I and section 5 ofhelix 7, each of which contains only three residues) to 90 (section 1 ofhelices VI and VII, each of which contains 10 and 9 residues, respectively). Therefore, we evaluated the distance conservation for each of
the intersection pairs by calculating the ratio of the number of Capairs found in the top ranked 1,000 to the total number of possible
pairs (ratio column in Table 1).
Within helix III, our data show that the intracellular side appears
to be more structurally conserved than the intracellular side. Direct
evidence for this can be seen when comparison of the values inTable 1 is made between section pairs 2–3 and 3–4. A higher numberof pairs from the 3–4 section are ranked in the top 1000, indicatingthat conservation of the distance from the center of helix III is more
pronounced on the intracellular side. Importantly, the results of thiscomparison would not be affected by how the residue range for thishelix was chosen; for example, a shorter length of this helix, excluding a few residues from both the intracellular and extracellular termini, should provide the same result.
Figure 4 also demonstrates that helices IV and VII are the mostvariable in the rhodopsin family; this is even more apparent when the
non-rhodopsin family receptors are included (Fig 5 and Fig S1).
Furthermore, the results of our analysis suggest that helical segments
judged as regular a-helices by DSSP12 in many of the receptors (helices I, III, and VI) tend to exhibit high intrahelical distanceconservation.
Interhelical distance conservation. With regard to the interhelicalcomponents, a significant number of Ca-Ca distances of the I–VI
helix pair were found in the higher ranked population of sets 0 and 1(Fig. 4). This finding was rather unexpected because no direct contact
could be found between any parts of these two helices. The positions
on helix I in the higher ranked I–VI pairs were found mostly insections 4 and 5, a region that spans from around the conserved
asparagine (BW no. 1.50) to the cytoplasmic end. On the other
hand, positions on helix VI involved in the higher ranked I–VI
pairs were found in sections 1 and 2, extending from theextracellular end towards the region near the conserved proline(BW no. 6.50).

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Figure 4 | Cumulative ratio of the number of Ca- Ca pairs in the top-ranked 1,000. A. Set 0 containing 59 rhodopsin family 7TM bundles. B. Set 1
containing 40 rhodopsin family 7TM bundles. Left: intrahelical pairs, Right: interhelical pairs. Intrahelical pairs are colored as follows; purple, helix I;
blue, helix II; cyan, helix III; green, helix IV; yellow, helix V; orange, helix VI; red, helix VII. Interhelical pairs are colored as follows; blue, I–II; cyan, I–III;
green, I–IV; purple, I–VI; yellow, III–V; red, other pairs.

Importantly, the conservation of distances between the cytoplasmic side of helix I and the extracellular side of helix VI was also
apparent in all of the sets containing non-rhodopsin family receptors(Fig. 5 and Fig. S1). This finding was surprising as the superimposition of either the class B CRF1 receptor or smoothened receptor withthe rhodopsin family receptors indicated a significant displacementof the extracellular side of helix VI, while the cytoplasmic side of helix
I matched rather well. These observations might indicate that thedeviation at the extracellular side of helix VI between the rhodopsin
family and other receptor families reflects the presence of somedirectional restraint within a 7TM bundle, resulting in a relatively
conserved distance from the cytoplasmic part of helix I.
Another interesting observation was the outstanding conservationof the distances between helices III and V in some of the sets containing non-rhodopsin family receptors (Fig. 5 and Fig. S1). Although
this feature was not evident in set 10 where class C receptor was
included (Fig. 5D), detailed examination indicated that there werestill some high score pairs between the cytoplasmic side of helix III
and the extracellular part of helix V.
We also noticed that a significant increase in population in thehigher-ranked interhelical distances of the non-rhodopsin familycontaining sets was attributable to the pairs between helices I and
IV (Fig. 5). Furthermore, I–III and I–II helix pairs contributed significantly to the higher-ranked interhelical distances in the case of set
10. These observations and the intrahelical distance conservation
data shown above suggest that positioning of helix I within the
7TM bundle is substantially conserved among all GPCRs.
The distance conservation results are summarized graphically in
Figure 6, which includes lines connecting the 5 higher-ranked CaCas in each of the major interhelical section pairs in set 10. All ofthese Ca pairs had scores higher than 50. Figure 7 shows the positionsof residues conserved among the receptors in set 10. In summary, thepresent results are consistent with the previous observation thatSCIENTIFIC REPORTS | 5 : 9176 | DOI: 10.1038/srep09176

positional deviations among rhodopsin-family receptors are small
on the intracellular side of helices I, II, III, and in the middle of helix
VI,6 and suggest that interhelical distance conservation correlate wellwith the presence of conserved amino acid type at some specificpositions in the GPCRs with known structure. While no single position in the TM bundle is 100% identical among the receptors examined (Fig. S3), our analysis suggests that four of the five conserved
residues, all of which are in section 4 (the middle–to–intracellularregion), contribute to determining the basic arrangement of thebundle, at least from helices I to III, mostly by hydrophobic interactions. This interpretation explains why the relative contribution of
the I–II and I–III pairs to the high score population increases as theratio of the non-rhodopsin-family chains increases in a set (Fig. 5).

Discussion
Statistical and quantitative analysis of the sequences and the threedimensional structures of a protein family provide insights into functionally relevant and evolutionary significant regions. In the case of
GPCRs, one of the most intriguing questions is how the common
7TM bundle core works for diverse members that convert a variety ofsignals, mostly captured at the middle to the extracellular part of thebundle, to the degree of coupling with the cognate heterotrimeric G
proteins at the cytoplasmic surface. Although the basic folding pattern is similar for the seven helices in all the receptors of known
structure, it is now increasingly evident that there are significant
variations in spacing and shape of the helices depending on thesubfamilies they belong.
Structural conservation and deviation in the 7TM region of
GPCRs has been examined mostly based on superimposition and
RMSD examination, and has shown little ambiguity as long as thereceptors belonged to the rhodopsin family. The Ca positions at ornear to the conserved residues (*.50 in BW numbering) exhibited
relatively small deviations among the receptors6. However, recent

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Figure 5 | Cumulative ratio of the number of Ca- Ca pairs in the top-ranked 1,000. A. Set 2 containing 40 rhodopsin family 7TM bundles and three nonrhodopsin family bundles. B. Set 8 containing 31 rhodopsin family 7TM bundles and three non-rhodopsin family bundles. C. Set 9 containing 16
rhodopsin family 7TM bundles and two non-rhodopsin family bundles. D. Set 10 containing 16 rhodopsin family 7TM bundles and three non-rhodopsin
family bundles. Left: intrahelical pairs, Right: interhelical pairs. Coloring of the plots is the same as in figure 4.

determination of non-rhodopsin family receptor structures presents
challenges concerning the reliable comparison and quantitation ofdifferences among the 7TM regions. In fact, superimposition could
vary, depending on how it was performed, and RMSD analysis does
not appear to provide any quantitative results, as the present study
SCIENTIFIC REPORTS | 5 : 9176 | DOI: 10.1038/srep09176

does, such as the remarkable conservation of the structure of helix III
and the I–VI, I–III distances.
Furthermore, rational interpretation of the increasing data on therelation between the type of bound ligand and the degree of conformational change requires understanding of common structural fea-

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Table 1 | Conserved pairs within helix III and between helices I and VI
Ca1

Ca2

Set 1

Set 2

Helix III

Helix III

Number of pairs

Number of pairs

sectionsection

top1000

all

ratio

top1000

all

ratio

1
1
1
1
2
2
2
3
3
4
4

2
3
4
5
3
4
5
4
5
4
5

4
10
34
36
3
42
41
14
21
3
12
0
220

64
32
56
56
32
56
56
28
28
21
49
83
561

0.06
0.31
0.61
0.64
0.09
0.75
0.73
0.50
0.75
0.14
0.24
0.00
0.39

8
13
37
44
7
44
46
17
22
7
16
2
263

64
32
56
56
32
56
56
28
28
21
49
83
561

0.13
0.41
0.66
0.79
0.22
0.79
0.82
0.61
0.79
0.33
0.33
0.02
0.47

otherstotal
Helix I

Helix VI

sectionsection

top1000

all

ratio

top1000

all

ratio

3
3
4
4
5
5
5

1
2
1
2
1
2
3

6
3
31
25
25
20
2
0
112

50
25
60
30
50
25
20
540
800

0.12
0.12
0.52
0.83
0.50
0.80
0.10
0.00
0.14

12
4
28
14
16
14
1
0
89

50
25
60
30
50
25
20
540
800

0.24
0.16
0.47
0.47
0.32
0.56
0.05
0.00
0.11

otherstotal

Number of pairstures that define the functional basis of this large family of membraneproteins. Here, we demonstrate that intramolecular distance conservation scoring provides a reasonable measure of evolutionary conserved basic architecture of GPCRs. The present results on distanceconservation are consistent with a separate examination of the backbone torsion angles (phi, psi) of set 10: the lowest deviations arefound in the sections I–4 (5.2u and 6.9u for phi and psi, respectively),
III–4 (5.4u and 8.2u), and VI–2 (5.1u and 7.8u).
Although the amount of available structural data is limited, ourresults clearly show which part of the seven helices is the most or the
least conserved in terms of the intra and interhelical distances(Fig. 5). Practically, such information should be useful in screening
from a set of automatically constructed model for a receptor of
unknown structure. Additionally, identification of conservation patterns will further evoke studies from evolutionary point of view. For
instance, it would be interesting to analyze microbial 7TM retinal
proteins with the current scoring method (see below).
The most conserved intrahelical distances are assigned at the spacing containing five turns approximately from 3.32 to 3.50. In fact,
close examination of this part shows no significant distortion for all
the receptors of known structure. Therefore, our result suggests thatthe regular and conserved shape of this helical segment is a key to thecommon functionality of GPCRs. Interestingly, the distances between
the extracellular part of this segment and the cytoplasmic part of helix
I is also conserved (Fig. 6). These findings, in conjunction with theintrahelical conservation of helix I and inter-helical conservation
between the cytoplasmic part of helix I and the part around 6.50 ofhelix VI, highlight previously unidentified intramolecular spacing thatmight form the critical conditions for the functionality of GPCRs.
Since the two regions described for helices III and VI affect the levelof activity, it is conceivable that their relative position to a distant point(e.g., the cytoplasmic part of helix I) appears to be conserved.
Distance matrix analysis has been used for detecting the conformational transitions between two states within a protein13. The
SCIENTIFIC REPORTS | 5 : 9176 | DOI: 10.1038/srep09176

Number of pairs

recent high-resolution structural models of GPCRs are valuable
sources of data for extracting structural information regarding theconservation and variation among the evolutionally remote members of protein families, by systematically analyzing the intramolecular distances. To our knowledge, the variety or the quantity ofstructural data required for this sort of analysis are not available
for other membrane protein families. The structures of a relatedfamily of 7TM proteins, the microbial retinal proteins, have been
extensively investigated14. While the sequence variation of these proteins is limited, application of the current approach to this family ofproteins appears to demonstrate distinct features from GPCRs in theintramolecular conservation patterns (unpublished data).
The statistical analysis presented makes use of only one snapshot ofthese inherently dynamic and fluctuating molecules15. Therefore, thequantity and the quality of available structural data would be expected
to significantly affect the reliability of the inferred results. Indeed, thepresent scoring and ranking were reasonably sensitive to the choice ofstructures in a given set, especially with regard to interhelical pairs. It is
possible that further structural data would improve the characterizationof each of the families within the whole GPCR superfamily.
Changes in the intramolecular interatomic distance of specificpairs of amino acids have been frequently examined when estimating
the trajectories for molecular dynamics simulations16,17. As a reference, our 50 ns simulation of the delta opioid receptor indicatedthat the scores calculated for the trajectory of the distances between
the Cas of BW numbers 1.59 and 6.50, one of the highest-ranked-pairin the present study (score approximately 87 in set 10), was 51. On
the other hand, the corresponding score between 3.50 and 6.30,
which was expected to be sensitive to the state of receptor activity,
was 12, which was comparable to that obtained in set 10 (score 5.6).
The present study focused only on the inactive-like structures thatwere obtained in the presence of antagonists, inverse-agonists ornegative allosteric modulator. However, the degree of conformational ‘‘inactiveness’’ might differ among the structures in a given

1236www.nature.com/scientificreports

Figure 6 | Graphical representation of conserved interhelical distances in
set 10 including 19 unique bundles from all GPCRs with known structure.
Upper: top view from the cytoplasmic side. Lower: side view from helices II
and IV. Interhelical section pairs of conserved distance are colored on the
200 residue 7TM backbone of bovine rhodopsin template (1U19-A) as
follows; purple, I–VI; cyan, I–III. All of the lines shown between the pairs
have scores of more than 50. For clarity, the pairs involving the residues of
BW no. 3.37 and 6.50 are shown by these lines.

set depending on the type of ligand bound to each of the receptors.
Therefore, the higher-ranked section pairs described may also be
structurally conserved in the active-like states. While a direct comparison between the results obtained from inactive-like and activelike sets would be difficult due to the limited variety of receptors in
the active-like set, further analysis of the active-like set is likely toprovide valuable insights into the activation mechanism of the rhodopsin family receptors.
Analysis of the conserved interhelical pairs in each set appears toprovide subfamily-specific information. In the case of set 4, where
adrenergic receptors were omitted, we observe an increase in highscore contribution from the III–V pairs. This result is consistent withthe fact that superimposition of the bundles clearly showed that helix
V of the b1 and b2 adrenergic receptors was shifted slightly upward(toward the extracellular side) from the other receptors while helix III
did not exhibit any features among them.
On the other hand, deviations can also be accommodated withoutchanging the interhelical distances. Previously reported significant
deviations of bovine rhodopsin from other receptors with respect tothe positioning of the extracellular part of helix III6 did not appear toaffect its distance to the cytoplasmic end of helix V. Similarly, considerable deviation of class B and smoothened receptors from rhodopsin family at the extracellular part of helix VI did not cause major
SCIENTIFIC REPORTS | 5 : 9176 | DOI: 10.1038/srep09176

Figure 7 | Graphical representation of conserved residues among 19
receptors in set 10. Upper: top view from the cytoplasmic side. Lower: sideview from helices II and IV. Five conserved positions in helices I, II, and III(Fig. S3) are shown as sticks on each of the 7TM backbone ribbons. These
residues correspond to BW no. of 1.52, 1.53, 2.46, 3.43, and 3.54.

distance changes from this region to the cytoplasmic end of helix I.
These observations suggest that our analysis also provides information on allowable directions along which some helices can deviate
without affecting the receptor’s functionality. Further accumulationof high-resolution structures could confirm whether the highly
diverse backbone organization in the extracellular regions of
GPCRs might still be limited by directional restrains that are required
to ensure the correct positioning of the cytoplasmic parts of the 7TM
bundle.
The most intriguing structural feature of GPCR activation is that avariety of stimuli likely evoke large movement of helix VI while theremaining 6 helices exhibit smaller degree of rearrangement11,18. For
such a mechanism to operate in diverse receptors, we suspect thatsome common and key intramolecular restraints have been conserved during evolution. In this respect, conservation of the distancesfrom the intracellular side of helix I to the middle/extracellular part ofhelices III and VI might be an essential requirement.

Methods
The data processing from the original PDB entries was as follows. Each of the polypeptides in an entry was used to extract the heptahelical bundle, consisting of 200
residues, as described previously6. For each polypeptide, Ca coordinates wereextracted by pdbset in ccp4i19. Temperature factor analysis was also performed using
these Ca files.
In total, 19,900 interatomic distances were calculated and tabulated, using thecoordinates of the 200 Ca atoms in each bundle for all the chains archived on our web

124

7

www.nature.com/scientificreports
site (www.gses.jp). In cases where two conformers were provided for a residue, frequently W(4.50) in the rhodopsin family receptors, only conformer A was considered
because the differences of the Ca coordinates between A and B were mostly negligible.
Alignment of CRF1 receptor in class B, mGluRs in class C, and smoothened receptor
was identical to that reported previously7–10 at the most conserved position in eachhelix. The 7TM bundle of the smoothened receptor (PDB ID: 4JKV) aligned torhodopsin-like receptors was assumed to lack a residue at the amino terminal of helix
IV (4.39). Therefore, in the sets containing the smoothened receptor, the total numberof Ca pairs was 19,701. Similarly, the 7TM bundle of the class C mGluR1 receptor was
assumed to lack two residues at the carboxyl termini of helix II (2.66 and 2.67) and VI(6.59 and 6.60), giving the total number of 18,915 for Ca pairs in set 10. Even among
the receptors of the rhodopsin family, the possible sequence gaps supposed to occur ina few regions (e.g. the extracellular side of helices II and IV) in some receptors20,21 were
not taken into account, and the BW numbering was serially made in both directions
from the *.50 position.
In addition to the criteria for bundle selection for each of the sets, we selectedbundles for the minimal sets 9 and 10 as the ones with the highest resolution andlower overall average temperature factor. For instance, chain B of 4AMJ was chosen
for the b1 adrenergic receptor because 4AMJ has the highest resolution for thisreceptor and the chain B has lower overall temperature factor for Cas than chain A.
In each of the sets, the average, standard deviation, maximum, minimum, and theinverse of coefficient of variation (score) were calculated for the 19,900 or 19,701/
18,915 Ca pairs. Then sorting was made according to the score. The distribution of theaverage distances were examined for all 19,900 and the top ranked 1,000 Ca pairs of
set 1. Two-dimensional plots showing the correlation between the score and the
distance were also prepared for set 1.
Molecular dynamics (MD) simulation runs were performed on the delta opioid
receptor, having complete inter-helical loops (PDB ID: 4N6H), in a lipid bilayer of 80
3 80 POPC molecules, with NAMD22 running on a GPU accelerated PC, using
particle-mesh Ewald electrostatics, 10 Å nonbond cut-off, switching function at 9 Å,
12 Å pair list cut-off and 1 fs time step. Briefly, the delta receptor polypeptide (fromG36 to G338) having a complete loop connection was inserted into the POPC bilayer,
hydrated, neutralized with 0.15 M NaCl by VMD23. The whole system was energy
minimized for 50 ps and the lipid and the solvent were equilibrated by MD for 0.5 ns
under NVT condition (310 K). Then, a 50 ns MD run was performed with norestraints under NPT at 1.0 bar and 310 K and the Ca-Ca distances in the trajectorywere analyzed with MD tools implemented in Chimera24. All the graphs and thegraphics were drawn with Igor Pro (WaveMetrix) and DS visualizer (Accelrys),
respectively.
Backbone torsion angles were obtained by DSSP12 and the deviations of phi and psi
angles among the receptor structures were calculated for each Ca position. Then, thevalues were averaged for each of the 35 sections (5 sections per helix).

10. Wu, H. et al. Structure of a class C GPCR metabotropic glutamate receptor 1bound to an allosteric modulator. Science 344, 58–64 (2014).
11. Nakamura, S., Itabashi, T., Ogawa, D. & Okada, T. Common and distinct
mechanisms of activation of rhodopsin and other G protein-coupled receptors.
Sci. Rep. 3, 1844 (2013).
12. Kabsch, W. & Sander, C. Dictionary of protein secondary structure: patternrecognition of hydrogen-bonded and geometrical features. Biopolymers 22,
2577–2637 (1983).
13. Edman, K. et al. Deformation of helix C in the low temperature L-intermediate ofbacteriorhodopsin. J. Biol. Chem. 279, 2147–2158 (2004).
14. Gushchin, I. et al. Structural insights into the proton pumping by unusual
proteorhodopsin from nonmarine bacteria. Proc. Natl. Acad. Sci. U.S.A. 110,12631–12636 (2013).
15. Nygaard, R. et al. The dynamic process of b(2)-adrenergic receptor activation. Cell
152, 532–542 (2013).
16. Spijker, P., Vaidehi, N., Freddolino, P. L., Hilbers, P. A. & Goddard 3rd, W. A.
Dynamic behavior of fully solvated beta2-adrenergic receptor, embedded in themembrane with bound agonist or antagonist. Proc. Natl. Acad. Sci. U.S.A. 103,4882–4887 (2006).
17. Shan, J., Khelashvili, G., Mondal, S., Mehler, E. L. & Weinstein, H. Liganddependent conformations and dynamics of the serotonin 5-HT(2A) receptor
determine its activation and membrane-driven oligomerization properties. PLoS
Comput. Biol. 8, e1002473 (2012).
18. Kruse, A. C. et al. Activation and allosteric modulation of a muscarinic
acetylcholine receptor. Nature 504, 101–106 (2013).
19. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta
Crystallogr. D67, 235–242 (2011).
20. Gonzalez, A., Cordomı́, A., Caltabiano, G. & Pardo, L. Impact of helix
irregularities on sequence alignment and homology modeling of G proteincoupled receptors. Chembiochem 13, 1393–1399 (2012).
21. Katritch, V., Cherezov, V. & Stevens, R. C. Diversity and modularity of G proteincoupled receptor structures. Trends Pharmacol. Sci. 33, 17–27 (2012).
22. Phillips, J. C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem.
26, 1781–1802 (2005).
23. Humphrey, W., Dalke, A. & Schulten, K. VMD - Visual Molecular Dynamics.
J. Molec. Graphics 14, 33–38 (1996).
24. Pettersen, E. F. et al. UCSF Chimera-a visualization system for exploratory
research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

Acknowledgments
Supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan.

1. Kroeze, W. K., Sheffler, D. J. & Roth, B. L. G-protein-coupled receptors at a glance.
J. Cell Sci. 116, 4867–4869 (2003).
2. Fredriksson, R. & Schiöth, H. B. The repertoire of G-protein-coupled receptors infully sequenced genomes. Mol. Pharmacol. 67, 1414–1125 (2005).
3. Kolb, P. et al. Structure-based discovery of beta2-adrenergic receptor ligands.
Proc. Natl. Acad. Sci. U.S.A. 106, 6843–6848 (2009).
4. Beuming, T. & Sherman, W. Current assessment of docking into GPCR crystal
structures and homology models: successes, challenges, and guidelines. J. Chem.
Inf. Model. 52, 3263–3277 (2012).
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three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. Methods Neurosci. 25, 366–428 (1995).
6. Okada, T. Comparative analysis of the heptahelical transmembrane bundles of G
protein-coupled receptors. PLoS One 7, e35802 (2012).
7. Wang, C. et al. Structure of the human smoothened receptor bound to an
antitumour agent. Nature 497, 338–343 (2013).
8. Hollenstein, K. et al. Structure of class B GPCR corticotropin-releasing factor
receptor 1. Nature 497, 438–443 (2013).
9. Siu, F. Y. et al. Structure of the human glucagon class B G-protein-coupled
receptor. Nature 497, 444–449 (2013).

SCIENTIFIC REPORTS | 5 : 9176 | DOI: 10.1038/srep09176

Author contributions
M.K. performed analysis, T.O. designed research, performed analysis, and wrote themanuscript.

Additional information
Supplementary information accompanies this paper at http://www.nature.com/
scientificreports
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Kinoshita, M. & Okada, T. Structural conservation among therhodopsin-like and other G protein-coupled receptors. Sci. Rep. 5, 9176; DOI:10.1038/
srep09176 (2015).

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to obtain permission from the license holder in order to reproduce the material. To
view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

8光の情報を受け取る分子メカニズムの解明
教授 岡本 治正助教 本郷 育子
[目的]
光情報を受け取る主要組織は眼である。その複雑な多層構造の発生過程、また機能発現の分子メカニズムについてはいまだ未知な部分も多いが、近年眼について多くの発生、機能関連遺伝子が FGF シグナルの制御下にあることが明らかにされつつある。他方、当研究室ではこれまで FGF シグナルの下流にある神経系関連遺伝子を網羅的にスクリーニングする系をアフリカツメガエル初期嚢胚の未分化な外胚葉細胞を用いて確立してきた。これまでにこの外胚葉細胞を低濃度の FGF で処理して得られるトランスクリプトームに、眼を含む前脳で発現する遺伝子産物が相対的に多く存在することが明らかになっている。そこで当該研究では眼の発生、機能に関連することが期待される遺伝子に焦点を絞り、上記トランスクリプトームからのスクリーニングを行った。発生関連では、1) 転写因子型、あるいは 2) シグナル伝達関連型の、また機能関連では 3) GPCR 型の遺伝子を選抜し in situ
hybridization により眼における発現の確認を目指した。
[結果と考察]当該研究で解析した以下 6 種の遺伝子はいずれも、アフリカツメガエル初期胚における時間的・空間的発現パターンについて、これまで詳細な報告の乏しいものである。
1) 転写因子型a) elf1 (E74-like factor 1 ): FGF/MAPK シグナル経路の下流で働く Ets 転写因子ファミリ－のメンバ－。ツメガエル初期胚での役割は不明。
神経管形成期（受精 20 時間後）に前脳領域を中心に発現が始まり、尾芽胚期（受精 32時間後）に眼胞（眼のもとになる原基構造）、特にその周縁部に発現が確認された（Fig.1a）。

126b) ctcf (CCCTC-binding factor ): Zinc-finger モチーフを DNA 結合モチーフとして持つ
転写因子ファミリ－のメンバ－。ツメガエル初期胚での役割は不明。
神経板誘導期（受精 16 時間後）に前脳領域を中心に発現が始まり、尾芽胚期（受精。
32 時間後）に眼胞での発現が確認された（Fig.1b）c) znf182 (Zinc-finger protein 182 ): Zinc-finger モチーフを DNA 結合モチーフとして持つ転写因子ファミリ－のメンバ－。ツメガエル初期胚での役割は不明。
神経板誘導期（受精 16 時間後）に前脳領域を中心に発現が始まり、尾芽胚期（受精32 時間後）に眼胞での発現が確認された（Fig.1c）。
2) シグナル伝達関連型a) birc2 (baculoviral IAP repeat containing 2 ): 免疫系細胞内のシグナル伝達分子とされているが、ツメガエル初期胚での役割は不明。
神経管形成期（受精 20 時間後）に前脳領域を中心に発現が始まり、尾芽胚期（受精32 時間後）に眼胞での発現が確認された（Fig.2a）b) frs2 (FGF receptor substrate 2 ): receptor docking protein ファミリ－のメンバ－。ツメガエル初期胚での役割は不明。
神経管形成期（受精 20 時間後）に前脳領域を中心に発現が始まり、尾芽胚期（受精32 時間後）に眼胞での発現が確認された（Fig.2b）3) GPCR 型

gpc160 (G protein-coupled receptor 160 ): G タンパク質共役型受容体ファミリ－のメン
バ－。ツメガエル初期胚での役割は不明。

神経管形成期（受精 20 時間後）に前脳領域を中心に発現が始まり、尾芽胚期（受精 32 時
間後）に眼胞での発現が確認された（Fig.3）

127

当該研究で解析された六つの遺伝子は、いずれも尾芽胚の眼胞（眼の原基）での発現が確認された。また発現の開始は、神経板誘導期ないし神経管形成期にまでさかのぼることも示された。今後は各遺伝子について、mRNA 過剰発現による gain of function 解析、またアンチセンスRNAによる loss of function 解析を行い、各遺伝子の眼の発生、また機能発現における働きを明らかにする必要がある。

あああああ

あああさああああああああああああああああああああああああ128青色光受容体 (FKF1)欠損変異体を利用した花成時期制御機構の解明
教授 清末 知宏
助教 高瀬 智敬[目的]
固着生活を営む植物にとって栄養成長から生殖成長への転換である花成を適切な時期に行うことは、繁殖戦略上極めて重要である。花成時期制御に関わる遺伝子の多くはモデル
植物シロイヌナズナを用いて明らかにされてきているが、花成が光・温度・養分などの外的要因と齢・植物ホルモンなどの内的要因とによって影響を及ぼされる複雑な制御を受けているため、未だ同定されていない花成時期制御遺伝子の存在が予想されている。本研究では、シロイヌナズナの光周性花成経路ではたらく花成促進因子の一つである青色光受容体 FKF1 の機能欠損変異体（遅咲き変異体）を変異原処理することで早咲きとなる新規変異体を単離し、その原因遺伝子の同定と早咲きとなる仕組みとを解析することで、植物の栄養成長から生殖成長への転換機構の分子的な基盤を明らかにすることを目的とする。
[結果と考察]fkf1 変異体をメタンスルホン酸エチル（EMS）処理し、M2 世代の植物を長日条件で育成して早咲き個体を得るというサプレッサースクリーニングによって 32 個体の早咲き変異体を得た。本研究ではこのうち２つの変異体 F0501、F4501 の解析を中心に行った。
[1] F0501 と F4501 の花成時期

F0501 と F4501 は早咲きの変異と fkf1 変異の両方を有している。そこで、早咲きの表現

型が fkf1 変異に依存するか否かを明らかにするために、交配によって fkf1 変異を野生型に置き換えた植物体 F0501mono と F4501mono を作出し、Col、fkf1、F0501、F0501mono、

F4501、F4501mono の花成時期を測定した。その結果、長日条件（16L8D、22℃）と短日条件（8L16D、22℃）のどちらでも、F0501、F0501mono、F4501、F4501mono は同時期に抽苔し、その時の葉の枚数も同程度であり、Col に比べて早咲きであった（図 1、２）。

従って、F0501 と F4501 の早咲き変異は fkf1 変異と独立していることが明らかとなった。

[2] 早咲き原因遺伝子の同定
染色体歩行を行い、F0501 と F4501 の早咲きの原因遺伝子がそれぞれ第 2 染色体の下腕の 14.2 kbp と 260 kbp の領域内に座乗していることを明らかにした（図３）。次世代シー
ケンサーを用いて F0501 と F4501 の核全ゲノム DNA 塩基配列を調べた結果、染色体歩行で絞り込んだ領域内に F0501 では 1 つ、F4501 では 4 つの１塩基置換があることがわかった。更に、F0501 と F4501 は共にファミリーB に属する DNA ポリメラーゼの 1 つである
DNA ポリメラーゼδを構成するアクセサリーサブユニットである POLD2 に変異が生じており、F0501 では POLD2 の翻訳開始点から数えて 1286 番目の塩基が G から A に、F4501
では POLD2 の翻訳開始点から数えて 323 番目の塩基が G から A に置換していることが明129

らかとなった。このことから、F0501 と F4501 の早咲きは POLD2 の変異が原因であることが示唆された。
以降、
F0501 を fkf1 pold2-1、
F0501mono を pold2-1、F4501 を fkf1 pold2-2、F4501mono を pold2-2 と表記する。

[3] 早咲き変異体の POLD2 cDNA の塩基配列とコードされているアミノ酸配列

pold2-1 と pold2-2 の葉から RNA を単離し、RT-PCR により POLD2 の cDNA を合成した後、その塩基配列を決定した。その結果、pold2-1 では第 6 イントロンがスプライシング

されず、通常より 71 bp、cDNA が長くなり、POLD2 タンパク質の 440 個のアミノ酸残基のうち 220 番目に終始コドンが生じて、219 番目で終わることがわかった。pold2-2 では第2 エクソンの途中でスプライシングが起こり、cDNA が通常より 93 bp 短くなることがわかった。しかし、リーディングフレームのずれは生じないため、32 から 62 番目までの 31 ア
ミノ酸残基は欠損するものの、32 番目以降の領域の POLD2 は正常に作られることがわかった。このことから、pold2-1 と pold2-2 では一部が欠損した POLD2 タンパク質が作られることが示唆された。
[4] POLD2 T-DNA 挿入変異体（GK762B02、GK820B06）の表現型

POLD2 の変異が原因で pold2-1 と pold2-2 とが早咲きになるのであれば、T-DNA 挿入

によって POLD2 の機能が失われた植物体も早咲きになると考えられる。そこで、POLD2に T-DNA が挿入されている 2 つのライン GK762B02 と GK820B06 をストックセンター（ABRC）から入手し、その表現型を観察した。入手した種子から育てた植物体から DNAを抽出し、PCR によって遺伝子型を確認した結果、GK762B02 と GK820B06 のどちらのラインからも T-DNA 挿入ホモ接合体は得られなかった。T-DNA 挿入ヘテロ接合体の自家受粉によって得られた種子をまき、育った植物体の遺伝子型を調べたが、やはり T-DNA 挿

入ホモ接合体は得られなかった。このことは、POLD2 の T-DNA 挿入ホモ接合体が致死となることを示唆している。この点を明らかにするために、 T-DNA 挿入ヘテロ接合体の莢を剥き、中の種子を観察した。その結果、野生型ではほとんどが緑色の正常な種子であったのに対し、T-DNA 挿入ヘテロ接合体では緑色の正常な種子と茶色く小さい異常な種子とが観察された。カイ二乗検定を行ったところ、正常な種子：異常な種子の割合が 3：1 であることが確かめられたので、T-DNA 挿入ホモ接合体は種子形成の段階で致死になることが示された。
次に、T-DNA 挿入ヘテロ接合体の雄蕊と pold2-1 の雌蕊を用いて交配を行った。交配によって得られた植物体のうち T-DNA を保有せず、pold2-1 変異をヘテロで有する植物体は野生型と同じ植物体の大きさを示したが、T-DNA 挿入と pold2-1 変異の両方をヘテロに有する植物体はとても小型になり、早咲きであった。
以上のことから、pold2-1 と pold2-2 では POLD2 タンパク質の一部が欠損しているものの、POLD2 の機能が部分的に残っているために致死とならずに早咲きとなったと考えられ、T-DNA 挿入と pold2-1 変異の両方をヘテロに持つ植物体では、部分的に機能が残っている
POLD2 を 1 コピーしか有していないため、非常に小型で早咲きになったと考えられる。
[5] fkf1 pold2-1 と fkf1 pold2-2 との交配で得られた F1 個体の花成時期

POLD2 が早咲きの原因遺伝子か否かを更に検証するために、fkf1 pold2-1 の雄蕊と fkf1

130

pold2-2 の雌蕊を用いて交配を行った。得られた F1 個体が自家受粉によるものではなく、交配によって得られたものであるか否かを調べるため、F1 個体の POLD2 の DNA 塩基配列を決定した。その結果、POLD2 の翻訳開始点から数えて 1286 番目の塩基には pold2-1 の置換した塩基（A）と正常な塩基（G）の 2 つが検出され、323 番目の塩基には pold2-2 の置換した塩基（A）と正常な塩基（G）の 2 つが検出されたため、交配は成功していたことがわかった。長日条件で花成時期を測定したところ、F1 個体は fkf1 と比べて早咲きであった。F1 個体の抽苔日と抽苔時の葉の枚数は、fkf1 pold2-1 と fkf1 pold2-2 のそれらと同程度であった。この遺伝的相補性は、POLD2 が pold2-1 と pold2-2 の早咲きの原因遺伝子であることを支持する結果の 1 つである。

[6] FT の経時的発現解析
シロイヌナズナでは、光周性経路、自律的経路、春化経路、ジベレリン経路の 4 つの花
成経路が存在し、前者の三つの経路の統合因子として花成ホルモン FT が知られている。そこで、FT の発現を経時的に調べたところ、Col の FT の発現量は ZT（Zeitgeber Time）4と ZT16 にピークを持ち、ZT16 で最大となった。fkf1 では Col と比べてどの時間帯でもFT の発現量は低くかった。pold2-1 と pold2-2 では、FT の発現量はどの時間帯でも Col
より高いことがわかった。fkf1 pold2-1 と fkf1 pold2-2 では、FT の発現量はどの時間帯でも fkf1 より高かった。更に、fkf1 pold2-1 と fkf1 pold2-2 では ZT8 など一部の時間帯で Colに比べて FT の発現量が増加するが、1 日を通しての発現量は pold2-1 や pold2-2 のようには大きく変わらなかった。これらのことから、FT の発現上昇と FT 以外の花成遺伝子の活性化によって pold2-1 と pold2-2 とが早咲きとなるのではないかと考えられた。
[7] DNA マイクロアレイ

野生型と pold2 変異体との間で発現が変化している遺伝子を網羅的に調べるため、DNA

マイクロアレイ解析を行った。マイクロアレイに用いた RNA は、長日条件で 10 日間育てた Col、pold2-1、fkf1、fkf1 pold2-2 の植物体全体から抽出し、サンプリングは ZT4 に行った。pold2-1 での発現が Col と比べて 2 倍以上であり、かつ fkf1 pold2-2 での発現が fkf1と比べて 2 倍以上だった遺伝子は 307 個あった（図４）。それらを DAVID で機能分類した

結果、17 個のクラスターに分類された。そのうち統計的に有意とされる Enrichment Score
が 1.3 以上（Huang et al., 2009）のクラスターは 4 個であり、クラスター2 には花成に関わる遺伝子群が含まれていた。その中でも reproductive developmental process に関わる遺伝子数が 22 個と最も多く、APETALA1（AP1）、SEPALLATA3（SEP3）、FRUITFULL（FUL）、AGAMOUS-LIKE24（AGL24）などが含まれていた。AP1 や SEP3、FUL はFT の下流にあり、花の器官形成に関わる遺伝子である（Smaczniak et al., 2012）。AGL24
も FT の下流にあり、AGL24 と SOC1 は相互作用することで花成促進にはたらき、AGL24を過剰発現させたシロイヌナズナは早咲きになることが知られている（Liu et al., 2008）。
AGL24 の発現は、pold2-1 では Col 比べて 3.31 倍、fkf1 pold2-2 では fkf1 に比べて 6.82倍に増加していたことから、AGL24 が pold2-1 と pold2-2 の早咲きに関わる候補遺伝子の1 つであると考えられる。

131

[まとめ]
本研究により、POLD2 がシロイヌナズナの新規花成抑制因子であることが示され、FTの発現上昇が pold2 変異体の早咲きの一因になりうること、FT 以外の遺伝子（AGL24 が候補）も早咲きに関わっていることが示唆された。
ポリメラーゼδと同じファミリーB に属する DNA ポリメラーゼは、シロイヌナズナでは
δの他に、α、ε、ζがある（Garcia-Diaz et al. 2007）。DNA ポリメラーゼαの触媒サブユニットをコードする INCURAVATA2（ICU2）の変異体と DNA ポリメラーゼεの触媒サ

ブユニットをコードする EARLY IN SHORT DAYS 7（ESD7）の変異体でも野生型に比べ早咲きになり、T-DNA が挿入されると種子形成の段階で致死になることが報告されている（Barrero et al., 2007, del Olmo et al., 2010）。ESD7 と ICU2 は FT の発現を抑制する

LIKE HETEROCHROMATIN PROTEIN 1（LHP1）/ TERMINAL FLOWER2（TFL2）と相互作用することが知られている（del Olmo et al., 2010,

Hyun et al., 2013）。

LHP1/TFL2 は H3K9me2 もしくは H3K9me3 と結合し、花成制御因子遺伝子である FTや FLC、花芽器官形成遺伝子である AGAMOUS や APETALA3 などを含む多くの遺伝子の転写を抑制する（Turck et al., 2007）。今後、POLD2 による花成抑制機構の解析を進め、ICU2、ESD7 による花成抑制機構との相違点・一致点を明らかにすることで、DNA ポリ
メラーゼによる植物の栄養成長から生殖成長への転換機構の全貌に迫ることができると考えている。

長日条件での花成時期（図１）

短日条件での花成時期（図２）染色体歩行による早咲き原因遺伝子の同定（図３）132

DNAマイクロアレイ（図４）[原著論文]1. Miyazaki, Y., Abe, H., Takase, T., Kobayashi, M., and Kiyosue, T. (2015)

Overexpression of LOV KELCH PROTEIN2 confers dehydration tolerance and isassociated with enhanced expression of dehydration-inducible genes in Arabidopsis

thaliana. Plant Cell Rep. 34, 843-852.

2. Miyazaki, Y., Takase, T., and Kiyosue, T. (2015) ZEITLUPE positively regulates
hypocotyl elongation at warm temperature under light in Arabidopsis thaliana.

Plant Signal. Behav. 10, e998540.

3. Saitoh, A., Takase, T., Kitaki, H., and Kiyosue, T. (2015) Gene expression profile ofArabidopsis plants that overexpress ZEITLUPE/ LOV KELCH PROTEIN1:
up-regulation of auxin-inducible genes in hypocotyls. Plant Biotech. 32, 257-261.

4. Saitoh, A., Takase, T., Kitaki, H., Miyazaki, Y., and Kiyosue, T. (2015) Gene

expression profile of zeitlupe/lov kelch protein1 T-DNA insertion mutants in

Arabidopsis thaliana: downregulation of auxin-inducible genes in hypocotyls. Plant
Signal. Behav. 10, e1071752.

5. Takase, T., Miyazaki, T., Yasuhara, M., Mitsui, S., and Kiyosue, T. (2015)

Pleiotropic phenotype of transgenic Arabidopsis plants that produce the LOV
domain of LOV KELCH PROTEIN2 (LKP2). Plant Biotech. 32, 273-280.

6. Miyazaki, Y., Jikumaru, Y., Takase, T., Saitoh, A., Sugitani, A., Kamiya, Y., andKiyosue, T. (2016) Enhancement of hypocotyl elongation by LOV KELCH
PROTEIN2 production is mediated by auxin and phytochrome-interacting factors in

Arabidopsis thaliana. Plant Cell Rep. 35, 455-467.
[著書]1.

清末知宏 (2016) ZTL/LKP2/FKF1 光受容体「光と植物の事典」（分担著）朝倉書店。

[口頭発表、ポスター発表]
1.

矢作道枝、高瀬智敬、清末知宏：「シロイヌナズナ fkf1 サプレッサースクリーニングによる花成時期変異体の単離と解析」、第 55 回日本植物生理学会年会、富山、2014
年 3 月 20 日。
2.

齋藤彩、宮崎裕士、木下久樹、木滝博之、高瀬智敬、清末知宏：「青色光受容体の過剰発現がシロイヌナズナの胚軸伸長に及ぼす影響」、第 37 回日本分子生物学会年会、横浜、2014 年 11 月 27 日。

3.

矢作道枝、高瀬智敬、清末知宏：「シロイヌナズナ fkf1 サプレッサー変異体（ef1）の原因遺伝子の特定と特徴づけ」、第 37 回日本分子生物学会年会、横浜、2014 年 11 月
27 日。
4.

齋藤彩、宮崎裕士、高瀬智敬、清末知宏：「青色光受容体 ZTL がシロイヌナズナの胚軸伸長に及ぼす影響」、第 56 回日本植物生理学会年会、東京、2015 年 3 月 20 日。

5.

矢作道枝、高瀬智敬、清末知宏：「２つのシロイヌナズナ fkf1 サプレッサー変異体について」、第 56 回日本植物生理学会年会、 東京、2015 年 3 月 20 日。

133

Plant Biotechnology 32, 273–280 (2015)
DOI: 10.5511/plantbiotechnology.15.0808b

Original Paper

Pleiotropic phenotype of transgenic Arabidopsis plants that
produce the LOV domain of LOV KELCH PROTEIN2 (LKP2)
Tomoyuki Takase1,2, Yuji Miyazaki 1,2, Masahiro Yasuhara2, Shunya Mitsui 2,
Tomohiro Kiyosue1,2,*
1
Department of Life Science, Faculty of Science, Gakushuin University, Toshima-ku, Tokyo 171-8588, Japan; 2 Gene ResearchCenter, Kagawa University, Kita-gun, Kagawa 761-0795, Japan* E-mail: tomohiro.kiyosue@gakushuin.ac.jp Tel: +81-3-3986-0221 ext. 3602 Fax: +81-3-5992-1029

Received July 8, 2015; accepted August 8, 2015 (Edited by M. Sekine)

Abstract LOV KELCH PROTEIN2 (LKP2) is a blue-light receptor protein composed of three functional domains: a light,
oxygen, or voltage (LOV) domain, an F-box motif (F), and Kelch repeats. LKP2 is postulated to be a component of an SCF
complex and function in ubiquitination of proteins that control the circadian clock and photoperiodic flowering. Transgenic
Arabidopsis plants that produce LOV, F, or a combination of LOV and F fused to green fluorescent protein (named GL,
GF, and GLF, respectively) were produced using constructs containing the Cauliflower mosaic virus 35S promoter. Under
continuous white light, the circadian rhythms of control and GF plants were similar, whereas those of GL and GLF plants
were shorter. Under continuous red light, the hypocotyl lengths of control and GF seedlings were similar, whereas that of
GL seedlings was longer. Late flowering and down-regulation of CONSTANS and FLOWERING LOCUS T were observed in
GL and GLF plants compared to GF and control plants under long-day conditions. These results suggest that the previously
reported pleiotropic phenotype of LKP2-overproducing plants, which show altered circadian rhythm, hypocotyl elongation,and photoperiodic flowering, is not only due to the promotion of ubiquitination and subsequent degradation of substrate
proteins of the SCFLKP2 complex but may also be due to the functional disruption of regulatory proteins that interact with
LKP2 LOV.
Key words:

Arabidopsis, circadian rhythm, flowering time, hypocotyl elongation, LOV KELCH PROTEIN2 (LKP2).

(SCF) E3 ligase complex (Demarsy and Fankhauser2009). The SCFZTL complex regulates the circadian clockby ubiquitin-dependent degradation of two circadianclock regulators, TIMING OF CAB EXPRESSION1(TOC1) and PSEUDO-RESPONSE REGULATOR5(PRR5) (Kiba et al. 2007; Kim et al. 2007). The SCFFKF1
complex regulates flowering time under long-day (LD)conditions by ubiquitin-dependent degradation of
CYCLING DOF FACTOR (CDF) proteins, which are
repressors of CONSTANS (CO) expression (Imaizumi etal. 2003, 2005; Sawa et al. 2007).
The LOV domains of FKF1/LKP2/ZTL family
members are involved in blue-light perception bybinding flavin mononucleotide, which functions as achromophore, and by interaction with several regulatory
proteins. The FKF1 LOV domain forms homodimers(Zikihara et al. 2006) and functions in the regulation of
blue-light-dependent ubiquitination of CDF1 (Imaizumi
et al. 2005). The degradation of ubiquitinated CDFs by

Plants perceive light not only as the energy source forphotosynthesis but also as an environmental stimulusthat controls plant growth and development. Multiple
photoreceptors including phytochromes, cryptochromes,
and phototropins perceive environmental light signals in
Arabidopsis (Kendrick and Kronenberg 1994; Nagatani2010). In addition to these photoreceptors, membersof the FLAVIN-BINDING KELCH REPEAT F-BOX1(FKF1)/LOV KELCH PROTEIN2 (LKP2)/ZEITLUPE(ZTL) family are blue-light photoreceptors and are
involved in regulation of the circadian clock, hypocotyl
elongation, and flowering time (Jarillo et al. 2001;
Kiyosue and Wada 2000; Miyazaki et al. 2011; Nelson etal. 2000; Schultz et al. 2001; Somers et al. 2000; Takase etal. 2011). Arabidopsis FKF1/LKP2/ZTL family proteins
have three functional domains—the light, oxygen, orvoltage (LOV) domain, F-box motif, and Kelch repeat—
and function in ubiquitination of target proteins ascomponents of the SKP1-Cullin-Rbx1-F-box protein

Abbreviations: ACT2, ACTIN2; 3AT, 3-amino-1H-1,2,4-triazole; CDF, CYCLING DOF FACTOR; CO, CONSTANS; FKF1, FLAVIN-BINDING
KELCH REPEAT F-BOX1; FT, FLOWERING LOCUS T; GFP, green fluorescent protein; GI, GIGANTEA; LD, long-day; LKP2, LOV KELCH
PROTEIN2; LOV, light, oxygen, or voltage; PRR5, PSEUDO-RESPONSE REGULATOR5; SCF, SKP1-Cullin-Rbx1-F-box protein; T-DNA, transfer
DNA; TOC1, TIMING OF CAB EXPRESSION1; ZT, Zeitgeber time; ZTL, ZEITLUPE.
This article can be found at http://www.jspcmb.jp/
Published online September 26, 2015

Copyright © 2015 The Japanese Society for Plant Cell and Molecular Biology

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Pleiotropic phenotype of LKP2 LOV-producing Arabidopsis

26S proteasomes leads to the activation of CO expression,
which triggers the expression of FLOWERING LOCUS
T (FT), a florigen gene (Imaizumi et al. 2005). Theinteraction of FKF1 with GIGANTEA (GI) throughFKF1 LOV is required for this activation of CO
expression (Sawa et al. 2007). FKF1 also interacts with
CO through its LOV domain in a blue-light-enhancedmanner; this interaction stabilizes CO in the LD
afternoon (Song et al. 2014).
ZTL interacts with TOC1 and PRR5 through its LOV
domain (Kiba et al. 2007; Más et al. 2003). The ZTL
LOV domain also interacts with GI. This interaction isblue-light dependent and stabilizes ZTL protein (Kimet al. 2007). Production of ZTL LOV in transgenic
Arabidopsis causes period lengthening under red- or
blue-light conditions, similar to ztl loss-of-function
mutations, and a decreased level of endogenous ZTL
protein compared to that in the control (Kim et al. 2013;
Somers et al. 2000). These results suggest that productionof ZTL LOV reduces endogenous ZTL protein stability
by interfering with the interaction between endogenous
ZTL and GI, and that the reduction in ZTL levels leads
to period lengthening. Furthermore, production of ZTL
LOV results in elongated hypocotyls under red- or bluelight conditions and in delayed flowering under LD
conditions, similar to overproduction of ZTL (Kiyosue
and Wada 2000; Somers et al. 2004). These phenotypes
are postulated to be caused by the inhibition of GI
functions in nuclei due to enhancement of cytosolic GI
distribution caused by the ZTL LOV–GI complex (Kimet al. 2013).
LKP2 shows high amino acid sequence similarity
to ZTL, and the tissue-specific expression pattern of
LKP2 overlaps largely with that of ZTL (Kiyosue andWada 2000; Yasuhara et al. 2004). LKP2-overexpressing
transgenic plants phenocopy ZTL-overexpressing
transgenic plants: both exhibit circadian rhythm
defects under continuous light or in the dark, elongatedhypocotyls under continuous white or red light, anddelayed flowering under LD conditions compared tocontrol plants (Nelson et al. 2000; Schultz et al. 2001).
Transfer DNA (T-DNA) insertion mutants of lkp2 are
phenotypically similar to wild-type plants (Baudry et
al. 2010; Takase et al. 2011), while ztl T-DNA insertion
mutants show slower circadian rhythms undercontinuous red or blue light or in the dark, shorter
hypocotyls under continuous red light, and earlier
flowering under non-inductive photoperiodic conditions
compared to control plants (Somers et al. 2004; Takase
et al. 2011). The phenotypic similarity of lkp2 plants
with wild-type plants is postulated to be due to the very
low expression of LKP2 relative to that of ZTL (Baudry
et al. 2010; Michael et al. 2008; Mockler et al. 2007).
Introduction of LKP2 cDNA controlled by the ZTL
promoter complemented the long-period phenotype ofa ztl mutant (Baudry et al. 2010). These results suggestthat LKP2 possesses ZTL-like functions with respect toregulation of the circadian clock, hypocotyl elongationand flowering time in Arabidopsis.
Similar to the overexpression of LKP2, production ofgreen fluorescent protein (GFP)-LKP2 in Arabidopsis
also causes arrhythmicity of the circadian clock,lengthens hypocotyls in continuous white light, anddelays flowering under LD conditions (Miyazaki et al.
2011). In this study, we analyzed the effects of productionof the GFP-tagged LOV and F-box of LKP2 on thecircadian clock, hypocotyl elongation, and flowering time
regulation in Arabidopsis, and discussed a LOV-mediatedfunction of LKP2.

Materials and methods
Plant materials and growth conditions
All plant material was generated using the Columbia accessionof Arabidopsis thaliana. Hypocotyl length was measured as
described previously (Takase et al. 2004). Arabidopsis seeds
were surface sterilized and placed on GM medium (Valvekens
et al. 1988) supplemented with 0.8% agar. Plates were kept at
4°C for 7 days and then transferred to continuous white lightat 22°C for 8 h to induce germination. After induction ofgermination, the plates were transferred to monochromatic
light or to darkness. Blue, red, or far-red light was generated bylight-emitting diodes at 450, 660, or 750 nm, respectively (NKSystems, Tokyo, Japan). Fluence rates were measured with aradiometer (model LI-189; LI-COR, Inc., Lincoln, NE, USA).
Hypocotyl length was measured with Scion Image software(Scion Corp., Frederick, MD, USA). To determine the flowering
time, Arabidopsis seeds were sown on vermiculite in pots. Pots
were cold-treated at 4°C for 3 days and then transferred to LD
conditions (16-h light/8-h dark) at 22°C. All statistical analyses
were performed using IBM SPSS Statistics for Windows,
version 20.0 (IBM Corp., Armonk, NY, USA).

Vector construction and production of transgenic
plantsThree constructs encoding the LKP2 LOV domain, F-box
motif, and a region that includes both the LOV domain andF-box motif were generated. PCR primers are listed in Table
S1. The PCR template was cDNA from A. thaliana accession
Columbia. Each PCR fragment was subcloned into pCR4TOPO (Invitrogen, Carlsbad, CA, USA). After the entire
inserts were sequenced, they were excised with BglII and
BamHI and subcloned into the BglII site of pCR4-TOPO
containing the S65T GFP coding sequence (Niwa et al. 1999).
The resulting vectors were then digested with BamHI, andall fragments (encoding S65T GFP and LKP2 domains) were
subcloned into the BamHI site of the pBE2113 binary vector(Mitsuhara et al. 1996). The resulting vectors were introduced
into Agrobacterium tumefaciens LBA4404 by triparental mating(Figurski and Helinski 1979) and subsequently into Arabidopsis

Copyright © 2015 The Japanese Society for Plant Cell and Molecular Biology

135

T. Takase et al.

by the floral-dip method (Clough and Bent 1998). Homogygous
T4 progeny lines were obtained and used for the experiments.

Immunoblot analysis
Protein extraction from rosette leaves and immunoblot analysis
were performed as described previously (Takase et al. 2011).
Immunodetection was performed using an anti-GFP antibody(Nakalai Tesque Inc., Kyoto, Japan) and an ECL Advancesystem (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA).
Chemiluminescence signals were captured using a light-capturesystem (model AE-6792; ATTO, Tokyo, Japan).

Fluorescence microscopy
GFP signals from stomatal guard cells in leaf epidermal peels
were observed using an IX71 microscope with a standard GFPfilter (Olympus, Tokyo, Japan). The images were captured using
MetaMorph imaging software (Universal Imaging Corporation,
West Chester, PA, USA). A transgenic Arabidopsis plant
expressing GFP with a nuclear localization signal of SV40 at
the N-terminus (NLS-GFP) (Chiu et al. 1996; van der Krol andChua 1991) was used as a control for nuclear localization.

Bioluminescence analysis
To measure circadian rhythms, plants from homozygous linesof CAB2:LUC and CCR2:LUC (seeds provided by Dr. S.A. Kay,
University of California, San Diego) were crossed to plantsfrom homozygous lines of GL, GF, GLF and GFP-producing
plants. The resulting F1 seedlings were grown on GM agar
plates at 22°C under 12-h light/12-h dark cycles for 4 days
and then transferred to continuous white light. An automatedluminometer was used to monitor luciferase activity (Okamoto
et al. 2005a), and circadian period length in the individualtransgenic plants was estimated by visual identification ofpeaks and troughs followed by analysis with a rhythm-analysis
program (Okamoto et al. 2005b).

Quantitative RT-PCR
Plates were incubated at 22°C under 16-h light/8-h dark for 10days, and then seedlings were harvested every 4 h over a 24-h
period. Total RNA from the whole seedlings was extracted and
500 ng was used to synthesize cDNA with a High-Capacity
cDNA Reverse Transcription Kit (Applied Biosystems, Foster
City, CA, USA) as previously described by Takase et al. (2011).
Quantitative RT-PCR was performed using Power SYBR GreenPCR Master Mix (Applied Biosystems) and an ABI PRISM
7000 sequence detection system (Applied Biosystems). The
expression levels of ACTIN2 (ACT2) were used to normalize
expression of the target genes between samples. The primer
pairs used in the PCR analysis are listed in Table S1.

Figure 1. Structure of the GL, GF, and GLF proteins and theirproduction in transgenic plants. (A) Schematic diagram of the GL(containing the LKP2 LOV domain), GF (containing the LKP2 F-box
motif), and GLF (containing the LKP2 LOV and F-box) proteinsused in this study. All of the domains were produced as C-terminal
fusions to S65T green fluorescent protein (GFP). Amino acid positions
correspond to those in the native LKP2 protein sequence. (B) Transgene
expression in Arabidopsis thaliana. GFP primers were used to detect
transgene expression by RT-PCR. Actin (ACT2) primers were used as
the control. (C) Accumulation of GFP, GL, GF, and GLF proteins intransgenic plants. These proteins were detected by immunoblotting(top panel) using an anti-GFP antibody. Ponceau S staining of themembrane (bottom panel) was used as the loading control. (D)Subcellular localization of GFP, GL, GF, and GLF proteins in stomatal
guard cells of the transgenic plants. Each panel shows a representative
GFP image from the transgenic plants and non-transgenic Columbia(Col) plants. An NLS-GFP plant (containing a nuclear localization
signal fused to GFP) was used as a control for the nuclear localization.
Scale bars=20 µm.

Yeast two-hybrid assay
Yeast two-hybrid assays were performed as reported previously(Yasuhara et al. 2004). The Columbia version of full-length
cDNA for GI was obtained by RT-PCR with ReverTra Dash
(Toyobo, Osaka, Japan) and GI primers listed in Table S1,Copyright © 2015 The Japanese Society for Plant Cell and Molecular Biology

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Pleiotropic phenotype of LKP2 LOV-producing Arabidopsis

Figure 2. Circadian rhythms of GFP, GL, GF, and GLF transgenic plants under continuous white-light conditions. Graphs show luciferase activity
driven by the CAB2 (A) or CCR2 (B) promoter under continuous white light (LL) conditions. All transgenic plants were entrained in 12-h light/12-hdark for 4 days and then transferred to LL conditions. White and gray boxes indicate subjective day and subjective night, respectively.

Two homozygous lines were examined for each construct(e.g., for GL, the lines are designated GL 1 and GL 2).
The expression of each transgene was confirmed by
RT-PCR (Figure 1B), and the accumulation of GL, GF,
and GLF proteins of the predicted sizes was detected byimmunoblotting (Figure 1C). The subcellular localizationof these proteins in the transgenic plants was analyzed
microscopically (Figure 1D). Fluorescence signals for GL,
GF, and GLF were detected in the nucleus and cytoplasmof stomatal guard cells, while those for GFP fused with anuclear localization signal at the N-terminus (NLS-GFP)
were detected only in the nucleus.

sequenced for verification, and then cloned into the BamHI siteof pGBKT7 (Clontech, Palo Alto, CA, USA), which encodes the
GAL4 DNA-binding domain (bait). Fragments encoding theLOV, F-box, and Kelch repeat of LKP2 were cloned individually
into pGADT7 (Clontech), which encodes the GAL4 activation
domain (prey). AH109 yeast cells (Clontech) were used for theassay. Yeast transformants were grown on synthetic complete(SD) medium that lacked leucine (L) and tryptophan (W).
Transformants were assayed on SD medium that lacked
adenine (A), histidine (H), L, and W but was supplemented
with 3-amino-1H-1,2,4-triazole (3AT) at 15 mM to repress thebasal activity of the HIS3 reporter gene. Yeast containing two
plasmids from Clontech, one plasmid encoding amino acids
72–390 of murine p53 protein (GenBank accession K01700)
and the GAL4 DNA-binding domain and the other plasmid
encoding the SV40 large T antigen (GenBank locus SV4CG)
and the GAL4 activation domain, was used as the positive
control. Interaction between GI and the LKP2 domain encodedby the prey vector was indicated by colony growth on the SDAHLW + 3AT medium.

Circadian rhythm phenotypes of transgenic plants

CAB2:LUC and CCR2:LUC reporters were used tomeasure the circadian rhythms of the transgenic plants(Figure 2 and Table S2). Both GL and GLF plants had
shorter circadian rhythms (e.g., GL 1: 21.3 h, GL 2: 22.9 h;
GLF 1: 23.3 h, and GLF 2: 22.7 h for CAB2:LUC) thancontrol plants (e.g., GFP 1: 24.2 h and GFP 2: 24.3 h for
CAB2:LUC) under continuous white light conditions,while the period lengths of GF plants were similar
to those of control plants (e.g., GF 1: 24.5 h and GF 2:
24.5 h for CAB2:LUC). A decrease in amplitude over time
was obvious in GL plants for both reporters and in GLF
plants for the CCR2:LUC reporter.

Results
Construction of GL, GF, and GLF transgenic plants

Three fusion genes that encode the LKP2 LOV domain(GL), the F-box motif (GF), or a combination of theLOV domain and F-box motif (GLF) attached to the
C-terminus of GFP were constructed (Figure 1A) and
expressed in transgenic Arabidopsis plants under thecontrol of the Cauliflower mosaic virus 35S promoter.

Hypocotyl elongation of transgenic plants

Hypocotyl lengths were measured under continuous red(cR), blue (cB), and far-red (cFR) light and in darkness

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(Figure 3 and Table S3). GFP fused to LKP2 (GLFK;Miyazaki et al. 2011) was used as a positive control. The
GL, GF, and GLF seedlings and one GLFK seedling did
not show significant differences in hypocotyl length
in darkness compared to control (GFP) seedlings.
Both GLFK lines showed longer hypocotyls under cR(0.052–1.005 W m−2), cB (0.052–0.225 W m−2) and cFR(0.103 W m−2) than control seedlings; in particular, the
GLFK hypocotyls were longer than those of any otherlines under cR at 0.052 and 0.204 W m−2. Both GLlines showed longer hypocotyls under cR at 0.099 and1.005 W m−2 than did control seedlings. By contrast, thehypocotyl lengths of GF or GLF seedlings (at least one

line per light treatment) were not significantly different
from those of control seedlings under any continuous
light condition examined.
Flowering phenotypes of transgenic plants under
LD conditions

Flowering times were measured under LD conditions(Figure 4). GF plants bolted after 1 month and did notshow significant differences in bolting time comparedwith control plants. The rosette leaf numbers at boltingof GF and control plants were also not significantly
different. By contrast, GL and GLF plants bolted later andhad more rosette leaves at bolting than the control plants.
The expression levels of four flowering-time genesthat function in the photoperiodic pathway (FT, CO,
CDF1, and GI) were examined in the transgenic plants
grown under LD conditions (Figure 5). Both FT and
CO expression patterns in GF and control plants weresimilar, while those in GL and GLF plants were different.
Specifically, the peak of CO expression at Zeitgeber
time (ZT)12 and the peak of FT expression at ZT16,
both of which were observed in GF and control plants,
were absent in GL and GLF plants. By contrast, thegene expression patterns of CDF1 and GI were similar
among the four genotypes. These results suggest thatthe late flowering of GL and GLF plants is due to thedown-regulation of CO at dusk and subsequent downregulation of FT.
Interaction of LKP2 LOV with GI

Interaction of ZTL LOV with GI is postulated as a reason
for the pleiotropic phenotype of ZTL LOV-producing
plants (Kim et al. 2013). Though interaction between
LKP2 and GI has been demonstrated in vitro and inyeast (Kim et al. 2007), the binding site in LKP2 has
not previously been reported. Therefore, two-hybrid

Figure 3. Hypocotyl length of GFP, GL, GF, GLF, and GFP-LKP2(GLFK) transgenic plants under continuous red (cR) light. Seedlings
were grown for 5 days under cR with various fluence rates (W m−2).
Each point represents the mean hypocotyl length of approximately 40
seedlings. Error bars represent standard error of the mean.

Figure 4. Flowering time of GFP, GL, GF, and GLF transgenic plants under LD conditions. Graphs show bolting time (left panel) and rosette leaf
number at bolting (right panel) in transgenic plants under 16-h light/8-h dark conditions. Values are means and standard errors (n=5). Different
letters indicate statistical differences detected by one-way analysis of variance (ANOVA) and Tukey–Kramer test (p<0.05).

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Pleiotropic phenotype of LKP2 LOV-producing Arabidopsis

Figure 5. Expression of flowering-time genes in GFP, GL, GF, and GLF transgenic plants under 16-h light/8-h dark conditions. Relative expression
levels of the flowering-time genes FT, CO, CDF1 and GI were normalized to ACT2 expression. Light and dark periods are indicated by white and blackboxes, respectively. Values are means and standard error (n=3).

interaction of LKP2 LOV with GI was examined in yeast(Figure 6). GI interacted with LKP2 LOV, but not with
the LKP2 F-box or Kelch repeat region.

is postulated to be due to instability of ZTL caused byinteraction between ZTL LOV and GI. In contrast to ZTL
LOV-producing plants, both GL and GLF plants showed
short-period phenotypes under continuous white lightcompared to the control (Figure 2 and Table S2). The
contrasting effects of ZTL LOV and LKP2 LOV in clock
regulation could be due to differences between some of
their interacting proteins and/or due differing affinities
to interacting proteins, even though ZTL LOV and LKP2
LOV interact with overlapping sets of proteins. BothZTL and LKP2 bind to GI (Kim et al. 2007), and bothZTL LOV and LKP2 LOV interact with two core clock
proteins, TOC1 and PRR5 (Baudry et al. 2010; Yasuhara
et al. 2004). The short-period phenotypes in both GL
and GLF plants are phenocopies of gi, toc1, and prr5
mutants (Mizoguchi et al. 2005; Nakamichi et al. 2005;
Somers et al. 1998). Therefore, production of LKP2 LOVDiscussion
A fusion protein consisting of GFP and LKP2 waspreviously shown to be localized in the nucleus andcytoplasm of Arabidopsis cells (Takase et al. 2011).
Consistently, the GL, GF, and GLF proteins were also
localized in the nucleus and cytoplasm (Figure 1D),suggesting that the differences in phenotype among
GL, GF, and GLF plants were not due to the subcellular
localization of these three proteins.
Kim et al. (2013) showed that production of LOV or
LOV-F of ZTL caused circadian rhythm lengthening
under red- or blue light. This long-period phenotype
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might disrupt the function of clock components that caninteract with LKP2 LOV and subsequently result in shortcircadian rhythm.
The elongated hypocotyl of GL plants is a phenocopyof that seen in LKP2 overproducers (Miyazaki et al.
2011). Hypocotyl elongation is enhanced under cR
and cB by ZTL LOV production (Kim et al. 2013). As
for LKP2 LOV, enhancement was obvious under cR
but not under cB (Figure 3 and Table S3), though one
GL line showed a significant difference from control
seedlings under cB (0.102 W m−2 [GL 2] and 0.225 W m−2[GL 1]; Table S3). Therefore, the effect of LKP2 LOV or
ZTL LOV production on hypocotyl length seems to befundamentally similar. The lower level of GLF protein
accumulation in GLF seedlings compared with that of GL
protein in GL seedlings might be a reason for the absenceof obvious enhancement of hypocotyl elongation in GLF
seedlings (Figure 1C).
Under LD conditions, production of LKP2 LOV
delayed flowering and down-regulated CO and FT
expression compared to control plants (Figures 4 and
5). Overproduction of LKP2 or production of LKP2
Kelch also delayed flowering and down-regulated
CO and FT expression (Takase et al. 2011). LKP2 and
LKP2 Kelch are postulated to capture FKF1 in thecytosol, which probably disturbs the ubiquitination andsubsequent degradation of CDFs, which are repressorsof CO and FT expression (Takase et al. 2011). Because
LKP2 LOV cannot capture FKF1 in the cytosol (Takase
et al. 2011), the mechanism for late flowering by LKP2
LOV production seems to be different from that by
LKP2 Kelch. Thus, the late flowering caused by LKP2
overproduction is probably caused not only by captureof FKF1 in the cytosol but also by a LOV-mediatedmechanism.
ZTL LOV facilitates cytosolic retention of GI protein
by interacting with it (Kim et al. 2013). GI binds to FKF1
in the nucleus in a blue-light-dependent manner, and the
GI–FKF1 complex induces expression of CO throughubiquitination of CDF1 (Sawa et al. 2007). Both LKP2and LKP2 LOV can interact with GI (Figure 6; Kim etal. 2007). Given the effects of ZTL LOV production on
cytosolic retention of GI (Kim et al. 2013), it is possiblethat the interaction between LKP2 LOV and GI enhances
cytosolic GI distribution, which interferes with theformation of the GI–FKF1 complex in the nucleus andresults in delayed flowering under LD conditions. Thishypothesis is consistent with down-regulation of CO
expression in both GL and GLF plants (Figure 4). GI
and FKF1/LKP2/ZTL family proteins are postulated toform interrelated complexes that regulate CO stability forphotoperiodic flowering (Song et al. 2014). Instability of
CO could be another reason for late flowering of GL andGLF plants, in addition to the down-regulation of CO.
In this paper, we have shown that production of LKP2

Figure 6. Yeast two-hybrid interactions between GI and three
LKP2 fragments: LOV (LKP2-L), F-box (LKP2-F), and Kelch repeat(LKP2-K). Each panel in the “GI” columns shows yeast AH109 cells
containing a GAL4 DNA-binding domain fusion of GI and either a
GAL4 activation domain fusion of the indicated LKP2 fragment or theempty GAL4 activation domain expression vector, pGADT7 (“Vector”).
The bait vector was a GAL4 DNA-binding domain expression vector,
pGBKT7. Colonies were grown on SD agar medium lacking leucine
and tryptophan (SD-LW; left) or lacking adenine, histidine, leucine,and tryptophan (SD-AHLW; right). The positive control (p53/SV40)
contained one plasmid encoding amino acids 72–390 of murine p53
protein and the GAL4 DNA-binding domain and another plasmid
encoding the SV40 large T antigen and the GAL4 activation domain.

LOV in transgenic Arabidopsis results in short circadianrhythm, elongated hypocotyl, and late flowering.
This result provides additional evidence for the LOVmediated functions of FKF1/LKP2/ZTL family proteinsin plant growth and development.
Acknowledgements
This work was partially supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Scienceand Technology of Japan. We thank Dr. S.A. Kay (University ofCalifornia, San Diego) for providing CAB2:LUC and CCR2:LUC
seeds. We send our special thanks to the previous members of ourformer laboratory at Kagawa University.

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DOI 10.1007/s00299-015-1896-4

ORIGINAL ARTICLE

Enhancement of hypocotyl elongation by LOV KELCH
PROTEIN2 production is mediated by auxin and phytochromeinteracting factors in Arabidopsis thaliana
Yuji Miyazaki1 • Yusuke Jikumaru2 • Tomoyuki Takase1 • Aya Saitoh1 •Asuka Sugitani1 • Yuji Kamiya2 • Tomohiro Kiyosue1

Received: 14 August 2015 / Revised: 12 October 2015 / Accepted: 3 November 2015
! Springer-Verlag Berlin Heidelberg 2015

Abstract
Key message Auxin and two phytochrome-interacting
factors, PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5, play crucial roles in theenhancement of hypocotyl elongation in transgenic
Arabidopsis thaliana plants that overproduce LOVKELCH PROTEIN2 (LKP2).
Abstract LOV KELCH PROTEIN2 (LKP2) is a positive
regulator of hypocotyl elongation under white light inArabidopsis thaliana. In this study, using microarray
analysis, we compared the gene expression profiles of
hypocotyls of wild-type Arabidopsis (Columbia accession),a transgenic line that produces green fluorescent protein(GFP), and two lines that produce GFP-tagged LKP2
(GFP-LKP2). We found that, in GFP-LKP2 hypocotyls,
775 genes were up-regulated, including 36 auxin-responsive genes, such as 27 SMALL AUXIN UP RNA (SAUR)
and 6 AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA)
genes, and 21 genes involved in responses to red or far-red
light, including PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5; and 725 genes were down-

regulated, including 15 flavonoid biosynthesis genes.
Hypocotyls of GFP-LKP2 seedlings, but not cotyledons orroots, contained a higher level of indole-3-acetic acid(IAA) than those of control seedlings. Auxin inhibitors
reduced the enhancement of hypocotyl elongation in GFPLKP2 seedlings by inhibiting the increase in cortical cellnumber and elongation of the epidermal and cortical cells.
The enhancement of hypocotyl elongation was completely
suppressed in progeny of the crosses between GFP-LKP2lines and dominant gain-of-function auxin-resistantmutants (axr2-1 and axr3-1) or loss-of-function mutants
pif4, pif5, and pif4 pif5. Our results suggest that theenhancement of hypocotyl elongation in GFP-LKP2 seedlings is due to the elevated level of IAA and to the upregulated expression of PIF4 and PIF5 in hypocotyls.
Keywords Hypocotyl elongation ! IAA ! LKP2 !
Microarray ! PIF ! SAUR

Introduction
In plants, light regulates many aspects of development andphysiological processes such as germination, de-etiolation,leaf and stem growth, flowering, entrainment of circadianrhythms, stomatal opening, chloroplast movement, andanthocyanin synthesis (Jiao et al. 2007; Kendrick andKronenberg 1994; Mancinelli 1990; Millar 2004). Hypocotyl elongation is used as a model system to elucidate how
light affects organ growth via cell expansion and division(Boron and Vissenberg 2014). In seedlings, an absence oflight induces hypocotyl elongation, whereas blue, red, or
far-red light inhibits it (Lau and Deng 2010). Numerous
genes and proteins, including photoreceptors, induce orinhibit hypocotyl growth (Nozue and Maloof 2006).

Communicated by P. P. Kumar.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00299-015-1896-4) contains supplementarymaterial, which is available to authorized users.
& Tomohiro Kiyosue
tomohiro.kiyosue@gakushuin.ac.jp
1

Department of Life Science, Faculty of Science, Gakushuin
University, 1-5-1 Mejiro, Toshima-Ku, Tokyo 171-8588,
Japan

2

Growth Regulation Research Group, RIKEN Plant Science
Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama,
Kanagawa 230-0045, Japan

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The circadian clock is another factor that affects hypocotyl growth in Arabidopsis immediately upon germination(Dowson-Day and Millar 1999). Under constant dim light,
hypocotyl elongation is fastest at subjective dusk, and stops
close to subjective dawn (Dowson-Day and Millar 1999).
Studies using clock mutants revealed the importance of thecircadian clock in hypocotyl growth; there is a strong
correlation between circadian and hypocotyl mutant phenotypes. Arrhythmic mutants tend to lack the daily arrest in
hypocotyl growth in continuous light, whereas shorter- or
longer-period mutants tend to have alterations in either theduration or the amplitude of the growth phase (Nozue andMaloof 2006). Since FKF1/LKP2/ZTL family proteins are
involved in circadian clock regulation, they may contributeto hypocotyl growth regulation via their circadian clockfunction.
To clarify the molecular mechanisms of the regulationof hypocotyl elongation in Arabidopsis by FKF1/LKP2/
ZTL family proteins, we analyzed hypocotyl elongation
caused by LKP2 production. Production of green fluorescent protein (GFP)-tagged LKP2 (GFP-LKP2) caused
hypocotyl elongation; hypocotyls had elongated epidermaland cortical cells and an increased number of cortical cells
in comparison with controls (Miyazaki et al. 2011). The
cells in GFP-LKP2 hypocotyls had larger nuclei withincreased DNA content (Miyazaki et al. 2011). Productionof the LOV domain of LKP2, which interacts with
GIGANTEA (GI), enhances hypocotyl elongation undercontinuous red light and delays flowering under long-day
conditions, suggesting the involvement of GI in LKP2mediated hypocotyl elongation (Takase et al. 2015).
However, the molecular mechanisms of the enhancementof hypocotyl elongation caused by LKP2 are largely
unknown.
In this study, we compared the gene expression profilesof hypocotyls of wild-type Arabidopsis Columbia accession (Col), a transgenic line that produces GFP, and two
GFP-LKP2 lines (Miyazaki et al. 2011). Using microarray
analysis, we found that 775 genes were up-regulated,
including 36 auxin-responsive genes, such as 27 SMALL
AUXIN UP RNA (SAUR) and 6 AUXIN/INDOLE-3ACETIC ACID (AUX/IAA) genes, and 21 genes involved in
responses to red or far-red light, including PIF4 and PIF5;
and 725 genes were down-regulated, including 15 flavonoid biosynthesis genes. Next, we measured indole-3acetic acid (IAA) levels in these seedlings and examinedthe effects of three auxin inhibitors—the polar auxin
transport inhibitor N-1-naphthylphthalamic acid (NPA), theputative anti-auxin p-chlorophenoxyisobutyric acid
(PCIB), and the auxin antagonist a-(phenylethyl-2-one)indole-3-acetic acid (PEO-IAA)—on hypocotyl elongation.
Finally, we examined the effects of two dominant gain-offunction auxin-resistant mutations (axr2-1 and axr3-1) and

The direct interaction of the red/far-red light receptors,
phytochromes, with transcription factors (TFs) in the
nucleus is an important step in phytochrome-dependent
regulation of hypocotyl elongation (Bauer et al. 2004;
Nozue and Maloof 2006). Red light activates phytochrome
B (phyB) and induces its translocation from the cytoplasm
to the nucleus, where it regulates gene transcription via
interaction with several TFs, including phytochrome-interacting factors (PIFs) and PIF-like proteins (PILs) (Baeand Choi 2008; Castillon et al. 2007). Light-activated phyB
induces proteasome-mediated degradation of two homologous basic helix–loop–helix TFs, PHYTOCHROMEINTERACTING FACTOR4 (PIF4) and PIF5, which
redundantly promote hypocotyl elongation; the degradationof these proteins results in the short hypocotyl under red or
white light (Duek and Fankhauser 2005; Nozue and Maloof
2006; Vandenbussche et al. 2005). Phytochromes also
regulate hypocotyl elongation by controlling the proteasomal degradation of photomorphogenesis-promoting TFs;
this degradation is mediated by the CONSTITUTIVEPHOTOMORPHOGENIC1 (COP1)-SUPPRESSOR OF
PHYA-105 (SPA) E3 ubiquitin ligase complex, and also bycontrolling the alternative splicing of transcripts for certain
regulatory genes (Lau and Deng 2012; Shikata et al. 2014).
The FKF1/LKP2/ZTL protein family is a group of blue
light receptors, which consists of FLAVIN-BINDING
KELCH REPEAT F-BOX 1 (FKF1), LOV KELCH PROTEIN1 (LKP1)/ZEITLUPE (ZTL), and LOV KELCH
PROTEIN2 (LKP2). Proteins of this family are highly
similar to each other and possess three functional domains,i.e., the light, oxygen, or voltage (LOV) domain, F-box
motif, and Kelch repeat region (Zoltowski and Imaizumi2014). They function in polyubiquitination of target proteinsas the components of the SKP1-Cullin-Rbx1-F-box protein(SCF) E3 ligase complex, and their E3 ligase activities are
regulated by blue light perceived by the LOV domains(Demarsy and Fankhauser 2009). The FKF1/LKP2/ZTL
family proteins regulate circadian expression of genes andphotoperiodic flowering, as well as hypocotyl elongation
under light (Baudry et al. 2010; Zoltowski and Imaizumi2014). Hypocotyls of fkf1 mutants are short under continuous blue or red light (Nelson et al. 2000). Both LKP2overexpressing seedlings and ZTL-overexpressing seedlings
produce long hypocotyls under continuous blue, red, or
white light (Kiyosue and Wada 2000; Nelson et al. 2000;
Schultz et al. 2001). Hypocotyls of ztl mutants are short
under continuous red light but are indistinguishable fromthose of wild-type plants under blue light (Somers et al.
2000). These results suggest that proteins of this family do
not function as blue light receptors in the context of the red
light-induced inhibition of hypocotyl growth. ZTL interacts
with phyB apoprotein; therefore, FKF1/LKP2/ZTL may
affect phytochrome-mediated signaling (Jarillo et al. 2001).

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image files. Microarray data are accessible through GEO
series accession number GSE37197 (http://www.ncbi.nlm.
nih.gov/geo/query/acc.cgi?acc=GSE37197). Putative candidate genes with a fold change of [2 or \-2 with P \ 0.01
were used for pathway analysis in DAVID 6.7 (http://david.
abcc.ncifcrf.gov/home.jsp) (Huang et al. 2009a, b). Geneannotations were retrieved from TAIR (http://www.arabi
dopsis.org/).

three loss-of-function mutations (pif4, pif5, and pif4 pif5)
on hypocotyl elongation.

Materials and methods
Plant materials and growth conditions
All plant materials had the Arabidopsis thaliana Columbia(Col) background. Transgenic plants 35S:GFP-LKP2(GFP-LKP2) and 35S:GFP (GFP) were described previously (Miyazaki et al. 2011). Seeds were surface-sterilized
and placed on plates with GM medium (Valvekens et al.
1988) supplemented with 0.8 % agar. Plates were kept at
4 "C for 3–7 days in the dark and then transferred tocontinuous white light at 22 "C (80 lmol m-2 s-1). Seedsof axr2-1 and axr3-1 mutants were obtained from the
ABRC stock center, and those of pif4-101 and pif5 (pil6-1)
came from Dr. J. N. Maloof at the University of California,
Davis (Nozue et al. 2011). The axr mutants were crossedwith the GFP- or GFP-LKP2 plants to produce F1 plants.
Plants homozygous for GFP or GFP-LKP2 and carrying pif
mutations were generated by crossing and self-pollination.

Semi-quantitative RT-PCR analysis
RNA for RT-PCR was isolated independently from thatused for microarray analysis. Total RNA (1 lg) fromhypocotyls of 5-day-old seedlings was primed with anoligo(dT) primer and reverse-transcribed using a HighCapacity cDNA Reverse Transcription Kit (Applied
Biosystems, Foster City, CA, USA). PCR amplification
was performed with AmpliTaq Gold (Applied Biosystems)
and gene-specific primer sets are listed in Table S1. PCRs
were carried out in a thermal cycler (GeneAmp PCR
System 2700, Applied Biosystems) with denaturation at
94 "C for 5 min, followed by a variable number of cycles(Table S1) at 94 "C for 30 s, 50 "C for 20 s, and 72 "C for
1 min. PCR products were separated on a 2.0 % (w/v)
agarose gel, stained with ethidium bromide, and visualized
under UV light. Gel images were captured with a Printgraph video capture device (AE-6911FX; Atto, Tokyo,
Japan). Each PCR fragment was sequenced directly toconfirm its identity.

Microarray analysis
Hypocotyls of 5-day-old GFP-LKP2 (two lines), GFP (oneline), and wild-type (Col) seedlings were used for analysis.
RNA was prepared using the RNeasy Plant Mini Kit (Qiagen,
Valencia, CA, USA). The microarray experiment was conducted by Miltenyi Biotec K.K. (Tokyo, Japan). RNA samples(100 ng) were used for the linear T7-based amplification step.
To produce Cy3-labeled cRNA, RNA was amplified andlabeled using the Agilent Low Input Quick Amp Labeling Kit(Agilent Technologies, Böblingen, Germany) following the
manufacturer’s protocol. The yields of cRNA and the dye
incorporation rate were measured using a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE,
USA). Hybridization was performed according to the Agilent
60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Cy3-labeled fragmented cRNA (1.65 lg) inhybridization buffer was hybridized overnight (17 h, 65 "C)with Agilent Whole Arabidopsis Genome Oligo Microarrays
4 9 44 K V4 (G2519F) in the hybridization chamber andoven recommended by Agilent. The microarray slides were
washed with Agilent Gene Expression Wash Buffer 1(1 9 1 min at room temperature), then with preheated Agilent Gene Expression Wash Buffer 2 (1 9 1 min at 37 "C),and finally with acetonitrile. Fluorescence signals weredetected using an Agilent Microarray Scanner System (Agilent Technologies), and the Agilent Feature Extraction Software (FES) was used to read out and process the microarray

IAA measurements
Cotyledons, hypocotyls, and roots from 80 to 100 five-dayold seedlings were cut with a surgical knife, weighed, andfrozen in liquid N2. IAA was extracted, purified, andquantified using a liquid chromatography–electrosprayionization tandem mass spectrometry (LC–ESI–MS/MS)
system as described previously (Yoshimoto et al. 2009).
Six or 7 biological replicates were performed.
Measurement of hypocotyl length
Images of the hypocotyls of 5-day-old seedlings grown on
agar plates (plates were kept vertically for auxin inhibitor
experiments and horizontally for other experiments) weretaken on a BX51 microscope (Olympus, Tokyo, Japan),and the cell length and cell number were determined withScion Image software (Scion Corp., Frederick, MD, USA).
In experiments with auxin inhibitors, agar contained 1 %DMSO (v/v, final concentration; mock), NPA (1 or 5 lM;
Wako Pure Chemical Industries, Ltd., Osaka, Japan), PCIB
(50 or 100 lM; MP Biomedicals, Santa Ana, CA, USA), or
PEO-IAA (50 or 100 lM; provided by Dr. K. Hayashi,

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Okayama University of Science). The top, middle, andbottom parts of hypocotyls were defined as reported previously (Miyazaki et al. 2011). In the case of embryonichypocotyls, seeds were soaked in water for 6 h, theembryos were surgically excised and stained with 0.1 %(w/v) Toluidine blue (Wako Pure Chemical Industries,Ltd.). Images were taken on a BX51 microscope, andepidermal cells on a 250-lm line along the long axis of the
hypocotyl at its thickest part were counted. Five independent embryos were used per line.

Results
Hypocotyl growth kinetics
GFP-LKP2 seedlings have elongated hypocotyls withlonger epidermal cells in comparison with controls(Miyazaki et al. 2011; Yasuhara et al. 2004). This difference in hypocotyl cell length appeared only after germination and was not observed in embryos (Figs. S1A, B). To
examine the growth kinetics of hypocotyls of GFP-LKP2
seedlings, we monitored the germination and growth of two
GFP-LKP2 lines and two controls (Col and GFP) for aweek (Fig. S2). All seeds germinated 2 days after incubation (DAI) under light. GFP-LKP2 hypocotyls became
longer than controls at 3 DAI and the difference in the
hypocotyl length was almost maximum at 5 DAI. At thattime, all seedlings had two expanded green cotyledons anda small true leaf, as reported previously (Miyazaki et al.
2011), suggesting that 5-day-old seedlings of GFP-LKP2
and controls were at the same growth stage, even though
their hypocotyl length was different. After 5 DAI, allhypocotyls still continued to grow for two more days
without considerable elongation.
Gene expression profile of hypocotyls of GFP-LKP2
seedlings
We performed microarray analysis to compare the gene
expression profiles of hypocotyls of two GFP-LKP2 lines
and controls. In GFP-LKP2, a total of 775 genes were upregulated (average fold change [2.0; P \ 0.01; Table S2)
and 725 genes were down-regulated (average fold change\-2.0; P \ 0.01; Table S3); the functional groups of the
up-regulated genes are shown in Table S4 and those ofdown-regulated genes in Table S5. The fact that LKP2
(AT2G18915; average fold change 68.864) was the most
up-regulated gene validated this analysis. The up- or downregulation of the following clock genes was observed in
GFP-LKP2 hypocotyls: PSEUDO RESPONSE REGULATOR7 (PRR7) (AT5G02810; 8.189), GIGANTEA(AT1G22770; 4.343 and 3.831 by two probes), PRR5

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(AT5G24470; 2.081), and EARLY FLOWERING4 (ELF4)(AT2G40080; -3.055 and -2.895). These observations are
consistent with the involvement of LKP2 in circadian clock
regulation. In the GFP-LKP2 hypocotyls, genes involved in
responses to hormone stimuli, responses to abiotic stimuli,
responses to blue light, external encapsulating structureorganization, regulation of transcription, and red or far-red
light signaling pathways were up-regulated, whereas those
involved in secondary metabolism, responses to organic
substances, flavonoid biosynthesis, and post-embryonicdevelopment were down-regulated. Among the up-regulated genes, 36 genes involved in the response to auxin had
the smallest P value (9.28E-15; Table S4). This group
included 27 SMALL AUXIN UP RNA (SAUR) (SAUR1, 7,
9, 12, 15, 16, 19, 20, 22–29, 50, 52, 61–66, 68, 73, and 75)
genes, 6 AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA)(IAA1, 2, 6, 14, 19, and 29) genes, a gene for putative GH3
indole-3-acetic acid-amido synthetase (DWARF IN
LIGHT2: DFL2), 1-aminocyclopropane-1-carboxylic acidsynthase 4 (ACS4), and AUXIN-REGULATED GENE
INVOLVED IN ORGAN SIZE (ARGOS). Based on thisgrouping and the result from the key word search for IAA,
SAUR, GH3, and auxin, we constructed Table 1A. Twentyone genes involved in responses to red or far-red light had
the second smallest P value (2.44E-10; Table S4). This
group included PIF4, PIF5, PRR5, PRR7, and PRR9
(Table 1B). The increased expression of auxin-inducible
genes suggested the elevation of IAA levels or sensitivity
to IAA in hypocotyl cells of GFP-LKP2 seedlings.
We also observed up-regulation of genes involved ingibberellin biosynthesis and brassinosteroid signaling:gibberellin 20-oxidase (GA5) (AT4G25420; average foldchange of 6.883), BRASSINOSTEROID INSENSITIVE1(BRI1) (AT4G39400; 2.999, 2.393, and 2.268 by threeprobes), and BR ENHANCED EXPRESSION1 (BEE1)(AT1G18400; 2.008). The expression of the genes involvedin cell wall loosening and expansion was also increased
in GFP-LKP2 hypocotyls: EXPANSIN3 (EXPA3)(AT2G37640; average fold change 6.750 and 2.015 by twoprobes), EXPANSIN11 (EXPA11) (AT1G20190; 5.021),
XYLOGLUCAN:XYLOGLUCOSYL
TRANSFERASE33(XTH33) (AT1G10550; 4.688), XTH8 (AT1G11545;
3.662), EXPA8 (AT2G40610; 4.333, 3.533, and 3.118),EXPANSIN-LIKE B1 (EXLB1) (AT4G17030; 2.568 and
2.179), EXPA5 (AT3G29030; 2.558, 2.423 and 2.203),
EXLA1 (AT3G45970; 2.422), EXPA12 (AT3G15370;
2.312), EXLA3 (AT3G45960; 2.101), and EXPA1(AT1G69530; 2.108) (Table S2).
Semi-quantitative RT-PCR confirmed some of the
microarray results, i.e., the expression of three auxin-inducible genes (IAA6, SAUR25, and ACS4), two PIF genes(PIF4 and PIF5), and one gene for a cell wall-modifying
enzyme (EXPA11) was higher in the hypocotyls of 5-day-

Plant Cell Rep
Table 1 Genes up-regulated in GFP-LKP2 hypocotyls under white light in comparison with those of controls (Col and GFP)
Array element

Gene locus

Gene name

Fold change

P value(A) Up-regulated genes involved in the response to auxin stimulus in GFP-LKP2 hypocotyls
A_84_P10124

AT4G14560

IAA1

8.516

4.98.E-07

A_84_P734158

AT3G23030

IAA2

2.031

1.50.E-04

A_84_P13379

AT1G52830

IAA6

6.303

2.56.E-14

A_84_P826648

AT4G14550

IAA14

2.395

4.10.E-17

2.079

5.71.E-13

A_84_P23363
A_84_P14556

AT3G15540

IAA19

2.805

4.19.E-14

A_84_P55550

AT4G32280

IAA29

2.349

8.00.E-05

A_84_P21449

AT4G34770

SAUR1

2.814

2.46.E-17

A_84_P21078

AT2G21200

SAUR7

4.076

1.00.E-25

A_84_P797355
A_84_P12944

AT4G36110

SAUR9

3.579
3.342

3.02.E-20
1.74.E-07

A_84_P23871

AT2G21220

SAUR12

3.509

6.20.E-12

A_84_P16734

AT4G38850

SAUR156.861

0.00.E?00

A_84_P17680

AT4G38860

SAUR16

5.534

1.05.E-27

A_84_P196504

AT5G18010

SAUR197.093

9.52.E-23

A_84_P147028

AT5G18020

SAUR205.068

3.04.E-31

A_84_P152798

AT5G18030

SAUR21

5.592

2.89.E-29

AT5G18080

SAUR24

A_84_P141269

AT5G18050

SAUR22

5.563

4.83.E-35

A_84_P94979

AT5G18060

SAUR235.332

1.00.E-26

A_84_P272980

AT5G18080

SAUR244.895

1.59.E-27

A_84_P21376

AT4G13790

SAUR259.767

4.62.E-12

8.781

3.61.E-20

A_84_P792091
A_84_P554482

AT3G03850

SAUR263.445

4.11.E-18

A_84_P18297

AT3G03840

SAUR27

2.747

2.47.E-18

A_84_P20189
A_84_P21135

AT3G03830
AT3G03820

SAUR28
SAUR29

3.083
2.672

7.35.E-21
4.62.E-19

A_84_P20503

AT4G34760

SAUR502.956

8.71.E-11

A_84_P832877

AT1G75590

SAUR52

3.091

1.69.E-18

2.905

3.00.E-05

A_84_P537376

AT1G29420

SAUR615.674

0.00.E?00

A_84_P10257

AT1G29430

SAUR62

3.989

3.05.E-21

A_84_P279980

AT1G29440

SAUR633.485

1.12.E-35

A_84_P846978

AT1G29450

SAUR644.002

5.37.E-13

3.425

1.96.E-25

A_84_P15930

AT1G29460

SAUR654.298

4.30.E-27

A_84_P11207

AT1G29500

SAUR663.055

2.00.E-25

A_84_P843096

AT1G29490

SAUR6810.956

6.25.E-11

A_84_P169803

AT3G03847

SAUR73

3.541

6.06.E-12

A_84_P563710

AT5G27780

SAUR75

3.772

3.63.E-18

A_84_P826972
A_84_P20399

AT2G47750
AT4G03400

GH3.9

3.087

1.20.E-04

DFL2

2.954

6.03.E-18

A_84_P17068

A_84_P19713

A_84_P17344

AT2G22810

ACS413.5869.85.E-12

A_84_P524502

AT3G59900

ARGOS

3.297

7.33.E-29

A_84_P179084

AT5G35735

Auxin-responsive family protein

2.124

3.33.E-07

A_84_P751876

AT1G11803

Pseudogene, auxin responsive protein

4.810

2.96.E-28

A_84_P769426

AT5G27771

Pseudogene of auxin-responsive family protein

3.311

6.89.E-07

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Table 1 continued
Array element

Gene locus

Gene name

Fold change

P value(B) Up-regulated genes involved in the response to red or far-red light in GFP-LKP2 hypocotyls
A_84_P21376

AT4G13790

SAUR259.767

4.62.E-12

A_84_P162983

AT2G43010

PIF4

9.301

1.24.E-12

A_84_P23852

AT2G42540

COR15A

8.899

5.47.E-08

A_84_P13379

AT1G52830

IAA6

6.303

2.56.E-14

A_84_P21494

AT5G02810

PRR7

5.330

4.93.E-15

A_84_P126821

AT2G46790

PRR9

5.101

4.90.E-04

A_84_P240805

AT2G05100

LHCB2.1

4.454

1.36.E-06

A_84_P14690

AT3G59060

PIF5

4.170

2.25.E-14

A_84_P18969

AT1G22770

GI

3.831

1.86.E-10

A_84_P521683

AT2G46970

PIL1

3.298

2.82.E-16

A_84_P14733

AT1G06040

STO

3.197

1.29.E-22

A_84_P19825

AT5G04190

PKS4

3.074

1.08.E-10

A_84_P17015

AT1G02340

HFR1

2.528

3.01.E-08

A_84_P21407

AT4G25260

Plant invertase/pectin methylesterase inhibitor superfamily protein

2.482

4.51.E-16

A_84_P23021

AT2G29090

CYP707A2

2.438

2.61.E-08

A_84_P55550

AT4G32280

IAA29

2.349

8.00.E-05

A_84_P137439

AT3G58850

PAR2

2.296

6.05.E-20

A_84_P13983
A_84_P10576

AT5G24120
AT1G14290

SIG5
SBH2

2.294
2.120

1.19.E-07
1.42.E-16

A_84_P16821

AT5G24470

PRR5

2.081

8.52.E-10

A_84_P10694

AT2G37640

EXPA3

2.015

3.70.E-04

Genes involved in (A) the response to auxin or (B) red or far-red light signaling were identified by the Gene Functional Classification Tool within
DAVID Bioinformatics Resources 6.7

hypocotyl growth in GFP-LKP2 seedlings, but the difference between GFP-LKP2 and control seedlings was still
significant (Fig. S4). By contrast, NPA (1 or 5 lM) or
higher concentrations of PCIB or PEO-IAA (100 lM each)strongly inhibited the enhancement of GFP-LKP2 hypocotyl elongation, and there was no significant difference
between the control(s) and at least one GFP-LKP2 line
(Figs. 3, S4).
Previously, we reported that enhanced hypocotyl elongation in GFP-LKP2 seedlings is caused by the expansionof cortical and epidermal cells, and the increased numberof cortical cells (Miyazaki et al. 2011). Therefore, we
examined the effects of auxin inhibitors on these parameters. Epidermal cell numbers in GFP-LKP2 hypocotyls
were not significantly different from those in controls
either in the absence (in line with our previous report;
Miyazaki et al. 2011) or in the presence of 5 lM NPA,
100 lM PCIB, or 100 lM PEO-IAA (Fig. 4a). By contrast,
cortical cell numbers in GFP-LKP2 hypocotyls were1.7–1.9 times those in controls, and this difference wasstrongly reduced or abolished by 5 lM NPA, 100 lM
PCIB, or 100 lM PEO-IAA (Fig. 4b). The epidermal andcortical cells were significantly longer in GFP-LKP2

old GFP-LKP2-1 and GFP-LKP2-2 seedlings than in control hypocotyls (Fig. 1).
IAA level is increased in GFP-LKP2 hypocotyls
In cotyledons or roots, the IAA levels were not significantly different between GFP-LKP2 and control seedlings(Fig. 2). In hypocotyls, the IAA levels were higher in GFPLKP2 seedlings than in control seedlings (Fig. 2), whichsuggested that the enhancement of hypocotyl elongation in
GFP-LKP2 seedlings is due to the elevated IAA levels intheir hypocotyls.
Hypocotyl elongation in GFP-LKP2 seedlings isinhibited by exogenous auxin inhibitors
To evaluate the contribution of IAA to hypocotyl elongation in GFP-LKP2 seedlings, we used NPA (a polar auxin
transport inhibitor; Bernasconi et al. 1996), PCIB (a putative anti-auxin; Oono et al. 2003), and PEO-IAA (an auxin
antagonist that binds to TIR1/AFBs auxin receptors;
Hayashi et al. 2012) (Fig. S3). Exogenous application of
PCIB (25 lM) or PEO-IAA (50 lM) partially inhibited

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axr2-1 and axr3-1 genes are dominant, we crossed theseaxr mutants with GFP-LKP2 or control plants and assessed
the hypocotyl length of F1 seedlings that had one copy ofaxr2-1 or axr3-1 and 35S:GFP-LKP2 (Fig. 6). The
enhancement of hypocotyl elongation in GFP-LKP2 seedlings was completely suppressed in the F1 seedlings. These
results support the involvement of IAA in the enhancementof hypocotyl elongation in GFP-LKP2 seedlings.
Hypocotyl elongation is suppressed in progenyof the cross between GFP-LKP2 lines and loss-offunction pif mutants
PIF4 and PIF5 redundantly promote hypocotyl elongation
by directly activating the expression of genes that are
involved in cell wall loosening and expansion, and pif4,
pif5, and pif4 pif5 mutants show short hypocotyls whengrown under light (Hornitschek et al. 2012; de Lucas andPrat 2014). To assess the contribution of PIF4 and PIF5 tohypocotyl elongation in GFP-LKP2 seedlings, we measured the hypocotyl length in progeny derived from crosses
between GFP-LKP2 seedlings and pif4, pif5, or pif4 pif5
mutants. The enhancement of hypocotyl elongation was
suppressed in all three groups, most strongly in the progeny
of GFP-LKP2 and pif4 pif5 mutant (Fig. 6). Therefore,
PIF4 and PIF5 are critical for the enhancement of hypocotyl elongation in GFP-LKP2 seedlings.

Fig. 1 Semi-quantitative RT-PCR analysis of IAA6, SAUR25, ACS4,
PIF4, PIF5, and EXPA11. Hypocotyls of 5-day-old control (Col and
GFP) and GFP-LKP2 seedlings (lines GFP-LKP2-1 and GFP-LKP22) were used. RT-PCR products were separated on a 2.0 % (w/v)
agarose gel and stained with ethidium bromide. ACTIN2 (ACT2) wasused as a control

hypocotyls than in controls in the bottom, middle, and top
hypocotyl areas, whereas the application of 5 lM NPA,
100 lM PCIB, or 100 lM PEO-IAA diminished thesedifferences (Fig. 5). These results support the idea thathigh levels of IAA promote hypocotyl elongation in GFPLKP2 seedlings.

Discussion
Hypocotyl elongation is suppressed in progenyof the cross between GFP-LKP2 lines and dominant
gain-of-function auxin-resistant mutants

In this study, we found that the early or primary auxininducible genes from the AUX/IAA, GH3, and SAUR
families (Hagen and Guilfoyle 2002; Woodward and Bartel2005) were up-regulated in GFP-LKP2 hypocotyls(Table 1A). Proteins of both AUX/IAAs and GH3 families
attenuate auxin responses (Staswick et al. 2005; Tiwari
et al. 2001). The expression of SAURs is usually associatedDominant gain-of-function mutants axr2-1 and axr3-1
show agravitropic root and shoot growth, short hypocotyls
and stems, and auxin-resistant root growth (Nagpal et al.
2000). Because the 35S:GFP-LKP2 transgene, and theFig. 2 IAA contents in
cotyledons, hypocotyls, androots of 5-day-old control (Coland GFP) and GFP-LKP2
seedlings (lines GFP-LKP2-1
and GFP-LKP2-2) quantified
using LC–ESI-MS/MS. Error
bars represent standard error of
the mean (n = 6 or 7 biologicalreplicates); different letters
indicate statistically significant
differences (P \ 0.05, Tukey’s
test)

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Fig. 3 Inhibition of hypocotyl elongation by auxin inhibitors.
Hypocotyl length of 5-day-old control (Col and GFP) and GFPLKP2 seedlings (lines GFP-LKP2-1 and GFP-LKP2-2) was measured
in the absence or presence of the indicated concentrations of N-1naphthylphthalamic acid (NPA), p-chlorophenoxyisobutyric acid

(PCIB), or a-(phenylethyl-2-one)-indole-3-acetic acid (PEO-IAA).
Error bars represent standard error of the mean (n = 7–15); different
letters indicate statistically significant differences (P \ 0.05, Tukey’s
test)

Fig. 4 Inhibition of the increase in hypocotyl cell number by auxin
inhibitors. Total numbers of (a) epidermal and (b) cortical cells in
5-day-old control (Col and GFP) and GFP-LKP2 seedlings (lines
GFP-LKP2-1 and GFP-LKP2-2) in the absence or presence of theindicated concentrations of N-1-naphthylphthalamic acid (NPA), p-

chlorophenoxyisobutyric acid (PCIB), or a-(phenylethyl-2-one)-indole-3-acetic acid (PEO-IAA). Error bars represent standard error of
the mean (n = 10); different letters indicate statistically significant
differences (P \ 0.05, Tukey’s test)with tissue elongation (Franklin et al. 2011; Roig-Villanova et al. 2007). Among SAURs, members of two subfamilies, SAUR19–24 and SAUR61–68, are considered to

be positive effectors of cell expansion (Chae et al. 2012;
Spartz et al. 2012, 2014). The sequences of SAUR19–24 arehighly similar to each other, and these six genes form a

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Fig. 5 Inhibition of hypocotylcell elongation by auxin
inhibitors. a Epidermal andb cortical cell length in the top,
middle, and bottom parts of the
hypocotyls of 5-day-old control(Col and GFP) and GFP-LKP2
seedlings (lines GFP-LKP2-1
and GFP-LKP2-2) in theabsence or presence of indicatedconcentrations of N-1naphthylphthalamic acid (NPA),
p-chlorophenoxyisobutyric acid
(PCIB), or a-(phenylethyl-2one)-indole-3-acetic acid (PEOIAA). Error bars represent
standard error of the mean(n = 20); different letters
indicate statistically significant
differences (P \ 0.05, Tukey’s
test)

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Fig. 6 Suppression of enhanced hypocotyl elongation in progeny of
the crosses between GFP-LKP2 lines and dominant gain-of-function
auxin-resistant mutants (axr2-1 and axr3-1) or loss-of-function
mutants (pif4, pif5, and pif4 pif5). The following seedlings wereused: GFP, GFP-LKP2 (lines GFP-LKP2-1 and GFP-LKP2-2), F1
progeny of the cross between GFP or GFP-LKP2 and axr2 or axr3,

and GFP and GFP-LKP2 carrying the indicated pif mutations.
Hypocotyl length is expressed relative to that of GFP seedlings. All
seedlings were 5 days old. Error bars represent standard error of themean (n = 5–29); different letters indicate statistically significant
differences (P \ 0.05, Tukey’s test)

cluster on chromosome 5, which suggests their functional
redundancy (Franklin et al. 2011; Hagen and Guilfoyle
2002). Plants overexpressing SAUR19 have longer hypocotyls than wild-type plants under continuous light, andplants producing GFP-SAUR19, 21, 23, or 24 fusion proteins have elongated hypocotyls under long-day conditions(Spartz et al. 2012). SAUR19 stimulates plasma membrane
H?-ATPase by inhibiting the activity of type 2C protein
phosphatases, which leads to cell expansion via an acidgrowth mechanism (Spartz et al. 2014). Therefore, auxinmediated induction of SAUR expression is a possible
mechanism for the enhancement of hypocotyl elongation in
GFP-LKP2 seedlings.
Gibberellins stimulate cell elongation in Arabidopsis
hypocotyls (Cowling and Harberd 2007), and brassinosteroids enhance light-grown hypocotyl elongation independent of, but co-operatively with, gibberellins andauxin(s) (Tanaka et al. 2003). Although genes for gibberellin biosynthesis and brassinosteroid signaling were
up-regulated in GFP-LKP2 hypocotyls, further studies areneeded to ascertain the contribution of these hormones tothe effect of LKP2 on hypocotyl elongation.
Expression of PIF4 and PIF5 was up-regulated in GFPLKP2 seedlings (Tables 1B, S2). PIF4 and PIF5 regulate
auxin sensitivity to control hypocotyl growth (Chapman
et al. 2012; Nozue et al. 2011). Their expression is regulatedby the circadian clock (Arabidopsis eFP Browser: http://bar.
utoronto.ca/efp/cgi-bin/efpWeb.cgi). Therefore, the up-regulation of PIF4 and PIF5 may be due to circadian clock
defects in GFP-LKP2 plants (Miyazaki et al. 2011). PIF4
and PIF5 accumulation is regulated by light (Nozue et al.
2007). Although PIF4 and PIF5 are stable in the dark but are
degraded under light, our data indicate their importance inhypocotyl elongation in GFP-LKP2 seedlings grown undercontinuous light. This is consistent with the up-regulation of
PIF4 target genes LONG HYPOCOTYL IN FAR-RED(HFR1) and IAA29 (Koini et al. 2009; Lorrain et al. 2008) in
GFP-LKP2 hypocotyls (Table 1).
The circadian clock regulates the expression and formation of the evening complex (EC), which consists of
ELF3, ELF4, and LUX ARRHYTHMO (LUX)/PHYTOCLOCK1 (PCL1) (Nusinow et al. 2011). The EC binds to
the PIF4 and PIF5 promoters to repress PIF4 and PIF5
transcription (Nusinow et al. 2011). ELF4 was down-regulated in GFP-LKP2 hypocotyls (Table S3). Thus, the upregulation of PIF4 and PIF5 may be due to the downregulation of ELF4 (fold change of *-3) in GFP-LKP2
hypocotyls.
TOC1 binds to the promoters and represses the expression of CCA1, LHY, PRR9, PRR7, TOC1, GI, LUX, andELF4 (Huang et al. 2012). PRR5 also binds to the promoters and represses the expression of CCA1, LHY, and
PRR5 (Nakamichi et al. 2010). LKP2 and ZTL bind to
TOC1 and PRR5 and are involved in ubiquitination andsubsequent 26S proteasome-dependent degradation of
TOC1 and PRR5 (Kiba et al. 2007; Más et al. 2003;
Yasuhara et al. 2004). Thus, the down-regulation of TOC1
and PRR5 proteins by LKP2 overproduction may explain
the up-regulation of PRR7 (*8-fold), GI (*4-fold), and
PRR5 (*2-fold) expression in GFP-LKP2 hypocotyls.
However, degradation of TOC1 and PRR5 proteins by
LKP2 does not provide a persuasive explanation for PIF4
and PIF5 up-regulation in GFP-LKP2 hypocotyls, because,
unlike the expression of PRR5, PRR7, and GI, that of ELF4
is down-regulated.

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Author contribution statement Conceived and designed
the experiments: YM, YJ, YK, TK. Performed the experiments: YM, YJ, AS, AS. Analyzed the data: YM, YJ, TT.
Contributed reagents, materials, and analysis tools: YM,
YJ, AS, AS. Wrote the paper: YM, TK.

LKP2 shows high amino acid sequence similarity to
ZTL (75 % identity), and the tissue-specific expression
pattern of LKP2 largely overlaps with that of ZTL(Kiyosue and Wada 2000; Yasuhara et al. 2004). LKP2overexpressing transgenic plants phenocopy ZTL-overexpressing transgenic plants: both exhibit circadian rhythm
defects under continuous light or in the dark, elongatedhypocotyls under continuous white or red light, anddelayed flowering under long-day conditions comparedwith control plants (Nelson et al. 2000; Schultz et al.
2001). Transfer DNA (T-DNA) insertion mutants of
LKP2 are phenotypically similar to wild-type plants(Baudry et al. 2010; Takase et al. 2011), whereas T-DNA
insertion mutants of ZTL show slower circadian rhythms
under continuous red or blue light or in the dark, shorter
hypocotyls under continuous red light, and earlier flowering under non-inductive photoperiod conditions thancontrol plants (Somers et al. 2004; Takase et al. 2011).
The phenotypic similarity of lkp2 plants to wild-type
plants is considered to be due to the very low expressionof LKP2 in wild-type plants relative to that of ZTL(Baudry et al. 2010; Michael et al. 2008; Mockler et al.
2007). Introduction of LKP2 cDNA controlled by the ZTL
promoter complemented the long-period phenotype of aztl mutant (Baudry et al. 2010). These results suggest that
LKP2 has ZTL-like functions with respect to the regulation of the circadian clock, hypocotyl elongation, andflowering time in Arabidopsis. Therefore, it is possiblethat ZTL also regulates hypocotyl elongation via auxin
and PIF4, PIF5, or both. Indeed, transgenic Arabidopsis
seedlings that overexpress ZTL accumulate higher levels
of mRNAs for auxin-inducible genes and PIF target
genes, and loss-of-function ztl mutants have lower levelsof these mRNAs than wild-type seedlings (Saitoh et al.
2015a, b). Enhancement of hypocotyl elongation in ZTLoverexpressing seedlings was abolished by NPA, PEOIAA, or yucasin (5-(4-chlorophenyl)-4H-1,2,4-triazole-3thiol, an auxin biosynthesis inhibitor) (Saitoh et al.
2015a). A loss-of-function fkf1 lkp2 ztl triple mutant has
shorter hypocotyls and a lower level of PIF4 mRNA thanwild-type seedlings (Fornara et al. 2015).
One major function of the FKF1/LKP2/ZTL protein
family is to regulate target protein stability via ubiquitination. Thus, our microarray analysis using stable transgenic lines with ectopic expression of LKP2 does notreveal its direct targets, but downstream effects of LKP2
function. In spite of this limitation, our study suggests that
LKP2-mediated hypocotyl growth is regulated at a transcriptional level mainly through auxin signaling (Fig. S5).
Investigations of the functional relationships between
FKF1/LKP2/ZTL family proteins and auxin, PIF transcription factors, or the EC complex would improve ourunderstanding of diurnal plant growth.

Acknowledgments This research was partially supported by a grantfrom the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Dr. K. Hayashi (Okayama University of
Science, Okayama, Japan) for kindly providing PEO-IAA, Dr. J. N.
Maloof (University of California, Davis, CA, USA) for kindly providing pif4 and pif5 seeds, and ABRC for providing axr2-1 and axr31 seeds.
Compliance with ethical standards
Conflict of interest

The authors declare no conflict of interest.

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V. 研究課題 3 「光を利用した生命機能の解明と発がん制御技術の開発」
本課題では、光を様々な形で利用して、生命現象の解明やがんの治療に役立てようとして研究を進めた。具体的には、がん治療法の一つである中性子照射の効率を高めるために「ホウ素脂質」を開発し、それを組み込んだナノカプセルをがん移植マウスに投与して、腫瘍の消失がみられた。また先進的な光学顕微鏡を独自に開発し、マイコプラズマの滑走運動を 3 次
元的に観測する手法を開発したり、最小の回転モーターである F1-ATPase のサブユニットの構造変化を明らかにした。一方、分裂酵母を用いて、細胞質分裂の in vitro 系を開発し、収縮環の働きを詳細に調べ、さらに超解像顕微鏡の利用によって収縮環の形成過程におけるミオ
シンとアクチン繊維の位置関係を明らかにする可能性を示した。さらにタンパク質分解酵素であるズブチリシンを阻害しうる 12 残基のペプチドを既にファージディスプレイ法で選別していたが、そのペプチドのアミノ酸を置換することにより、より強く結合するペプチドを探索し、ズブ
チリシンとの複合体の結晶構造を解こうとした。以下、これらの研究について、それぞれの研究担当者からの解説と業績リスト、そして代表的な論文２編ずつを記載する。

155

光を利用した生命機能解明とがん治療
教授 中村 浩之
助教 佐藤 伸一
[目的]
生きた細胞内で標的タンパク質を化学修飾することは、生体機能の解明だけでなく、新しい機能を付加できることから、最近盛んに研究されている。これまでは、遺伝子工学的に標的タンパク質遺伝子に蛍光タンパク質の遺伝子を導入し、細胞内で発現させる方法が一般的であったが、この手法では時間がかかるだけでなく、この導入する蛍光タンパク質
が立体的に大きく、標的タンパク質が本来の機能を果たさないなどの問題点があった。それに対して、化学的分子修飾（ケミカルラベリング）によるタンパク質への機能付加は細胞内に既に存在するタンパク質を標的とし、遺伝子導入により強制的にタンパク質を発現させる分子生物学的手法とは一線を画す手法であり、ケミカルラベリングにしか成し得ないメリットがある。タンパク質中のアミノ酸残基への分子修飾法の開発は、生物学、生化学、ケミカルバイオロジーといった幅広い研究分野で有用な新たな実験手法の提供を目的とするものである。これまで用いられてきたタンパク質化学修飾法のほとんどは求電子的試薬を使って、主に Cys, Lys といった求核的アミノ酸残基との間に共有結合を形成するものであった。標的タンパク質選択的な分子修飾という点に着目すれば、求電子剤の反応性の高さゆえの非特異的結合が問題となることも多い。近年、光の特性を利用した生命機能の制御技術が精力的に研究され、飛躍的な進展を遂げている。本研究では、光を利用した生命機能解明を１つの研究課題とし、光応答性一電子酸化触媒による新規タンパク質修飾法を開発し、標的タンパク質選択的ケミカルラベリング法へと発展させることを目的とする。
また、光を用いたがん治療研究として、中性子捕捉療法のための新しいホウ素薬剤開発を目的として研究を進めた。中性子捕捉療法は、低エネルギーである熱・熱外中性子がホウ素との核反応により生ずる強力な粒子線を用いてがん細胞を殺傷する治療法である。がん部位へホウ素デリバリーと中性子線のダブルターゲティングが可能であることから、正常組織へのダメージもきわめて低く、治療後の患者への負担が極めて小さい特徴をもつ。
我が国では、世界で初めて BNCT 用小型加速器の開発に成功し、2012 年からホウ素薬剤
BPA と加速器を用いた脳腫瘍ならびに頭頚部がんを対象とした第１相臨床試験が始められ、2016 年からは第２相臨床試験が計画されている。一方で、BPA 非感受性がん患者も多いことから、こういった患者への適応拡大を目的に、次世代ホウ素デリバリーシステムの構築を目的とする。
[結果と考察]１．光を利用した生命機能解明
本研究では、標的のタンパ光触媒としてルテニウムービピリジル錯体に着目した。Ru(bpy)3
錯体は光照射刺激依存的に一電子酸化反応を触媒し、チロシルラジカルを生成することが知られている。そこで、光増感触媒に Ru(bpy)3 錯体を用いることとし、標的タンパク質に選択的に結合するリガンドにこの光増感触媒を導入すれば、この光増感触媒（local

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environmental single electron transfer catalyst (LESC)と名付けた）によって標的タンパ
ク質の Tyr 残基上に選択的にラジカルを発生させることができる。そのチロシルラジカルと選択的に反応する分子（演者らは tyrosyl radical trapper (TRT)と名付けた）を設計する
ことで、標的タンパク質を化学修飾することができる。種々の TRT 構造をスクリーニング

した結果、N,N-dimethyl amino-N’-acyl phenylenediamine 構造（図 1）が Tyr 残基と光触媒の活性化条件において効率的に縮合反応を起こすこと見い出した。本ラベル化反応は精製タンパク質のラベル化にも適用できることが分かったので、次に標的タンパク質の選択的ラベル化反応について検討した。リガンド連結型の Ru 錯体を用いて一電子酸化触媒となる Ru 錯体をリガンド親和性標的タンパク質に近接させることで、短寿命のチロシルラジ
カル生成‐トラップ反応を標的近傍で選択的に触媒することができると考えた。炭酸脱水酵素（CA）のリガンドであるスルホン酸アミドを光増感触媒 Ru(bpy)3 錯体に結合した
LESC およびビオチンを結合させた TRT を用いて、マウス赤血球中のタンパク質混在系の中で CA を選択的にラベル化することに成功した（図 2）1)。

図 1. 光増感触媒の設計と生細胞内標的タンパク質修飾の戦略

図 2.マウス赤血球中における炭酸脱水酵素（CA）の選択的ラベル化

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また、LESC を用いることにより、標的タンパク質を選択的に酸化させて不活性化させる「タンパク質ノックダウン法」の開発にも成功した 2)。
２．放射光を利用したがん治療：中性子捕捉療法
中性子捕捉療法への有効なホウ素デリバリーシステムとして、本研究ではリポソームを用いる戦略に着目した 3)。リポソームとは, 細胞などの生体膜を構成している成分であるリン脂質から形成された脂質二分子膜の人工ナノカプセルである。がん組織にある新生血管は、正常組織の血管に比べて物質透過性が高いため、200 nm より小さい粒子はより多くがん組織に透過・移行し、さらに、がん組織ではリンパ管による薬剤の回収機構が不完全なため、高分子化合物はがん組織内に滞留する。この現象は“Enhanced permeation andretention effect (EPR 効果)”と呼ばれ、1986 年に松村・前田（熊本大学）によって発見された 4)。現在のナノ粒子を用いたドラッグデリバリーシステム（DDS）の根幹となっている多面体構造のホウ素イオンクラスター内封リポソーム製剤を用いると、高い治療効果を得られる可能性が示されてきた。その一方で、リポソームに内封されるホウ素薬剤濃度は、浸透圧の問題から限界があると同時に、リポソーム膜安定性の問題が生じた。我々は、これらの問題点に対し、２つの戦略により克服を試みた。１つは、ホウ素脂質リポソームの開発である。リポソームの脂質二分子膜は、分子間相互作用により自己集合化しているため密度が高いため、この二分子膜へホウ素分子を導入できれば、非常に高濃度でホウ素をデリバリーできると考えられる。そこで、生体膜を構成しているリン脂質の構造を基に二本鎖ホウ素イオンクラスター脂質を分子設計・合成し（図３）、世界初のホウ素脂質リポソームの開発に成功している 5)。本研究では、さらにリポソーム膜内にもホウ素化合物を導入させることで、ホウ素高集積化に成功し、大腸がん細胞移植マウスを用いた実験では、リ
ポソーム投与量を BSH 内封リポソームの５分の１においても、高い BNCT 抗腫瘍効果が
得られ
た６)。

図 3.ホウ素脂質の化学構造

もう１つの戦略は、BSH をより高濃度で内封する技術の開発である。筆者らは、BSH が負電荷をもつイオンクラスターであるため、リポソームの構成成分であるリン脂質の正電荷をもつコリン部位と相互作用し、このことによってリポソーム膜が不安定化され、内封効率を下げていると考えた。そこで、BSH のカウンターカチオンを種々検討したところ、ナトリウムからスペルミジニウムカチオンに置き換えたときに、非常に効率よく BSH が内封されることを見出した（図 4）。大腸がん細胞移植マウスを用いた実験では、リポソーム
投与量を BSH 内封リポソームの７分の１においても、高い BNCT 抗腫瘍効果を得ることに成功した 7)。現在、これらのホウ素高集積化リポソームを用いて、実用化に向けた研究を進めている。

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図４.ナトリウムカチオン（左）およびスペルミジウムカチオン（右）ホウ素クラスター内封リポソームの電子顕微鏡写真[まとめ]
光触媒による生命機能解明のための新しいケミカルプローブの開発研究では、標的タン
パク質内のチロシンへの新たなラベル化法の開発ならびに局所環境での活性酸素誘導によるタンパク質ノック段法へ展開できる成果を得た。また、ホウ素高集積化ナノカプセルを用いることで、中性子捕捉治療によりがん移植マウスのがん増殖を完全に抑制する成果を得た。今後、ホウ素高集積化ナノカプセルの中性子捕捉療法への実用化に向けた研究開発
を医療機関と連携して引き続き進める。
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Metals J. Syn. Org. Chem. Jpn. 72(6), 654-665.
C. Verdiá-Báguena, A. Alcaraz, V. M. Aguilella, A. M. Cioran, Tachikawa, S., Nakamura, H., F.
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(2014) A novel photodynamic therapy for drug resistant prostate cancer cells using porphyrus
Envelope as a novel photosensitizer Photodiagnosis and Photodynamic Therapy 11(1), 48-54.
Sato, S., and Nakamura, H. (2013) Ligand-directed Selective Protein Modification Based on Local
Single Electron Transfer Catalysis
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Nakamura, H., Yasui,Y., and H. S. Ban (2013) Synthesis and Biological Evaluation ofortho-Carborane Containing Benzoxazole as an Inhibitor of Hypoxia Inducible Factor(HIF)-1Transcriptional Activity
J. Organomet. Chem. (special issue) 747, 189-194.
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1. Nakamura, H. (2015) C(sp3)–H versus C(sp3)–C(sp) in Activation of Propargylic Amines
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中村浩之（監訳兼）、岩本武明、斎藤雅一、柴田高範、田中健、長澤和夫、西林仁
昭、共訳(2015) ウェイド「有機化学」ソリューションマニュアル、丸善出版
中村浩之（監訳兼）、岩本武明、斎藤雅一、柴田高範、田中健、長澤和夫、西林仁
昭、共訳(2014) ウェイド「有機化学」上下巻、丸善出版
中村浩之、柳衛宏宣 (2013) 第 7 章 7.1 中性子捕捉療法にける薬剤送達システム
医学物理の理工学（下巻）上坂充、中川恵一、金井達明、西尾禎治、共編養賢堂
153-164 .
Nakamura, H. (2013) Boron Cluster Lipid Liposomes as New Vehicles for Boron Delivery
System of Neutron Capture Therapy Future Med. Chem. 5(6), 715-730.
Nakamura, H. (2013) Development of High Boron Content Liposomes and Their
Promising Antitumor Effect for Neutron Capture Therapy Yakugaku Zasshi, 133(12)1297-1306.
Nakamura, H., and H. Minegishi. (2013) HSP60 as a drug target, Curr. Pharm. Des. 19,
441-451.

[口頭発表]
1.

北村 捷、京極 信輔、Manjusha Joshi、中村 浩之、岩田 耕一 ピコ秒時間分解けい光分光法で観測された単一成分脂質二重膜における不均一構造 (学習院大理・東工大資源研)
日本化学会第 96 回年会（2016 年 3 月 24 日-27 日、同志社大学）
2. 佐藤 伸一、中村 公亮、中村 浩之 ルミノール誘導体を用いたチロシン残基選択的な化学修飾法開発
日本化学会第 96 回年会（2016 年 3 月 24 日-27 日、同志社大学）
3. 羽田野 兼資、佐藤 伸一、中村 浩之 光触媒を用いた Tyr 残基修飾反応における一電子移動範囲の解明 日本化学会第 96 回年会（2016 年 3 月 24 日-27 日、同志社大学）
4. 御舩 悠人、田中 浩士、中村 浩之、布施 新一郎 マイクロフローアミド結合形成法を基盤とするフェグリマイシンの全合成 (東工大資源研・東工大院理工)日本化学会第 96
回年会（2016 年 3 月 24 日-27 日、同志社大学）
5. 佐藤 伸一、中村 公亮、中野 洋文、中村 浩之 チロシン残基選択的化学修飾法を用いたチロシンホスファターゼ活性スクリーニング法の開発 日本化学会第 96 回年会（2016年 3 月 24 日-27 日、同志社大学）
6. 中村浩之 外部エネルギーと局所増感型ナノデバイスによる低侵襲がん治療 LSC 研究発
表会、特別講演（2016 年 3 月 18 日、神戸学院大学）
7. Nakamura, H., Development of Efficient Boron Delivery Nano Carriers for Neutron Capture
Therapy , IAEA-Southern Tohoku Hospital Group Joint Workshop on BNCT, Invited Lecture（2016 年 3 月 7 日、郡山）
8. 中村浩之 ホウ素を使ってがんに挑む ～ホウ素創薬の最前線～ 第８回「国際的キャリア展望に向けた生命化学研究者による講演会」招待講演（2016 年 1 月 27 日、岡山大学）
9. 佐藤伸一、中村公亮、中村浩之 ヘムを触媒とする酸化的チロシン修飾法開発とその応用
新学術領域「酸素生物学」＆「ダイイングコード」合同若手会議（2016 年 1 月 26-28 日、千葉）
10. Sato, S., Nakamura, K., and Nakamura, H., Heme-Catalyzed Tyrosine Click Reaction The 8th
Takeda Science Foundation Symposium on PharmaSciences（2016 年 1 月 20-22 日、大阪）
11. Kikuchi, S., Kanoh, D., Sato, S., and Nakamura, H. Use of serum albumin as an efficient boron

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delivery carrier for neutron capture therapy, The International Chemical Congress of Pacific Basin
Societies 2015 (Pacifichem 2015), (2015 年 12 月 15-20 日、Honolulu, Hawaii)
Nakamura, H., Discovery of carboranylphenoxyacetanilides as inhibitors of heat shock protein(HSP) 60 chaperone activity, The International Chemical Congress of Pacific Basin
Societies 2015,Invited Lecture, (Pacifichem 2015), (2015 年 12 月 15-20 日、Honolulu, Hawaii)
Nakamura, H. Development of boronated liposomes as efficient boron delivery vehicles for neutron
capture therapy, The International Chemical Congress of Pacific Basin Societies 2015
(Pacifichem 2015), (2015 年 12 月 15-20 日、Honolulu, Hawaii)
Sato, S., Nakamura, K., and Nakamura, H. Tyrosine-selective chemical modification using
single-electron-transfer catalyst The International Chemical Congress of Pacific Basin Societies
2015 (Pacifichem 2015), (2015 年 12 月 15-20 日、Honolulu, Hawaii)
Fuse, S., Mifune, Y., Tanabe, N., H. Nakamura., and Takahashi, T. Utilization of microflow technology
for the site-selective modification of multifunctionalized molecules, The International Chemical
Congress of Pacific Basin Societies 2015 (Pacifichem 2015), (2015 年 12 月 15-20 日、
Honolulu, Hawaii)
Morita, T., Fuse, S., Matsumura, K., Sugiyama, H., Kobayashi, D., Tanaka, H., Nakamura, H., andTakahashi, T. Rapid synthesis of tri/tetraaryl-substituted pyrazoles via [3+2] cycloadditon and
C-H direct arylation approaches, The International Chemical Congress of Pacific Basin Societies
2015 (Pacifichem 2015), (2015 年 12 月 15-20 日、Honolulu, Hawaii)
Nakamura, H., Overview and Prospect of Boron Agents for Neutron Capture Therapy, 1st
Academic meeting for Taiwan’s Society of Neutron Capture Therapy, Invited Lecture,( 2015 年
12 月 5 日、Taipei)
小竹佑磨、布施新一郎、御舩悠人、中村浩之、田中浩士 マイクロフローリアクター内での四連続反応によるαアリールカルボニル化合物合成法の開発、第 70 回記念有機合成化学協会関東支部シンポジウム−新潟（長岡）シンポジウム（2015 年 11 月 21 日-22 日、長岡工業高等専門学校）
Nakamura, H., Anticancer Drug Design Using the Element Boron, Hankuk University of Foreign
Studies, Invited Seminar,（2016 年 11 月 19 日、Korea)
Nakamura,H., Sato, S., and Morita., K. Target Protein-Selective Labeling and Inactivation Using
Ligand-Directed Ru(bpy)3 Photocatalysts, 1st Asian Conference on Chemosensors &; Imaging
Probes (2015 年 11 月 16 日-18 日、Korea)
Sato,S., Nakamura, K., and Nakamura, H., Heme-Catalyzed Tyrosine Click Reaction with Luminol
Derivatives, 1st Asian Conference on Chemosensors & Imaging Probes, Invited Lecture ,( 2015 年
11 月 16 日-18 日、Korea)
盛田大輝、田中浩士、中村浩之、布施新一郎 ４連続の直接アリール化による４置換ピラゾールの合成とその発光特性 第 108 回有機合成シンポジウム（2015 年 11 月 5 日-6 日、早稲田大学）
Nakamura, H., Highly Boronated Liposomes as Efficient Boron Delivery Vehicles for Neutron
Capture Therapy
ZING Conferences, Invited Lecture,( 2015 年 10 月 4 日-7 日、Spain)
御舩悠人、布施新一郎、田中浩士、中村浩之、高橋孝志 カルボン酸の迅速かつ強力な活性化を基盤とするマイクロフローペプチド合成法の開発とフェグリマイシンの合成研究 第32 回有機合成化学セミナー（ 2015 年 9 月 15 日-17 日、湯河原）
中村浩之 ホウ素をつかってがんに挑む ～ホウ素創薬の最前線～ 工学院大学 化学応用
学特論 A、(集中講義）（2015 年 9 月 8 日、東京）
盛田大輝、田中浩士、中村浩之、布施新一郎 連続的 C−H 結合直接アリール化による４置
換ピラゾールの合成 第 62 回有機金属化学討論会（2015 年 9 月 7 日−9 日、関西大学、吹田）
中村浩之 何故新しいホウ素薬剤は開発されないのか？ ―これからの Boron Delivery
System を考える― 第 12 回日本中性子捕捉療法学会学術大会(招待講演）（ 2015 年 9 月 4日−5 日神戸学院大学、神戸）
菊地 俊介、佐藤 伸一、中村 浩之 血清アルブミンを用いた新しいホウ素送達法の開

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発 第 12 回日本中性子捕捉療法学会学術大会（2015 年 9 月 4 日−5 日、神戸学院大学、神戸）立川将士、佐藤伸一、間久直、金田安史、粟津邦男、 中村浩之 ポルフィリン脂質リポソームの細胞内局在と PDT 効果 第 25 回日本光線力学学会 学術講演会（2015 年 7 月 10 日
-11 日、新宿）
中村浩之、Clara Vinas、鈴木実、小野公二、立川将士 ホウ素高集積化リポソームを指向した内封ホウ素薬剤開発 第 31 回日本 DDS 学会学術集会（2015 年 7 月 2 日-3 日、新宿）
中村浩之 光を使って生命機能を探る・操る 有機合成化学協会関東支部ミニシンポジウ
ム（招待講演）（2015 年 6 月 27 日-28 日、神奈川）
中村浩之 インデノピラゾール骨格を基軸とした創薬研究 平成 27 年度 環境調和材料・
デバイスプロジェクトグループ(G4)分科会（2015 年 6 月 19-20 日、千歳）李 廣哲、峯岸 秀充、佐藤 伸一、中村 浩之 カルボラン骨格を有する新規 Hsp60 阻害剤の開発 第 19 回日本がん分子標的治療学会学術集会（2015 年 6 月 10 日-12 日、松山）佐藤伸一、中村公亮、中村浩之 ルミノール誘導体を用いたチロシン残基に選択的な共有結合形成反応の開発 日本ケミカルバイオロジー学会第 10 回年会（2015 年 6 月 10 日-12日、東北大学、仙台）
中村浩之、峯岸秀光、室井誠、二村友史、川谷誠、長田裕之 高い細胞増殖阻害を有する
インデノピラゾールの発見とプロファイリング法による阻害機構の解明 新学術領域研究「天然物ケミカルバイオロジー」第 8 回公開シンポジウム（2015 年 6 月 8 日-9 日、東北大学、仙台）Nakamura, H., Development of Liposomes for Efficient Boron Neutron Capture Therapy, 15th
International Congress of Radiation Research (ICRR2015), Invited Lecture, (2015 年 5 月 25-29
日、Kyoto)中村公亮、佐藤伸一、中村浩之 Luminol 誘導体を用いた鉄触媒存在下での Tyr 残基高選択的ラベル化反応 第 69 回有機合成化学協会関東支部会シンポジウム（2015 年 5 月 16 日、横浜）
中村浩之 高い細胞増殖阻害を有するインデノピラゾール化合物の発見とその作用機序解
明 平成 26 年度アライアンス成果報告会（2015 年 4 月 21 日、九州大学、福岡）
峯岸秀充、中村浩之 ベンゾフロピラゾール化合物の合成と HIF-1 転写活性への影響 日本
化学会第 95 回年会（2015 年 3 月 26 日-29 日、船橋）
大内俊明、峯岸秀充、中村浩之 イミダゾピリジン骨格を有する新規 HIF-1α阻害剤の開発
日本化学会第 95 回年会（2015 年 3 月 26 日-29 日、船橋）
菊地俊介、加納大輔、佐藤伸一、中村浩之 中性子補足療法のための血清アルブミンを用いた新しいホウ素送達法の開発 日本化学会第 95 回年会（2015 年 3 月 26 日-29 日、船橋）
中村公亮、佐藤伸一、中村浩之 ルミノール誘導体を用いた鉄触媒存在下での Tyr 残基特
異的タンパク質ラベル化反応 日本化学会第 95 回年会（2015 年 3 月 26 日-29 日、船橋）
Nakamura, H., Development of Liposomes for Efficient Boron Neutron Capture Therapy Invited
Lecture 15th International Congress of Radiation Research (ICRR2015), （2015 年 3 月 25 日-29
日、Kyoto）
佐藤伸一、森田耕平、中村浩之 リガンド連結型 Ru 光触媒を用いたタンパク質ノックダウ
ンとラベル化の制御 日本薬学会第 135 回年会（2015 年 3 月 25 日-28 日、神戸）
Nakamura, H., Development of Chemical Tools for Target Protein-Selective Identification and
Selective Modification Invited Lecture at Indian Institute of Science, Bangalore, Bangalore (2015
年 3 月 10 日、India)
Nakamura, H., Development of Chemical Tools for Target Protein-Selective Identification and
Selective Modification Invited Lecture at Jawaharl Nehru Centre for Advanced Scientific
Research(JNCASR) Bangalore (2015 年 3 月 9 日、India)
Sato, S., Morita, K., and Nakamura, H., Development of Ligand-directed Ru(bpy)3 Photocatalysts
for Target Protein Knockdown and Labeling An International Symposium on Recent Advances in
Chemistry (REACH-2015), Plenary Lecture, Shillong (2015 年 3 月 3-5 日、India)
Nakamura, H., Development of Chemical Tools for Target Protein-Selective Identification and

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Modification Invited Lecture at Indian Institute of Technology, Guwahati (2015 年 3 月 2 日、India)
中村浩之 DDS によるがん治療の最前線 川崎医科大学川崎医学会セミナー、招待講演（2015 年 2 月 26 日、倉敷）
中村浩之 ホウ素でがんに挑む 第一三共株式会社品川研究開発センターセミナー、招待
講演（2015 年 2 月 20 日、東京）
Inai, M., Yamauchi, M., Honda, N., Hazama, H., Tachikawa, T., Nakamura, H., Kaneda,Y., and Awazu,
K., Therapeutic effect of porphyrus envelope-mediated photodynamic therapy: a novel therapeutic
approach for the treatment of hormone antagonistic prostate cancer SPIE Photonics West BiOS
(2015 年 2 月 7-12 日、San Francisco)
中村浩之 がん細胞内部で放射線を発生させる次世代治療法ホウ素中性子捕捉療法 第３回次世代がん治療推進専門家養成プランシンポジウム（2015 年 1 月 25 日、東京）
Nakamura, H., Albumin-bound closo-Dodecaborate as a Promising Boron Carrier to Tumor The
2nd International Workshop on BNCT, Invited Lecture (2014 年 12 月 9ー10 日、京 都)
中村浩之 外部エネルギーによる生体内局所増感型ナノデバイスの開発? QOL の高い低侵
襲がん治療を目指して 第 14 回東北大学多元物質科学研究所研究発表会、招待講演 （2014年 12 月 5 日、仙台）
中村浩之 標的タンパク質のラベル化を指向した一電子酸化的チロシン残基修飾反応の開発佐藤伸一、第 68 回有機合成化学協会関東支部シンポジウム（新潟シンポジウム）（2014
年 11 月 29-30 日、新潟）
山内 将哉、稲井 瑞穂、本多 典広、間 久直、立川 将士、中村 浩之、金田 安史、粟津 邦
男 非ウイルスベクターを用いた薬剤耐性前立腺がんへの PDT における最適な薬剤調製条件の検討第 35 回 日本レー ザー医学会総会 （2014 年 11 月 29 日）
山内将哉、稲井瑞穂、 本多典広、 間久直、 立川将士、 中村浩之、 金田安史、粟津邦男
ホルモン拮抗ヒト前立腺がん細胞株(PC-3)における新規光感受性物質 porphyrus envelope
細胞内局在の検討 第 35 回 日本レーザー医学会総会 （2014 年 11 月 29 日）
峯岸秀充、中村浩之 ベンゾフロピラゾール化合物による HIF-1 転写活性への影響 第 12回がんとハイポキシア研究会（2014 年 11 月 21-22 日、佐賀）
Nakamura, H., Photoaffinity Labeling Molecules as Tools for Identification and Modification of
Target Proteins Vietnam Malaysian International Chemical Congress (VMICC)2014, Invited
Lecture, Hanoi (2014 年 11 月 7-9 日、Vietnam)
Sato, S., and Nakamaura, H., Target-selective Protein Modification Based on Local Environmental
Single Electron Transfer Catalysis 18th Malaysian International Chemical Congress (18MICC),
Invited Lecture, Kuala Lumpur(2014 年 11 月 3-5 日、Malaysia)
Nakamura, H., Development of liposomes for efficient boron neutron capture therapy International
Symposium on Integrated Molecular/Materials Science and Engineering (IMSE2014), Invited
Lecture, 南京（2014 年 11 月 1-3 日、中国）
中村浩之 Target-selective Protein Modification Based on Local Environmental Single ElectronTransfer Catalysis 新学術領域第 3 回国際シンポジウム（招待講演）（2014 年 10 月 28-29
日、大阪）
中村浩之 効率的ホウ素デリバリーシステムの構築を目指して 平成 26 年度京都大学原子炉実験所専門研究会「BNCT の新展開−特殊な療法から一般的な療法への移行を目指して−」（2014 年 9 月 29-30 日、大阪）
Yamauchi, M., Honda, N., Hazama, H., Tachikawa, S., Nakamura, H., Kaneda, Y., and Awazu, K.,A
Photodynamic Therapy for Hormone Antagonistic Human Prostate Cancer Cells Utilizing
Protoporphyrin IX Lipid Delivered by Hemagglutinating Virus of Japan Envelope (HVJ-E) as a
Non-Viral Vector 16th International Congress on Photobiology (2014 年 9 月 8-12 日、Argentina)
Nakamura, H., Tazaki, L., Kanoh, D., Sato, S., and H. S. Ban., Diaryl-substituted carboranes as
inhibitors of hypoxia inducible factor-1 transcriptional activity
XV Internationl Meeting of Boron
Chemistry, Key Note Lecture (2014 年 8 月 24-28 日、Prague, Zech)
Nakamaura, H., and Sato, S., Target-selective Protein Modification Using Ligand-conjugated
Ru(bpy)3 Catalyst XXVI International Congress of Organometallic Chemistry (ICOMC-XXVI)

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（2014 年 7 月 13-18 日、札幌）
立川将士、佐藤伸一、間久直、粟津邦男、金田安史、中村浩之 がん光線力学的治療のためのポルフィリン脂質の開発とナノキャリアへの応用 創薬懇親会 2014 （2014 年 7 月 10-11日、岐阜）
東礎代子、李廣哲、佐藤伸一、中村浩之 カルボラン骨格を有する新規 Hsp60 阻害剤の開発
創薬懇親会 2014 （2014 年 7 月 10～11 日、岐阜）
Nakamura, H., H. S. Ban, Shimizu, K., Minegishi, H., and Sato, S., Design of Photoaffinity Proble
Molecules for Identification and Modification of Target Proteins The 31st International
Conference of Photopolymer Science and Technology Materials & Processes for Advanced
Microlithography, Nanotechnology and Phototechnology (ICPST-31) 招待講演（2014 年 7 月
8-11 日、千葉）
加納大輔、立川将士、佐藤伸一、近藤夏子、鈴木実、桜井良憲、小野公二、中村浩之 生
体高分子をキャリアとしたホウ素デリバリーシステム：マレイミドドデカボレート（MID）の開発と生物活性評価 第 11 回日本中性子捕捉療法学会学術大会（2014 年 7 月 5-6 日、大阪）
立川将士、小金井逸人、菊池俊介、加納大輔、鈴木実、小野公二、中村浩之 リポソーム
内へのホウ素高集積化を可能とするデンドリマー型新規リポソーム薬剤の開発 第 11 回日
本中性子捕捉療法学会学術大会 （2014 年 7 月 5～6 日、大阪）
野々口直助、宮武伸一、古瀬元雅、頼経英倫那、宮田とも、東保太一郎、藤田貢、中村浩之、川端信司、 梶本宜永、田村陽史、辻求、鳴海善文、黒岩敏彦 脳放射線壊死の病態解析と、治療実験（前臨床試験）を実施するためのラット脳放射線壊死も出るの開発 第 11回日本中性子捕捉療法学会学術大会（2014 年 7 月 5-6 日、大阪）
中村浩之 光増感分子を設計しタンパク質を探る・操る、そして創薬へ挑む 第 49 回天然物
化学談話会 招待講演（2014 年 7 月 2-4 日、岡山）
中村浩之 脱 SUMO 化酵素 SENP1 阻害剤 第 18 回日本がん分子標的治療学会学術集会（2014 年 6 月 26 日、仙台）
山内将哉、本多典広、間久直、立川将士、中村浩之、金田安史、粟津邦男 非ウイルスベ
クターとプロトポルフィリン IX 脂質を用いた薬剤耐性前立腺がん細胞への新規光線力学療法の検討 第 24 回日本光線力学学会 学術講演会（2014 年 6 月 28 日～29 日、浜松）
Kanoh, Daisuke., Tachikawa, Shoji., Sato, Shinich., and H, Nakamura., Development ofAlbumin-bound closo-Dodecaborate and its Promising Boron Delivery Efficacy to Tumor 16th
International Congress on Neutron Capture Therapy（2014 年 6 月 14 日-19 日、Finland）
佐藤伸一、森田耕平、中村浩之リガンド連結型 Ru(bpy)3 触媒を用いた標的選択的タンパク
質光分解とラベル化反応の制御日本ケミカルバイオロジー学会 第 9 回年会（2014 年 6 月
11 日、-13 日、大阪）
中村公亮、佐藤伸一、中村浩之 Luminol 誘導体を用いた鉄触媒存在下での Tyr 残基高選択的ラベル化反応日本ケミカルバイオロジー学会 第 9 回年会（2014 年 6 月 11 日-13 日、大阪）中村浩之 光を用いて生命機能を探り操る 平成２６年度 アライアンス G4 分科会（2014年 5 月 29 日、大阪）
佐藤伸一、中村浩之 光触媒を利用した生体内標的タンパク質ラベル化法の開発 新学術
領域研究「天然物ケミカルバイオロジー～分子標的と活性制御～」 第６回公開シンポジウ
ム（2014 年 5 月 28-29 日、名古屋）
峯岸秀充、深代真司、佐藤伸一、中村浩之 インデノピラゾール化合物による低酸素応答
および細胞増殖に対する効果 文科省新学術領域研究・がん支援「化学療法基盤支援活動」
第 3 回シンポジウム （2014 年 5 月 12 日、沖縄）
佐藤伸一、安井友香、丸山美菜子、潘鉉承、中村浩之 カルボラン骨格を基盤とした新規
Hsp60 阻害剤の開発東礎代子、文科省新学術領域研究・がん支援「化学療法基盤支援活動」
第 3 回シンポジウム （2014 年 5 月 12 日、沖縄）
峯岸秀充、深代真司、潘鉉承、佐藤伸一、中村浩之 インデノピラゾール化合物による HIF-1
阻害活性および細胞増殖抑制作用機序の解明 日本薬学会第 134 年会（2014 年 3 月 27-30

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日、熊本）
84. 立川将士、佐藤伸一、山内将哉、間久直、 粟津邦男、金田安史、 中村浩之 がん光線力
学的療法のための PpIX 脂質導入ナノキャリアの開発とその効果 日本薬学会第 134 年会（2014 年 3 月 27-30 日、熊本）
85. 佐藤 伸一、中村 浩之 一電子酸化反応に基づくチロシン残基修飾法の開発と標的タンパ
ク質選択的ラベル化法への応用 日本薬学会第 134 年会（2014 年 3 月 27-30 日、熊本）
86. 中村浩之 次世代低侵襲放射線治療：中性子捕捉療法用ホウ素デリバリーシステム開発
川崎スマートライフケア COI 拠点キックオフシンポジウム、招待スピーチ 、（2014 年 3 月
19 日、東京）
87. 峯岸秀充、深代真司、潘鉉承、中村浩之 インデノピラゾール化合物による HIF 阻害活性
および細胞増殖抑制作用機序の解明 第 11 回がんとハイポキシア研究会 （2013 年 12 月
13-14 日、仙台）
88. Nakamura, H., Design of Photoaffinity Labeling Molecules: Tools for Identification and
Modification of Target Proteins
International Conference on Emerging Trends in Chemical
Sciences (IETC 2013)
Plenary Lecture( 2013 年 12 月 5 日-7 日、India)
89. 松川卓也、佐藤伸一、山乙教之、広野修一、中村浩之 ジフェニルウレア骨格を有する SENP1阻害剤の開発 第 66 回有機合成化学協会関東支部シンポジウム（2013 年 11 月 30 日、東京）
90. 山内将哉、本田典広、間久直、立川将士、中村浩之、金田安史、粟津邦男 非ウィルスベ
クターとプロトポルフィリン IX 脂質を用いた薬剤耐性前立腺がん細胞への新規光線力学療法の検討 34 回日本レーザー医 学会総会（2013 年 11 月 9-10 日、東京）
91. 中村浩之 １人のがん患者を目の前にして有機化学者ができることとは何か？を考えたと
き… 早稲田大学先進理工学部「実践的化学知セミナー」招待講演 （2013 年 11 月 8 日、
東 京）
92. 中村 浩之 ホウ素創薬の最前線 CBI 学会 2013 年大会、基調講演（2013 年 10 月 31 日、
東京）
93. Sato, S., and Nakamura, H., Target-selective Protein Labeling Based on Local Single ElectronTransfer Catalysis The 2nd International Symposium on Chemical Biology of Natural Products:
Target ID and Regulation of Bioactivity （2013 年 10 月 28-29、横浜）
94. 峯岸秀充、深代真司、潘鉉承、中村浩之 インデノピラゾール骨格を有する新規 HIF-1 阻害剤の開発と作用機序 第 57 回日本薬学会関東支部大会（2013 年 10 月 26 日、東京）
95. Sato, S., and Nakamura, H., Ligand-directed Selective Protein Modification Based on Local
Environmental Single Electron Transfer Catalysis International Chemical Biology Society 2nd
Annual Conference ICBS2013 (2013 年 10 月 7-9 日、京都)
96. Koganei, H., Tachikawa, S., Suzuki, M., Ono, K., and Nakamura, H., Development of high boron
content molecules and their liposome encapsulation for neutron capture therapy. 7th Young
Researchers In Boron Neutron Capture Therapy Meeting. Granada （2013 年 9 月 23－26 日、
スペイン）
97. 佐藤 伸一、中村 浩之 標的指向性 Ru(bpy)3 錯体をもちいた局所環境での一電子酸化反応の制御と標的タンパク質選択ラベル化法への応用 第 60 回有機金属討論会（2013 年 9 月
12-14 日、東京）
98. 佐藤 聡、伊藤 祐、齋藤総一郎、藤森浩彰、平井崇久、今堀良夫、伊丹 純、村上康文、中村 浩之、鈴木 実、小野公二、増永慎一、 益谷美都子 ホウ素中性子線捕捉反応に対するがん細胞の応答機構のプロテオーム解析 第 10 回日本中性子捕捉学会（2013 年 9 月 7-8 日、
岡山）
99. 伊藤祐、佐藤 聡、齋藤総一郎、藤森浩彰、平井崇久、新井康仁、今堀良夫、伊丹純、村上康文、中村 浩之、鈴木 実、小野 公二 、増永慎一郎、益谷美都子 口腔がん細胞株
SAS におけるホウ素中性子線捕捉反応後の遺伝子発現解析 第 10 回日本中性子捕捉学会（2013 年 9 月 7-8 日、岡山）
100. 小金井 逸人、三好 達郎、鈴木 実、小野公二、中村 浩之 ホウ素ナノチューブ（BNNT）の中性子捕捉治療用薬剤としての可能性 第 10 回日本中性子捕捉学会（2013 年 9 月 7-8 日、
岡山）

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101. 加納大輔、立川将士、佐藤伸一、中村 浩之 生体高分子への簡便なホウ素導入法の開発と DDS への展開 第 10 回日本中性子捕捉学会（2013 年 9 月 7-8 日、岡山）
102. 中村 浩之 がんを治す薬 ―人々の夢実現に向けて― 読売サイエンスフォーラム ―学
習院大学理学部の研究力― ここまで来た！がん研究最前線 招待講演（2013 年 7 月 27
日、東 京）
103. 森田耕平、峯岸秀充、佐藤伸一、中村浩之 小分子化合物標的タンパク同定のための温和な条件下で切断可能なリンカー開発とその応用 日本ケミカルバイオロジー学会第 8 回年会（2013 年 6 月 19-21 日、東京）
104. 佐藤伸一、中村浩之 局所環境での一電子酸化反応を利用した標的タンパク質選択的ラベル化法の開発 日本ケミカルバイオロジー学会第 8 回年会（2013 年 6 月 19-21 日、東京）
105. 松川卓也、峯岸秀充、中村浩之 ジアリールカルボラン骨格を有する HIF-1 阻害剤の開発
第 17 回日本がん分子標的治療学会学術集会（2013 年 6 月 12-14 日、京都）
106. 立川将士、佐藤伸一、中村浩之 がん光線力学的治療のためのポルフィリン脂質の開発とナノキャリアーへの応用 第 23 回日本光線力学学会学術講演会 (2013 年 6 月 7-8 日、旭川)
107. Yamauchi, M., Honda, N., Hazama, H., Tachikawa, S., Nakamura, H., Kaneda, Y., and Awazu, K.,
Evaluation of protoporphyrin IX combined with lipid chains as a novel photosensitizer The 14th
World Congress of the International Photodynamic Association (2013 年 5 月 28 日-6 月 1 日、韓国)108. 中村 浩之、松川卓也、佐藤 伸一 Manassantin 骨格を模倣したカルボラン含有低酸素誘導因子阻害剤の開発新学術領域「天然物ケミカルバイオロジー～分子標的と活性制御～」第 4回公開シンポジウム（2013 年 5 月 28-29 日、つくば ）109. 森田耕平、峯岸秀充、佐藤伸一、中村浩之 小分子化合物標的タンパク同定のための温和な条件下で切断可能なリンカー開発とその応用 新学術領域「天然物ケミカルバイオロジ
ー～分子標的と活性制御～」第 4 回公開シンポジウム（2013 年 5 月 28-29 日、つくば）
110. Nakamura, H., Boron-Based Drug Design: Discovery of HIF-1α Degradation Pathway Induced by
Inhibiting HSP60 under Hypoxia XVth Conference on Heterocycles in Bio-organic Chemistry(Invited Lecture) (2013 年 5 月 27－30 日、ラトビア)

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DOI: 10.1002/anie.201303831

Protein Modification

Ligand-Directed Selective Protein Modification Based on Local SingleElectron-Transfer Catalysis**
Shinichi Sato and Hiroyuki Nakamura*
Techniques for the visualization of target proteins in living
systems are highly important to investigate the function,
dynamics, localization, and crosstalk of individual proteins.[1]For this purpose, GFP fusion tags and monoclonal antibodies[2] are widely used for conventional protein labeling inmolecular biology. Although these are useful methods for the
real-time monitoring of target proteins in living systems,
genetic manipulation is often necessary, and the conjugationof these proteins with relatively large labeling groups sometimes results in improper biophysical functions owing toundesirable interactions with other molecules.
The chemical modification of proteins with small-molecule probes has received much interest as an alternative
powerful method for the study of individual proteins in theirnative environments. The key to this chemical modification isa bioorthogonal chemical reaction that enables the rapid andselective bioconjugation of proteins with nonnatural functional groups under physiological conditions. The most widely
utilized bioorthogonal chemical reactions rely on electrophilic reagents that target nucleophilic amino acids, such as
lysine and cysteine. In addition, the lysine-specific reductive
alkylation using an iridium catalyst,[3] the conversion ofcysteine into dehydroalanine,[4] and the radical addition
reaction of cysteine with an alkene[5] were recently developed.
Furthermore, remarkable attention has been paid to themodification of aromatic amino acids, such as tyrosine andtryptophan, by transition-metal-mediated processes,[6–8] thethree-component Mannich reaction,[9] the click-like ene-type
reaction,[10] the oxidative modification using cerium(IV)ammonium nitrate,[11] and the use of formylbenzene diazonium reagents.[12]Although various bioorthogonal chemical reactions havebeen reported, there are still few reports regarding selective
chemical modifications of native target proteins. Popp andBall recently reported the site-specific protein modification ofaromatic side chains with dirhodium metallopeptide catalysts.[13, 14] Hamachi and co-workers pioneered a liganddirected protein labeling method in which an electrophilic
phenylsulfonate ester[15] or imidazole[16] group conjugated[*] Dr. S. Sato, Prof. H. Nakamura
Department of Chemistry, Faculty of Science, Gakushuin University
Mejiro, Tokyo 171-8588 (Japan)
E-mail: hiroyuki.nakamura@gakushuin.ac.jp[**] This work was partially supported by a Grant-in-Aid for Scientific
Research on Innovative Areas “Chemical Biology of Natural
Products” from The Ministry of Education, Culture, Sports, Scienceand Technology (Japan).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201303831.
Angew. Chem. Int. Ed. 2013, 52, 8681 –8684

with ligand molecules specifically reacted with the nucleophilic amino acid side chains on the surfaces of target proteinsthrough an SN2-type reaction with the concomitant release of
the ligand molecules. Furthermore, they were the first to
demonstrate the catalytic target-selective modification using
ligand-tethered N,N-dimethylaminopyridine (DMAP) catalysts (Figure 1 A), which allowed highly efficient transfer of

Figure 1. Ligand-directed selective protein modification. A) Acyl-transfer DMAP catalyst promotes the acylation of the nucleophilic regionsof the target protein through an SN2-type reaction. B) Local SET
catalyst (LSC; this work) promotes the generation of tyrosyl radicals
that react with tyrosyl radical trapping agents (TRTs) in the local
environment of the probe on the target protein in the presence ofvisible light.

acyl donor probes to the nucleophilic regions of the targeted
protein through an SN2-type reaction.[17, 18] Herein, we report
the development of a ligand-directed selective protein
modification method based on local single-electron transfer
catalysis (Figure 1 B). In this method, a single-electron transfer (SET) with a photocatalyst,[19, 20] such as the ruthenium
tris(2,2’-bipyridyl) complex ([Ru(bpy)3]2+), is essential for thegeneration of tyrosyl radicals[21, 22] that react with tyrosyl
radical trapping agents (TRTs) containing an N’-acyl-N,Ndimethyl-1,4-phenylenediamine through a catalytic oxidative
radical addition reaction.

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We first examined TRTs suitable for the SET-based
addition reaction using angiotensin II as the model peptide.
Among the compounds examined, N’-acetyl-N,N-dimethyl1,4-phenylenediamine (1; Table 1) was found to be the mostsuitable TRT for this reaction, and the monoadduct toangiotensin II was obtained in 50 % yield in the presence ofthe [Ru(bpy)3]Cl2 complex in Tris buffer (10 mm, pH 4.2) andTable 1: Optimization of the addition reaction of angiotensin II with
N’-acetyl-N,N-dimethyl-1,4-phenylenediamine (1).[a]Entry [Ru(bpy)3]Cl2 BufferpH

Irradiation
time [min]Additive

Yield [%]
(mono-/
bisadduct)

1
2
3
4

1 mm
10 mm
1 mm
1 mm

6.0
7.4
7.4
7.4

15
5
1
1

55 (1:0)70 (9:1)
95 (3:2)
0

5
6
7

1 mm
–
1 mm

7.4
7.4
7.4

–[b]
–[b]
–[b]

–
1 mm APS
1 mm APS
1 mm APS
10 mm DTT
1 mm APS
1 mm APS–

10 (1:0)
10 (1:0)0[a] Reaction conditions: angiotensin II (100 mm) and 1 (500 mm) in MES(10 mm) buffer. All reactions were quenched with DTT (10 mm) andanalyzed by using MALDI-TOF MS. Each reaction was repeated several
times and the average ratios of the mono- and bisadducts are indicated.
[b] Incubated for 5 min without irradiation.

under irradiation with light (Table S1 in the Supporting
Information). Then, we tested different buffers and additives
for the addition reaction with TRT 1 under mild conditions(pH 6.0–7.4) to prevent proteins from denaturation (Table 1
and Table S2 in the Supporting Information). The addition
reaction proceeded preferentially in 2-morpholinoethanesulfonic acid (10 mm, MES) buffer under irradiation with visible
light for 15 min (Table 1, entry 1). It was reported that SET ofthe excited state *RuII to RuIII was accelerated in the presenceof oxidants, such as ammonium persulfate (APS).[21] Therefore, we examined the effects of APS on the addition reactionof angiotensin II (100 mm) with 1 (500 mm). The reaction
proceeded smoothly at pH 7.4 in the presence of APS (1 mm)
and a catalytic amount of [Ru(bpy)3]Cl2 (10 mm) underirradiation with visible light for 5 min, giving the monoadductand the bisadduct (9:1 ratio) in 70 % yield (Table 1, entry 2).
The use of 1 mm of [Ru(bpy)3]Cl2 accelerated the reaction
rate and both mono- and bisadducts were obtained in 95 %yield in a 3:2 ratio even under irradiation with visible light for
1 min (Table 1, entry 3). The reaction was abolished byaddition of dithiothreitol (DTT), a radical scavenger(Table 1, entry 4). Both irradiation with visible light and the[Ru(bpy)3]Cl2 complex were essential for the addition reaction (Table 1, entries 5–7),[23] thus suggesting that the reac-8682

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tions proceeded through the photoinduced oxidative SET
radical mechanism proposed in Scheme S1 in the Supporting
Information. MS/MS analysis of the mono- and bisadducts of
1 to angiotensin II indicated that all modifications were
implemented on tyrosine residues (Figures S1 and S2 in the
Supporting Information). To understand the binding mode
between 1 and tyrosine residues, the addition reaction of
1 with ethyl-N-acetyl-tyrosine amide, a model substrate, wasinvestigated. Structural analysis revealed that a carbon–
carbon bond was formed between the ortho-carbon atom of
the phenolic oxygen of ethyl-N-acetyl-tyrosine amide and theortho-carbon atom of the dimethylamino group of 1 (Figures S3 and S4 in the Supporting Information). We also
examined the reactivity of 1 to the tryptophan residue by
using the model peptide melittin. The observed oxidation of
the tryptophan residue with oxygen proceeded prior to theaddition with 1.[24–26] These results suggest that this method
can be used for the specific modification of tyrosine residues
in the target protein.
With the suitable conditions established, we applied thistyrosine-residue-specific addition reaction to the modificationof a purified protein. We designed and synthesized fluorescent TRT 2 (Scheme S3 in the Supporting Information).
Bovine serum albumin (BSA), the model protein, was treatedwith 2 under several conditions (Figure 2). Whereas BSA wasnot modified in the presence of [Ru(bpy)3]Cl2 without 2 or
APS (lanes 1 and 2), successful modification was observed in
the presence of [Ru(bpy)3]Cl2 (10 mm), 2 (500 mm), and APS(1 mm) under irradiation with visible light for 5 min (lane 3).
A slight modification of BSA with 2 was observed without
irradiation and [Ru(bpy)3]Cl2 (lanes 4 and 5), thereby
revealing that APS caused a photocatalyst-independent
modification reaction, which was considered to be one of
the undesired background reactions. This modification proceeded without APS, although an excess amount of [Ru-Figure 2. Modification of BSA with fluorescent TRT 2 under various
conditions. Fluorescence images and coomassie brilliant blue (CBB)stained images of SDS-PAGE gels, with the conditions for each lane
given above. [a] The exposure time for fluorescence detection in lanes
6–10 was longer (3.0 s) than that in lanes 1–5 (0.3 s; also see
Figure S7 in the Supporting Information).

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Angewandte

ChemieFigure 4. Selective modification of CA in mouse erythrocyte lysates.
A) Structure of biotin-conjugated TRT 5. B) The reaction for theselective modification of CA was performed in MES (10 mm, pH 6.0)
using erythrocyte lysates from 6-week-old female mice. The left image
shows visualization of biotinylated proteins with a streptavidin–HRP
conjugate. The right image shows CBB staining of proteins in mouseerythrocyte lysates.

Figure 3. Selective modification of carbonic anhydrase (CA) promotedby the CA-ligand-conjugated ruthenium complex 3 as LSC in a mixtureof BSA and bovine CA. A) Structures of 3 and CA-binding competitor
4. B) Protein modification reactions were performed in a mixture of
BSA (5 mm) and CA (5 mm) in MES buffer (10 mm; pH 6.0) without
APS. Fluorescence images of modified proteins (upper) and wholeproteins with CBB stain (lower) are shown.

(bpy)3]Cl2 (1 mm) was needed. [Ru(bpy)3]Cl2 and irradiation
with light were indispensable in this case (lanes 6–10). To
evaluate the biocompatibility of this reaction, double modification experiments of BSA and streptavidin were carriedout using PEG-conjugated TRT and tetramethylrhodamineconjugated maleimide or N-hydroxysuccinimide (NHS). The
electrophilic modifications proceeded even after the PEG
modification, thus suggesting that this SET-based tyrosinetargeting method is compatible with the electrophilic methods targeting cysteine and lysine residues (see Figure S8 in the
Supporting Information).
To clarify whether this addition reaction based on local
SET is applicable to selective protein modification, we
designed and synthesized a ligand-conjugated ruthenium
complex as an LSC. Carbonic anhydrase (CA) and benzenesulfonamide were chosen as the model of the target protein
and its ligand, respectively.[27] When the SET-based radical
modification reaction was carried out in the presence of[Ru(bpy)3]Cl2 using a mixture of BSA and CA, the extent ofmodification of BSA was higher than of CA, because it hada larger number of accessible tyrosine residues (Figure 3,
lanes 2 and 3). Meanwhile, benzenesulfonamide-conjugatedruthenium complex 3, which was designed as a CA-targeting
LSC, promoted the selective modification of CA (lane 4) with
Angew. Chem. Int. Ed. 2013, 52, 8681 –8684

more than 100 times higher efficiency than BSA modification(lane 3; 1 mm [Ru(bpy)3]Cl2 versus lane 4; 10 mm 3).[28] The
selective modification of CA was inhibited by the CA-binding
competitor 4, which was present in an amount that was 100
times that of LSC 3 (lane 5), thereby revealing that thisreaction was accelerated by the target protein–ligand interaction between CA and 3.[29] No protein modification of both
BSA and CA was observed in the absence of irradiation withvisible light or 2 (lanes 6 and 7).
Finally, we applied this target-selective protein modification to native CA proteins in cell lysate using 3. CA
modification was performed in mouse erythrocytes thatoriginally expressed CAs. However, the lysates of erythrocytes contained a large amount of hemoglobin (14 kDa) andthe fluorescence of hemoglobin interfered with the fluorescence detection of CA modified with 2. Therefore, wedeveloped biotin-conjugated N,N-dimethyl-1,4-phenylenediamine 5 as a TRT (Figure 4 A) to detect the target protein byusing a streptavidin–horseradish peroxidase(HRP) conjugate.
No biotinylated proteins were detected with streptavidin–
HRP in the absence of 3, biotin-conjugated TRT 5, or
irradiation (Figure 4 B, lanes 1–4). Those proteins, however,were observed at 29 kDa, which corresponded to CA from thecell lysate, in the presence of 3 and 5 under irradiation withlight for 15 min (lane 5), and this modification was inhibitedby the addition of 4 (lane 6).[30] These results clearly indicatedthat SET-based protein modification using 3 and 5 specificallyoccurred on the target protein CA despite the coexistence ofvarious kinds of proteins in the cell lysate. We also performed

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this modification reaction in intact erythrocytes in MESbuffered saline (10 mm MES (pH 6.0), 150 mm NaCl) andagain obtained similar results to those from the cell lysate(Figure S11 in the Supporting Information), thus suggestingthat 5 and 3 could permeate the erythrocyte cell membrane.[31]Therefore, this target-selective protein modification based on
local SET catalysis is applicable to the modification of native
proteins in an intracellular environment.
In conclusion, we have developed a target-selective
protein modification strategy based on local SET catalysis.
In cell lysates, tyrosyl radicals were selectively generated on
the target protein by SET from the photocatalyst in the local
environment of the ligand, and the radicals were trapped by
TRTs through the specific radical addition to tyrosine
residues. Although target-selective protein modifications incell lysates were achieved before with electrophilic reagentsusing ligand-tethered DMAP catalysts, those methods werelimited to modifying the nucleophilic residues of the targeted
proteins, such as lysine and cysteine. The current method is
not only an alternative to the strategy using electrophilic
agents but can also be utilized for the identification ofproteins interacting with the target protein through a tyrosine–tyrosine SET addition reaction between the target
protein and its interaction partners induced by the LSCbased generation of tyrosyl radicals. Studies with respect tofurther applications are in progress.
Received: May 4, 2013
Published online: July 3, 2013.

Keywords: photocatalysts · protein modifications ·radical reactions · single-electron transfer · tyrosine[1] T. Hayashi, I. Hamachi, Acc. Chem. Res. 2012, 45, 1460 – 1469.
[2] A. M. Wu, P. D. Senter, Nat. Biotechnol. 2005, 23, 1137 – 1146.
[3] J. M. McFarland, M. B. Francis, J. Am. Chem. Soc. 2005, 127,
13490 – 13491.
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Cite this: DOI: 10.1039/c4cc04344h
Received 9th June 2014,
Accepted 21st August 2014

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Spermidinium closo-dodecaborate-encapsulating
liposomes as eﬃcient boron delivery vehicles forneutron capture therapy†
Shoji Tachikawa,ab Tatsuro Miyoshi,b Hayato Koganei,b Mohamed E. El-Zaria,bc
Clara Viñas,d Minoru Suzuki,e Koji Onoe and Hiroyuki Nakamura*ab

DOI: 10.1039/c4cc04344h
www.rsc.org/chemcomm

closo-Dodecaborate-encapsulating liposomes were developed as borondelivery vehicles for neutron capture therapy. The use of spermidinium
as a counter cation of closo-dodecaborates was essential not only forthe preparation of high boron content liposome solutions but also foreﬃcient boron delivery to tumors.

Boron neutron capture therapy (BNCT) has been attracting growinginterest as one of the minimally invasive cancer therapies.1
Mercaptoundecahydrododecaborate (Na2[B12H11SH]; Na2BSH)
and L-p-boronophenylalanine (L-BPA) have been used in BNCT
for many years. L-BPA, in particular, has been widely used for thetreatment of not only melanoma but also brain tumor2 and headand neck cancer3 because it can be taken up selectively by tumor
cells through an amino acid transporter.4 The accelerator-based
BNCT is now undergoing phase I clinical study for the treatmentof brain tumor and head and neck cancer patients in Japan.5,6
In recent years, liposomal 10B carriers have attracted attention
as some of the eﬃcient boron delivery systems in BNCT.7–10
Several efficient in vivo BNCTs have been reported. Yanagië andcoworkers demonstrated the first antitumor effect of Na2BSHencapsulating liposomes conjugated with a monoclonal antibody
specific for the carcinoembryonic antigen.9a,b Maruyama andco-workers developed transferrin-conjugating Na2BSH-encapsulating
liposomes.9d,10 Although they succeeded in completely suppressing
tumor growth in mice after neutron irradiation, the concentration of
inner 10B of liposomes was limited in preparation due to osmotic
reasons. For this reason, boron lipids embedded within the liposomeaChemical Resources Laboratory, Tokyo Institute of Technology,
4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan.
E-mail: hiro@res.titech.ac.jp; Fax: +81 45 924 5244b
Department of Chemistry, Faculty of Science, Gakushuin University, Mejiro,
Toshima-ku, Tokyo 171-8588, Japanc
Department of Chemistry, Faculty of Science, Tanta University, 31527-Tanta, EgyptdInstitut de Ciència de Materials de Barcelona (ICMAB-CSIC), Campus U.A.B.,
08193 Bellaterra, Barcelona, SpaineParticle Radiation Oncology Research Center, Kyoto University Research Reactor
Institute, Asashiro-nishi, Kumatori-cho, Sennan-gun, Osaka 590-0494, Japan† Electronic supplementary information (ESI) available: Experimental details anddata. See DOI: 10.1039/c4cc04344h

This journal is © The Royal Society of Chemistry 2014

bilayer have been studied.10–12 Hawthorne and coworkers developed
liposomes incorporating Na3[1-(2-B10H9)-2-NH3B10H8] into the internalaqueous core and K[nido-7-CH3(CH2)15-7,8-C2B9H11] into the bilayer
membrane to increase the boron content in liposomes.8b,13
We previously developed Na2BSH-encapsulating 10% distearoyl
boron lipid (DSBL)11b liposomes that have high boron content withexcellent boron delivery eﬃcacy to tumors.14 In this communication,
we studied the eﬀects of the counter cations of boron clusters on
liposome formation to develop high boron content liposomes for
BNCT by overcoming osmotic pressure limitations.
We selected three closo-dodecaborates, Na2[B12H12],Na2[B12H11OH]15 and Na[B12H11NH3]16 in addition to Na2BSH(Fig. 1). We first tested the cytotoxicity of the closo-dodecaborates
toward colon 26 cells. The closo-dodecaborates are relatively nontoxic and the GI50 values of Na2[B12H12], Na2BSH, Na[B12H11NH3],
and Na2[B12H11OH] are 5.1, 2.1, 32.9, and 7.7 mM, respectively(Table S1 in the ESI†). Liposomes containing the closododecaborates were prepared from DSPC, cholesterol, and
DSPE-PEG2000 by the reverse phase evaporation method withsizes of approximately 100 nm in diameter. The results are
summarized in Table 1. The final boron and phosphorus concentrations of liposome solution containing Na2[B12H12] were3438  2.0 and 2864  18.3 ppm, respectively, and the B/P ratio
was 1.2 (entry 1). The higher B/P ratio indicates the higher boron
content in liposomes. The liposome yield was 58% based on the
total phospholipids used in the preparation. The B/P ratio of theliposome containing Na[B12H11NH3] was 2.2 (entry 2), which wasslightly higher than those of liposomes containing Na2BSH,
Na2[B12H11OH], and Na2[B12H12] (1.2–1.6, entries 1, 3, and 4).
In the case of Na[B12H11NH3], an ammonium ion group served
as one of the two counter cations of the closo-dodecaborate

Fig. 1

173

Structures of closo-dodecaborates used for encapsulation in liposomes.

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Table 1 Physical characteristics of liposomes containing closo-dodecaborates
associated with sodium and various ammonium cationsaPublished on 21 August 2014. Downloaded by Tokyo Institute of Technology on 03/09/2014 11:46:28.

Entry Boron cluster
1
2
3
4
5
6
7
8
9

B conc.b,c(ppm)

Na2BSH
3438  2.0
4072  22.8
Na[B12H11NH3]2635  184.2
Na2[B12H11OH]
3133  10.3
Na2[B12H12]2874  47.7
(n-C3H7NH3)2BSH(H3NC4H8NH3)BSH 4711  17.4
spd-BSH
13 867  185.8(Sperminium)BSH
9759  139.6
13 970  216.5
spd-[B12H11NH3]

P conc.
(ppm)

B/P

Yieldd(%)

2864  18.3
1835  38.5
1600  99.0
1932  13.3
2225  15.8
1833  43.4
4046  18.3
3559  44.53943  43.4

1.2
2.2
1.5
1.6
1.3
2.6
3.4
2.7
3.5

58
44
39
47
54
44
98
87
95

b,c

Fig. 2 Eﬀect of the amount of spd cation on spd-BSH encapsulation inliposomes. Boron/phosphorus (B/P) ratios of (spd)x-closo-dodecaborateencapsulating liposomes are shown in the vertical axis.

a

In all cases, liposomes were prepared from DSPC, cholesterol, and DSPEPEG2000 (1 : 1 : 0.11, molar ratio) by the REV method. b Data are expressed
as means  standard deviation (SD). c Boron and phosphorus concentrations of liposome solution were determined by ICP-AES. d Liposome yields
were calculated from the phosphorus concentration of liposome solution
based on the total phospholipids used in preparation.

dianion. We speculated that ammonium counter cations wouldaﬀect the encapsulation of closo-dodecaborates in liposomes.
Recently, Gabel and coworkers reported that Na2BSH induces
aggregation and membrane rupture, increasing wall thickness of
the liposome and triggering the release of liposome contents.17
Indeed, it is known that tetramethylammonium (TMA) salts ofcloso-dodecaborates are insoluble in water, and the ion-exchange
from Na2BSH to (TMA)2BSH proceeds readily, whereas the ionexchange from (TMA)2BSH to Na2BSH is not easy. We predictedthat encapsulation as well as liposome yield would be increased
if we could reduce this interaction in the preparation of closododecaborate-encapsulating liposomes. Thus we prepared various
ammonium salts of BSH and examined their encapsulation into
liposomes (entries 5–9 in Table 1). The B/P ratio of the liposome
containing the n-C3H7NH3+ salt of BSH was similar to that of theliposome containing Na2BSH (entries 1 vs. 5). The H3N+C4H8NH3+
cation increased the B/P ratio (2.6) and boron concentration(4711 ppm). Interestingly, the B/P ratio dramatically increased to
3.4 when the spermidinium (spd) cation was employed (entry 7).
In addition, the liposome yield was markedly increased to 98% andthe final boron concentration of the liposome solution reached
13 867 ppm. In contrast, the B/P ratio of liposome containing(sperminium) BSH dropped to 2.7, although the liposome yield
was still high (87% yield) and the final boron concentration of theliposome solution was high at 9759 ppm (entry 8). Liposome
containing spd-[B12H11NH3] showed the highest B/P ratio (3.5); theboron concentration of the liposome solution reached 13 790 ppm
and the liposome yield was 95% (entry 9).
We examined whether the formation of high boron content
liposomes is aﬀected by the viscosity of the closo-dodecaborate
solutions. However, the spd cation of [B12H12]2 does not aﬀect theviscosity of internal aqueous solution of liposomes (Table S3, ESI†).
We measured liposome yields and B/P ratios under the condition ofvarious ratios of BSH to spd cations (Na2BSH: [spermidine + HCl] = 1:
X, X = 0, 0.25, 0.5, 1, and 2). As shown in Fig. 2, the B/P ratio

Chem. Commun.

reached a maximum of 3.4 when the BSH : spd ratio was 1 : 1.
Liposome yields showed a similar tendency to B/P ratios. The highest
liposome yield was observed at the BSH to spd cation ratio of 1 : 1.
Transmission electron microscopy (TEM) analysis of spd-BSHencapsulating liposomes and Na2BSH-encapsulating liposomes
was also carried out using Cryo-TEM (Fig. 3). It is notable thatthe liposomes interacted with each other in the case of Na2BSHencapsulating liposomes, whereas the liposomes dispersed insolution without interacting with each other in the case of spdBSH-encapsulating liposomes.
We next examined boron distribution of the spd-closododecaborate-encapsulating liposomes in colon 26 tumor-bearing
mice.18 The liposomes were injected at doses of 15, 30, and 100 mg[B] kg 1 body weight via the tail veins. Na2BSH- and Na2[B12H11NH3]encapsulating liposomes were also injected at a dose of
30 mg [B] kg 1 as control experiments. The time courses of boron
distribution in each organ are shown in Fig. 4. Blood boron
concentrations of 460.7, 104.0, and 33.2 ppm were detected 24 hafter injection of spd-BSH-encapsulating liposomes (100, 30, and
15 mg [B] kg 1), respectively (Fig. 4a). Blood boron concentration inmice injected with 100 mg [B] kg 1 of spd-BSH-encapsulating
liposomes did not decrease notably during the 48 h period, whereas
those in mice injected with 30 and 15 mg [B] kg 1 of spd-BSHencapsulating liposomes gradually decreased in a time-dependent
manner. The time courses of boron concentrations in liver, kidneys,
and spleen are shown in Fig. 4b–d, respectively. Boron concentrations of 528.5, 144.2, and 74.4 ppm in liver were observed 48 h after
the injection of 100, 30, and 15 mg [B] kg 1 of spd-BSHencapsulating liposomes, respectively. In the meantime, maximum

Fig. 3 TEM images of Na2BSH-encapsulating liposomes (left) and spdBSH-encapsulating liposomes (right). Scale bar represents 200 nm.

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Fig. 4 Time courses of distribution of spd-BSH- and Na2BSH-encapsulating
liposomes (n, spd-BSH: 15 mg [B] kg 1; B, spd-BSH: 30 mg [B] kg 1; J,
spd-BSH: 100 mg [B] kg 1; m, Na2BSH: 15 mg [B] kg 1). Each liposome
was injected into tumor-bearing mice via the tail vein. Data are expressed asmeans  SD (n = 5).

tumor boron concentrations of 202.7 and 82.4 ppm were achieved
36 h after injection at doses of 100 and 30 mg [B] kg 1, respectively.
Even at the low boron dose of 15 mg [B] kg 1, the tumor boron
concentration was 34.0 ppm at 36 h after injection (Fig. 4e). We
also demonstrated the boron distribution of Na2BSH-encapsulating
liposomes in tumor-bearing mice for comparison. Althoughblood and liver boron concentrations after injection of Na2BSHencapsulating liposomes at a dose of 30 mg [B] kg 1 were similar
to those after injection of spd-BSH-encapsulating liposomes at adose of 30 mg [B] kg 1, kidney and spleen boron concentrations
after injection of Na2BSH-encapsulating liposomes were lower
than those after injection of spd-BSH-encapsulating liposomes
up to 48 h. The tumor boron concentration at 36 h after injecting
Na2BSH-encapsulating liposomes was 31.9 ppm, although theclearance of Na2BSH-encapsulating liposomes was slow (Fig. 4e).
A similar tendency was observed in spd-[B12H11NH3]-encapsulating
liposomes (Fig. S1, ESI†). Blood, kidney, and spleen boron concentrations gradually decreased after injection. Maximum tumor boron
concentrations of 242.2, 88.7, and 35.4 ppm were achieved 6 h after
injection at doses of 100, 30, and 15 mg [B] kg 1, respectively.
Interestingly, significant tumor boron accumulation was also observedin the case of Na[B12H11NH3]-encapsulating liposomes.
Finally, we examined the antitumor eﬀect of liposomes
containing spd closo-dodecaborates in colon 26 tumor bearing
mice exposed to thermal neutron irradiation. Thermal neutron
irradiation of the tumor-transplanted left thighs of mice wascarried out 36 h after injection. The tumor growth curves ofmice are shown in Fig. 5 (and in Fig. S2, ESI†). ‘‘Hot control(–K–)’’ and ‘‘Cold control (––)’’ represent tumor volumes of mice
injected with saline with and without thermal neutron irradiation,
respectively. Tumor growth was significantly suppressed in micetreated with spd-[10BSH]- and spd-[10B12H11NH3]-encapsulating
liposomes at doses of 15, 30, and 100 mg [10B] kg 1 and exposedto thermal neutron irradiation. The tumor completely disappeared
within three weeks even when a dose of 15 mg [10B] kg 1 was
employed. Liposomes containing Na2[10BSH] and Na[10B12H11NH3]This journal is © The Royal Society of Chemistry 2014

Fig. 5 Tumor volumes in mice (Balb/c, female, six weeks old, 14–20 g)bearing colon 26 solid tumor, exposed to thermal neutron irradiation (hot)for 50 min (1.3–2.2  1012 neutrons per cm2) or not exposed to thermal
neutron irradiation (cold). Irradiation was performed 36 h after injection ofliposomes containing spd-[10BSH] (n, 15;B, 30; J, 100 mg [10B] kg 1) andNa2[10BSH] (E, 30 mg [10B] kg 1), or 1 h after injection of Na2[10BSH]
solution (’, 100 mg [10B] kg 1). K, hot control; , cold control. **P o 0.01,
compared with hot control.

also inhibited tumor growth at a dose of 30 mg [10B] kg 1, and thetumor was completely controlled three weeks after thermal neutron
irradiation. Tumor growth was suppressed in mice treated with
Na2[10BSH] solution (100 mg [10B] kg 1) during the two weeks afterthermal neutron irradiation. However, the tumor started to growthereafter (Fig. 5). In contrast, tumor growth was not suppressed inmice treated with Na[10B12H11NH3] solution (100 mg [10B] kg 1)even after thermal neutron irradiation (Fig. S2, ESI†).
Fig. 6 shows the survival curve of tumor-bearing mice afterthermal neutron irradiation. All untreated mice without thermal
neutron irradiation died within two weeks. Thermal neutron irradiation enhanced mouse survival and all mice exposed to thermal
neutron irradiation died within 78 days. Prolonged survival wasobserved in mice injected with spd-[10BSH]- and spd-[10B12H11NH3]encapsulating liposomes; 72% of the mice that received a dose of
15 mg [10B] kg 1 survived up to 100 days after the thermal
neutron irradiation. Furthermore, a remarkable antitumor eﬀect

Fig. 6 Survival curve of tumor-bearing mice after thermal neutron irradiation.
The irradiation was performed 36 h after injection of closo-dodecaborates(TT, spd-BSH: 30 mg [10B] kg 1; E, spd-BSH: 15 mg [10B] kg 1; B,
spd-10B12H11NH3: 15 mg [10B] kg 1) for 50 min (1.3–2.2  1012 neutrons percm2). , cold control; K, hot control. Mice were sacrificed when their tumor
volumes reached B3000 mm3.

175

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was observed in the mice treated with spd-[10BSH]-encapsulating
liposomes at a dose of 30 mg [10B] kg 1; 100% of the micesurvived up to 100 days after the thermal neutron irradiation.
We succeeded in the preparation of high boron content liposomes. The use of spd as a counter cation of closo-dodecaborates was
essential to obtain the liposomes with high yields and high B/Pratios. All of the mice injected with 30 mg [10B] kg 1 of spd-[10BSH]encapsulating liposomes were completely cured while five of sevenmice injected with 15 mg [10B] kg 1 of spd-[10BSH]- andspd-[10B12H11NH3]-encapsulating liposomes were cured 100 days
after thermal neutron irradiation. The results indicate that the
total amount of phospholipids could be reduced to less than
one-seventh of those used to prepare Na2[10BSH]-encapsulating
liposomes.19 We believe that the spd-closo-dodecaborateencapsulating liposomes are promising candidates for clinicaluse in BNCT.
We thank Dr S. Kondoh (Teijin Pharm. Co. Ltd) for themeasurement of osmotic pressures and Professor K. Iwata(Gakushuin University) for the measurement of the viscosity.
This work was supported by Health and Labor Sciences Research
Grants for research on Nanotechnical Medical from the Ministryof Health, Labor and Welfare (20100201) and the Ministry of
Education, Science, Sports, Culture and Technology, Grant-inAid for Scientific Research (B) (No. 23390018) from Japan.

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Chem., 2007, 18, 1287; (c) X. Pan, G. Wu, W. Yang, R. F. Barth,
W. Tjarks and R. J. Lee, Bioconjugate Chem., 2007, 18, 101.
13 P. J. Kueﬀer, C. A. Maitz, A. A. Khan, S. A. Schuster, N. I. Shlyakhtina,
S. S. Jalisatgi, J. D. Brockman, D. W. Nigg and M. F. Hawthorne,
Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 6512.
14 H. Koganei, M. Ueno, S. Tachikawa, L. Tasaki, H. S. Ban, M. Suzuki,
K. Shiraishi, K. Kawano, M. Yokoyama, Y. Maitani, K. Ono and
H. Nakamura, Bioconjugate Chem., 2013, 24, 124.
15 T. Peymann, C. B. Knobler and M. F. Hawthorne, Inorg. Chem., 2000,
39, 1163.
16 W. R. Hertler and M. S. Raasch, J. Am. Chem. Soc., 1964, 86, 3661.
17 (a) D. Gabel, D. Awad, T. Schaﬀran, D. Radovan, D. Daraban,
L. Damian, M. Winterhalter, G. Karlsson and K. Edwards, ChemMedChem, 2007, 2, 51; (b) D. Awad, L. Damian, M. Winterhalter,
G. Karlsson, K. Edwards and D. Gabel, Chem. Phys. Lipids, 2009,
157, 78.
18 All protocols were approved by the Institutional Animal Care and
Use Committee of Gakushuin University.
19 Conventionally, a preinjection of liposome at a high dose was given for
an achievement of targeting through saturation of liver’s scavenging
capacity (see; Y. Y. Kao and R. L. Juliano, Biochim. Biophys. Acta, 1981,
677, 453). Such a high liposome dose may cause possible liver toxicity,
since the liver’s normal scavenging function is impaired. Indeed, an
important character of an approved liposomal formulation (doxil)
encapsulating an anticancer drug doxorubicin is the very high concentration of the encapsulated drug (also see; P. G. Tardi, N. L. Boman and
P. R. Cullis, J. Drug Targeting, 1996, 4, 129).

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This journal is © The Royal Society of Chemistry 2014

生体防御タンパク質の構造及び機能解析教授 小島 修一
助教 津田 岳夫
[目的]
プロテアーゼやキナーゼはシグナル伝達や細胞周期制御などの様々な生命現象に関与しており、これらの異常はヒトの発がんと深く関係しており、またこれらのタンパク質の活性は、タンパク質やペプチド、さらに低分子化合物等によって巧みに調整されている。本
研究では、それらのタンパク質の活性を制御しうる化合物を創製すべく、それらに対して特異的に結合するタンパク質やペプチド、そして阻害剤との相互作用を中心に生化学及び構造生物学的解析を行なうことにする。具体的にはプロテアーゼ（タンパク質分解酵素）であるズブチリシンを対象として用い、ファージディスプレイ法によってこの酵素に特異的に結合して阻害する 12 残基のペプチドを出発物質とした。そのペプチドに変異を導入して、阻害活性の増強を試みることにした。一方、ペプチドに 20 種類以外のアミノ酸を導入することによって新機能を有するペプチドの創製を目指して研究を進めている枯草菌由来の L-アミノ酸リガーゼについては、明らかにした立体構造に基づいて、この酵素への変異導入によって、基質であるアミノ酸の特異性の改変の可能性を検討した。

[結果と考察]１．ファージディスプレイ法によって得られた 12 残基のズブチリシン阻害ペプチドへの変異導入による阻害活性の増強
ファージディスプレイ法は、バクテリオファージの表面に異種タンパク質やペプチドを提示させる研究手法であり、提示された様々なアミノ酸配列を持つタンパク質やペプチドの中から、ある機能を持った新規なタンパク質あるいはペプチドを選別することができる。
この手法を用いてタンパク質間相互作用を調節する新規ペプチドを創製する研究の一環として、12 残基のペプチドをコードするライブラリーから、ズブチリシンというセリンプロ
テアーゼに親和性を示す SerAspPheSerCysLeuSerGluGlyCysArgThr というアミノ酸配列を持つ新規なペプチドを見出した。その後、阻害の中心が Leu6-Ser7 であると予想されること、そしてジスルフィド結合の形成に必要な Cys5、Cys10 および Gly9 を除く各アミノ酸を 1 個ずつ Ala に置換した変異ペプチドの中では、C 末端の Thr12 あるいは Ser7 をAla に置換したペプチドが野生型より阻害活性が上昇することを明らかにした。このことは、
ファージディスプレイ法によって選別されたペプチドへの変異導入により、さらに効果的な阻害剤が創製できることの可能性を示している。
そこで本研究では、12 残基のペプチドの Ser7 および Thr12 を性質の異なる他の数種類のアミノ酸に置換して、ズブチリシンに対する阻害活性の増強を試みた。具体的には大きさの異なる疎水的なアミノ酸や、正あるいは負の電荷を持つアミノ酸などへ置換した。合177

わせて、阻害活性を上昇させることが明らかになった
Ser7 あるいは Thr12 へのアミノ酸置換を共に持つような二重変異体についても検討を行なった。これらの変異
合成ペプチドのズブチリシ
ンに対する阻害活性を図 1 にまとめた。
この図より、Ser7 については、Ala への置換は阻害活を上昇させたが、Lys や Val、
Phe に置換すると阻害活性が

図1

極端に低下することから、7 番目でのある程度の疎水性はズブチリシンとの相互作用に好ましいものの、それ以上の大きな側鎖は荷電基同様、ズブチリシンとの相互作用を弱めることがわかった。一方、Thr12 については、Leu への置換は野生型と変わらないものの、Lys や Arg への置換は阻害活性を上昇させることがわかり、12 番目については正電荷の存在がズブチリシンとの強い相互作用に有効であることが明らかとなった。そこで、Ser7
を Ala、Thr12 を Lys に置換した二重変異体（S7A＋T12K）を合成し、ズブチリシンに対する阻害活性を測定したところ、図１に示すように、これまで合成された変異ペプチドの中で最も強い阻害活性を示すことがわかった。
この二重変異体（S7A＋T12K）とズブチリシンの相互作用を分子レベルで明らかにするために、X 線結晶構造解析によって両者の複合体の立体構造を決定しようとした。ズブチリ
シンに対して過剰量の二重変異体（S7A＋T12K）を加え、ゲルろ過によって複合体を精製し、様々な溶液条件にて結晶化を試みた。しかしながら現時点では、構造解析ができるだ
けの結晶の生成には至っていない。

２．L-アミノ酸リガーゼの結晶構造解析ならびに特異性の改変の試み
遺伝暗号に基づいてリボソーム上で生合成されたタンパク質に対して翻訳後修飾によって側鎖の構造が変わったり、アミノ酸あるいはその誘導体がアミノ酸リガーゼによって連結されることによって、20 種類以外のアミノ酸がタンパク質あるいはペプチドに導入され、新機能を発揮している例が多く報告されている。タンパク質あるいはペプチドに 20 種類以
外のアミノ酸を取り込ませて新機能を持たせるため、枯草菌由来の L-アミノ酸リガーゼである YwfE タンパク質に注目し、立体構造解析およびそれに基づく変異導入による基質特異性の改変を試みている。YwfE タンパク質は非天然アミノ酸であるアンチカプシンと Ala
を ATP を用いて連結する反応を触媒する。このタンパク質と GST との融合タンパク質を大腸菌を用いて大量発現させ、アフィニティークロマトグラフィーで精製後、プロテアー
ゼで切断して得られた YwfE タンパク質を用いて結晶化を試みた。その結果、ADP と、リン酸あるいは L-Ala 存在下、PEG3350 を含む溶液条件で結晶が得られた。

178

そこで本研究では、結晶化条件の最適化によって高分解能での X 線結晶構造解析を行ない、それによって得られた立体構造に基づいて
YwfE タンパク質に変異を導入し、触媒反応に関わるアミノ酸残基の同定や、基質特異性の改変を試みた。
結晶化条件の最適化の結果、ADP とリン酸、
ADP と L-Ala 存在下での立体構造を、それぞれ
1.9Åおよび 2.0Åで決定することができた。図 2には ADP とリン酸を結合した YwfE タンパク質の全体の立体構造を示す。この立体構造から
YwfE タンパク質は A、B および C の 3 個のドメインから構成されており、ADP は B および C
ドメイン間に位置し、ATP－grasp superfamily
図2で保存されているアミノ酸残基の側鎖によって認識されていることがわかった。

また図 3 は、ADP と、リン酸あるいは L-Ala を結合している状態の基質結合部位近傍の構造を表している。これらの構造から、
Arg328 が基質である L-Ala のカルボキシル基の認識や、
触媒反応に強く関与していることが示唆された。
そこで、この Arg328
を他のアミノ酸に置換したところ、酵素活性は消失することがわかり、Arg328 の重要性が示された。
一方、この YwfE タンパク質がジペプ
チドを合成する際の N 末端側のアミノ酸として L-Ala を認識するのは、図 3 の基
質結合部位の構造から、Trp332 という側鎖の大きいアミノ酸が存在しているためであると考えられた。そこで Trp322 を側鎖の小さい Ala に置換し、様々なアミノ
図3酸を用いた時の酵素活性を測定した。その結果、
図 4 において△で示されたように、L-Ala が存在していなくても、L-Phe が存在しているだけで酵
素活性が検出された。このことは、N 末端側のアミノ酸として L-Phe が認識されたことを示している。実際に L-Phe-L-Phe のジペプチドが合成
図4

されることも確認した。

179そこで Trp332→Ala 変異体についても結晶化を行い、立体構造を決定した。得られた立体構
造の基質結合部位近傍の構造を図 5 に示す。その結果、N 末端側の基質として L-Phe を収容できるだけの大きな空隙が存在することがわかり、この空隙の出現によって、Trp332→Ala 変異体
において基質特異性が変化したものと考えられた。
さらに Trp332 を Ala 以外のアミノ酸に置換したところ、L-Phe のみが存在している時の酵素活性は、Ala 以外では Ser に置換した変異体のみに検出され、それ以上の大きさの側鎖のアミノ酸への置換では、Phe を基質として結合できないことがわかった。
以上のように、L-アミノ酸リガーゼである
YwfE タンパク質について、ADP、L-Ala などの基質存在下での立体構造を決定した。それに基づいて、基質の N 末端側の特異性を L-Ala に限定していると考えられる Trp332 を Ala に置換することにより、L-Ala だけでなくより大きな L-Phe を認識できるよう、基質特異性の改図5変に成功した。このことは、この酵素がさまざまなアミノ酸の連結反応に利用できうる可能性を示している。

[まとめ]
生体防御に関わるプロテアーゼに結合して活性を調整しうるペプチドを創製する方法としてのファージディスプレイ法によって得られた、ズブチリシンを阻害する 12 残基のペプ
チドについて、さらなる変異導入によって、より結合の強いペプチドの合成に至った。また、ペプチドに 20 種類以外のアミノ酸を導入しうる L-アミノ酸リガーゼである YwfE タン
パク質については、決定した立体構造に基づいて、L-Ala の認識に関わる Trp332 を Ala に置換することによって、より大きなアミノ酸を認識できるよう、基質特異性の改変に成功した。これらの方法を組み合わせることによって、新機能を有するペプチドの創製の可能性が広げられると考えられる。
[参考文献]
Tsuda, T., Suzuki, T. and Kojima, S. Crystallization and preliminary X-ray diffraction
analysis of Bacillus subtilis YwfE, an L-amino-acid ligase. Acta Crystal. F68, 203-206(2012).

180

[原著論文]1. Takei, T., Hasegawa, K., Imada, K., Namba, K., Tsumoto, K., Kuriki, Y.,
Yoshino, M., Yazaki, K., Kojima, S., Takei, T., Ueda, T. and Miura, K. Effects ofchain length of an amphipathic polypeptide carrying the repeated amino acidsequence (LETLAKA)n on α-helix and fibrous assembly formation. Biochemistry,
52, 2810-2820 (2013)
2. Tsuda, T., Asami, M., Koguchi, Y. and Kojima, S. A single mutation alters thesubstrate specificity of L-amino acid ligase. Biochemistry, 53, 2650-2660 (2014)
3. Takei, T., Tsumoto, K, Yoshino, M., Kojima, S., Yazaki, K., Ueda, T., Takei, T.,
Arisaka, F. and Miura, K. Role of positions e and g in the fibrous assembly
formation of an amphipathic α-helix-forming polypeptide. Biopolymers (Peptide
Science), 102, 260-272 (2014)
4. Takei, T., Tsumoto, K., Okonogi, A., Kimura, A., Kojima, S., Yazaki, K., Takei,
T., Ueda, T. and Miura, K. pH responsiveness of fibrous assemblies ofrepeat-sequence amphipathic α-helix polypeptides. Protein Science, 24, 883-894(2015)[口頭発表]

1.

2.
3.

津田岳夫、浅見真奈、小口孔明、小島修一、L-アミノ酸リガーゼ YwfE のアミノ酸置換による基質特異性の改変、第 66 回生物工学会大会、（2014 年 9 月 9－11 日、札幌）
津田岳夫、浅見真奈、小口孔明、小島修一、変異導入による L-アミノ酸リガーゼの基質特異性の改変、BMB2015、（2015 年 12 月 1－4 日、神戸）
林佳恵子、津田岳夫、小島修一、枯草菌ホスホエノールピルビン酸ムターゼ RhiHの結晶構造、BMB2015、（2015 年 12 月 1－4 日、神戸）

181Articlepubs.acs.org/biochemistry

Single Mutation Alters the Substrate Speciﬁcity of L‑Amino Acid
LigaseTakeo Tsuda,* Mana Asami, Yoshiaki Koguchi, and Shuichi Kojima
Department of Life Science, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, JapanS Supporting Information*

ABSTRACT: L-Amino acid ligase (Lal) catalyzes theformation of a dipeptide from two L-amino acids in an ATPdependent manner and belongs to the ATP-grasp superfamily.
Bacillus subtilis YwfE, the ﬁrst identiﬁed Lal, produces the
dipeptide antibiotic bacilysin, which consists of L-Ala and Lanticapsin. Its substrate speciﬁcity is restricted to smaller
amino acids such as L-Ala for the N-terminal end of the
dipeptide, whereas a wide range of hydrophobic amino acids
including L-Phe and L-Met are recognized for the C-terminal
end in vitro. We determined the crystal structures of YwfE withbound ADP-Mg2+-Pi and ADP-Mg2+-L-Ala at 1.9 and 2.0 Å resolutions, respectively. On the basis of these structures, we
generated point mutants of residues that are considered to participate in the recognition of L-Ala and measured their ATPase
activity. The conserved Arg328 is suggested to be a crucial residue for L-Ala recognition and catalysis. The mutation of Trp332 toAla caused the enzyme to hydrolyze ATP, even in the absence of L-Ala, and the structure of this mutant protein appeared to show
a cavity in the N-terminal substrate-binding pocket. These results suggest that Trp332 plays a key role in restricting the substratespeciﬁcity to smaller amino acids such as L-Ala. Moreover, Trp332 mutants can alter the substrate speciﬁcity and activity
depending on the size and shape of substituted amino acids. These observations provide suﬃcient scope for the rational design of
Lal to produce desirable dipeptides. We propose that the positioning of the conserved Arg residue in Lal is important forenantioselective recognition of L-amino acids.

M

Scheme 1

icroorganisms produce a wide range of biologically
active peptide compounds such as antibiotics, antitumors, and siderophores.1 Most of these compounds areproduced via two distinct biosynthetic strategies. The ﬁrst
strategy uses ribosomal synthesis of linear precursor peptides
that undergo posttranslational modiﬁcation and proteolysis.2
The second strategy uses nonribosomal peptide synthetase(NRPS), a large enzyme composed of multiple modules.3,4
Independent of these two pathways, several L-amino acid ligases(Lals), which are enzymes catalyzing the ATP-dependent
formation of an α-peptide bond between two L-amino acids,
have been identiﬁed in certain microorganisms.5−8 For
example, Bacillus subtilis YwfE (also referred to as BacD), theﬁrst identiﬁed and well-studied Lal,5 produces a dipeptide
antibiotic called bacilysin that consists of L-Ala at the Nterminus and the nonproteogenic amino acid, L-anticapsin, at
the C-terminus9 (Scheme 1). The substrate speciﬁcity of YwfE
is restricted to smaller amino acids such as L-Ala, for the Nterminal end of the dipeptide, whereas a wide range ofhydrophobic amino acids, such as L-Phe and L-Met, arerecognized for the C-terminal end. Despite the recognition of anonproteogenic L-amino acid as a substrate by YwfE, thisenzyme is not able to synthesize D-amino acid-containing
dipeptides (Scheme 1).
Lal belongs to the ATP-grasp superfamily of enzymes, whichcatalyze the formation of an amide bond between a carboxylatecompound and an amine compound in an ATP-dependent© 2014 American Chemical Society

manner.10,11 ATP-grasp enzymes share a similar overall foldcomposed of three domains (A, B, and C) and similar binding
modes for nucleotides. However, they accept a wide range ofcarboxylate and amine substrates, and the sequence andstructural conservation of the binding sites for these substrates
is much lower. The catalytic mechanism of the enzymes in thissuperfamily is considered to involve the initial activation of the
carboxyl group of a substrate by ATP-phosphorylation to form
an acylphosphate intermediate. This is followed by anucleophilic attack by the amine group of a second substrateon the acylphosphate to yield a tetrahedral transition state andthe release of phosphate by the tetrahedral intermediate to yielda product.
Received: January 22, 2014
Revised: April 4, 2014
Published: April 4, 2014
2650

182

dx.doi.org/10.1021/bi500292b | Biochemistry 2014, 53, 2650−2660

Biochemistry

Articlethe cells were harvested by centrifugation and suspended inphosphate-buﬀered saline (PBS) containing 1 mM dithiothreitol (DTT) and lysed by sonication. The lysate was centrifuged
at 10,000g for 20 min, and the supernatant was loaded onto a
Glutathione Sepharose FF column (GE Healthcare) preequilibrated with PBS containing 1 mM DTT. The GST-fused YwfE
was eluted with 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1
mM DTT, and 15 mM reduced glutathione. The GST tag wascleaved using PreScission protease (GE Healthcare), and the
reduced glutathione was removed by extensive dialysis against
50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA, and 1
mM DTT. Furthermore, the cleaved GST tag was subsequently
removed by chromatography on a Glutathione Sepharose FF
column (GE Healthcare). After concentration by ultraﬁltration
using a Vivaspin-20 10,000 MWCO (Sartorius), the ﬂowthrough fraction was loaded onto a HiLoad 16/60 Superdex 75
pg column (GE Healthcare) preequilibrated with 50 mM TrisHCl (pH 8.0) and 100 mM NaCl. Fractions containing pure
YwfE were pooled and dialyzed against 10 mM Tris-HCl (pH8.0) containing 1 mM DTT and concentrated to approximately
20 mg/mL by ultraﬁltration using a Vivaspin-20 10,000
MWCO (Sartorius). The protein concentration was determined by the BCA method (Pierce) using bovine serum
albumin as a standard.
Crystallization and Data Collection. Crystallization
screening was performed by the hanging-drop vapor-diﬀusion
method at 20 °C.16 Drops were prepared by manually mixing 2
μL of deletion mutant (Lys4−Tyr468) of YwfE protein
solution (8 mg/mL) containing 10 mM ADP, 10 mM MgCl2(or 5 mM MnCl2), and 10 mM sodium phosphate (pH 7.4)
and 2 μL of reservoir solution containing 16%−18% (w/v)
PEG 3350, 0.3 M NaCl, 0.1 M HEPES-NaOH (pH 7.0−7.25),
5% (v/v) ethylene glycol, and 1 mM DTT and were
equilibrated against 500 μL of reservoir solution. Crystals of
the complex with ADP-Mg2+-L-Ala were grown using the same
method except that 100 mM L-Ala was added instead of sodium
phosphate. The crystallization conditions of the W332A mutant(Lys4−Tyr468) were the same as those for the wild type.
Diﬀraction data were collected at the BL5A, AR-NE3A, and
AR-NW12A beamlines at the Photon Factory (PF; Tsukuba,
Japan). Ethylene glycol (20%, v/v) was used as a cryoprotectant
for data collection under cryogenic conditions. The crystals
were soaked in cryoprotectant-containing mother liquor andﬂash-cooled in nitrogen stream at 100 K. Mn2+-anomalous dataof the Mn2+-derivative crystals were collected at a wavelength of
1.2 Å. The diﬀraction data sets were processed using the
HKL2000 program.17 All of the crystals belonged to the spacegroup P6522, with one molecule in the asymmetric unit.
Structure Determination and Reﬁnement. Preparationof the SeMet-labeled crystals and data collection using a singlewavelength anomalous dispersion (SAD) method werepreviously described.16 To determine the structure of YwfE,
SAD phasing, density modiﬁcation, and automatic modelbuilding were performed at 2.8 Å using the CRANK program18
from CCP4.19 All of the eight selenomethionine sites in theasymmetric unit were identiﬁed with suﬃcient quality. An
initial solvent-ﬂattened electron density map for the polyalanine
chains was modeled with the automated model building
program ARP/wARP,20 followed by manual ﬁtting using
Coot.21 Reﬁnement was performed by alternating cycles ofmanual rebuilding using Coot and reﬁnement using CNS22 andRefmac5 including TLS reﬁnement.23 All crystal structures
were determined starting from the atomic model obtainedSome of the ATP-grasp enzymes are considered to beimportant targets for the development of new antibacterial
drugs because of the critical nature of these enzymes and lack ofhuman orthologues. Although Lal is not a clinical target, it has
the potential to become an interesting target for the industrial
production of useful dipeptides. Dipeptides have unique
physicochemical properties and physiological functions. For
example, the artiﬁcial sweetener aspartame is L-aspartyl-Lphenylalanine methyl ester (L-Asp-L-Phe-OMe). L-Ala-L-Gln isused as a component of patient infusions because of its higher
solubility than L-Gln. L-Asp-L-Phe, a precursor of aspartame,
was biosynthesized by hybrid NRPS; however, the turnover rate
was extremely low (0.7 min−1).12 Compared with the NRPS
system, Lal is a smaller soluble enzyme and participates in a
simpler reaction to combine two amino acids. An L-Ala-L-Gln
has reportedly been produced via fermentative processes using
YwfE-transformed bacteria.13 However, biochemical andstructural studies of Lal are extremely limited compared withthose of other members of the ATP-grasp superfamily.
The crystal structures of two Lals have recently beenreported. The ﬁrst determined structure was that of YwfE withbound ADP and phosphinophosphate analogue (P-analogue),
which was believed to mimic the tetrahedral transition state.14
The structure showed that YwfE binds the dipeptide in thereverse orientation to that observed in Ddl, suggesting that thesubstrate entry mode diﬀers between them. The other structure
was BL00235 from Bacillus licheniformis, which was bound to
ADP and Ca2+ ions.15
Here, we describe new YwfE crystal structures with bound
ADP-Mg2+-Pi and ADP-Mg2+-L-Ala that are reﬁned to 1.9 and2.0 Å resolutions, respectively. The results suggest that Trp332
plays a key role in N-terminal substrate speciﬁcity for smaller
amino acids such as L-Ala. Substitution with other residues at
position 332 altered the substrate speciﬁcity depending on thesize and shape of the incorporated residues. These observations
aﬀord the possibility of a rational design of YwfE for the
production of desirable dipeptides. Furthermore, we proposethat based on a structural comparison between Lal and Ddl, thepositioning of Arg328 is important for enantioselective
recognition of L-amino acids.

■

EXPERIMENTAL PROCEDURES
Protein Preparation. Cloning, expression, and puriﬁcationof Bacillus subtilis YwfE were performed as previously
described,16 with slight modiﬁcations. The gene encoding thefull-length protein (Met1−Val472) and deletion mutant(Lys4−Tyr468) of YwfE were ampliﬁed using PCR from B.
subtilis genomic DNA. The ampliﬁed PCR products werecloned into the pGEX-6P-1 expression vector (GE Healthcare)with BamHI and XhoI sites to produce the fusion protein with
N-terminal GST. Mutations were introduced according to the
QuikChange site-directed mutagenesis procedure using oligonucleotide primers, except that KODplus (TOYOBO)
polymerase was used for PCR. The DNA sequence of theexpression plasmid was conﬁrmed by automated DNA
sequencing.
The Escherichia coli strain ArcticExpress RIL cells (DE3)(Stratagene), containing the expression plasmid, were grown at
37 °C in a Luria−Bertani medium containing 50 μg/mL
ampicillin. When the optical density at 600 nm of the culture
reached approximately 0.8, expression was induced by theaddition of isopropyl β-D-thiogalactopyranoside to a ﬁnal
concentration of 0.3 mM. After an 18-h cultivation at 25 °C,
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ArticleTable 1. Diﬀraction Data and Reﬁnement Statistics of YwfE Crystalsa
crystalwavelength (Å)
beamlinespace groupcell a = b (Å)
c (Å)
resolution (Å)
completeness (%)
redundancy
I/σ (I)
Rmerge (%)
resolution (Å)
number of reﬂections
Rwork/Rfree (%)
number of atoms
proteinligands
water
overall B-factor (Å2)
proteinligandswater
rmsd
bond lengths (Å)
bond angles (deg)Ramachandranb (%)
PDB codewild-typewild-type

W332A mutant

ADP-Mg2+-Pi

ADP-Mg2+-L-Ala

ADP-Mg2+-L-Ala

1.000
PF BL-5A
P6522
90.58
252.6520−1.9 (1.95−1.90)
88.9 (96.5)
10.6 (11.1)
53.5 (9.9)
0.069 (0.312)
20−1.941 60619.90/24.30
4056
3626
34
396
49.6
49.7
43.1
49.2

1.000
PF AR-NW12A
P6522
90.02
249.8020−2.0 (2.05−2.00)
98.3 (99.4)
9.0 (9.3)
53.5 (8.3)
0.068 (0.350)
20−2.038 65619.74/23.59
3872
3626
35
211
47.0
47.4
42.2
40.7

1.000
PF AR-NW12A
P6522
91.13
258.7620−2.30 (2.35−2.30)
98.1 (100.0)
7.6 (7.7)
55.6 (7.6)
0.038 (0.322)
20−2.324 08922.36/26.14
3745
3623
35
87
73.9
74.3
93.6
51.3

0.012
1.3
90.1/9.7/0.2/0
3WNZ

0.011
1.3
89.4/10.4/0.2/0
3WO0

0.011
1.3
88.0/12.0/0/0
3WO1

aValues in parentheses are for the highest-resolution shells. bFractions of residues in most favored/additionally allowed/generously allowed/
outliners of the Ramachandran plot according to Procheck24 in the CCP4 suite.19

using the SAD data set. Procheck24 was used for Ramachandrananalysis. Statistics of the diﬀraction data and reﬁnement arelisted in Table 1 and Supporting Information, Table S1. Allprotein structure ﬁgures were prepared using PyMol.25
Measurement of ATPase Activity. ATPase activities ofwild-type and mutated YwfE were measured using the Fiske−
Subbarow method26 to colorimetrically quantify the liberated
Pi. Standard ATPase assays were performed at 37 °C in 100 μLof the assay buﬀer containing 10 mM L-Ala; 30 mM L-Phe (or50 mM L-Met); 100 mM Bicine-NaOH (pH 9.0); 10 mM
MgCl2; 10 mM ATP; and 2.5, 5.0, 7.25, 10, or 15 μg/mL
puriﬁed full-length YwfE in a 96-well assay plate. Reaction times(between 10 and 45 min) and concentrations of protein(between 2.5 μg/mL and 15 μg/mL) used in the assay werevaried depending on the degree of activity. Reactions werestarted by the addition of 10 μL of 100 mM ATP and stopped
by the addition of 50 μL of stop solution containing 10% TCA
and 0.1% SDS. Furthermore, 100 μL of the molybdate solution(0.75% ammonium molybdate in 0.675 M H2SO4) followed by
10 μL of reducing solution (0.1% 1-amino-2-naphthol-4sulfonic acid, 1.2% NaHSO3, and 1.2% Na2SO3) were added
and incubated at 37 °C for 10 min. The amount of released Pi
was determined by measuring absorbance at 665 nm using a
BiotracII plate reader (GE Healthcare) in a 96-well format. The
absorbance of a blank, measured in the condition without
protein, was subtracted. Each ATPase activity of the wild-type
protein in the presence of 10 mM L-Ala or 30 mM L-Phe alone
exhibited activity similar to the background. Pi standards wereprepared by dissolving KH2PO4 in the buﬀer. To determine thekinetic parameters, the velocity versus substrate concentration

data were subjected to nonlinear least-squares analysis using the
Michaelis−Menten equation, v = Vmax [S]/([S] + Km), using
the SigmaPlot software. All data are presented as the mean ±standard deviation of at least three independent experiments.
Analysis of Dipeptide by HPLC. The reactions were
performed at 37 °C in 500 μL of the assay buﬀer containing 50
mM L-Phe; 100 mM Bicine-NaOH (pH 9.0); 10 mM MgCl2;
10 mM ATP; and 10 μg of puriﬁed full-length W332A mutant.
After an 18-h incubation, the reactions were stopped by theaddition of 0.1% triﬂuoroacetic acid, and 250 μL of the reaction
mixtures were subjected to a reverse-phase HPLC (C18 column:
250 mm × 4.6 mm; Kaseisorb LC ODS-120−5, Tokyo
Chemical Industry, Japan) with a gradient of acetonitrile in
0.1% triﬂuoroacetic acid at a ﬂow rate of 0.8 mL/min (elution
program: 0 to 15 min, 0% of CH3CN; 15 to 39 min, a linear
increase in CH3CN from 0% to 32%; 39 to 40 min, a linear
increase in CH3CN from 32% to 80%). Absorbance wasmonitored at 230 nm. An authentic sample of L-Phe-L-Phe(Sigma-Aldrich) was used as a standard.

■

RESULTS AND DISCUSSION
Crystal Structures of YwfE in Complex with ADPMg2+-Pi and ADP-Mg2+-L-Ala. To elucidate the substratebinding mode and reaction mechanism of L-amino acid ligase(Lal), we prepared native and Se-Met derivative crystals of B.
subtilis YwfE in the presence of ADP, Mg2+, and L-Ala-L-Gln
dipeptide.16 Although no electron density corresponding to thedipeptide was observed, an unexplained tetrahedral-shaped
piece of weak electron density appeared over the β-phosphategroup of bound ADP. We assumed this to be an inorganic
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ArticleFigure 1. Crystal structure of YwfE from Bacillus subtilis. (A) Overall structure of YwfE with bound ADP-Mg2+-Pi. ADP and Pi molecules arepresented as a stick model. Two Mg2+ ions are presented as green spheres. (B) Oligomeric state of YwfE with bound ADP-Mg2+-Pi. The purple
broken circles represent the extended loop region in the A-domain. (C) Close up view of the dimer interface. Two protomers are colored yellow andgreen. Side chains involved in important interactions in the dimer interface are presented as stick models. The blue broken lines represent theelectrostatic interactions between protomers. The ﬁgure was prepared using PyMol.25

the interface was stabilized by electrostatic and hydrophobic
interactions of several residues in the α2-helix, the β3-strand,
and the extended loop between the β3-strand and α3-helix.
This type of β-strand-mediated dimer interaction was observedin the human, yeast, and Trypansoma brucei glutathione
synthetase (GS) structures, which belong to the ATP-grasp
superfamily.27−29 The dimer interface of T. brucei GS was
calculated to cover 7.5% of the total surface area of a
protomer.29
Active Site Structures of YwfE. A Fourier diﬀerence (|Fobs| − |Fcalc|) map before the introduction of ADP, Mg2+, andPi into the atomic model clearly showed the bound nucleotide
in the cleft between the B-domain and C1-subdomain (Figure2A). ADP is recognized by conserved residues in a manner
similar to that observed in other ATP-grasp enzymes. To
conﬁrm the location and number of bound Mg2+ ion(s), an
Mn2+ derivative was prepared by cocrystallization with MnCl2
instead of MgCl2 (Table S1, Supporting Information). An
anomalous Fourier map clearly showed the location of two
Mn2+ beside the β-phosphate group of ADP. Therefore, we
modeled two Mg2+ ions in the ADP-Mg2+-Pi-bound structure.
One of the Mg2+ ions labeled “Mg1” in Figure 2 is coordinatedby oxygen atoms of the α- and β-phosphate groups of ADP,
Glu311, Glu324, Pi, and one water molecule (Figure 2B and
C). The other Mg2+ labeled “Mg2” in Figure 2 is coordinatedby oxygen atoms of the β-phosphate group of ADP, Glu324, Pi,
and two water molecules. The bidentate carboxyl group of
Glu324, which is the highly conserved residue in ATP-grasp
enzymes, bridges two Mg2+ ions, suggesting that it plays acrucial role in catalysis. For both Mg2+ ions, the metal−ligand
distances range from 1.8 and 2.4 Å. In the ADP-Mg2+-L-Alabound structure, the location and number of bound Mg2+ ionswere also conﬁrmed by the Mn2+ derivative. Two Mg2+ ions areoctahedrally coordinated by the same ligands as observed in thephosphate ion (Pi), which may possibly be derived from the
puriﬁcation procedure. To obtain clear electron density, we
attempted to crystallize YwfE in the presence of ADP, Mg2+,
and Pi instead of L-Ala-L-Gln. The structure of YwfE withbound ADP, Mg2+, and Pi was determined and reﬁned to 1.9 Å
resolution (Table 1). In addition, we determined the structureof YwfE with bound ADP, Mg2+, and L-Ala at 2.0 Å resolution.
However, we have not yet determined the crystal structure withbound L-Ala-L-Gln. The YwfE protein used in this study
consists of residues Lys4−Tyr468, which lack the N- and Cterminal short regions, so as to improve the quality of crystals,
as previously reported.16 The overall structure consists of threelarge A-, B-, and C-domains from the N terminus, and the Cdomain is further divided into C1- and C2-subdomains ﬂanked
by a large antiparallel β-sheet (Figure 1A). The two structureswith ADP, Mg2+, and Pi or L-Ala were extremely similar, with aroot-mean-square deviation (rmsd) of 0.23 Å for all Cα atoms.
These structures were similar to those of the earlier full-length
YwfE (Glu2−Val472) with bound ADP and phosphorylated
phosphinate L-Ala-L-Phe analogue (P-analogue)14 with an rmsd
of 0.48 Å for 464 Cα atoms, indicating that the deletion ofshort peptides at both termini exerts little inﬂuence on theoverall fold. Moreover, the present structures were similar toanother available structure of Lal, BL00235, from B. licheniformis15 with an rmsd of 2.75 Å for 365 structurally equivalentCα atoms.
Size exclusion gel ﬁltration analysis under low ionic strength
conditions has shown dimer formation of YwfE in a solution,16
and two protomers in the crystals formed a dimer through acrystallographic 2-fold axis (Figure 1B). Because the buried
surface area of the dimer interface was calculated to cover only
6% of the total surface of the protomers, either protomer wastightly connected by extension of the central β-sheet over theadjacent protomer in the A-domain (Figure 1C). In addition,
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ArticleFigure 2. Details of the ligand-binding sites of YwfE. (A) Side chains important for ligand binding are presented as stick models. The cyan net
represents a simulated omit map contoured at 2.5σ of ADP-Mg2+-Pi. The red net represents an anomalous Fourier diﬀerence map for Mn2+ of ADPMn2+-Pi-bound form contoured at 10σ. Two Mg2+ ions are labeled with Mg1 and Mg2. The blue broken lines represent hydrogen bonds. (B) Stereo
view of the Mg2+-Pi-binding site. The red spheres represent Mg2+-coordinating water molecules. The green broken lines represent the coordinationof Mg2+ ions. The close-up views of (C) ADP-Mg2+-Pi-; (D) ADP-Mg2+-Ala-; and (E) ADP-Mg2+-P-analogue (PDB entry 3VMM)-bound structuresare shown in the same view. The cyan net in D represents a simulated omit map contoured at 5σ of L-Ala.

ADP-Mg2+-Pi-bound structure except for substitution of oxygen
atoms from a water molecule with those from Pi (Figure 2C).
In contrast, in the earlier P-analogue-bound YwfE structure,
Mg2 was located at a diﬀerent position and coordinated by themain-chain carbonyl group of Leu182 and the side chain of
Glu10914 (Figure 2E). A water molecule occupied the same
position in our structure (Figure 2C and D). In addition, it
should be noted that the present structure showed the side
chain of Tyr75 pointed on the opposite side into the active site,
although the earlier study suggested that Tyr75 may function asa catalytic base for the deprotonation of the amino group of Lanticapsin, which is a physiological C-terminal end substrate of
YwfE14 (Figure S1, Supporting Information).
The bound Pi is stabilized by hydrogen bonds with the mainchain amide group of Ser184 in the B-domain and the side
chains of Glu311, Glu324, and Arg328 in the C1-subdomain(Figure 2B); therefore, Pi bridges these domains. In the ADPMg2+-L-Ala-bound structure, the omit map clearly showed thatthe bound L-Ala was extensively recognized by the YwfE
protein (Figure 2D). Both the oxygen atoms of the bound L-Ala
are involved in bidentate electrostatic interactions with theguanidinium group of the side chain of Arg328, and one of theoxygen atoms is hydrogen bonded with the main-chain amidegroup of Gly331. The nitrogen atom of L-Ala has electrostaticinteractions with the side chains of Glu273, His309, andGlu311. In addition, His309 makes van der Waals contacts withcarbon and oxygen atoms of L-Ala. These residues are highly
conserved among Lal, suggesting an important role in catalysis.

The Cβ atom of L-Ala makes van der Waals contacts with
Glu273, Trp332, and Met324. It is likely that these threeresidues determine the substrate speciﬁcity of YwfE for aminoacid with the small side chain in the N-terminal substratebinding pocket.
Functional Analysis by Site-Directed Mutations. It has
been reported that YwfE prefers L-Ala for the N-terminal end
and hydrophobic L-Phe or L-Met for the C-terminal end.5,14 In
the functional assay, we used the full-length YwfE (Met1−
Val472) protein, and ATPase activity was measured in the
combination of L-Ala and L-Phe or L-Met. For the wild-type
enzyme, the ATPase activities increased with increasing
concentrations of each amino acid, and the obtained curves
were well ﬁtted with the Michaelis−Menten equation (Figure
S2, Supporting Information). The apparent Km and kcat values
for Ala (1.64 ± 0.45 mM and 9.32 ± 0.89 s−1), Phe (5.23 ±1.59 mM and 10.4 ± 1.19 s−1), and Met (11.3 ± 2.69 mM and
8.77 ± 0.96 s−1) are similar to those previously reported.14 The
presence of either L-Ala or L-Phe did not show any activity. Theaﬃnity for L-Ser was 15-fold lower compared with that of L-Ala,and total activity was decreased to approximately half, which
conﬁrms that L-Ala is a suitable substrate for the N-terminal
end.
Furthermore, on the basis of the present crystal structure,mutational analysis was performed to elucidate the contributionof each amino acid residue to substrate recognition andcatalysis. ATPase assays were performed using the full-lengthmutants YwfE (Met1−Val472), and the obtained kinetic
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ArticleTable 2. Eﬀect of Mutations on ATPase Activitya
YwfE

kcat (s−1) for Ala

Km (mM) for Ala

kcat /Km (mM −1s −1) for Ala

kcat (s−1) for Phe

Km (mM) for Phe

kcat /Km (mM −1s −1) for Phe

wild-type
E109A
E109D
E273A
E273D
H276A
H309A
H309R
E311A
E311D
R328A
R328 K
W332A
M334A

9.32 ± 0.89
<0.01
3.19 ± 1.43
NDb
0.52 ± 0.26
6.63 ± 1.34
ND
ND
ND
<0.01
ND
ND
4.32 ± 0.39
2.62 ± 1.16

1.64 ± 0.45

6.07 ± 1.80

5.23 ± 1.59

2.13 ± 0.58

7.12 ± 3.19

0.48 ± 0.14

2.28 ± 0.96

0.91 ± 0.39

17.06 ± 9.37
7.72 ± 1.87

0.03 ± 0.00
0.90 ± 0.26

7.71 ± 2.58
8.50 ± 0.70

0.08 ± 0.05
0.51 ± 0.05

2.12 ± 0.31
2.96 ± 1.82

2.07 ± 0.26
0.95 ± 0.17

10.4 ± 1.19
<0.01
1.83 ± 0.20
ND
0.49 ± 0.11
4.36 ± 0.66
ND
ND
ND
<0.01
ND
ND
4.83 ± 0.55
2.33 ± 0.41

15.9 ± 2.35
3.70 ± 1.12

0.31 ± 0.08
0.65 ± 0.08aReactions were performed as described in the Experimental Procedures section. The kinetic parameters were determined by the varying
concentrations of either L-Ala or L-Phe. All kcat and Km values are shown as the mean ± standard deviation of at least three independent experiments.
b
ND means not detectable.

Ala. The activity at 50 mM L-Phe alone increased toapproximately 20% of that in the presence of both L-Ala and
L-Phe (Figure 3B). This L-Ala-independent activity was reduced
at lower concentrations of L-Phe. Surprisingly, the ATPase
activity increased with increasing concentrations of L-Met toalmost the same levels regardless of L-Ala. The activity curve
did not ﬁt the Michaelis−Menten equation in the absence of LAla but did so in the presence of L-Ala. This observationsuggests that L-Met is bound within two distinct binding
pockets with diﬀerent aﬃnities in the absence of L-Ala. It wasdiﬃcult to determine the apparent Km value of each aﬃnity.
To conﬁrm the formation of an L-Phe-L-Phe productcatalyzed by the YwfE W332A mutant, we performed reversephase HPLC analysis on a C18 column. The W332A mutant(10 μg) was incubated with 50 mM L-Phe in the presence of 10
mM ATP and 10 mM MgCl2, and the reaction product in 250
μL was identiﬁed using a reference standard of an authentic LPhe-L-Phe (2.5 μmol). The HPLC analysis showed that theW332A mutant enzyme produced about 1.4 μmol of the L-PheL-Phe dipeptide (Figure 4A and B). If all of the ATP moleculesin the reaction mixture were consumed in order to produce thedipeptide, 2.5 μmol of the dipeptide would be detected by the
HPLC analysis. The peaks that appeared before elution byacetonitrile corresponded to ATP (or ADP) and L-Phe (Figure
S3A and B, Supporting Information). When the reaction was
performed in the absence of ATP, the dipeptide was notobserved (Figure S3C, Supporting Information). Thus,
substitution of Trp332 with Ala will alter the substratespeciﬁcity of YwfE to allow the binding of larger amino acids,
such as L-Phe, in the N-terminal substrate-binding pocket, andcatalyzes the formation of the L-Phe-L-Phe dipeptide.
Crystal Structure of the W332A Mutant. To elucidate
the role of Trp332 and the eﬀects of its mutation on the
structure of the substrate-binding pocket, we determined the
crystal structure of the YwfE mutant W332A (Lys4−Tyr468)
with bound ADP, Mg2+, and L-Ala at 2.3 Å resolution (Table 1).
The overall structure of the mutant was similar to that of thewild type with an rmsd of 1.2 Å for all Cα atoms. In addition,
the mutant binds ADP and two Mg2+ in the same mode as thatobserved in the wild type. These ﬁndings indicate that mutation
does not inﬂuence the overall fold or the nucleotide binding
property. However, a large cavity seems to appear in theparameters are listed in Table 2. The mutation of each Glu273,
His309, Glu311, and Arg328 residue to Ala almost completely
diminished the activity, which is consistent with the structurebased predictions. An E273D mutation partially restored theactivity, whereas H309R, E311D, and R328K enzymes
remained inactive despite the similar charge-conserved
mutations. These ﬁndings indicate that His309, Glu311, andArg328 are the crucial residues for catalysis. It appears that theside chains of His309 and Arg328 along with the main-chain
amide group of Gly331 form an oxyanion hole to stabilize thephosphorylated intermediate during the reaction cycle. The
enzyme with substitution of Glu109 by Ala showed no activity,
whereas that with substitution by Asp reduced the activity byapproximately 30% compared with that of the wild type. The
carboxyl group of Glu109 forms a hydrogen bond with the Nεatom of Arg328 (Figure 2C and D). In addition, at the opposite
side, the main-chain carbonyl group of Phe329 forms ahydrogen bond with Arg328. These observations suggest thateach residue plays an important role in ﬁxing the side chain of
Arg328 in the optimal conformation for L-Ala binding andcatalytic reaction. An H276A mutation largely decreased theapparent Km value for L-Ala approximately 5-fold but exerted
little inﬂuence on the apparent Km value for L-Phe, suggestingthat His276 can contribute to L-Ala recognition, although it
does not directly contact L-Ala. Mutation of Trp332 to Ala
caused a decrease in catalytic eﬃciency (kcat/Km) for both L-Ala
and L-Phe compared with that by the wild type. In addition,
M334A mutation decreased the activity approximately 3-foldbut did not strongly aﬀect the apparent Km value for eachamino acid. Although Met334 is located in contact with the side
chain of bound L-Ala, it is unlikely that this residue is essential
for L-Ala binding.
L-Ala-Independent ATPase Activity of Trp332 Mutants. The W332A mutant appeared to retain considerable
ATPase activity even in the absence of L-Ala (Figure 3A). In
contrast, the addition of L-Ala alone to the reaction mixture
showed no activity. It suggests that the mutant could accept LPhe as an N-terminal end and may produce an L-Phe-L-Phe
dipeptide, albeit at an extremely low turnover rate. To
investigate the details of the L-Ala-independent ATPase activity
observed in this W332A mutant, we measured ATPase activity
at varying concentrations of L-Phe or L-Met in the absence of L2655

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ArticleFigure 4. HPLC chromatograms. (A) 250 μL of the reaction productsof the W332A mutant were separated by HPLC. The absorbance wasmonitored at 230 nm (left axis). The dotted lines indicate a linear
gradient of acetonitrile (right axis). (B) An L-Phe-L-Phe standard (2.5μmol) was separated by HPLC under the same conditions. The peakarea values of the enzymatic reaction (A) and the standard (B) were
estimated to be 38.23 AU × s and 66.56 AU × s, respectively, by using
Clarify Lite software (DataApex).

Figure 3. ATPase activity of W332A mutant. (A) The L-Ala
concentration dependency of the ATPase activity of the W332A
mutant was measured in the presence of 30 mM L-Phe (●). The curve
was ﬁtted to the Michaelis−Menten equation using the SigmaPlot
software. The errors correspond to the standard deviation of at least
three independent experiments. (B) The L-Phe (▲, △) or L-Met (■,□) concentration dependency of the ATPase activity was measured in
the presence (closed symbols) or absence (open symbols) of 10 mM
L-Ala. The curves could be ﬁtted to a Michaelis−Menten equation
when the measurements were performed in the presence of both L-Ala
and L-Phe or L-Met.

The structural and functional properties of W332A mutant
YwfE remind us of those of BL00235. The crystal structure of
BL00235 with bound ADP-Ca2+ showed a large hydrophobic
cavity within a putative N-terminal substrate-binding pocketthat is formed by the side chains on both C1- and C2subdomains15 (Figure 5C). The substrate speciﬁcity ofBL00235 is entirely diﬀerent from that of YwfE. It prefers LMet and L-Leu for the N-terminal end and small substrates suchas L-Ala, L-Ser, L-Thr, and L-Cys for the C-terminal end. It canbe assumed that introduction of a large residue, such as Trp,
into the entrance of the cavity of BL00235 would alter thesubstrate speciﬁcity for small amino acids, as is the case for
YwfE. We have not yet obtained the crystal structure of the LMet-bound form of the W332A mutant, although crystals wereobtained in the presence of 10 mM ADP, 10 mM Mg2+, and100 mM L-Met. In the mutant crystal, the electron density
corresponding to the expected side chain portion of L-Met was
extremely weak, suggesting that the side chain of L-Met wasdisordered and did not interact strongly with the enzyme. This
result is probably because of the lower aﬃnity of the mutatedenzyme for L-Met than for L-Ala, as expected from thefunctional assay.
Eﬀect on the Substrate Speciﬁcity of Mutations at
Position 332. To determine whether the size and shape ofmutant, due to the absence of the large indole ring of Trp332(Figure 5A). This cavity is formed by the side chains of aminoacid residues not only on the C1-subdomain (His276, Ala332,
and Met334) but also on the C2-subdomain (Asp376, His378,
and Tyr380). A Fourier diﬀerence (|Fobs| − |Fcalc|) map before
the introduction of L-Ala into the atomic model showed that
the L-Ala bound in the same mode as that observed in the wild
type. The model shows a large space over the Cβ atom ofbound L-Ala, suggesting that this space is large enough toaccommodate a larger amino acid such as L-Met. Thus, the large
and rigid indole ring of Trp332 appears to restrict the substratespeciﬁcity for smaller amino acids such as L-Ala in the Nterminal substrate-binding pocket. The orientation of His378
was rotated by approximately 90° compared with that of thewild-type structure. This is because the planar surface of
Trp332 is almost parallel to the imidazole ring of His378 via
stacking interactions in the wild type (Figure 5B).
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Articledecreased the activity approximately 70% to 40% comparedwith that of the wild type (black bars, Figure 6A). However,

Figure 6. Eﬀect of mutations at Trp332 on the L-Ala-independent
ATPase activities. (A) The black and white bars represent 50 mM LPhe-induced ATPase activities in the presence and absence of 10 mM
L-Ala, respectively. The errors correspond to the standard deviations ofat least three independent experiments. (B) The same measurements
were performed except that 50 mM L-Met was used instead of L-Phe.

mutation of Trp332 to an aromatic Phe largely reduced theactivity to >10% compared with that observed in the wild type.
It is likely that a 6-member phenyl ring introduced at position
322 collides with the surrounding residues, such as Glu273 andHis276, and inﬂuences the local structure within the substratebinding pocket. When the same measurements were performedin the presence of L-Met instead of L-Phe, the results obtained
were similar (black bars, Figure 6B). These ﬁndings indicatethat the Trp residue at position 332 is not directly essential forthe catalytic reaction.
Furthermore, to test each mutation on L-Ala-independent
ATPase activity, the measurements were performed in thepresence of 50 mM L-Phe or L-Met alone. Although the wildtype enzyme hardly showed any activity in the presence of 50
mM L-Phe alone, W332A and W332S retained activities ofapproximately 20% and 30%, respectively, compared with thosein the presence of both L-Ala and L-Phe (white bars, Figure 6A).
In contrast to these mutants, each mutant with substitution by
Val, Leu, His, or Phe did not show any L-Ala-independent
activity. When L-Met was used instead of L-Phe, even the wildtype enzyme retained approximately 6% of the L-Ala-dependent

Figure 5. Molecular surface representation of the N-terminal
substrate-binding site. (A) Structure of the W332A mutant of YwfE
with bound ADP-Mg2+-L-Ala. The cyan net represents a simulated
omit map contoured at 3σ of L-Ala. (B) Structure of the wild-type
YwfE with bound ADP-Mg2+-Ala. The atom-type and orange nets
represent van der Waals surfaces of bound L-Ala and the side chain of
Trp332, respectively, in the wild-type ADP-Mg2+-L-Ala-boundstructure. (C) The structure of the expected N-terminal substratebinding region of BL00235 with bound ADP-Ca2+ (PDB entry
3VOT). The numbers in parentheses represent the corresponding
residues in YwfE.

amino acid residues at position 332 inﬂuence the L-Alaindependent ATPase activity, we prepared ﬁve additional
Trp332 mutants, replacing Trp332 with Ser, Val, Leu, His, orPhe. First, we compared the eﬀect of mutations on the ATPase
activity under standard conditions (the presence of L-Ala and LPhe). Substitutions of Trp332 with Ala, Ser, Val, Leu, or His
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Biochemistry

Articlecarbonyl carbon of the acylphosphate intermediate to form atetrahedral transition state intermediate, which is stabilized by
the positive charge environment formed with His309, Arg328,
and the main-chain amide group of Gly331. Arg328 and thetwo Mg2+ ions assist the phosphate transfer and compensate
the resulting negative charge in each intermediate. The
phosphate is ﬁnally released from the acylphosphate to yield
the L-Ala-L-Phe dipeptide (bacilysin in vivo), and ADP is also
released from the nucleotide-binding site.
Role of Conserved Arg. In the catalysis and L-Ala
recognition, the crucial residue appears to be Arg328. This
Arg is conserved among the other members of the ATP-grasp
enzymes. For example, the crystal structure of E. coli biotindependent carboxylase (BC) shows that bound bicarbonate isrecognized by bidentate interactions with the side chain of
Arg292.30 The structure of E. coli PurK, one of the purine
biosynthesis enzymes, in complex with ADP-Mg2+-Pi, showsthat conserved Arg242 recognizes the Pi molecule.31 In the E.
coli PurT structure, which is an enzyme participating in thepurine biosynthetic pathway, Arg283 is located in theequivalent position to Arg242 in PurK.32 In addition, thepresent study reveals that Arg328 plays an important role in thebinding of L-Ala and Pi molecules. Amino acid sequencealignment with these enzymes indicates that the Mg1coordinating Glu residue, which is also highly conserved inthis family, is located four residues upstream from these Arg
residues (Figure 8A). Although Ddl contains an Arg residue,
which is crucial for D-Ala recognition and catalysis, this residue
lies at a diﬀerent position compared with those in other ATPgrasp enzymes. In E. coli DdlB,33 Arg255 lies two residues
downstream of another conserved Asp or Glu residue thatcoordinates Mg2. This Arg position is highly conserved among
Ddl family members. YwfE and BL00235 contain important His
residues, although the other members of the ATP-grasp
superfamily carry uncharged residues at this position.
To compare the locations of the conserved Arg residues
between Lal and Ddl, we superimposed the crystal structures of
ADP-Mg2+-L-Ala-bound YwfE and ADP-Mg2+-D-Ala-bound Ddlfrom Thermus thermophilus HB8.34 The structures were similar
to an rmsd of 2.70 Å for structurally equivalent 255 Cα atoms.
The model also showed that ADP and two Mg2+ occupy similar
positions in each structure. As expected from the sequencealignment, the model clearly indicates that each Arg residue lies
in a diﬀerent position (Figure 8B). Both side chains of Arg328
in YwfE and Arg268 in TtDdl recognize each carboxyl group of
bound L-Ala or D-Ala from the opposite side. As a result, the Ala
molecules are bound by the enzyme in nearly reverse
orientations. The diﬀerent positioning of conserved Arg
residues between Lal and Ddl will consequently cause thediverse orientation of the N- and C-terminal substrate-binding
pockets in each enzyme. Despite the diﬀerence in theorientation of bound Ala, the two oxygen atoms of the
carboxyl group of each Ala molecule exist in extremely similar
positions. Superimposition of the structures of ADP-Mg2+-Pibound YwfE and ADP-Mg2+-phosphorylated phosphinatebound E. coli DdlB33 showed that the ADP, Mg2+, andphosphate moieties in each structure occupied considerably
similar positions (not shown). These observations suggest thatthe formation of an acylphosphate intermediate will occur inthe same place independent of the diﬀerent orientations ofbound substrate amino acid. As mentioned above, L-Ala isstrictly recognized by multiple electrostatic interactions withthe side chains of Glu273, His309, Glu311, Arg328, and theactivity (white bars, Figure 6B). This result is consistent withthe ﬁnding that YwfE has the ability to produce the L-Met-LMet dipeptide.5 In addition to W332A and W332S mutants,
both W332V and W332L mutants also retained activities atalmost the same levels, independent of the presence or absenceof L-Ala. Furthermore, the enzyme with a mutation of Trp332
to His retained approximately 25% of the L-Ala-independent
activity. These results indicate that each mutant prefers L-Met
to the bulky and rigid L-Phe as a substrate. These functional andstructural studies of Trp332 mutants allow us to propose thatthe size and shape of residues at position 332 of YwfE greatly
inﬂuence substrate speciﬁcity in the N-terminal substratebinding pocket.
Proposed Catalytic Mechanism. The reaction scheme of
the ATP-grasp family is believed to proceed through theformation of an acylphosphate intermediate, which is attacked
by a nucleophile, leading to a tetrahedral intermediate that
ultimately collapses to form the product.10,11 The superpositionof the wild-type YwfE structure of ADP-Mg2+-Pi and ADPMg2+-L-Ala-bound forms demonstrated that one of the oxygen
atoms from Pi was located very close to the position of the
carboxyl oxygen from L-Ala (Figure 7). Thus, this model

Figure 7. Model of an acylphosphate intermediate. Superimposition ofmodels for the ADP-Mg2+-L-Ala-bound form (atom color) and ADPMg2+-Pi-bound form (green) of wild-type YwfE are shown. Side chains
involved in important roles in the active site are presented as stickmodels. Three water molecules of the ADP-Mg2+-L-Ala-boundstructure are shown in red spheres.

appears to mimic the acylphosphate intermediate. In
comparison with a P-analogue-bound model of YwfE, theacylphosphate portion in each model occupies a nearly identical
position, suggesting that binding of phosphorylated L-Ala isﬁrmly stabilized in the enzyme during the reaction cycle.
These considerations lead us to propose a scenario for thecatalytic mechanism of YwfE enzyme. First, an ATP molecule isbound within the highly conserved nucleotide-binding cleft, andan L-Ala is recognized within the N-terminal substrate-binding
pocket. Furthermore, Arg328 ﬁxes the position of the carbonylgroup of L-Ala to orient the positioning of one of the oxygen
atoms and polarize a C−O bond suitable for the oxygen atom
to attack the γ-phosphate group of ATP to produce an
acylphosphate intermediate. An L-Phe (L-anticapsin in vivo) is
subsequently bound within the C-terminal substrate-binding
pocket, and its amino group makes a nucleophilic attack on the
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Biochemistry

Articlespeciﬁcity for L-Asp, the introduction of a positively charged
residue within the substrate-binding pocket of YwfE may be aneﬀective strategy. One example of a successful change ofsubstrate speciﬁcity was reported for subtilisin, a serine
protease, in which electrostatic interactions between charged
substrates and complementary charged amino acid residues inthe substrate-binding pocket were engineered.35 We are
currently attempting to create a mutant YwfE with improved
properties of substrate speciﬁcity and activity for L-Asp with theaim of engineering protein for the production of aspartame.
Most recently, enzymatic production of L-Asp-L-Phe, aprecursor of aspartame, by a two-step synthesis with a
combination of Lal and yeast N-terminal amidase, given thatno Lal has been identiﬁed that can produce a dipeptide carrying
an acidic residue at the N terminus, has been reported.36 In theﬁrst step, Lal catalyzed the formation of L-Asn-L-Phe from L-Asn
and L-Phe, and subsequently, N-terminal amidase deamidated
Asn to Asp to produce L-Asp-L-Phe. Several Lal’s wereidentiﬁed in bacterial genomes by in silico screening, and each
recombinant protein showed diﬀerent substrate speciﬁcity.
Detailed structural and functional studies of several other Lal’swith bound amino acid substrates are desirable for elucidating
the relationship between the substrate-binding pockets andamino acid substrate speciﬁcity. On the basis of this structural
information, systematical introduction of point mutations,insertion, or deletion of amino acids in or near the substratebinding pocket of Lal will allow the synthesis of modiﬁed Lal’swith novel substrate-binding properties.
In conclusion, in this study, we determined new crystal
structures of YwfE and performed mutagenesis studies. The
results demonstrate that Trp332 plays a crucial role inrestricting the substrate speciﬁcity for smaller amino acids
such as L-Ala at the N-terminus and that a single mutation of
YwfE can alter substrate speciﬁcity. To the best of ourknowledge, this is the ﬁrst demonstration of changing thesubstrate speciﬁcity of Lal by site-directed mutagenesis. Theseobservations raise the possibility of engineering a suitable
enzyme for the biosynthesis of dipeptides useful for industrial
production. Taken together, our results provide useful
guidelines for the rational design of Lal.

Figure 8. Conserved Arg residue in the ATP-grasp superfamily. (A)Bs, Bacillus subtilis; Bl, Bacillus licheniformis; Ec, Esherichia coli; Tt,
Thermus thermophilus HB8. A structure-based sequence alignment ofresidues of conserved Arg (cyan), Mg2+-coordinating Glu or Asp (red),and conserved His among only Lal (green) is shown. All residue
numberings are shown on both sides of each line. (B) Superimpositionof models for YwfE (atom color) and Ddl from Thermus thermophilus
HB8 (green) with bound ADP-Mg2+-L-Ala and ADP-Mg2+-D-Ala,respectively. Mg2+ ions are represented by green (YwfE) and cyan(Ddl) spheres.

■main-chain amide group of Gly321. Among these amino acids,
the bidentate binding of the carboxyl group of L-Ala by Arg328
is important for the proper orientation of L-Ala. In addition, we
propose that the diﬀerent positioning of conserved Arg residues
in Lal and Ddl is crucial for distinguishing the enantioselectivity
in recognition of L- and D-amino acids.
Toward Rational Design of Lal. It has been proposed that
Lals are potentially desirable tools for the enzymatic productionof dipeptides composed of L-amino acids.6 In the case of YwfE,
an artiﬁcial sweetener aspartame (L-Asp-L-Phe-OMe) is likely to
be a suitable and attractive candidate for a dipeptide that is
produced by an engineered enzyme because L-Phe presents thehighest aﬃnity for the C-terminal substrate among the 20
proteogenic L-amino acids. However, restricted substratespeciﬁcity for smaller amino acids such as L-Ala at the Nterminus is one of the important issues that should be resolved
for aspartame production. At present, the Trp332 mutant YwfE
is a promising candidate for resolving this issue. We accordingly
measured the ATPase activity of W332A mutant in thepresence of L-Asp and L-Phe but observed that the activity was
similar to that of the background, although L-Asp is smaller
than L-Met or L-Phe. This result may be because of the negative
charge of the side chain of L-Asp. To increase aﬃnity and

ASSOCIATED CONTENT* Supporting Information
S

Diﬀraction data statistics of Mn2+-derivative crystals; orientation of Tyr75; ATPase activity of wild-type YwfE; and HPLC
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org.
Accession Codes

The atomic coordinates and structure factors (codes 3WNZ,
3WO0, and 3WO1) have been deposited in the Protein Data
Bank, Research Collaboratory for Structural Bioinformatics,
Rutgers University, New Brunswick, NJ (http://wwwrscb.org/).

■

AUTHOR INFORMATION

Corresponding Author*Tel: 83-3-3986-0221. Fax: 83-3-5992-1029. E-mail: takeo.
tsuda@gakushuin.ac.jp.
Funding

This work was supported in part by a grant from JGC-S
Scholarship Foundation to T.T.
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Biochemistry

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catalytic mechanism. J. Biol. Chem. 284, 11690−11697.
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Structural analysis of the active site geometry of N5-Carboxyaminoimidazole ribonucleotide synthetase from Escherichia coli. Biochemistry47, 13346−13353.
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The authors declare no competing ﬁnancial interest.

■

ACKNOWLEDGMENTS
We would like to thank the beamline scientists at the Photon
Factory for assistance with data collection. We thank Enago(www.enago.jp) for English language review.

■

ABBREVIATIONS
BC, biotin-dependent carboxylase; BSA, bovine serum albumin;
Ddl, D-alanyl-D-alanine ligase; DTT, dithiothreitol; HPLC, highperformance liquid chromatography; Lal, L-amino acid ligase;
NRPS, nonribosomal peptide synthetase; PCR, polymerase
chain reaction; PDB, protein data bank; PEG, poly(ethylene
glycol); Pi, inorganic phosphate■

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Saito, J., Komori, H., Shibata, N., Yonetani, Y., and Higuchi, Y. (2012)
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細胞分裂構造の制御メカニズムの解明
教授 馬渕 一誠
助教 柏崎 隼[目的]細胞の分裂は細胞の増殖や生物体の成長にとって必須の過程である。全ての細胞において細胞分裂は細胞の外部環境や細胞周期によって制御されており，制御システムに異常が生じると細胞は無限に増殖し、これが多細胞生物において起こると癌となる。したがって細胞の分裂がどのように制御されているのかを知ることは重要である。細胞分裂は染色体DNA が複製された後に２極に分離する核分裂と細胞自体が 2 分する細胞質分裂が連続的に起こることによって完了する。動物細胞の細胞質分裂においては分裂位置が染色体分離を担った分裂装置によって決定され、分裂位置の細胞表層に収縮環と呼ばれる構造が形成される。収縮環は細胞骨格タンパク質アクチンの繊維によって構成され，モータータンパク
質ミオシン II をも含んでいる。私たちのこれまでの研究により収縮環はミオシンとアクチ
ン繊維の相互作用によって収縮して細胞を２分すると考えられている(1)。同じ様式の分裂は原始的な真核細胞であるモデル生物としても研究されている菌類（酵母など）でも行われていることが分かってきた。本研究は分裂酵母，ウニ卵、カエル卵をもちいて、収縮環の形成と収縮がどのような分子機構により制御されているかを明らかにすることを目的とする。
[結果と考察]1． 分裂酵母の細胞質分裂の in vitro 研究系の開発と成果

分裂酵母 Schizosaccharomyces pombe は単細胞かつ半数体で栄養増殖する子嚢菌の一種であり、細胞周期や細胞分裂の研究のモデル生物として用いられている。分裂期にはいると細胞の中央部に F アクチンリングと呼ばれる環状構造を形成し、リングの径が減少すると同時に隔壁が形成され、侵入してきて分裂する。このリングにはミオシンをはじめ(2)、動物細胞の収縮環の成分と共通のいくつかのタンパク質成分が含まれていることが分かっているが、このリングが自力で収縮するかどうかは分かっていなかった。
本研究では F アクチンリングが動物細胞の収縮環に相当する構造であると考え、分裂酵
母細胞を透過性にして F アクチンリングの収縮を研究できる実験系を開発することにした。
多数の F アクチンリングを観察する必要があるので細胞周期を同調するために cdc25

分裂変異株を用いた。さらにリングを蛍光可視化するために、cdc25 株にミオシン調節

軽鎖—GFP 融合タンパク質を染色体から発現させた。細胞壁を lysing enzyme 処理によって除いた。細胞を制限温度処理によって同調させ，F-アクチンリング（観察しているのはミオシンリング）が形成された事を確認して、界面活性剤により細胞膜を破壊した。
こうして得られた「細胞ゴースト」にはミオシンリングは残っていたが、細胞質は消失していた。
細胞ゴースト中のミオシンリングは ATP を与えることによって速やかに収縮した。
各種ヌクレオチドの中で ATP が最も良い収縮を起こし、ATP の非水解性アナログでは

193

収縮が起こらなかった。ATP による収縮速度は動物細胞の収縮環の収縮速度に匹敵し、それは生細胞のミオシンリングの収縮速度の 20-30 倍であった。この実験結果は、ミオ
シンリング（F-アクチンリング）がまさに収縮環であることを初めて証明したのみならず，生細胞ではその収縮を制御するシステムが存在することを示唆した。細胞ゴーストの収縮環の収縮はミオシン ATPase 活性の阻害剤によって阻害された。またミオシンの温度感受性変異株を用いて作製したゴーストは制限温度下では収縮しなかった。従って，動物細胞同様、ミオシン II が収縮に働いていることが分かった。一方、動物細胞、分裂酵母の収縮環は収縮しながらアクチンを失って行き(3)，収縮後にはアクチン繊維が分裂位置から消失している。一方、アクチン繊維安定化剤を加えると収縮は停止する(4)。
これらの研究から、アクチンの脱重合が収縮環の収縮には必須なのではないかと考えられてきた。
しかしこの in vitro 収縮系での実験から、収縮中にアクチンは脱重合するが、それは収縮そのものには必須ではないことが証明された。また、収縮環には IQGAP,

α-actinin(5), fimbrin(6)などのアクチン繊維架橋タンパク質が存在して収縮環構造を安定化していると考えられている。本研究では細胞ゴーストにこれらの精製タンパク質を加えたところ、加える量を増やすに伴って収縮は阻害された。このことから，収縮には適正量のアクチン繊維架橋タンパク質が必要であることが分かった。以上の研究は論文公表した。
2． 収縮環の形成とアクチン調節タンパク質、ミオシン
収縮環はアクチンが分裂位置で再編成される、あるいは重合することによって形成されると考えられている。そしてこれらの過程には様々なアクチン調節タンパク質が働いていると考えられている。本研究ではまず分裂酵母のアクチン脱重合因子 Adf1 の働きを調べた。
そのため我々が単離した adf1 の温度感受性変異株 adf1-1 (7)を用いた。
adf1-1

変異を起こすには制限温度下で数分を要した。この性質をもちい、細胞周期の様々な段
階で Adf1 を失活させて分裂に対する影響を調べた。その結果、G2 期の終わりから、分裂後期 B の初めまでに Adf1 を失活させると、収縮環の形成が妨げられるか，異常になることが分かった。この結果から、収縮環の形成には Adf1 によるアクチンの再編成が必要であることが分かった。この研究は論文を執筆中である。
一方，収縮環アクチンを重合させると考えられている formin の一種 Diaphanous の局在をウニ卵を用いて調べた。ウニ Diaphanous（uDia）の抗体をモルモットで作製し、間接蛍光抗体法でその局在を調べた結果，分裂溝に点状に分布し、収縮環アクチン繊維とほぼ同レベルに存在していた。この研究は超解像顕微鏡を用いて継続中である。
また、ウニ卵を超解像蛍光顕微鏡で観察することにより，アクチン繊維とミオシンの位置関係を解析した。超解像蛍光顕微鏡の一つ SIM で観察すると、収縮環アクチン繊維の 1 本 1 本が確認でき、その間にミオシンがおそらく繊維状に存在していることがわかった。この系を用いてアクチン繊維とミオシン繊維の位置関係，収縮環形成過程での位置関係の変化を研究継続中である。
3． 収縮環の in vitro 形成系の開発

収縮環を in vitro で形成させることができれば，形成過程を簡単に人為的に制御することができるため、細胞分裂の理解は著しく進展すると思われる。このような系を開発

194

する目的で、アフリカツメガエルの卵母細胞の細胞質を人工脂質膜に封入する研究を行った。リポソームに細胞質を封入することも可能だが，リポソームの安定性が悪いので、オイル中で細胞質を含む脂質膜小胞を作製した。すると細胞質中に X-body と命名した
小塊が形成され、脂質膜から X-body に向かってアクチンの流れが起こった。この流れは脂質膜でのアクチンの重合と X-body での脱重合がカップルして起こることで維持されていることがわかった。また流れにはミオシンの ATPase 活性が必要だった。さらに，
アクチンの流れは脂質膜小胞そのものの運動を誘発した。これらの現象は生きた卵母細胞中では観察されていないが、魚の鱗のケラトサイトや培養繊維芽細胞の葉状仮足で見られる有名な現象で，運動性が見られていない卵母細胞の抽出液で観察されたことは大変興味深い。ここまでの研究成果の論文を執筆中である。今後は脂質膜小胞中に人工的に収縮環を形成させる方向で研究を発展させる。
[まとめ]

１．分裂酵母の細胞質分裂の in vitro 研究系の開発と成果

分裂酵母の細胞から、収縮環の in vitro 収縮系の開発に成功し，分裂酵母の収縮環が動物細胞のそれと同等であること、収縮にミオシンが必須であること、細胞内には収縮を制御するシステムがあること，アクチン脱重合は収縮とともに起こるが収縮には必須でないこと、収縮には適正量のアクチン繊維架橋タンパク質が必要であること、などを明らかにした。
2．収縮環形成過程の研究
アクチン脱重合因子 ADF が収縮環形成におけるアクチンの再編成に働いていることを解明できた。また、収縮環の形成過程におけるミオシンとアクチン繊維の位置関係
を分子レベルで構造の上から解明できる糸口をつかんだ。
3．収縮環の in vitro 形成系の開発
本研究はまた始まった段階だが、カエル卵母細胞の抽出液にアクチン重合によって駆動される新しい運動能力があることを発見するという，大きな成果が得られている。
[参考文献]
1. Mabuchi, I., and Okuno, M. (1977). The effect of myosin antibody on the division of
starfish blastomeres. J. Cell Biol. 74, 251-263.
2. Motegi, F, Nakano, K., and Mabuchi, I. (2000). Molecular mechanism of myosin-II
assembly at the division site in Schizosaccharomyces pombe. J. Cell Sci. 113,
1813-1825.
3. Kamasaki, T., Osumi, M., and Mabuchi, I. (2007). Three-dimensional arrangementof F-actin in the contractile ring of fission yeast. J. Cell Biol. 178, 765-771.
4. Hamaguchi, Y. and Mabuchi, I. (1982). Effects of phalloidin microinjection andlocalization of fluorescein-labeled phalloidin in living sand dollar eggs. Cell Motility
2, 103-113.
5. Mabuchi, I., Hamaguchi, Y., Kobayashi, T., Hosoya, H., Tsukita. S. and Tsukita, S.
(1985). Alpha-actinin from sea urchin eggs: biochemical properties, interaction
with actin, and distribution in the cell during fertilization and cleavage. J. Cell
Biol. 100, 375-383.
6. Nakano, K., Sato, K., Morimatsu, A., Ohnuma, M., and Mabuchi, I. (2001).
Interactions among a fimbrin, a capping protein, and an actin-depolymerizing factor
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7. Nakano, K., and Mabuchi, I. (2006). Actin-depolymerizing protein Adf1 is required
for formation and maintenance of the contractile ring during cytokinesis in fission
yeast. Mol. Biol. Cell 17, 1933-1945.
[原著論文]
1. Yano, K., Uesono, Y., Yoshida, S., Kikuchi, A., Kashiwazaki, J., Mabuchi, I., andKikuchi, Y. (2013). Mih1/Cdc25 is negatively regulated by Pkc1 in Saccharomyces
cerevisiae. Genes Cells 18, 425-441.
2. Mishra, M., Kashiwazaki, J., Takagi, T., Srinivasan, R., Huang, Y.,
Balasubramanian, M. K., and Mabuchi, I. (2013).
In vitro contraction ofcytokinetic ring depends on myosin II but not on actin dynamics. Nat. Cell Biol. 15,
853-859.
3. Nakase, Y., Nakase, M., Kashiwazaki, J., Murai, T., Otsubo, Y., Mabuchi, I.,
Yamamoto, M., Takegawa K., and Matsumoto, T. (2013). Fission yeast Any1, aβ-arrestin-like protein, is involved in TSC-Rheb signaling and regulates amino acidtransporters. J. Cell Sci. 126, 3972-3981.
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柏崎隼、高木智子、馬渕一誠. (2013). In vitro における収縮環の収縮はミオシン IIに依存するがアクチンのダイナミクスには依存しない。ライフサイエンス新着論文レ3.

ビュー 7443.
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学 64, 524-525 .
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4.

柏崎隼、馬渕一誠. (2014). 収縮環のin vitro 収縮系の開発。生物物理 54, 201-205.

5.

馬渕一誠、柏崎隼. (2014). 細胞質分裂における収縮環の収縮：in vitro系の開発。

2.

学・医学賞受賞 Paul Nurse 博士。学習院大学講演録。 細胞工学 33, 182.

細胞工学 33, 660-665.
6.

Mabuchi, I., Kashiwazaki, J., and Mishra, M. (2016). In vitro reactivation of thecytokinetic contractile ring of fission yeast cells. Methods in Cell Biol. (Elsevier),in press.
[口頭発表]
1. 柏﨑隼、Mishra, M., Balasubramanian, M., 馬渕一誠： 分裂酵母ゴーストを用いた収縮環の in vitro 再活性化。第 65 回日本細胞生物学会大会シンポジウム（2013.6.19-21,名古屋）招待講演。
2. Mishra, M., Kashiwazaki, J., Takagi, T., Srinivasan, R., Huang, Y.,
Balasubramanian, M., and Mabuchi, I. 2013.: In vitro contraction of cytokinetic ringdepends on myosin II but not on actin dynamics. EMBO Conference on Fission
Yeast: Pombe 2013 (24-29 June 2013, London). 招待講演。
3. Balasubramanian, M., Tao, E. Y., Mishra, M., Kashiwazaki, J., Huang, Y., Huang, J.
J., Takagi, T., Subramanian, D., Xie, T., and Mabuchi, I. : Cytokinesis in vitro andin vivo. EMBO Conference in Fission Yeast (24-29 June 2013, London). 招待講演。

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4. Balasubramanian, M., Mishra, M., Kashiwazaki, J., Tao, E. Y., Huang, Y., Huang, J.
J., Takagi, T., Subramanian, D., Xie, T., Padmanabhan, A., Haochen, J.,
Wedlich-Soldner, R. and Mabuchi, I.: Cytokinesis in vitro and in vivo. Cell Biology of
Yeasts, (Cold Spring Harbor, NY, 2013.11.5-9). 招待講演。
5. Kashiwazaki, J., and Mabuchi, I. Actin cable motility during interphase in fission
yeast. Cell Biology of Yeasts, (Cold Spring Harbor, NY, 2013.11.5-9)
6. 馬渕一誠: In vitro 実験系の開発による細胞質分裂の研究。NAIST シンポジウム「細胞を創る操る」（奈良、2013.11.28）、 招待講演。
7. 馬 渕 一 誠 、 柏 崎 隼 、 Mishra,M., 高 木 智 子 、 Srinivasan, R., Huang, Y., andBalasubramanian, M. 収縮環の in vitro 実験系の開発による収縮機構の研究。第 36回日本分子生物学会年会 (神戸、2013.12.3-6)、 招待講演。
8. 柏崎隼、Mishra, M., 高木智子、Balasubramanian, M., and 馬渕一誠：分裂酵母収縮
環の in vitro 再活性化。第 36 回日本分子生物学会年会 (神戸、2013.12.3-6)
9. Kashiwazaki. J., Mishra, M., Balasubramanian, M., and Mabuchi, I. In vitro
contraction system of the fission yeast contractile ring. 2013 ASCB Annual
Meeting. (New Orleans, 2013.12.14-18)
10. Mishra, M., Kashiwazaki, J., Takagi, T., Srinivasan, R., Huang, Y.,
Balasubramanian, M. K., and Mabuchi, I.: In vitro contraction of cytokinetic ringdepends on myosin II but not on actin dynamics. 58th Annual Meeting of the
Biophysical Society (15-19 Feb. 2014, San Francisco). 招待講演。
11. Mabuchi, I.: In vitro reactivation of contractile ring in fission yeast cell ghost. 7th
APOCB Congress and ASCB Workshop (24-27 Feb. 2014, Singapore). 招待講演。
12. Mabuchi, I.: Contraction of the contractile ring as studied by using an in vitro
system. International Symposium on the Diversity of Cell Division System inEukaryotes. (24-25 March 2014, Tokyo). 招待講演。
13. 植田英一、柏崎隼、馬渕一誠：分裂酵母の収縮環形成における ADF/cofilin とフィンブ
リンの機能。 第 66 回日本細胞生物学会大会（2014.6.11-13, 奈良）
14. 野田直紀、馬渕一誠：Actin dynamics in Xenopus egg extract encapsulated in a lipid
membrane. Xenopus 卵抽出液を封入した脂質膜小胞中でのアクチンのダイナミクス。
日本生物物理学会第 52 回年会（2014.10.25-27 札幌）
15. 野田直紀、馬渕一誠：Xenopus 卵抽出液を封入した脂質膜人工小胞中でのアクチン動態。
第 37 回日本分子生物学会年会（2014.11.25-27, 横浜）、招待講演。
16. Mabuchi, I. Novel approaches to study mechanism of cytokinesis in animal cells.
OIST Seminar (2015.4.22 沖縄科学技術大学院大学)、招待講演。
17. Kashiwazaki, J., Mishra, M., and Mabuchi, I. Behavior of proteins during thecontractile ring contraction in the S. pombe cell-ghost. The 18th International
Fission Yeast Meeting (2015.6.21-26, 三宮)
18. 野田直紀、馬渕一誠：Xenopus 卵抽出液を封入した脂質膜小胞はアクチンの動態と共役して運動する。第 67 回日本細胞生物学会大会 (2015.6.30-7.2 船堀)

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19. 関根彩子、小林礼、柏崎隼、植田英一、馬渕一誠： 分裂酵母 Cyclase-associated protein(CAP) の低グルコース環境への適応における役割。第 67 回日本細胞生物学会大会(2015.6.30-7.2 船堀)
20. Noda, N., Mabuchi, I. Actin flows in Xenopus egg extract confined in oil andgenerates a force for migration of the extract. 第 53 回生物物理学会年会シンポジウム(2015. 9. 13-15, Kanazawa) 招待講演。
21. 細谷夏実、馬渕一誠： ウニ卵 formin/Diaphanous の細胞質分裂における局在。日本動物学会第 86 回大会 (2015.9.17-19, 新潟)

198

LETTERS

In vitro contraction of cytokinetic ring depends on
myosin II but not on actin dynamics
Mithilesh Mishra1,5 , Jun Kashiwazaki2,5 , Tomoko Takagi2 , Ramanujam Srinivasan3 , Yinyi Huang3 ,Mohan K. Balasubramanian1,3,4,6 and Issei Mabuchi2,6
Cytokinesis in many eukaryotes involves the contraction of anactomyosin-based contractile ring1,2 . However, the detailed
mechanism of contractile ring contraction is not fully
understood. Here, we establish an experimental system to study
contraction of the ring to completion in vitro. We show that thecontractile ring of permeabilized fission yeast cells undergoes
rapid contraction in an ATP- and myosin-II-dependent mannerin the absence of other cytoplasmic constituents. Surprisingly,
neither actin polymerization nor its disassembly is required forcontraction of the contractile ring, although addition ofexogenous actin-crosslinking proteins blocks ring contraction.
Using contractile rings generated from fission yeast cytokinesis
mutants, we show that not all proteins required for assembly of
the ring are required for its contraction in vitro. Our work
provides the beginnings of the definition of a minimal
contraction-competent cytokinetic ring apparatus.
The understanding of biological processes benefits from the combined
use of genetic analyses and in vitro reconstitution. Early studies inmetazoan cells and embryos have established that an actomyosinbased contractile ring drives cytokinesis3,4 . Isolation of the cleavage
furrow containing the contractile ring has provided some structural
information for the mechanism of cytokinesis5 , but the molecularcomponents responsible for the supramolecular organization andfunction are not fully understood. Genetic analysis in various
model organisms has succeeded in the identification of mutants
defective in contractile ring assembly, characterization of which inturn has led to the identification of components of the contractile
ring1,2,6 . However, mutants defective specifically in ring contraction
have not been identified, possibly because proteins involved inring assembly might contribute to its contraction. As a result, themechanism for ring contraction and disassembly is poorly understood.
To circumvent the gaps in genetic analysis and to generate athorough understanding of ring contraction we have sought toestablish an in vitro system.
The fission yeast Schizosaccharomyces pombe divides by the use of
a contractile ring and an invaginating septum7,8 . Given the wealth ofcytokinesis-defective mutants and the ease of using live-cell microscopy,
we chose to establish the in vitro ring contraction system in thisorganism (Fig. 1a and Methods). We first removed the cell wallby enzymic digestion to obtain spheroplasts. Time-lapse imaging
of mCherry–tubulin- and Rlc1p–3xGFP (myosin-II regulatory lightchain)-expressing spheroplasts revealed that, like intact cells, spheroplasts assembled normal-appearing contractile rings9 (Supplementary
Fig. S1a,b). Then spheroplasts were permeabilized with 0.5% NP-40.
The resultant cell ghosts retained a contractile ring (Fig. 1b) surroundedby plasma membrane full of holes as seen by FM4-64 staining andby thin-sectioning electron microscopy, but were devoid of any cytoplasmic structures (Fig. 1b,c). Most proteins necessary for contractile
ring formation were contained in the contractile ring in cell ghosts(Fig. 1d–g and Supplementary Fig. S2a and Table S1) but other cytoplasmic components were not (Supplementary Fig. S2b and Table S1).
Biochemical analysis showed that the ring components myosin-II,actin and tropomyosin Cdc8p were enriched in the ghost preparations,whereas tubulin was not (Fig. 1h and also Supplementary Fig. S2b).
To determine whether these rings were contractile, we treated cellghosts with 0.5 mM ATP. Rings underwent rapid contraction on ATP
addition (Fig. 2a,b and Supplementary Video S1). It has been reported
that contractile rings isolated from spheroplasts of budding yeast
disappeared on ATP addition, although the mechanisms were notexplored10 . As for the intact cells11–13 , the rate of contraction of rings
from fission yeast cell ghosts was almost constant from initiation tocompletion, and was 0.22 ± 0.09 µm s−1 (n = 37; Fig. 2b). Contraction
with a slightly reduced rate was observed even when the ATPconcentration was as low as 0.01 mM (Fig. 2c and Supplementary VideoS2), but not in the presence of the non-hydrolysable ATP analogue

1

Temasek Life Sciences Laboratory, The National University of Singapore, 1 Research Link, Singapore 117604, Singapore. 2 Department of Life Sciences, Faculty of
Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo, 171-8588, Japan. 3 Mechanobiology Institute, The National University of Singapore, 117543,
Singapore. 4 Department of Biological Sciences, The National University of Singapore, 117411, Singapore. 5 These authors contributed equally to this work.
6
Correspondence should be addressed to M.K.B. or I.M. (e-mail: mohan@tll.org.sg or issei.mabuchi@gakushuin.ac.jp)
Received 13 August 2012; accepted 9 May 2013; published online 16 June 2013; DOI: 10.1038/ncb2781

NATURE CELL BIOLOGY VOLUME 15 | NUMBER 7 | JULY 2013199

© 2013 Macmillan Publishers Limited. All rights reserved.

853

LETTERS
a cdc25-22 rlc1–3GFPb

Rlc1–3GFP

DIC

FM4-64

FM/Rlc1

Spheroplastingc

Ring formationh
1x 4xSphe CGPM
Ring filaments

Permeabilization

Rlc1
–3GFP

1x 4x
Sphe CG M (K)
r
250
150
100
75

α-tubulin

Cell ghost

Ring filaments

50
42
37

Actin

+ATP

25

Cdc8

20

PM

d

F-actin

Rlc1–3Re

Myo2–G15

f

Rlc1–3R

Anti-Cdc4

Rlc1–3Gg

Cdc8–3V

Rlc1–3R

Figure 1 Contractile rings in cell ghosts. (a) Schematic illustrationof procedure to obtain cell ghosts. (b) Maximum projection image of
deconvolved sections of Rlc1–3xGFP and the plasma membrane stainedwith FM4-64 in a cell ghost. (c) A thin-section electron micrograph ofthe mid region of a cell ghost that is not parallel to the ring plane. Actin
filaments in the ring were decorated with skeletal heavy meromyosin. The
plasma membrane (PM) showed numerous gaps as a result of the detergent
treatment. The area surrounded by a dotted rectangle is magnified below.
(d) Maximum projection images of deconvolved sections of Rlc1–3xmDsRed
and F-actin stained with BODIPY-FL–phallacidin in the cell ghosts.

(e) Maximum projection images of deconvolved sections of Myo2–GFP
and Rlc1–3xmDsRed. (f) Maximum projection images of deconvolved
sections of Cdc4 stained with anti-Cdc4 antibody and anti-rabbit IgGlabelled with tetramethylrhodamine after permeabilization and Rlc1–3xGFP.
(g) Maximum projection images of deconvolved sections of Cdc8–3xVenus
and Rlc1–3xmDsRed. (h) Western blot analysis of several proteins in thewhole-cell lysate of spheroplasts (Sphe) or cell ghosts (CG). The density ofcell ghosts was fourfold higher than that of spheroplasts. The right panel
shows Coomassie-stained gels of total protein in spheroplasts and cell ghosts.
Scale bars, 5 µm (b,d–g) and 1 µm (c).

AMP-PNP (Fig. 2d and Supplementary Video S3). The rate of ring
contraction in cell ghosts in the presence of 0.5 mM ATP was more than
20 times faster than that in wild-type cells (Fig. 2e). The measurementof fluorescence of ring components over time revealed that whereas thetotal amount of most contractile ring proteins reduced only marginally,
Cdc8p was rapidly depleted from the constricting ring (Fig. 2f and
Supplementary Table S2). The fast rate of ring contraction in vitro may
be a result of delinking of ring contraction from membrane and division
septum assembly in cell ghosts as the associated membranes did notinvaginate as the ring contracted (Fig. 2g and Supplementary Video
S4). Although slow contraction was observed in the presence of othernucleoside triphosphates, the lowest Km and highest Vm were observedfor ATP (Fig. 2h,i). Thus, ring contraction in fission yeast requires
ATP hydrolysis. Maximal ring contraction was observed at pH 8.0–8.5
with contraction being slower below pH 7 (Supplementary Fig. S3a).
The optimal calcium concentration for ring contraction was pCa 8–9
and free Ca2+ lowered the contraction rate (Supplementary Fig. S3b),suggesting a Ca-sensitive step in ring contraction in fission yeast.
In time-lapse imaging of spheroplasts, contractile rings wereroutinely observed to initiate from a single location and occurred
through what appeared as a bi-directionally growing leading cable14(Supplementary Fig. S1b). We established that this mode of bidirectional cable assembly was not a peculiarity of spheroplasts, because

fission yeast cells rendered spherical also exhibited a similar ringassembly process (Supplementary Fig. S1c). Whereas fully formed rings
underwent rapid ATP-dependent contraction, incomplete actomyosinarcs were unable to contract (Fig. 2j and Supplementary Video S5)
although the fluorescence decayed significantly in the presence of ATP,which is probably due to disassembly (Supplementary Fig. S3c). This
result is consistent with the observation that the forming ring consistsof two semicircles of predominantly parallel F-actin filaments15 , which
would not support ring contraction.
As the motor protein myosin-II hydrolyses ATP during its walking
along an actin filament, we examined whether myosin-II activity wasessential for ring contraction. Although myosin-II–actin interaction has
been shown to be essential for cytokinesis16 , its molecular function incytokinesis is not fully understood. We incubated cell ghosts in the presence of ATP and the myosin-II ATPase inhibitor blebbistatin (0.1 mM;
ref. 17). Whereas rings underwent rapid contraction in the absenceof blebbistatin, ring contraction was abolished in the presence of
blebbistatin (Fig. 3a, n = 8). We also found that incubation of cell ghosts
with dehydroxestoquinone (DXQ; 2.5 mM), an inhibitor of skeletal
muscle myosin II (ref. 18), and kinesin and dynein ATPases blocked
ring contraction (Supplementary Fig. S3d,e). Treatment of cell ghosts
with 0.1 mM sodium vanadate (an inhibitor of dynein and kinesin
ATPases, but not myosin II ATPase) or microtubule-depolymerizing

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Time (s)90fd +0.5 mM AMP-PNP

0s

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6
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15

18

21

g +0.5 mM ATP

24

27

30

Rlc1-3GFP/FM4-64

1.0

0.5

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80 100Time affter ATP addition (s)
0s

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Rlc1–3GFP (n = 10)
Myo2–GFP (n = 9)
Myp2–GFP (n = 9)
Cdc8–3Venus (n = 7)
Rng2–GFP (n = 9)
Cdc15–3Venus (n = 6)

ATP

0.3

ITP
CTP
GTP

0.2

i

Km
Vm
(mM) (µm s–1)

R2

ATP

0.032

0.23

0.50

ITP

1.6

0.23

0.48

CTP

3.2

0.20

0.75

GTP

1.9

0.037

0.23

0.10
(µm s–1)

Contraction rate (µm s–1)

h

0.1

0.05

0

0
0

1

2
3
Concentration (mM)

4

0

5

0.005
(mM)

0.010

j +0.5 mM ATP
0s

30

60

90

120

150

180

210

240

Figure 2 ATP stimulates rapid ring contraction in vitro. Experiments weredone at 25 ◦ C. Fluorescence micrographs of Rlc1–3xGFP are shown as
maximum projections of Z -stacks. (a,b) Ring contraction is stimulatedon ATP addition in vitro. 0 s indicates time of ATP addition. Typicalexamples of time course of ring contraction are shown. Perimeters were
measured after three-dimensional rotation (Methods). Each measured value
is represented. (c) Ring contraction is slower in the presence of 0.01 mM
ATP. (d) The ring does not contract in the presence of 0.5 mM AMP-PNP.
(e) Ring contraction in a cylindrical wild-type fission yeast cell. Fluorescencemicrographs of Rlc1–3xGFP are shown as 30◦ -tilted maximum projectionsof Z -stacks. (f) Disassembly of each ring component after 0.5 mM ATP
addition. Fluorescence intensity is shown as the relative value to that in theabsence of ATP to eliminate the effect of photobleaching. Each mean value

is represented. For the values at 100 s, unpaired two-tailed t -tests were
done. Each P value is shown in Supplementary Table S2. (g) The plasma
membrane of Rlc1–3xGFP-expressing cell ghosts stained with FM4-64
during contraction in vitro in the presence of 0.5 mM ATP. (h,i) Nucleotide
specificity for ring contraction in vitro. Actual values are shown in
Supplementary Table S3. Each curve was fitted to the Michaelis–Menten
equation. The small graph shows the rates at low concentrations of ATP.
Km and Vm were estimated using Prism-5. R 2 should be close to 1.0 if
the curve is well fitted. Statistics source data for Fig. 2h can be found in
Supplementary Table S6. (j) Incomplete contractile ring does not undergo
ATP-driven contraction in vitro. ATP (0.5 mM) was added at 0 s. See
Supplementary Videos S1–S5. Time is indicated in seconds (a,c,d,g,j) or
minutes (e). Scale bars, 5 µm (a,c–e,g,j). Error bars represent s.d. (f,h).

drugs (methyl benzimidazol-2-yl-carbamate (MBC) and thiabendazole (TBZ)) did not affect ring contraction (Supplementary Fig. S4a).
The inhibition of ring contraction by DXQ, but not by vanadate, MBC
or TBZ, taken together with our results with blebbistatin strongly

suggested that the ATPase activity of myosin II was essential forring contraction.
We independently confirmed the role of myosin-II in ring
contraction by preparing cell ghosts from myosin-II-mutant strains.

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c

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+blst

0s

0s

20

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0.5 mM ATP, 36 °C

0.4

P = 0.003

Control

Contraction rate (μm s–1)

+0.5 mM ATP

P = 8.0 × 10–7

a

0.3
0.2
0.1
0

b

30

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myo2-E1

f

myp2Δ

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e cdc4-8, 36 °C 40 min

d rng3-65

0s

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-8

60

c4

30

cd

0s

W

T

(n

(n

=

=

11

)

18
)

myo2-E1
myp2 Δ0s

40

0.5 mM ATP, 36 °C

P = 5.3 × 10–7

20

m

10

Contraction rate (μm s–1)

0s

yo

W

T

WT

(n

=
212
E1
)
(n
=
m
yp
12
2Δ
)
(n
=
9)

+0.5 mM ATP

40

80

120

0s

40

80

120

160

200

240

Figure 3 Myosin-II drives ring contraction in vitro. ATP concentrationin each experiment was 0.5 mM. Fluorescence micrographs of
Rlc1–3xGFP are shown as maximum projections of Z -stacks. (a) Effectof blebbistatin on ring contraction in vitro. Rings were pre-incubatedin 0.1 mM blebbistatin for 3 min before perfusion with ATP (time0 s) at 25 ◦ C. (b) Rings in wild-type or myosin-II mutant cell ghosts.
Ring contraction after ATP addition was monitored at the permissive
temperature, 25 ◦ C. (c) Contraction rate (µm s−1 ) of contractile
rings in myosin-II mutant cell ghosts at the permissive temperature.

Each spot represents the rate of contraction of an individual ring.
Medial horizontal lines and error bars represent mean values ands.d., respectively. Two-tailed t -test was used for statistical analysis.
(d) Ring behaviour in rng3-65 mutant cell ghosts after ATP additionat the permissive temperature of 25 ◦ C. (e) Ring contraction in
cdc4-8 mutant cell ghosts 40 min after the temperature shift to 36 ◦ C.
(f) Contraction rate (µm s−1 ) of rings in cdc4-8 mutant cell ghosts at
the restrictive temperature. The graph is represented as in c. All scalebars, 5 µm. See Supplementary Videos S6–S8.

There are two genes that encode myosin-II heavy chain in fission yeast,
essential myo2+ (ref. 19) and non-essential myp2+ (also known as
myo3+ ; refs 20,21). Spheroplasts were prepared from the temperaturesensitive (ts) mutant myo2-E1 (ref. 22), the myp2-null mutant (myp21)
and the myo2-E1 myp21 double-mutant grown at the permissive
temperature of 25 ◦ C. Mutant Myo2-E1p has been shown to havea reduced ATPase activity and an associated reduction in its motor
activity even at the permissive temperature23 . Rings in myo2-E1 or
myp21 cell ghosts showed slower contraction even at 25 ◦ C (Fig. 3b,cand Supplementary Videos S6 and S7). Most of the rings in myo2-E1
myp21 cell ghosts were either fragmented or deformed and theserings did not show any ATP-dependent contraction (Fig. 3b andSupplementary Video S8), suggesting that the contractile ring was
not in a proper organization. Taken together, these experiments
established that ATP hydrolysis by myosin-II powers the fission yeast
ring contraction in vitro. Contractile rings in cell ghosts isolated
from rng3-65ts (defective in the UCS-domain-containing myosin-II
activator and chaperone Rng3p; refs 23,24) were also unstable anddid not undergo perceptible contraction even at 25 ◦ C (17 out of

20 rings, Fig. 3d) even though they did retain all of the essential
contractile ring proteins tested (Supplementary Table S4). Rings
prepared from the myosin-II essential light chain mutant (cdc4-8ts )underwent complex behaviour, ranging from very slow contraction toring disintegration, possibly owing to the multiple roles performed by
Cdc4p in cytokinesis25 (Fig. 3e,f).
Previous studies in yeasts and animal cells have proposed therequirement for actin depolymerization and/or severing in ring
contraction3,26 . We first established that F-actin fluorescence wasalmost completely lost during ring contraction in cell ghosts (Fig. 4a,b).
Using biochemical assays we found that on ATP addition to cell ghosts,
actin was released from the ghosts (Fig. 4c,d). Myosin-II behaved
differently, as most of it remained in the cell ghosts after ATP addition(Fig. 4e). This is consistent with the presence of myosin in a large spot
following ring contraction on ATP addition (Figs 2a,f and 4a,b).
We next investigated whether this loss of F-actin was essential forring contraction in vitro. To this end we first treated the cell ghosts
with an F-actin-stabilizing drug jasplakinolide (Jasp). We found thaton ATP addition ring contraction in cell ghosts occurred at normal

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ControlDIC

0.1 mM Jasp
TMR
–Lifeact

Rlc1–3GFP

DIC

TMR–Lifeact

Rlc1–3GFP

–ATP

+ATP

b

d

P = 7.3 × 10–7

Actin

–ATP

+ATP

+Jasp
+ATP

S

S

S

6,000

2,000
P = 4.2 × 10–3

0
Rlc1–3GFP
3,000

P = 0.59

P

P

1.0

P = 3 × 10–16

P = 0.061

P = 1.6 × 10–4

0.5

0

e

2,000

P = 2.1 × 10–7

)
=

11

10
)

P = 2.9 × 10–15 P = 9.1 × 10–4 P = 2.9 × 10–5

1.0
0.5
0

f

+J
as
p,

+J

as

p

+A
TP

(n

(n

=

=

+A
TP

(n

(n

=

10

9)

)

0

–A
TP

Rlc1
–3GFP

Relative amount

1,000

+Jasp
+ATP

+ATP
G

c

P

Actin

Relative amount

Fluorescence intensity (a.u.)

4,000

F

S

P

G

F

S

P

Cell ghost suspension
Actin+drug, 3 min+ATP +drug, 5 min

Relative amount

15,000g
Cell ghost(P)

Supernatant(S)
200,000g

Pellet(F-actin)

1.0
0.5
0

Supernatant(G-actin, short F-actin)

Figure 4 Contribution of actin dynamics to ring contraction in vitro. (a) Actinstabilization by Jasp does not block ring contraction. Actin was stainedwith 10 µg ml−1 TMR–Lifeact before or after contraction. White arrows
indicate the positions of contracted rings. Scale bar, 5 µm. (b) Fluorescenceintensity of TMR–Lifeact or Rlc1–3xGFP in contractile rings before andafter ATP addition. Each spot represents the integrated intensity of actin or
Rlc1–GFP in each contractile ring. Medial horizontal lines and error bars
represent mean values and s.d., respectively. (c) Schematic flow chart ofactin disassembly assay (Methods). (d,e) Western blot analysis of actin or
Rlc1 disassembly on ATP addition in the absence or presence of Jasp. S,

supernatant; P, pellet. Amount of pellet loaded is threefold lower than thatof the corresponding supernatant. Each relative amount was calculated fromthe total intensity (S + P × 3). Statistics source data for d,e can be found in
Supplementary Table S6. (f) Sedimentation assay of released actin on ATP
addition in the absence or presence of Jasp. G, G-actin and short F-actin; F,
F-actin. Amount of pellet loaded is one-and-a-half times lower than that ofits supernatant. Each relative amount was calculated from the amount of
actin in the pellet without ATP. Two-tailed t -test was used for all statisticalanalyses. Statistics source data for f can be found in Supplementary Table
S6. All error bars represent s.d.

kinetics in the presence of 0.1 mM Jasp (Supplementary Fig. S4aand Video S9). Other F-actin stabilizers, phalloidin or phallacidin at
0.1 mM, also did not affect the rate of ring contraction (data not shown).
Unlike in the contractile rings of untreated cells, actin filaments werereadily visible in rings treated with Jasp, phalloidin or phallacidin(Fig. 4a,b and Supplementary Fig. S4c).
We reconfirmed the effect of Jasp, phalloidin or phallacidin
treatment on actin stabilization by sedimentation assays. When ghosts

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a+0.5 mM ATP
cdc8-110,
36 °C 15 min
0s

30

b

90

Alexa 568
Rlc1–3GFP –phalloidin

7 min

e

0.1 mM ATP, 25 °C
P = 0.0048

0.10
0.05

g2
rn

)

)

)
10
=
(n
T
W

-D

5

T

10
=

(n

(n

=

=

9)

0
(n

0

0.15

1Δ

0.1

P = 1.8 × 10–5

0.20

fim

0.2

Contraction rate (μm s–1)

P = 0.21

9)

Contraction rate (μm s–1)

0.1 mM ATP, 36 °C

(n

d

16

180

=

90

Rlc1
/F-actin

1Δ+Fim1, 0.1 mM ATP

0.3

Rlc1
/F-actin

7 min

180

c

0s

90

ai
n

0s

60

Alexa 568
Rlc1–3GFP –phalloidin+Rng2Ns, 0.1 mM ATP

W

were treated with ATP and Jasp, most actin was still retained in thecell-ghost pellet, and a small fraction was released in the supernatant(Fig. 4c,d). By performing further sedimentation assays, we found that
actin released into the supernatant in the presence of Jasp was largely
composed of F-actin (Fig. 4c,f). This contrasts with ghosts treated with
ATP alone, in which most actin detected in the supernatant might bemonomeric (Fig. 4f). Similar results were obtained when phalloidinor phallacidin-treated cell ghosts were biochemically characterized(Supplementary Fig. S4d). These results suggest that actin filament
depolymerization is not required for ring contraction in vitro.
The small amount of actin filaments released during ring contraction
in vitro suggested that, although actin filament depolymerizationand monomer release were not required for this process, actin
severing might be important. We therefore examined whether theF-actin-severing protein ADF/cofilin Adf1p, which is known tofunction in actin turnover and contractile ring formation27 , plays anessential role in ring contraction. Rings in adf1-1ts cell ghosts contracted
at rates comparable to those in the wild type at 36 ◦ C (Supplementary
Fig. S4b and Video S10). These results implied that actin severingby ADF/cofilin was not required for ring contraction in vitro. The
experiments with Jasp and the adf1-1ts mutant collectively establishedthat neither actin severing nor depolymerization was essential forring contraction in vitro.
It has been proposed that actin polymerization itself might generate
the force required for ring contraction12 . We directly examined whether
actin polymerization was required for contractile ring contraction by
pre-incubation of cell ghosts with the actin polymerization inhibitor
latrunculin A (LatA) or cytochalasin A (CytA) before ATP addition.
These rings contracted at a rate comparable to that of the control(Supplementary Fig. S4a and Video S11). The formin Cdc12p isan actin nucleator essential for cytokinesis28,29 . The F-BAR domain
protein Cdc15p recruits Cdc12p to the division site30 . Contractile ringsin cell ghosts of both cdc12-112ts mutants and cdc15-140ts mutants
incubated at the restrictive temperature underwent normal contraction(Supplementary Fig. S4b and Videos S12 and S13). Contractile ringin cell ghosts of cdc3-124ts cells (defective in the formin-binding
protein profilin)7 also contracted at wild-type rates (Supplementary
Fig. S4b). Taken together these experiments demonstrated that actin
polymerization was not essential for ring contraction.
We then tested investigated whether yeast tropomyosin Cdc8p
is required for ring contraction. Cdc8p is essential for cytokinesis
and is known to regulate actin stability and modulate actomyosininteraction, possibly by competing with fimbrin Fim1p and/or Adf1p
for binding to actin filaments27,31,32 . Contractile rings in cdc8-110ts cellghosts were capable of undergoing ATP-dependent contraction at thepermissive temperature (n = 5, data not shown). Although these rings
retained most essential contractile ring components at the restrictive
temperature (Supplementary Table S4) they became discontinuous anddid not contract to completion at the restrictive temperature (8 out of 9
rings, Fig. 5a and Supplementary Video S14). Thus, we concluded that
Cdc8p is required for ring integrity during contraction.
The contractile ring contains F-actin-crosslinking proteins α-actinin
Ain1p, the IQGAP Rng2p and Fim1p (refs 33–35), which are essential
for proper organization of the actin bundles in the ring. We investigatedwhether an excess of actin crosslinker(s) impeded ring contraction
by addition of these actin-crosslinking proteins to the cell ghosts

Figure 5 Effect of tropomyosin and actin-crosslinking proteins on ring
contraction in vitro. (a) Ring behaviour in cdc8 -110 mutant cell ghosts
15 min after the temperature shift to 36 ◦ C. Fluorescence micrographsof Rlc1–3xGFP are shown as maximum projections of Z -stacks. ATPconcentration was 0.5 mM. (b,c) Addition of purified amino-terminal regionof IQGAP (Rng2Ns, 1 µM) or fimbrin (Fim1, 0.9 µM) blocks ring contraction
in vitro. ATP concentrations were 0.1 mM. F-actin was stained with Alexa
568–phalloidin 7 min after ATP addition. (d) Contraction rate of rings inrng2 -D5 mutant cell ghosts 40 min after the temperature shift to 36 ◦ C.
(e) Contraction rates of rings in fim11 and ain11 cell ghosts at 25 ◦ C.
All scale bars, 5 µm. Each spot represents the rate of contraction of anindividual ring. Medial horizontal lines and error bars represent mean values
and s.d., respectively. Two-tailed t -test was used for statistical analysis. See
Supplementary Videos S9–S16.

before ATP addition. When cell ghosts were pre-incubated withpurified Rng2Ns possessing the actin-bundling activity35 , contraction
was blocked in a dose-dependent manner with a complete block at
1 µM (n = 4, Fig. 5b and Supplementary Fig. S5a and Video S15), a
concentration that promoted robust actin filament bundling in vitro(Supplementary Fig. S5c). Phalloidin staining of these rings showedthat actin filaments remained in the ring (Fig. 5b). Fim1p showed asimilar dose-dependent inhibition of ring contraction in vitro with
0.9 µM Fim1p completely blocking ring contraction (n = 3, Fig. 5cand Supplementary Fig. S5b and Video S16). To determine whether
a defect or absence of each crosslinker caused an increase in ring
contraction rate, we prepared ghosts from rng2-D5ts mutant cells
or cells deleted for fim1+ or ain1+ . Curiously, rings from rng2-D5ts

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spheroplasts contracted at a normal rate at 36 ◦ C, and in the absence of
Fim1p or Ain1p rings showed a slower rate of contraction (Fig. 5d,e).
These experiments suggested that a balance of activity of crosslinking
proteins was essential for proper ring contraction.
We have established an in vitro contractile ring activation system
in which we have shown that fully formed contractile rings, but notpartially formed arcs, undergo ATP-dependent contraction. The factthat the rings in cell ghosts undergo rapid contraction establishes thatneither a constant supply of cytoplasmic material nor the concomitantingression of septum is required for the mechanics of ring contraction
in fission yeast. Our analysis, using this in vitro approach, therefore
leaves us with the minimal requirements for ring contraction; namely,
F-actin, myosin-II ATPase and the appropriate actin-crosslinking. 
METHODS
Methods and any associated references are available in the online
version of the paper.
Note: Supplementary Information is available in the online version of the paper
ACKNOWLEDGEMENTS
We thank D. McCollum (University of Massachusetts Medical School, USA),
K. Gould (Vanderbilt University, USA), J-Q. Wu (Ohio State University, USA),
V. Simanis (ISREC, Switzerland), J. Bähler (University College London, UK),T. D. Pollard (Yale University, USA), T. Toda (Cancer Research, UK), I. Hagan(Paterson Institute, UK), F. Chang (Columbia University, USA), D. Mulvihill(University of Kent, UK), P. Perez (CSIC, Spain), Y. Hiraoka (Osaka University,
Japan), K. Nakano (University of Tsukuba, Japan), S. Oliferenko (TLL, Singapore),and M. Sato (Waseda University, Japan) and the Yeast Genetic Resource Center
Japan for providing plasmids and strains, Y. Oba and M. Ojika (Nagoya University,
Japan) for their kind gift of DXQ, Y. Toyoshima (University of Tokyo, Japan) for her
kind gift of kinesin, R. Amikura for help in electron microscopy, and S. Oliferenko,
S. Bulchand and D. Subramanian for critical reading of this manuscript. We
thank K. Gull (Oxford University, UK) for antibodies. We thank M. Sevugan fortechnical assistance. This work was supported by a Japan Society for Promotion of
Science (JSPS) grant-in-aid for scientific research (I.M., #22247031); JSPS research
fellowships for young scientists (J.K.); NUS JSPS collaborative grant (I.M. and M.B.),
Temasek Life Sciences Laboratory and Singapore Millennium Foundation (M.B.
and M.M.); visiting scientist fellowship from the Gakushuin University (M.M.) andMechanobiology Institute (M.B., R.S. and Y.H.).
AUTHOR CONTRIBUTIONS
I.M. (in vitro activation and biochemistry) and M.K.B. (establishment of ringassembly in protoplasts and mutant studies) conceived the study, I.M., M.M., M.K.B.
and J.K. designed the experiments. I.M., J.K., M.M., T.T., R.S. and Y.H. conducted
the experiments. J.K., I.M. and M.M. analysed the data. M.K.B., M.M., J.K. and I.M.
wrote the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at www.nature.com/doifinder/10.1038/ncb2781
Reprints and permissions information is available online at www.nature.com/reprints
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19. Kitayama, C., Sugimoto, A. & Yamamoto, M. Type II myosin heavy chain
encoded by the myo2 gene composes the contractile ring during cytokinesis in
Schizosaccharomyces pombe. J. Cell Biol. 137, 1309–1319 (1997).
20. Motegi, F., Nakano, K., Kitayama, C., Yamamoto, M. & Mabuchi, I. Identification of
Myo3, a second type-II myosin heavy chain in the fission yeast Schizosaccharomyces
pombe. FEBS Lett. 420, 161–166 (1997).
21. Bezanilla, M., Forsburg, S. L. & Pollard, T. D. Identification of a second myosin-II in
Schizosaccharomyces pombe: Myp2p is conditionally required for cytokinesis. Mol.
Biol. Cell 8, 2693–2705 (1997).
22. Balasubramanian, M. et al. Isolation and characterization of new fission yeast
cytokinesis mutants. Genetics 149, 1265–1275 (1998).
23. Lord, M. & Pollard, T. D. UCS protein Rng3p activates actin filament gliding by
fission yeast myosin-II. J. Cell Biol. 167, 315–325 (2004).
24. Wong, K. C., Naqvi, N. I., Iino, Y., Yamamoto, M. & Balasubramanian, M. K. Fission
yeast Rng3p: An UCS-domain protein that mediates myosin II assembly during
cytokinesis. J. Cell Sci. 113, 2421–2432 (2000).
25. McCollum, D., Balasubramanian, M. K., Pelcher, L. E., Hemmingsen, S. M. & Gould,
K. L. Schizosaccharomyces pombe cdc4+ gene encodes a novel EF-hand protein
essential for cytokinesis. J. Cell Biol. 130, 651–660 (1995).
26. Mendes Pinto, I., Rubinstein, B., Kucharavy, A., Unruh, J. R. & Li, R.
Actin depolymerization drives actomyosin ring contraction during budding yeast
cytokinesis. Dev. Cell 22, 1247–1260 (2012).
27. Nakano, K. & Mabuchi, I. Actin-depolymerizing protein Adf1 is required for formationand maintenance of the contractile ring during cytokinesis in fission yeast. Mol. Biol.
Cell 17, 1933–1945 (2006).
28. Kovar, D. R., Kuhn, J. R., Tichy, A. L. & Pollard, T. D. The fission yeast cytokinesis
formin Cdc12p is a barbed end actin filament capping protein gated by profilin. J.
Cell Biol. 161, 875–887 (2003).
29. Chang, F., Drubin, D. & Nurse, P. cdc12p, a protein required for cytokinesis in fission
yeast, is a component of the cell division ring and interacts with profilin. J. Cell Biol.
137, 169–182 (1997).
30. Roberts-Galbraith, R. H. et al. Dephosphorylation of F-BAR protein Cdc15 modulates
its conformation and stimulates its scaffolding activity at the cell division site. Mol.
Cell 39, 86–99 (2010).
31. Balasubramanian, M. K., Helfman, D. M. & Hemmingsen, S. M. A new tropomyosin
essential for cytokinesis in the fission yeast S. pombe. Nature 360, 84–87 (1992).
32. Stark, B. C., Sladewski, T. E., Pollard, L. W. & Lord, M. Tropomyosin and myosin-II
cellular levels promote actomyosin ring assembly in fission yeast. Mol. Biol. Cell 21,
989–1000 (2010).
33. Eng, K., Naqvi, N. I., Wong, K. C. & Balasubramanian, M. K. Rng2p, a protein
required for cytokinesis in fission yeast, is a component of the actomyosin ring andthe spindle pole body. Curr. Biol. 8, 611–621 (1998).
34. Nakano, K., Satoh, K., Morimatsu, A., Ohnuma, M. & Mabuchi, I. Interactions among
a fimbrin, a capping protein, and an actin-depolymerizing factor in organization of
the fission yeast actin cytoskeleton. Mol. Biol. Cell 12, 3515–3526 (2001).
35. Takaine, M., Numata, O. & Nakano, K. Fission yeast IQGAP arranges actin filaments
into the cytokinetic contractile ring. EMBO J. 28, 3117–3131 (2009).

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METHODS

DOI: 10.1038/ncb2781

METHODS
Strains, genetic techniques, chemicals and spheroplasting. S. pombe strainsused in this study are listed in Supplementary Table S5. Standard procedures
for S. pombe genetics were used36 . Blebbistatin and Jasp were purchased from
Calbiochem and CytA, MBC and TBZ were purchased from Sigma. Strains
expressing epitope-tagged proteins were constructed using a PCR-based approach37 .
Spheroplasts were obtained as described previously9,38 . The use of a synchronous
cell culture of cdc25-22 cells carrying Rlc1–3xGFP enhanced the proportion of
spheroplasts and cell ghosts with a contractile ring. Cells were first grown at thepermissive temperature of 25 ◦ C to early log phase in minimal medium containing
0.5% glucose. These cells were then shifted to the restrictive temperature of 36 ◦ C
for 4 h before spheroplasting. The time for cell wall digestion was different among
various strains and varied from 20 min to a 1 h. Typically more than 90% of
the cells were spheroplasted in all experiments. These spheroplasts were thengrown in minimal medium with 0.8% sorbitol at 25 ◦ C with slow shaking (80r.p.m.). Contractile ring formation was monitored by fluorescence microscopy.
For a typical experiment, at the point of spheroplasting Rlc1–3xGFP signal was
detected in a single bright dot-like structure in 100% of the spheroplasts andactomyosin arcs or contractile rings were not seen. After 5 h of incubation at
24 ◦ C, actomyosin arcs could be detected (in 3–7% of cells) and 25–35% of thespheroplasts had a fully formed contractile ring. At this point spheroplasts werecollected for permeabilization and the analysis of the contraction competenceof arcs and rings is described in the manuscript. For preparation of cell ghosts
from various cytokinesis mutants, mutant cells expressing Rlc1–3GFP or Rlc1–GFP
were grown at the permissive temperature of 25 ◦ C overnight in minimal medium
containing 0.5% glucose and spheroplasted at 25 ◦ C. To determine the time taken
for inactivation of various ts mutant proteins, intact mutant cells were shifted to therestrictive temperature of 36 ◦ C and their inability to compact the contractile ring
was monitored.

Permeabilization of spheroplasts and preparation of cell ghosts. Spheroplasts
were washed twice with wash buffer (0.8 M sorbitol, 2 mM EGTA, 5 mM MgCl2
and 20 mM PIPES–NaOH, at pH 7.0) and incubated for 5 min in isolationbuffer (0.16 M sucrose, 50 mM EGTA, 5 mM MgCl2 , 50 mM potassium acetate,
50 mM PIPES–NaOH, at pH 7.0, 0.5% NP-40, 10 µg ml−1 leupeptin, 10 µg ml−1
aprotinin, 10 µg ml−1 pepstatin, 0.5 mM phenylmethylsulphonyl fluoride and
1 mM dithiothreitol) on ice. Spheroplasts were homogenized with a Teflon/glass
homogenizer. Cell ghosts were washed twice with reactivation buffer (0.16 M
sucrose, 5 mM MgCl2 , 50 mM potassium acetate, 20 mM MOPS–NaOH, at
pH 7.0, 10 µg ml−1 leupeptin, 10 µg ml−1 aprotinin, 10 µg ml−1 pepstatin, 0.5 mM
phenylmethylsulphonyl fluoride and 1 mM dithiothreitol). About 30% of theresultant cell ghosts retained intact contractile rings and another 5–10% had
actomyosin arcs or discontinuous or fragmented contractile rings. Most intact fully
formed contractile rings were competent for in vitro contraction on ATP addition.

Reactivation was performed by perfusion with reactivation buffer containing anappropriate concentration of ATP. Most intact contractile rings in wild-type cellghosts underwent ATP-mediated contraction.
Conventional fluorescence microscopy, three-dimensional reconstruction andtime-lapse observations were made using a DeltaVision system (Applied Precision)
attached to an Olympus IX-70 wide-field inverted fluorescence microscope
equipped with an Olympus UplanSApo ×100 oil-immersion objective lens (NA1.4, Olympus), and a Photometrics CoolSNAP HQ camera (Roper Scientific).
Temperature was controlled by both the thermo-plate and a lens heater (MATS55RAF20, MATS-LH; TOKAI HIT). More than 14 optical sections (0.5 µm
spaces) were acquired every 10 s. Images were captured and processed by iterative
constrained deconvolution using SoftWoRx (Applied Precision), and analysed by
SoftWoRx and ImageJ (W. S. Rasband, National Institutes of Health, Bethesda,
MD) incorporated with the McMaster Biophotonics Facility ImageJ for Microscopy
collection of plugins (T. Collins, McMaster Biophotonics Facility, Hamilton,
Ontario, Canada). For measurement of the contraction rate, stacks of opticalsections were reconstructed in a horizontal direction using the three-dimensionalrotation tool of SoftWoRx and the perimeter of each ring was measured. Eachcontraction rate was determined by using values of the most linear phase ofcontraction. For measurement of fluorescence intensity in Fig. 4b, sum projectionsof six deconvolved sections were used. Integrated intensity was calculated frommean value and area, after subtracting the background mean value of the same area.
Graphical images were established using Prism-5 (Graphpad Software).

Actin disassembly assay and western blotting. Cell ghosts suspended inreactivation buffer were split into three. A half-volume of reactivation buffer
containing 15% dimethylsulphoxide or 0.3 mM Jasp was added and gently mixed.
After three minutes, a one-quarter volume of reactivation buffer containing 5%
dimethylsulphoxide, 5% dimethylsulphoxide and 2 mM ATP, or 0.1 mM Jasp and
2 mM ATP was added and gently mixed to induce ring contraction. After fiveminutes incubation at 25 ◦ C, suspensions were centrifuged at 16,000g for 5 min
at 4 ◦ C. The pellet was frozen at −80 ◦ C. Supernatant with 0.02% deoxycholate
added was left for 30 min on ice followed by 10% trichloroacetic acid additionand overnight incubation on ice. Precipitates were washed with cold acetone
and dried. The pellet and supernatant were analysed by SDS–PAGE and western
blotting. The volume of pellet loaded was one-third that of the supernatant. For the
sedimentation assay, half of the supernatant was centrifuged at 200,000g for 15 min
at 4 ◦ C. As primary antibody, mouse anti-actin monoclonal antibody MAB1501(1:1,000, Millipore), rabbit anti-GFP polyclonal antibody 598 (1:2,000, MBL), rabbit
anti-Cdc8 polyclonal antibody (1:250; ref. 40) or mouse anti-α-tubulin monoclonal
antibody TAT-1 (1:1,000, gift from K. Gull) was used. Appropriate HRP-conjugatedsecondary antibody was used at 1:5,000. Western blots were developed using
SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific,
Waltham) and luminoimage analyser LAS3000 (Fujifilm). Each integrated intensity
was calculated from the mean value and the area measured using ImageJ.

Protein localization in cell ghosts and immunofluorescence microscopy.
Cell ghosts were prepared from strains carrying the protein of interest fused
to fluorescent proteins as described above. Indirect immunofluorescence
was used to label Cdc4p and Adf1p. F-actin was stained with BODIPYFL–phallacidin, Alexa 568–phalloidin (Molecular Probes-Invitrogen) or synthesized
tetramethylrhodamine-labelled Lifeact peptide (Operon Biotechnologies). For immunofluorescence microscopy, cell ghosts were fixed with 3.8% paraformaldehyde
in PEM buffer (1 mM EGTA, 1 mM MgCl2 and 0.1 M PIPES–NaOH, at pH 6.9)
for 30 min. After washing with PEM buffer, cell ghosts were blocked in PEM
buffer containing 1% BSA, 0.1% NaN3 and 10 mM l-lysine-HCl for 1 h at room
temperature. Rabbit anti-Cdc4 polyclonal antibody25 (1:100) and rabbit anti-Adf1(ref. 27) polyclonal antibody (1:500) were used as a primary antibody for Cdc4p andAdf1p detection, respectively. Tetramethylrhodamine-conjugated anti-rabbit IgG
was used as a secondary antibody at 1:1,000.

Measurement of ATPase activity. Motor proteins used were a mixture of

Electron microscopy. Cell ghosts for electron microscopy were prepared as

36. Moreno, S., Klar, A. & Nurse, P. Molecular genetic analysis of fission yeast
Schizosaccharomyces pombe. Methods Enzymol. 194, 795–823 (1991).
37. Bähler, J. et al. Heterologous modules for efficient and versatile PCR-based genetargeting in Schizosaccharomyces pombe. Yeast 14, 943–951 (1998).
38. Kobori, H., Yamada, N., Taki, A. & Osumi, M. Actin is associated with the formationof the cell wall in reverting protoplasts of the fission yeast Schizosaccharomyces
pombe. J. Cell Sci. 94, 635–646 (1989).
39. Kamasaki, T., Arai, R., Osumi, M. & Mabuchi, I. Directionality of F-actin cables
changes during the fission yeast cell cycle. Nat. Cell Biol. 7, 916–917 (2005).
40. Arai, R., Nakano, K. & Mabuchi, I. Subcellular localization and possible functionof actin, tropomyosin and actin-related protein 3 (Arp3) in the fission yeast
Schizosaccharomyces pombe. Eur. J. Cell Biol. 76, 288–295 (1998).
41. Fiske, C. H. & Subbarow, Y. The Colorimetric determination of phosphorus. J. Biol.
Chem. 66, 375–400 (1925).

described previously39 . Actin filaments were decorated with skeletal heavy
meromyosin for easy visualization. Sections were examined with a JEM1400 electron
microscope (JEOL) at 80–100 kV.

Reactivation of contractile ring and microscopy. The reactivation of thecontractile ring in the cell ghost was assessed with a hand-made perfusion chamber.
The base of the chamber was constructed with a large coverslip (Matsunami
Glass, 24 × 36 mm, No.1) coated with 0.01% poly-l-Lysine (Sigma, P1524) anddouble-sided tape (Nichiban; NW-5S, 60 µm wall thickness). Cell ghosts insuspension were adhered to the base of the chamber and then covered with asmall coverslip (Matsunami, 18 × 18 mm, No. 1). The volume in the chamber
was around 10 µl. Cell ghosts were washed with reactivation buffer by perfusion.

rabbit skeletal muscle myosin chymotryptic S1 (0.18 mg ml−1 ) and rabbit skeletal
muscle F-actin (0.89 mg ml−1 ), sea urchin sperm axonemal dynein (Tris–EDTA
extract, 0.21 mg ml−1 ), and recombinant rat brain kinesin-1 motor domain (relative
molecular mass 430,000, 0.17 mg ml−1 , gift from Y. Toyoshima). These wereincubated in 0.1 M KCl, 2.8 mM MgCl2 , 10 mM TES (pH 7.0) and 2.6 mM ATP
at 25 ◦ C. EDTA (0.55 mM) was also included for dynein and kinesin. Inorganic
phosphates released were measured by the method of ref. 41 after removing proteinsas trichloroacetic-acid-induced precipitates.

Statistical analysis. Statistical comparisons of mean values were made using theunpaired two-tailed t -test for two data sets and one-way analysis of variance formultiple data sets. The actual P values for each test are represented in each figure.

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NATURE CELL BIOLOGYS U P P L E M E N TA R Y I N F O R M AT I O N
DOI: 10.1038/ncb2781

Supplementary Figures:Supplementary Figures S1

Figure S1 Examples of contractile ring formation in intact wild type cell, spheroplast and spherical mutant. (a) Maximum projections of deconvolved sectionsof Rlc1-3xGFP and mCherry-Atb2 images in an intact fission yeast cell. White doted lines indicate the cell outline. (b) Maximum projections of deconvolved
sections of Rlc1-3xGFP and mCherry-Atb2 images in an intact spheroplast. White dotted line represents the outline of the spheroplast. (c) Maximum
projections of contractile ring formation in orb3/nak1 null cell (orb3D) expressing Rlc1-3xGFP at 25˚C. Contractile ring initiates from one predominantlocation on the surface of the spheroplast (shown by arrowhead at time 0 min) and grows in a bidirectional manner (shown by yellow arrows) to form a maturering. Scale bars, 5 mm.

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S U P P L E M E N TA R Y I N F O R M AT I O N
Supplementary Figures S2

Figure S2 Cell-ghosts contain essential contractile ring proteins butlack other cytoplasmic components. (a) Essential components of thecontractile ring are retained in cell-ghosts. Images are shown as maximum
projections of Z-stacks. Spheroplasts expressing fluorescent-tagged
proteins as indicated on each image were permeabilised before imaging.
Adf1 was stained with anti-Adf1 antibody and anti-rabbit IgG labeled withtetramethylrhodamine after permeabilisation of spheroplasts expressing
Rlc1-3xGFP. (b) Most cytoplasmic components are not retained in cellghosts. Fluorescence images are shown as maximum projections ofdeconvolved sections. Spheroplasts expressing fluorescent-tagged proteinsas indicated were imaged before and after permeabilisation. Scale bars,
5 mm.

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Supplementary Figures S3
S U P P L E M E N TA R Y I N F O R M AT I O N

Figure S3 General features and myosin ATPase dependency of ring contraction in vitro. Experiments were done at 25˚C. (a) pH sensitivity of ring contraction
in vitro. ATP concentration was 0.25 mM. Each spot represents the rate of contraction of an individual ring. Medial horizontal lines and error bars represent
mean values and standard deviations, respectively. One-way analysis of variance (ANOVA) was used for statistical analysis. See Table S6 for statistics source
data. (b) Ca++ sensitivity of ring contraction in vitro. ATP concentration was 0.25 mM. Statistical analysis in panel b was done as in panel a. See Table S6
for statistics source data. (c) Reduction of relative fluorescence intensity after 0.5 mM ATP addition. Integrated fluorescence was calculated from meanvalues of area of region of interest and were normalised to the value at time 0. It is apparent that the photobleaching and/or spontaneous loss of Rlc1-3GFP
from contractile ring or from the arc in the absence of ATP are relatively minor.3(d) Effect of dehydroxestoquinone (DXQ) on ATPase activity of motor proteins.
DXQ has been reported to be an ATPase inhibitor that inhibits ATPase activity of muscle myosin II. Since DXQ had not been tested on other ATPase-motors,
we tested its effects on kinesin (from brain), dynein (flagella), and myosin II S1 fragment (skeletal muscle) as a control. DXQ showed strong inhibition of
ATPase activity of myosin II and dynein and a weaker inhibition of kinesin. Mean values of two measurements are plotted. See Table S6 for statistics source
data. (e) Ring contraction was inhibited by the addition of 2.5 mM DXQ. Vanadate, which at 100 mM inhibits kinesin and dynein ATPases, did not affect ring
contraction. Fluorescent images of Rlc1-3xGFP are shown as maximum projections of Z-stacks (0.5 mm spaces). ATP concentration was 0.5 mM for DXQ or
0.1 mM for vanadate. Scale bar, 5 mm.
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Supplementary Figures S4

S U P P L E M E N TA R Y I N F O R M AT I O N

Figure S4 Neither actin polymerization nor depolymerization are required
graphical representation of the data is shown. (c) Phalloidin and phallacidin
for ring contraction in vitro. (a) The effects of drugs (Jasp, 0.1 mM
prevent release of actin from contractile ring after ATP addition. Actin was
jasplakinolide; Cyt A, 0.1 mM cytochalasin A; Lat A, 10 mM latrunculin A;
stained with 10 mg/ml TMR-Lifeact before or after contraction. Fluorescent
MBC+TBZ, 50 mg/ml MBC and 50 mg/ml TBZ) on ring contraction in vitro.
images of Rlc1-3xGFP and TMR-Lifeact are shown as maximum projections
5% dimethyl sulfoxide (DMSO) was used for control experiment. Each spotof Z-stacks (0.5 mm steps). Scale bar, 5 mm. (d) Western blot analysis of actin
represents the rate of contraction of an individual ring. Medial horizontal lines
disassembly upon ATP addition in the presence of phalloidin or phallacidin.
and error bars represent mean values and standard deviations, respectively.
S, supernatant; P, pellet; G, G-actin and short F-actin; F, F-actin. Each(b) 0.5 mM ATP was added after appropriate incubation at 36˚C (cdc3-6ts,
relative amount was calculated from the amount of actin in the pellet without
4 ATP. Each spot represents relative amount in individual experiment. See Table
20 min.; cdc12-112ts cdc15-140ts and adf1-1ts, 15 min). Wild-type cellghosts were incubated at 36˚C for 30-40 minutes before ATP addition. A
S6 for statistics source data. See also Fig. 4c, f.

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S U P P L E M E N TA R Y I N F O R M AT I O N
Supplementary Figures S5

Figure S5 Dose dependent inhibition of ring contraction by purified actinbundling proteins in vitro. (a-b) Cell-ghosts were incubated in the presenceof purified proteins at the indicated concentrations for 3 minutes before
addition of 0.1 mM ATP. Experiments were done at 25˚C. Fluorescent imagesof Rlc1-3xGFP are shown as maximum projections of Z-stacks (0.5 mmsteps). Each spot represents the rate of contraction of an individual ring.
Medial horizontal lines and error bars represent mean values and standard

deviations, respectively. See Table S6 for statistics source data. (c) Actincross-linking activities of purified actin- bundling proteins. Skeletal actin (3mM) was polymerised in the reactivation buffer including purified proteinsfor 30 minutes at room temperature. F-actin was stained with BODIPYFL-phallacidin. Both, 1 mM Rng2Ns and 0.5 mM Fim1 show robust actinbundling activity. Images are deconvolved from single focal planes. Scalebars, 5 mm.

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S U P P L E M E N TA R Y I N F O R M AT I O N

Supplementary Figures S6

Figure S6 Full scans of original blots. (a-b) Full scan of blot shown in Fig. 1 h. (c) Full scan of blot shown in Fig. 4 d-e. (d) Full scan of blot shown in Fig. 4 f.
(e) Full scan of blot shown in Fig. S4d.

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S U P P L E M E N TA R Y I N F O R M AT I O N

Supplementary Table Legends:
Table S1. Proteins in the contractile rings. Intact cells and cell-ghosts expressing fluorescent-tagged proteins as indicated were imaged. Indirect
immunoflurosence was used to label Cdc4p and Adf1p. Actin was detected by Alexa 568- phalloidin.
Table S2. P values for comparison of relative intensities of contractile ring components in Fig. 2f. Values at 100 seconds were compared. Unpaired two-tailed
t-test was used for this analysis. P values less than 0.05, are represented in bold.
Table S3. Contraction rates in various nucleotide concentrations. Mean±SD was indicated. These datasets were used for the graph shown in Fig. 2h andcalculation of Km and Vm in Fig. 2i.
Table S4. Contractile ring components in the mutant rings. Cell-ghosts were prepared from the mutant cells expressing the indicated fluorescent-tagged
proteins. Cell-ghosts were incubated at restrictive temperature to inactivate the mutant protein before imaging. Indirect immunofluorescence was used tolabel Cdc4p
Table S5. List of strains used in this study. *YGRC, Yeast Genetic Resource Center Japan. (http://yeast.lab.nig.ac.jp/nig/index_en.html).

Supplementary Video Legends:
Video S1. ATP drives ring contraction in vitro. The video of a contracting ring in the presence of 0.5 mM ATP at 25˚C corresponds to the frames shown in Fig.
2a. X-Y and Y-Z images were shown as non-tilted and 90˚-tilted maximum projections of Z-stacks, respectively.
Video S2. ATP drives ring contraction. The video of a contracting ring in the presence of 0.01 mM ATP at 25˚C corresponds to the frames shown in Fig. 2c.
X-Y and Y-Z images were shown as non-tilted and 90˚-tilted maximum projections of Z-stacks, respectively.
Video S3. AMP-PNP does not induce ring contraction in vitro. Experiment was done at 25˚C. The video corresponds to the frames shown in Fig. 2d. X-Y and
Y-Z images were shown as non-tilted and 90˚-tilted maximum projections of Z-stacks, respectively.
Video S4. The plasma membrane of Rlc1-3xGFP expressing cell-ghosts stained with FM4-64 during contraction in vitro. Experiment was done at 25˚C. The
membrane did not ingress as the ring contracts. The video corresponds to the frames shown in Fig. 2g.
Video S5. Incomplete actomyosin arcs do not show ATP-dependent contraction in vitro. The GFP fluorescence decays the presence of 0.5 mM ATP but theactomyosin arc do not show any contraction at 25˚C. The video corresponds to the frames shown in Fig. 2j. X-Y and Y-Z images were shown as non-tilted and90˚-tilted maximum projections of Z-stacks, respectively
Video S6. Contractile ring of myo2-E1ts undergoes slow in vitro contraction even at the permissive temperature of 25˚C. The video corresponds to the frames
shown in Fig. 3b.
Video S7. Contractile ring of myp2 null mutant (myp2D) in vitro. Experiment was done at 25˚C. The video corresponds to the frames shown in Fig. 3b.
Video S8. Contractile ring contraction of the double mutant of myo2 and myp2 (myo2-E1ts myp2D) in vitro. These rings did not show any contraction andinstead fragmented and disassembled in presence of ATP at 25˚C. The video corresponds to the frames shown in Fig. 3b.
Video S9. Addition of Jasplakinolide does not affect ring contraction in vitro. Experiment was done at 25˚C.
Video S10. ADF/cofilin (Adf1p) is not required for ring contraction in vitro. Cell-ghosts from adf1-1ts were held at 36˚C for 15 minutes before ATP addition.
Video S11. LatA has minimal effect on ring contraction in vitro. Experiment was done at 25˚C.
Video S12. Formin (Cdc12p) is not required for ring contraction in vitro. Experiment was done at 36˚C. Cell-ghosts from cdc12-112ts were held at 36˚C for
15 minutes before ATP addition.
Video S13. F-BAR protein (Cdc15p) is not required for ring contraction in vitro. Experiment was done at 36˚C. Cell-ghosts from cdc15-140ts were held at
36˚C for 15 minutes before ATP addition.
Video S14. Tropomyosin (Cdc8p) is required for integrity and contraction of ring in vitro. Cell-ghosts from cdc8-110ts were held at 36˚C for 15 minutes before
ATP addition. The video corresponds to the frames shown in Fig. 5a.
Video S15. Addition of 1 mM purified N-terminal region of IQGAP (Rng2Ns) completely blocks ring contraction in vitro. Experiment was done at 25˚C. The
video corresponds to the frames shown in Fig. 5b.
Video S16. Addition of 0.9 mM purified fimbrin (Fim1p) completely blocks ring contraction in vitro. Experiment was done at 25˚C. The video corresponds tothe frames shown in Fig. 5c.

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光学顕微鏡技術を駆使した生体分子の動態の可視化

教授

西坂

崇之

[目的]
ほぼすべての微生物は運動する機能を持ち、生存のため適した環境に移動する手段として、その機能を巧みに用いている。バクテリア（真正細菌、bacteria）とアーキア（古細菌ある
いは始原菌、archaea）は、真核生物で知られている従来の分子モーターとは異なった多様な運動装置を有しており、生物学のみならず、それらの未知の動作原理は物理の視点からも注目すべき研究対象となっている。
我々のグループでは、広く知られているべん毛回転モーターとは異なり、ユニークな運動装置を持つ幾つもの種を培養して研究を進めている。独自に開発した先進的な光学顕微鏡を駆使し、モーターという観点から装置の動作メカニズムについて明らかにすることを目的としている。本課題では、分子レベルから集団レベルまでのマルチスケールにおいて多面的な研究を展開し、微生物の様々な新しい運動様式を発見し、詳細に解析した。ステップ状の運動から集団運動に至るまで、これまでの成果の概要について以下に記す。

[Mycoplasma mobile のゴーストモデルによるステップ状運動]

Mycoplasma mobile（以下 M. mobile）は遊泳能を持たず、基板の上を一方向に滑走運動を行う。その滑走速度はバクテリアの中で最大であり、運動に関与するタンパク質は同定されているものの、基本的な動作原理は仮説の域を出ていない。
膜を部分的に透過性にしたゴーストモデルに、高精度の位置検出顕微鏡を応用することで、

M. mobile の滑走装置は 70 nm の変位を行いながら運動を実現していることを明らかにした（参考文献１）。バクテリアの運動装置におけるステップ状運動を観察したのは、べん毛回転モーターの報告に引き続き２例目である。
観測されたステップの大きさ自体は、「巨大な脚状タンパク質の屈曲による運動」に矛盾はしないものの、既に報告のあるタンパク質の柔軟性を考えると、繰り返し正確なステップを行う機構がまったく説明できないことになる。新しいモデルとして、膜内にある回転モーターが別のタンパク質の回転を誘起し、その運動が未知の機構により直線運動へと変換される「回転⇔並進 変換モデル」を提案している。

[超解像顕微鏡による M. mobile の詳細な運動の定量化]
基板表面に可視光の波長より小さい金属のパターンを蒸着し、そこに全反射照明を行うこ

222

とで、表面近傍に「異常光学透過（EOT）」という特徴的な電磁波の分布を作ることができる。EOT の分布は、AFM や電子顕微鏡で決定したパターンの構造と、詳細なシミュレー
ションを組み合わせることにより推定できる。この分布を参照パターン（kernel）とし、一定速度で運動する蛍光バクテリアからのシグナルを kernel で再構成することによって、滑走中のバクテリア（M. mobile）の位置を３次元的に高精度で決定する新しい実験方法を提案した（参考文献２）。

[Flavobacterium johnsoniae の集団運動]桿菌である Flavobacterium johnsoniae（以下 F. johnsoniae）も M. mobile 同様、基板の上で滑走運動を行うが、その運動機構はまったく異なる仮説が提唱されている。細胞表面から突き出た接着タンパク質がらせん状に配置したレールの上を動き、この流れが細胞全体の一方向の運動を生み出すと考えている。我々は F. johnsoniae が飢餓状態において、特徴的な渦運動を生み出すことを見出し、研究機関の初年度に基本的なデータの収集を終えた。次年度ではこの機構を解明するため、渦の形成過程を長時間にわたり安定して記録するシステムの構築に成功した。

[アーキア回転モーターのステップ状回転]
アーキアの遊泳運動の研究は、試料調製の難しさからバクテリアと比べ未踏の領域であると言える。我々は Hanobacterium salinarum を対象として、再現性良く運動を観察する
実験系の構築に成功した。さらに ATP 駆動によるモーターのステップ状運動の計測にも成功した。この結果については生物物理学会のシンポジウム、および Pachifichem 2015 において招待講演として発表し、まだ原著論文は発表されていないものの（参考文献３）既に高い評価を得ている。

[参考文献]

1. Kinosita, Y., Nakane, D., Sugawa, M., Masaike, T., Mizutani, K., Miyata, M.,
and Nishizaka, T. (2014). Unitary step of gliding machinery in Mycoplasma mobile. Proc
Natl Acad Sci USA 111, 8601-8606.
2. Lee, W., Kinosita, Y., Oh, Y., Mikami, N., Yang, H., Miyata, M., Nishizaka, T., and Kim,
D. (2015). Three-Dimensional Superlocalization Imaging of Gliding Mycoplasma mobile
by Extraordinary Light Transmission through Arrayed Nanoholes. ACS Nano 9,10896-10908.
3. Kinosita, Y., Uchida, N., Nakane, D., and Nishizaka, T. (2016). Direct observation ofrotation and steps of the archaellum of swimming halophilic archaea. (submitted)

223

[原著論文]1. Kinosita, Y., Nakane, D., Sugawa, M., Masaike, T., Mizutani, K., Miyata, M.,
and Nishizaka, T. (2014). Unitary step of gliding machinery in Mycoplasma mobile. Proc
Natl Acad Sci USA 111, 8601-8606.
2. Kurushima, J., Nakane, D., Nishizaka, T., and Tomita, H. (2015). Bacteriocin protein
BacL1 of Enterococcus faecalis targets cell division loci and specifically recognizes
L-Ala2-cross-bridged peptidoglycan. Journal of bacteriology 197, 286-295.
3. Lee, W., Kinosita, Y., Oh, Y., Mikami, N., Yang, H., Miyata, M., Nishizaka, T., and Kim,
D. (2015). Three-Dimensional Superlocalization Imaging of Gliding Mycoplasma mobile
by Extraordinary Light Transmission through Arrayed Nanoholes. ACS Nano 9,10896-10908.
4. Yamaguchi, S., Saito, K., Sutoh, M., Nishizaka, T., Toyoshima, Y.Y., and Yajima, J.
(2015). Torque generation by axonemal outer-arm dynein. Biophys J 108, 872-879.
5. Sugawa, M., Okazaki, K., Kobayashi, M., Matsui, T., Hummer, G., Masaike, T.,
and Nishizaka, T. (2016). F1-ATPase conformational cycle from simultaneous
single-molecule FRET and rotation measurements. Proc Natl Acad Sci USA (in press).
[総説等]
1.

Katoh, T.A., Fujimura, S., and Nishizaka, T. (2015). 3-D Single Particle Tracking Using
Dual Images Divided by Prism: Method and Application to Optical Trapping. In Handbookof Photonics for Biomedical Engineering.

[口頭発表]
1.

Itou, T., Y. Kinosita, D. Nakane, H. Wada, W. Wang, T. Nishizaka (2015).
Spiroplasma eriocheiris において観察されたキンクの伝搬によって生じるらせん型ス
クリューの向きと回転速度の変化

Change in handedness and rotational speed ofhelical screw of Spiroplasma eriocheiris driven by kink propagation. 第53回日本生物物理学会年会, 金沢大学 角間キャンパス 自然科学本館, September 13-15, 2015.
2.

Iwase, T., M. Suegara, R. Nakayama, T. A. Katoh, M. Setou, T. Nishizaka, K.
Ikegami, T. Masaike (2015). 三次元で見た ATP 依存的な繊毛運動の Ca2+ による制御

Regulation of ATP-dependent ciliary motility by Ca2+ observed in three

dimensions. 第53回日本生物物理学会年会, 金沢大学 角間キャンパス 自然科学本館,
September 13-15, 2015.
3.

Mizutani, M., Y. Kinosita, T. Nishizaka, M. Miyata (2015). Mycoplasma mobile が捕捉状態で発生する力

Force generation of Mycoplasma mobile at trapped state.

日本マイコプラズマ学会第42回学術集会, 慶應義塾大学薬学部 芝共立キャンパス,May 22-23.
4.

Mizutani, M., Y. Kinosita, T. Nishizaka, M. Miyata (2015). Mycoplasma mobile のストール力

Stall force of Mycoplasma mobile. 第53回日本生物物理学会年会, 金沢

大学 角間キャンパス 自然科学本館, September 13-15, 2015.

224

5.

Mizutani, M., Y. Kinosita, T. Nishizaka, M. Miyata (2015). Mycoplasma mobile のストール力

Stall force of Mycoplasma mobile. 第88回日本細菌学会総会, 長良川国際会議場, March 26-28.
6.

下澤東吾, 橋本優, 井合健太郎, 宗行英朗, 政池知子, 西坂崇之 (2013). 回転軸をねじったF1-ATPase変異体の回転特性解析. 日本生体エネルギー研究会
静岡県コンベンションアーツセンター

7.

第39回討論会,

グランシップ, December 18, 2013.

加藤孝信, 岩瀬寿仁, 瀬藤光利, 池上浩司, 西坂崇之, 政池知子 (2013). マウス気管繊毛の３次元運動解析と力測定. 新学術研究領域「シリア・中心体による生体情報フローの制御」第2回領域会議プログラム, ルブラ王山（名古屋）, November 28, 2013.

8.

加藤孝信, 岩瀬寿仁, 政池知子, 池上浩司, 瀬藤光利, 西坂崇之 (2013).
Three-dimensional motion of an isolated murine tracheal cilium under load 負荷をかけた状態での単離マウス気管上皮シリアの三次元運動. 第51回日本生物物理学会
年会, 国立京都国際会館, October 28, 2013.

9.

加藤孝信, 岩瀬寿仁, 政池知子, 池上浩司, 瀬藤光利, 西坂崇之 (2014). 負荷をかけた状態での単離マウス気管上皮シリアの三次元運動. 生体運動研究合同班・2014年班会
議, 千葉大学西千葉キャンパス, January 10, 2014.

10. 加藤孝信, 池上浩司, 岩瀬寿仁, 政池知子, 瀬藤光利, 西坂崇之 (2014). 単離マウス気管上皮繊毛が生み出す力の三次元顕微計測

Force measurement of individual

isolated mouse tracheal cilia using three-dimensional optical trapping. 第52回日本
生物物理学会年会, 札幌コンベンションセンター, September 25-27, 2014.
11. 加藤孝信, 池上浩司, 岩瀬寿仁, 政池知子, 瀬藤光利, 西坂崇之 (2014). 単離繊毛非対称運動における発生張力の三次元顕微計測. 第5回繊毛研究会, 浜松市研修交流セン
ター, May 24-25, 2014.
12. 加藤孝信, 池上浩司, 岩瀬寿仁, 政池知子, 瀬藤光利, 西坂崇之 (2015). マウス気管繊毛の非対称運動メカニズム -軸糸の変形の視点から-. 生体運動研究合同班・2015年班会議, 学習院大学, January 7-9, 2015.
13. 加藤孝信, 池上浩司, 岩瀬寿仁, 政池知子, 瀬藤光利, 西坂崇之 (2015). マウス気管繊毛の非対称運動メカニズム -軸糸の変形の視点から-. 第67回日本細胞生物学会大会,
東京都 タワーホール船堀, June 30-July 2, 2015.
14. 加藤孝信, 池上浩司, 内田就也, 岩瀬寿仁, 政池知子, 瀬藤光利, 西坂崇之 (2015). マ
ウス気管繊毛の非対称運動メカニズム －軸糸の変形の視点から－

Mechanism ofasymmetric beating of mouse tracheal cilia - from the perspective of the axoneme
bending -. 第53回日本生物物理学会年会, 金沢大学 角間キャンパス 自然科学本館,
September 13-15, 2015.
15. 岩瀬寿仁, 加藤孝信, 瀬藤光利, 西坂崇之, 池上浩司, 政池知子 (2014). Ca2+依存的な運動性を持つ気管繊毛の三次元解析. 第5回繊毛研究会, 浜松市研修交流センター,
May 24-25, 2014.
16. 久留島潤, 林幾江, 中根大介, 西坂崇之, 菅井基行, 富田治芳 (2014). 腸球菌バクテリ
オシンBac41による殺菌メカニズムの解析

225

Functional analysis of bacteriocin Bac41

produced by Enterococcus faecalis. 第87回日本細菌学会総会, 東京都江戸川区船堀・
タワーホール船堀, March 26-28,2014.
17. 三上渚, 政池知子, 須川光弘, 西坂崇之 (2013). "Advanced TIRF microscopy to detect
single-molecule conformational changes in both azimuth and axial axis using
polarization switching 方位と倒れの構造変化を１分子レベルで検出する偏光スイッ
チングを用いた新しいTIRFM." 第51回日本生物物理学会年会 October 28, 2013.
18. 三上渚, 政池知子, 西坂崇之 (2014). 全反射型蛍光顕微鏡における受像偏向と偏光変
調 －F1-ATPase の構造変化検出への応用－

Emitter modulation and polarization

switching under TIRF illumination: Application for detection of conformationalchange in F1-ATPase. 第52回日本生物物理学会年会, 札幌コンベンションセンター,
September 25-27, 2014.
19. 三上渚, 西坂崇之 (2015). １分子の複数状態モニタリングにより明らかとなった
F1-ATPase におけるヌクレオチド周辺の局所環境と化学状態の相関

Correlation

between local environment around nucleotide and chemical state in F1-ATPase
revealed by single-molecule modes monitoring. 第53回日本生物物理学会年会, 金沢
大学 角間キャンパス 自然科学本館, September 13-15, 2015.
20. 三上渚, 中根大介, 西坂崇之 (2014). Polarization- and emitter- Modulation TIRF
Microscopy to Detect 3-D Oriental of Single Protein. International Symposium on
Mechanobiology 2014, 岡山大学鹿田キャンパス, May 20-23, 2014.
21. 三上渚, 内藤達也, 下澤東吾, 政池知子, 宗行英朗, 西坂崇之 (2014). Detection ofConformational Change of the Cylinder in Single F1-ATPase and its Interaction
with the Shaft under Advanced Optical Microscopes. Tokyo ATPase Workshop, 東京大学本郷キャンパス, June 2-3, 2014.
22. 山口真, K. Saito, M. Sutoh, 西坂崇之, 豊島陽子, 矢島潤一郎 (2014). 軸糸外腕ダイニンによるトルク発生

Torque generation by axonemal outer-arm dynein. 第52回日

本生物物理学会年会, 札幌コンベンションセンター, September 25-27, 2014.
23. 山口真, 西坂崇之, 豊島陽子, 矢島潤一郎 (2015). 微小管コークスクリュー運動の三次元計測による軸糸外腕ダイニン及びそのサブユニットの運動特性の定量. 生体運動研究合同班・2015年班会議, 学習院大学, January 7-9, 2015.
24. 小高祥子, 中根大介, 西坂崇之 (2013). "Three-dimensional tracking of tank-likemotility apparatus of the gliding bacterium 滑走するバクテリアの戦車のような運動装置を三次元で追跡する." 第51回日本生物物理学会年会 October 28, 2013.
25. 小高祥子, 中根大介, 西坂崇之 (2014). バクテリアの集団運動による巨大渦パターン
形成

Large-scale vortex pattern emerging from bacteria collective motion. 第52回

日本生物物理学会年会, 札幌コンベンションセンター, September 25-27, 2014.
26. 小高祥子, 中根大介, 西坂崇之 (2014). バクテリアの集団運動による巨大渦パターン
形成

Large-scale vortex pattern emerging from bacterium collective motion. 第87回日本細菌学会総会, 東京都江戸川区船堀・タワーホール船堀, March 26-28, 2014.
27. 小高祥子, 中根大介, 西坂崇之 (2014). 滑走するバクテリアがつくる巨大渦パターン.
生体運動研究合同班・2014年班会議, 千葉大学西千葉キャンパス, January 10, 2014.

226

28. 小高祥子, 中根大介, 西坂崇之 (2015). 滑走するバクテリアの自己集合により形成される巨大渦の一方向性回転

Directional rotation of large-scale vortex made ofself-assembly of gliding bacteria. 第53回日本生物物理学会年会, 金沢大学 角間キャンパス 自然科学本館, September 13-15, 2015.
29. 須河光弘, 山口真, 柴田桂太郎, 藤. 政池知子, 豊島陽子, 矢島潤一郎, 神隆, 西坂崇之
(2013). "受像偏向蛍光イメージングによる分子モーターの運動計測

生体運動研究合同班・2013年班会議: January 13, 2013.
30. 須河光弘, 小林大, 松井貴志, 政池知子 (2013). "Analysis of the ATP-waiting form of
F1-ATPase by single-pair FRET measurement 1分子FRET計測によるF1-ATPaseのATP結合待ち構造の解析." 第51回日本生物物理学会年会: October 28, 2013.
31. 須河光弘, 小林大, 松井貴志, 政池知子, 西坂崇之 (2014). CONFORMATIONALCHANGES IN THE β SUBUNITS OF F1-ATPase REVEALED BY FRETMEASUREMENTS DURING ROTATION OF THE γ SUBUNIT. BiophysicalSociety Annual Meeting 2014, San Francisco, California, February 15-19, 2014.
32. 須河光弘, 政池知子, 山口真, 柴田桂太郎, 豊島陽子, 矢島潤一郎, 神隆, 西坂崇之
(2013). "受像偏向蛍光顕微鏡による分子モーターの運動計測

Development ofcircular orientation fluorescenceemitter imaging（COFEI）." 日本顕微鏡学会第69回学術講演: May 21, 2013.
33. 政池知子 (2013). "タンパク質の動きを解き明かす１分子計測の最前線." 日本物理学会 第68回年次大会 領域12, 11合同シンポジウム

March 26, 2013.

34. 政池知子, 西坂崇之 (2013). "蛋白質の動きとはたらきを直接見て調べる." 第22回 日本バイオイメージング学会学術集会･公開講座: september 14-16,2013.
35. 政池知子, 池上浩司, 瀬藤光利, 鈴木裕, 西坂崇之 (2013). "1個から数個の分子が引き起こす運動と酵素反応のイメージング." 第51回日本生物物理学会年会･シンポジウム:
Octorber 28-30,2013.
36. 政池知子, 池上浩司, 瀬藤光利, 鈴木裕, 西坂崇之 (2013). "蛋白質1分子の構造変化と化学反応を可視化する." 2013年度 若手イメージング研究会: september 7-8,2013.
37. 政池知子, 鈴木裕, 西坂崇之 (2013). "プローブの向きと位置から蛋白質の局所構造変化を検出する." 日本顕微鏡学会第57回シンポジウム: November 15-16,2013.
38. 西坂崇之 (2015). Mechanics of Motor Proteins and Supermolecular Motility
Machinery Revealed under Advanced Optical Microscopes. WPI-next international
workshop ~~High resolution cell biology~~, E & S building, Nagoya Univ., 1st Oct.
2015.
39. 西坂崇之 (2015). シンポジウム「べん毛・繊毛が織りなす多様な生命現象に挑む～

分子から個体まで～ Diverse functions of flagella and cilia; -From molecules tomammalian development-」
40. 先進光学顕微鏡により明らかになる繊毛とアーキアべん毛の力学機能

Mechanicalfunction of cilia and archaeal flagella resolved by advanced optical microscopes. 第53回日本生物物理学会年会, 金沢大学 角間キャンパス 自然科学本館, September
13-15, 2015.

227

41. 西坂崇之, D. Kim, 須河光弘, 政池知子 (2013). "Imaging of Function andOrientation of Single Motor Proteins Under Advanced Optical Microscopy." The 8th
Asian Biophysical Association (ABA) Symposium: May 27, 2013.
42. 西坂崇之, 加藤孝信, 木下佳昭 (2014). Analysis of three-dimensional motion of
motor proteins and supermolecular motility machinery. The 6th International
Symposium on Aero Aqua Bio-Mechanics, Hawaii Tokai International College,
Honolulu, Hawawii, USA, November 13-16, 2014 (Nov 14).
43. 西坂崇之, 須河光弘, 木下佳昭, 政池知子 (2013). "1 細胞や1 分子の動態を直感的かつ定量的に検出するIntuitive but quantitative approach to image singlecells andmolecules." 日本顕微鏡学会第69回学術講演会: May 22, 2013.
44. 西坂崇之, 中根大介 (2013). "Single-Molecule Imaging of Protein Conformations
under Advanced Optical Microscopes." Protein Dynamics: August 5, 2013.
45. 西坂崇之, 中根大介 (2014). Mechanics of novel motors revealed under advanced
optical microscopes. The 45th NIPS International symposium “Cutting-edge
approaches towards the functioning mechanisms of membrane proteins”, 岡崎コン
ファレンスセンター, November 25-28, 2014

(26 Nov).

46. 西坂崇之, 中根大介 (2014). Single-molecule imaging of protein conformations
under advanced optical microscopes. 1st International Conference on
Nano-Bio-Medicine Convergence (NBMC-2014), Pusan Nat’l University, 24-25
February 2014

(25 Feb).

47. 池上浩司, 政池知子, 西坂崇之, 瀬藤光利 (2014). 繊毛と鞭毛におけるチューブリン
ポリグリシン化の異なる寄与度. 第5回繊毛研究会, 浜松市研修交流センター, May
24-25, 2014.
48. 池上浩司, 政池知子, 西坂崇之, 瀬藤光利 (2015). 軸糸チューブリンポリグリシン化
消失による鞭毛繊毛運動の亢進. 生体運動研究合同班・2015年班会議, 学習院大学,
January 7-9, 2015.
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Unitary step of gliding machinery in
Mycoplasma mobile
Yoshiaki Kinositaa,1, Daisuke Nakaneb,1,2, Mitsuhiro Sugawaa, Tomoko Masaikea,c,3, Kana Mizutania, Makoto Miyatab,4,and Takayuki Nishizakaa,4aDepartment of Physics, Faculty of Science, Gakushuin University, Toshima-ku, Tokyo 171-8588, Japan; bDepartment of Biology, Graduate School of Science,
Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan; and cPrecursory Research for Embryonic Science and Technology, Japan Science and Technology
Agency, Kawaguchi, Saitama 332-0012, Japan

Edited* by Howard C. Berg, Harvard University, Cambridge, MA, and approved April 28, 2014 (received for review June 22, 2013)

bacteria tracking
F1-ATPase| gliding ghost | leg protein | pairwise distance function |T

he fastest of the Mycoplasma species is Mycoplasma mobile(M. mobile); they glide with a speed of 2.0–4.5 μm·s−1 (1, 2).
Under an optimal-growth condition, cultivated single M. mobile
cells are flask-shaped (Fig. 1A) and glide smoothly across a substrate covered with surface-immobilized sialylated oligosaccharides(3) in the direction of protrusion at a constant speed (Movie S1).
Genomic sequencing and analysis have revealed that the mechanism must differ from other forms of motor protein systems andbacterial motility, because M. mobile lacks genes encoding conventional motor proteins in eukaryotes, such as myosin, kinesin,and dynein, in addition to lacking other motility structures inbacteria, such as flagella and pili (4). So far, three proteins
have been identified as a part of the gliding machinery (Fig. 1B,
Bottom): Gli123 (5), Gli521 (6), and Gli349 (7). The machinery
units localize around the cell neck, and their number has been
estimated to be ∼450 (2, 5, 8). Gli349 extends out from the cell
membrane and shows a rod structure, ∼100 nm in total, with two
flexible hinges when isolated (9). Notably, the machinery isdriven by hydrolysis of ATP to ADP and inorganic phosphate,caused by an unknown ATPase (10). Because of the large sizeand characteristic structure of Gli349, and a series of studies withmutants and inhibitory antibodies (2, 11), it has been hypothesized that Gli349 works as a “leg” by binding to and releasingfrom a substrate covered with randomly arranged sialylated oligosaccharides (2) consuming the chemical energy of ATP. In
addition, the pivoting movement of an elongated cell suggeststhat there are units working not simultaneously but rather independently to propel the cell forward (12). To test this hypothesis and identify conformational changes of a key part of
the gliding machinery, we here designed an assay to detect
the movement of M. mobile by high precision colocalization
microscopy. In the presence of an excess number of binding
targets in the solution, which decreased the number of active
legs, stepwise displacement was shown for the first time, to ourknowledge, to occur in gliding bacteria.

Results
Construction of an Assay for Tracking a Ghost with High Accuracy. To

track M. mobile with nanometer-scale precision, its cell surface
was stained with a fluorescent probe, Cy3, and its position was
localized by an EMCCD camera with a time resolution of 2 ms.
Note that the shape of the cell is mostly symmetric and its size
is less than 1 μm, which is close to the wavelength of visible light(Fig. 1 C and D). These two features made it possible to track
the movements of the bacteria with nanometer-scale precision byfitting the intensity profile of a fluorescent cell with a 2D
Gaussian function (Fig. 1E and Fig. S1), a technique (13) thathas been successfully applied to submicron-scale plastic probes
to detect the rotation of a single F1-ATPase, the smallest rotarymolecular motor made of a single molecule (14, 15). Because
motility at the cellular scale likely involves a large number ofmotors, isolating unitary conformational changes is essential for
a complete understanding of the gliding mechanism. To dissect
the movement, we used a permeabilized ghost model (10), in
which the cell membrane was damaged with Triton X-100 and,thus, we could regulate the activity of ATPase by changing[ATP] (Fig. 1 B and F and Fig. S2). Finally, to reduce thenumber of functional gliding machinery units, sialylated oligosaccharides, which have been identified as the binding target of
SignificanceThe mechanism of movement of bacteria shows extensive diversity, and some bacteria glide on the substrate surface via an
unknown process. Mycoplasma mobile is one of the fastest,
exhibiting smooth gliding movement with a speed of 2.0–4.5 μm/s.
By applying the modified in vitro ghost model of Mycoplasma
mobile to high precision colocalization microscopy, steps of theregular size, ∼70 nm, were detected for the first time in bacteria,
to our knowledge. The binding target of the gliding machinery,
sialylated oligosaccharides, was expected to be randomly oriented
on the surface and, thus, our results suggest that the machinery
can drive the steps with a cycle of attachment and detachment
even if there is no periodic structure on the substrate.
Author contributions: Y.K., D.N., M.M., and T.N. designed research; Y.K. and D.N.
performed research; M.S. and T.M. contributed new reagents/analytic tools; Y.K. and D.N.
analyzed data; Y.K., D.N., M.M., and T.N. wrote the paper; M.S. developed a frameworkof step fitting algorithm and applied it to data; T.M. verified the observation system; K.M.
initially designed the assay to observe gliding through a fluorescently labeled M. mobile;and T.N. constructed the optical setup, microscope, and analysis software.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1

Y.K. and D.N. contributed equally to this work.

2

Present address: Department of Physics, Gakushuin University, Toshima-ku, Tokyo 1718588, Japan.

3

Present address: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda City, Chiba 278-8510, Japan.

4

To whom correspondence may be addressed. E-mail: takayuki.nishizaka@gakushuin.ac.jp
or miyata@sci.osaka-cu.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1310355111/-/DCSupplemental.

PNAS | June 10, 2014 | vol. 111 | no. 23 | 8601–8606

www.pnas.org/cgi/doi/10.1073/pnas.1310355111

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MICROBIOLOGY

Among the bacteria that glide on substrate surfaces, Mycoplasma
mobile is one of the fastest, exhibiting smooth movement witha speed of 2.0–4.5 μm·s−1 with a cycle of attachment to and detachment from sialylated oligosaccharides. To study the gliding
mechanism at the molecular level, we applied an assay with a fluorescently labeled and membrane-permeabilized ghost model,
and investigated the motility by high precision colocalization microscopy. Under conditions designed to reduce the number of motor interactions on a randomly oriented substrate, ghosts took
unitary 70-nm steps in the direction of gliding. Although it remains
possible that the stepping behavior is produced by multiple interactions, our data suggest that these steps are produced by a unitary gliding machine that need not move between sites arranged
on a cytoskeletal lattice.

Fig. 1. Nanometer-scale tracking of Mycoplasma gliding. (A) A dark-field
image of M. mobile. The image was captured with center-stop optics tomaintain the high numerical aperture of the objective, which enabled a highspatial resolution (35). (Scale bar: 1 μm.) (B, Upper) Illustration of the fluorescent ghost. The gliding machinery was distributed around the neck portion, but only the active machinery bound to the glass is shown for simplicity.
(Bottom) A construction model of the gliding machinery comprising threeproteins: Gli123, Gli521, and Gli349. See the review by Miyata (2) for moredetail. (C) A fluorescent image of the labeled ghost was acquired with a time
resolution of 2 ms. (Scale bar: 1 μm; pixel size: 240 nm.) (D) The intensity
profile of C. The XY area is 5 × 5 μm. (E) Gaussian fitting to D. Nanometerscale tracking is achieved by positioning the peak of the 2D Gaussian function fitting to the intensity profile of the ghost. (F, Left) The speed of gliding
ghosts at different [ATP]s in the solution (n = 129). The cyan curve shows
speed
a fit with Michaelis–Menten kinetics; Vmax
and Km are 2.6 μm·s−1 and 61 μM,
respectively. The dotted cyan curve shows a fit with the kinetics including
speed
the Hill coefficient; Vmax
, [ATP50] and n are 2.2 μm·s−1, 43 μM, and 2.4,
respectively. (Right) The speed of living cells with no ATP in the solution(2.1 ± 0.1 μm·s−1; n = 22). (G) Effect of SL on the gliding velocity of the ghost
at saturated [ATP]s, 0.3–1.0 mM (n = 50).

Gli349 (3, 16) and serve as a scaffold for M. mobile gliding, wereadded to the solution in the form of N-acetylneuraminyllactose(sialyllactose, SL). Notably, the gliding speed became slower
with higher concentrations of SL (Fig. 1G), and the ghosts finally became unbound from the glass when [SL] was higher
than 1.0 mM, presumably because the number of legs acting on
the sialylated oligosaccharides on the glass was decreased as free
SL was added to the solution. To optimize the conditions of the
motility assay, 0.1% methylcellulose was also added to the solution to suppress the detachment of ghosts. Because the gliding speed did not alter much with or without methylcellulose(Fig. S3), we judged that the viscous drag against the gliding
movement caused by a 0.1% concentration of methylcellulose
was negligible.
8602 | www.pnas.org/cgi/doi/10.1073/pnas.1310355111

Detection of Steps. We recorded sequences of the gliding of ghosts
under various [ATP]s at [SL] = 1.0 mM, and carefully observed
their traces. Typically, the ghost glided with repetition of pauses
and sudden displacements as shown in dense dots in Fig. 2A. Thistendency was also confirmed at the high concentration of ATP(Fig. 2B), suggesting that the ghosts move not smoothly but ratherintermittently under the condition where the number of acting
legs was expected to be decreased. To check whether the displacements included a regular dispersion, we analyzed 937 ghosts
at various ATP concentrations in the range of 6–1,330 μM. The
period in which the ghost moved in a nearly straight line (e.g., theblack portion in Fig. 2A) was further extracted. Although mostruns showed creeping displacement and few intermittent pausesin the irregular manner, 196 ghosts showed more than two stepwise displacements. Among them, 134 records in 105 ghosts at[ATP] = 8–500 μM showed more than four clean steps. We also
extracted 70 time courses in 60 ghosts that showed instantaneous
stepping motion and could successfully be fitted with a step
function by the step-finding algorithm as described in a latersection.
An example of multiple steps is shown in Fig. 2C, Left. The
unfiltered trajectory along the displacement direction showedclean steps with regular intervals, indicating that the movement
driven by the gliding machinery of M. mobile may involve unitary
steps. The existence of a step becomes apparent when the pairwise distance function (PDF) is applied to each run. As exemplified in Fig. 2C, Right, the intervals between pauses appeared
as peaks of the PDF. The position of the peaks was precisely
correlated with multiples of the size of a single step, suggestingthat the gliding machinery repeats the stroke cycle for movementwith high precision. The tendency was barely shown even in theabsence of SL (Fig. S4); however, stepping was not instantaneous
but gradual, possibly because the number of functional legs was
still too large under this condition (Discussion). Displacementswith regular steps were observed under a variety of [ATP]s (Fig.
2D), and particularly at a high [ATP], under which condition thedwells awaiting ATP binding were expected to be negligible fromthe estimation based on Fig. 1F. Fig. 2E shows a histogram of thestep value determined from the PDF. The size of the step underthese conditions was 72 ± 14 nm, assuming that the histogram
comprises a single peak.
Characteristics of Dwell Time. As reported for other molecularmotors, the dwell time allows us to better understand themechanism of motors at the molecular level in terms of chemomechanical coupling, i.e., the relationship between a chemical
reaction (nucleotide binding and subsequent hydrolysis) andmechanical event (conformational change(s) of a motor). We
applied a “step-finding algorithm” that was originally developed
to analyze the position of tips of microtubules growing in a regular manner (17) to quantify the frequency and position of
pauses. The fitting was performed to minimize the variation
based on the algorithm (for details of the analysis, see Materials
and Methods). As shown by the rectangles in Fig. 2 C and D,
moments when stepping occurred were roughly estimated fromthe raw trajectory in each run. Histograms of the dwell time atvarious [ATP]s, 8–500 μM, were thus constructed from the fitting(Fig. 3A).
Note that, in our assay at this point, we were not able to determine the exact number of legs interacting with the surface,although steps of the regular size were successfully detected ineach run. It may be possible that dwells with several or multiple
motor units are incorporated in the histograms, which makes theinterpretation of results quite complicated, as considered below
in Discussion. The average of the dwell times at various [ATP]s issummarized in Fig. 3B. The maximum rate was estimated to be
24 s−1 at [ATP] = 500 μM, at which concentration the binding
rate was expected to be almost saturated (Fig. 1F). A small increase in the dwell time was observed at the low [ATP]s of 8
and 16 μM, compared with that at 500 μM (arrows in Fig. 3A;
Kinosita et al.

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MICROBIOLOGY

Fig. 2. Steps of single ghosts of M. mobile driven by ATP. (A) A typical example of the XY trace of the single gliding ghost. Black points in the trace show therange in which we judge that the ghost moved in a nearly straight line and were subsequently analyzed with the pairwise distance function in C. (B) Examples of
the trace at various concentrations of ATP. Red, blue, green, black, magenta, and orange represent [ATP] = 8, 16, 55, 90, 285, and 500 μM, respectively, and theircolors coincide with those of the time course of the displacement in C and D. (C, Left) A typical example of the displacement along the gliding direction. Opencircles are unfiltered data points, and rectangles in the displacement time course were the lines fitted with the step-finding algorithm. (Right) Histograms of the
pairwise distance function (PDF) of the run. The green solid line is fitted to the sum of eight Gaussians. (D) Other examples of the displacement time course atvarious [ATP]s. (E) Histogram showing the distribution of step sizes estimated from the PDF analysis (n =70 runs in 60 ghosts; each run contains 4–9 steps)
separated by 10-nm bins for clarity. The green solid curve shows the Gaussian distribution with 72 ± 14 nm.

P = 8.4 × 10−6 and 1.0 × 10−4, respectively, < 0.05 by Welch’st test). Notably, the dwell time was merely doubled in the presenceof SL at [ATP] ∼10 μM (Fig. 3B), whereas, in the absence of SL,
the average gliding speed was ∼10 times lower compared with thatat saturated [ATP] (Fig. 1F). This discrepancy may suggest thatlegs in the holding state could increase drag and, thus, reduce thespeed of the movement driven by the leg in the power-stroke state
only in the case that the number of legs was huge, while they
immediately detached from the surface when they were fewer innumber. This interpretation coincides with the observation of
stepping without SL, in which the creeping displacement between steps was observed as shown in Fig. S4B, Inset.

to detect the rotation of a single F1-ATPase (15). As the speed
decreased, the gliding showed intermittent pauses and, finally,
stepwise movement appeared when 4–8 mM SL was added(Fig. 4C), although creeping was not eliminated during stepping.
PDF analysis showed clear peaks being precisely correlated withmultiples of the size of a single step (e.g., Fig. 4D). The size of stepwas estimated as 69 ± 11 nm (Fig. 4E; n = 35 runs in 23 cells),
which was similar to the case of the ghost assay (P = 0.377 > 0.05
by Welch’s t test). We conclude that the gliding machinery moves
in unitary steps even when it operates under an intact condition
where it is buried in the nontreated cell membrane, and the size ofthe step is ∼70 nm for both the ghosts and intact cells.

Steps of Intact M. mobile. To check whether the properties of steps
in the ghost, especially their average size, were specific to theintact gliding machinery, we also analyzed the movement of
M. mobile that was not treated with the detergent. Note that inthis assay, we were not able to control the concentration of ATP
inside the cell, and so we only reduced the number of legs byadding SL to the solution. As expected from the results of theghost assay (Fig. 1G), the fraction of cells binding to the glass
also depended on the concentration of SL in the solution (Fig.
4A). Gelatin was added to increase the solution viscosity, tomake the fraction higher, and to maintain a sufficient number ofcells on the surface for recording. The gliding speed decreased as
SL was added to the solution (Fig. 4B), suggesting that the
number of legs was successfully minimized. A single fluorescentparticle was nonspecifically attached to the surface of the celland was tracked with a nanometer-scale precision based on atechnique for individual fluorescent probes (13) that has also
been successfully applied to submicrometer-scale plastic probes

Discussion
In this study, we established an assay in which the gliding motionof M. mobile was localized with nanometer-scale precision underan optical microscope. Cells were stained with a fluorescentprobe and subsequently permeabilized on the glass to prepare
the ghost model. Although the ghost showed frequent pauses
and the histogram of PDF barely showed peaks of multiple
numbers (Fig. S4), the steps were not clear because the stepping
between pauses was not instantaneous but rather gradual (Fig.
S4B, Inset). We further modified the motility assay system. The
addition of free SL to the solution hindered the attachment ofeach leg to the sialylated oligosaccharides on the glass. Inversely,a high concentration of methylcellulose in the solution suppressed the detachment of M. mobile. By balancing these two
conditions, stepwise displacement was finally achieved for the
first time in gliding bacteria. Note that the concentration of SL
approached that at which the ghost fully detaches from thesurface. In fact, with the addition of SL, the average speed of
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movement of the ghost. Such conformational change itself could
fluctuate largely and, thus, an additional mechanism would beneeded to explain the occurrence of precise repetitive steps likethose in Fig. 2. For example, it may be that the tip of the leg canbind to the target only when it makes a certain angle to thesurface, as proposed for the mechanism of myosin (21, 22).
If the gliding machinery of M. mobile is a processive motor in
the manner of kinesin-1 and myosin V, each of which has twofunctional domains in a single molecule and walks in a handover-hand manner by alternately using them (23, 24), then thedwell time between steps would be equal to the inverse of therate of a rate-limiting chemical step of the single motor unit. In
contrast, in the case that multiple active motors independently
interact with the surface-immobilized sialylated oligosaccharides
to move the ghost forward, the apparent dwell time would be
expected to be shorter, because, in this case, one motor would
stochastically induce the displacement before the rate-limiting
chemical step in the other motor completed. With the above
arguments in mind, we cannot evaluate the histograms in Fig. 3
without an assumption.
The increase in the dwell time was barely observed at low[ATP] (compare 8 and 16 μM), suggesting that the histograms

Fig. 3. Dwell-time analysis of displacements. (A) Histograms of dwell times
under various concentrations of ATP. Arrows indicate the average in eachgraph, and dashed lines indicate the average at 500 μM ATP. There wasa slight, barely detectable increase at 8 and 16 μM, relative to that at 500 μM
ATP, as shown by the gaps between the arrows and dashed lines. (B)Average of the dwell times (filled circles). The dashed line is the value at
500 μM ATP.

ghosts decreased by about one-fourth (Fig. 1G). In the case thatthe duty ratio–the fraction of binding time over the single ATPaseturnover–of the motor is low, it is generally considered that the
average speed should decrease as the density of the motor
decreases (18, 19). This model coincides with our observations.
Although the number of gliding machines involved in forward
movement during the unitary step was not conclusively determinedin our assay, we can infer a possible mechanism of the gliding
machinery from the fact that the ghost showed clean and repetitive
steps, as depicted in Fig. 2 C and D. One simple interpretation isthat each 70-nm stepwise displacement corresponds to a single
turnover in a single motor unit. In conventional linear motors suchas myosin V, the size of a step is determined by the conformationalchange and additional Brownian search to land the next bindingsite on its track (20). However, the scenario including the trackwith a periodic structure is not the case for the gliding machineryof M. mobile, because its binding target is randomly oriented
on the substrate surface. There might be unique and inherent
characteristics that realize the huge but precise size of the steps.
Probably, the specific conformational change in one of three (ormore) machinery proteins directly determines the forward 70-nm
8604 | www.pnas.org/cgi/doi/10.1073/pnas.1310355111

Fig. 4. Steps of intact M. mobile without the detergent treatment. (A)Binding activity of intact M. mobile at various [SL]s. (B) Gliding speed. In A
and B, filled and open circles show the data with or without, respectively,
gelatin in the solution (n = 40); 100% means the number of cells bound to
the glass surface at [SL] = 0 mM. (C) Gliding of intact M. mobile at various[SL]s. The displacement appeared smooth at 0 mM, whereas it became rather
stepwise at 8 mM. (D) An example of histograms of the PDF analysis. The
black line is fitted to the sum of seven Gaussians. (E) Histogram showing the
distribution of step sizes of intact M. mobile from PDF analysis separated by
10 nm bins (n = 35 runs in 23 cells). The black curve shows the Gaussian
distribution with 69 ± 11 nm.

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configuration of Gli349 to the glass. In addition, to consistently
maintain the regular steps, the detachment process of a leg isalso important and could be regulated in terms of load dependence and directionality. This point, which is essential for
elucidating the gliding mechanism in greater detail, will be
addressed by measuring the unbinding force of a single leg ofa ghost as in the case of myosin (30, 31), by using an opticaltrapping of the bead applied to the cell surface (32).
Materials and Methods
Preparation of the Fluorescent M. mobile. A mutant strain of M. mobile, theP476R gli521 mutant, which binds to sialylated oligosaccharides more tightly
than the wild-type strain (10, 11), was used in this study. Cells were cultured
in Aluotto medium [2.1% (wt/vol) heart infusion broth, 0.56% yeast extract,
10% (vol/vol) horse serum, 0.025% thallium acetate, and 0.005% ampicillin]at 25 °C (33) until the absorbance at 600 nm reached 0.06–0.10. Cells in a
culture with 1 mL of volume were collected by centrifugation at 12,000 × g
for 4 min at 25 °C, resuspended in Aluotto medium, and subsequently incubated for 30 min before labeling. Cells were incubated in buffer A (75 mM
sodium phosphate at pH 7.4 and 68.4 mM NaCl) containing 50 mM glucose
and Cy3-NHS ester (GE Healthcare) for 2 h at room temperature and subsequently cleaned by two rounds of centrifugation after suspending with
500 and 200 μL of buffer A containing glucose.
Assay for Tracking the Gliding of Ghosts. The flow chamber was comprised of
two coverslips (No. 1, 0.12–0.17 mm thickness; Matsunami Glass) with different sizes (24 × 24 and 24 × 36 mm). Two pieces of double-sided tape,which were cut approximately 5 mm wide and 30 mm in length, were usedas the spacers between coverslips (7). The resulting flow chamber had aninternal volume of ∼7 μl. Aluotto medium was infused to the chamber and,thus, sialylated oligosaccharides included in the serum in the medium wasnonspecifically bound to the glass surface. Fluorescent cells were infused
into the chamber from one side and kept for 30 min. Unbound cells were
removed with 20 μL of buffer B (buffer A containing 2 mM MgCl2, 1 mM
DTT, and 1 mM EGTA). The solution in the chamber was replaced with 10 μL
of buffer B containing 0.01% Triton X-100 (MP Biomedicals). Gliding cells
were permeabilized with the surfactant and their movement was stopped
and, thus, ghosts were prepared (10), typically within 1 min. The membrane
permeabilization was judged by eye as the cytosol was ejected from thebody and, thus, the density of the cells was reduced. At the moment when
their density was reduced in the observation field, the solution was immediately replaced with 10 μL of buffer C [10 mM Tris·HCl at pH 7.5, 50 mM
NaCl, 1 mM DTT, 1 mM EGTA, 2 mM MgCl2, and 0.5 mg/mL bovine-serum
albumin (Sigma-Aldrich)] containing 1 mg·ml−1 DNase (Sigma-Aldrich). The
chamber was subsequently washed with 10 μL of buffer C containing 0.1%
methylcellulose (M0512, 4,000 cps at 2% solution; Sigma-Aldrich). Note thatthe drag force on the ghost from the viscosity of the surrounding fluid was
calculated to be Fd = 2πηb/(ln(2b/a) – 0.5) × vglid = 0.008–0.07 pN and, thus,
negligible (34), where the viscosity η is 1–5 cps for 0.1% methylcellulose
solution; the half length of the ghost b is 0.5 μm; half of the size of the waistof the ghost a is 0.3 μm; and the gliding velocity vglid is 2–3 μm·s−1. The valueof 5 cps was estimated from an extrapolation of the product information,
and was used as the maximum viscosity of 0.1% methylcellulose solution,
whereas 1 cps, equivalent to water, was used as the minimum viscosity in thecase that M. mobile was too small to apply the macroscopic viscosity. Finally,
to activate ghosts and observe their gliding, the solution was replaced with
30 μL of buffer C containing a desired amount of ATP (Sigma-Aldrich),
0.2 mg·mL−1 creatine kinase (Roche), 0.8 mg·mL−1 creatine phosphate (Roche),
1 mM N-acetylneuraminyllactose (SL; Sigma-Aldrich), and 0.1% methylcellulose. After the fluorescent image of the moving ghosts was captured, theirphase-contrast image was also recorded to check whether each cell was
successfully permeabilized and converted into a ghost; the density of ghosts
was clearly lower than that of the living cells.
Gliding Assay of M. mobile with Fluorescent Beads. After the infusion of unlabeled cells into the flow chamber, fluorescent beads of 100-nm diameter(Invitrogen) were infused. An individual bead could be nonspecifically attached to the cell membrane by chance, and the bead could be easily detectedduring its continuous movement on the glass surface when the flow chamber
was observed under a fluorescence microscope.
Microscopy. Fluorescent M. mobile was visualized under an inverted fluorescence microscope (Ti-E; Nikon Instruments) equipped with a 100× objective (Fluor 100 with Ph and 1.3 N.A.; Nikon Instruments), a filter set (TxRed-

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MICROBIOLOGY

may include a component that depends on [ATP]. Additionally,at high [ATP], the dwell time reached 42 ms, indicating the existence of a rate-limiting step, apart from ATP binding, on theorder of 20 s−1 at most. The estimation of the average speed,(step size) × (rate) = (∼70 nm) × (∼20 s−1) = 1.4 μm·s−1,
roughly coincides with the maximum speed of M. mobile. Additionally, if we assume that the number of legs is single, then kon of
ATP and kcat of ATPase are estimated to be 15 × 10−6 s−1·M−1
and 15 s−1, respectively (Fig. S5, red curves), by the global fitting
through histograms. Two chemical steps may be included atsaturated [ATP] (Fig. S5, blue curve at 500 μM).
To address whether these estimations are proper, a reconstructed in vitro motile system, in which the number of gliding
machineries is known to be one, is inevitable, as in cases of
conventional motors such as myosin, kinesin, and dynein. The
assay has not been established yet for the gliding machinery of
M. mobile, because at least four proteins are needed and the legs
are assumed to protrude from the membrane. In the future,some additional techniques, which incorporate protein arrangements in nanodiscs or liposomes, will be needed to reconstruct
the unit as a whole active motor.
Because we cannot be certain about the number of active units
when clean steps were detected in our present assay, one could
argue that 70 nm is not the step size driven by the single assemblyof the gliding machinery, but the sum of smaller steps driven bymultiple motors. One possible scenario is that the clean 70-nm
displacement is comprised of cooperative and sequential displacements of smaller steps. For example, repetitive small steps
without an intermission longer than the time resolution of theobservation system may appear as a large step, although strong
cooperativity would be needed to explain the clear peaks in thehistogram of PDF (Fig. 2C). Alternatively, the unbinding processof legs holding the ghost may directly determine the apparent
size of each step. If the leg(s) that has just finished the conformational change still attaches to the ghost, it is probable that theleg subsequently works as a load when the other leg strokes andinduces pauses at the halfway point of each step. This scenario
would be similar to that reported for kinesin, in which the step
sizes half those of a single motor were found as the result of force
balance between two motors (25). To examine the above possibilities, again, a reconstructed in vitro motile system will be neededin the near future; in addition, it should be checked whether the
motor in M. mobile can function as a processive motor.
From the structural point of view, what possible mechanisms
can explain the regular step size of ∼70 nm? For the 8-nm step ofkinesin and 36-nm step of myosin V, the sizes are determined by
the repetitive structure of their substrate filaments, i.e., the microtubule and actin filaments, respectively. In contrast, M. mobile
glides on a surface on which the binding target (16) is distributed
randomly rather than being arranged in a regular manner. Additionally, because the leg protein, Gli349, is expected to beflexible rather than rigid (9), a simple stroke model like that ofskeletal myosin (26), in which the lever part executes a stroke ata regular angle, is unlikely to explain the clear peaks of the PDF
observed in Figs. 2C and 4D. Here, we propose a model in which
stepwise movement is induced not by the tilting of the leg protein, but by the movement of the protein that connects the leg toATPase. Gli521, which is assumed to work as a crank, is a rodlike structure with a size of 120 nm (27). The unidirectional
movement and the large size of Gli521, in either the lateral,tilted, or rotating direction, could explain the step we observed.
Because a protein similar to the catalytic subunit of F1-ATPaseis located at the cytoskeleton-like structure that may arrange
Gli123, Gli521, and Gli349 (28, 29), we hypothesize a model inwhich Gli521 converts a rotational motion of ATPase to a directed
linear motion, much like a connecting rod between the wheelsof a steam train. The movement of Gli521 may be effectivelytransmitted to Gli349 to make Gli349 pull against sialylated
oligosaccharides on the glass. The broad distribution shownin Fig. 2E might be attributable to the variation of the binding4040B; Semrock), an EMCCD camera (iXon+ DU860; Andor), a highly-stablecustomized stage (Chukousha), and an optical table (RS-2000; Newport).
Projection of the image to the camera was made at 240 nm per pixel. Phasecontrast images of ghosts were also taken with another CCD camera(CS8420i; Toshiba TELI) to determine whether the cells were permeabilized
by a surfactant. To acquire the image in Fig. 1A, a custom-made dark-fieldmicroscope (35) was used with a 100× objective (Plan Apo TIRF 1.4 N.A.;
Olympus) and a CCD camera (CCD-300-RCX; Dage-MTI) under an inverted
microscope (IX71; Olympus). In the gliding assay of M. mobile with a fluorescent bead, images were recorded by using a high-speed CCD camera
system (HDR1600; Digimo).
Data Analyses. Sequential images of the gliding of fluorescent ghosts were
captured as 14-bit images with an EMCCD camera under 2-ms resolution andconverted into a sequential TIF file without any compression. The position of
single ghosts, (xc, yc), was determined by fitting each image with a 2D Gaussian
including the cross-correlation term, cor, as I0 + A exp½−ððx − xc Þ2 =2σ2x +ðy − yc Þ2 =2σ2y − 2corðx − xc Þðy − yc Þ=σx σy Þ=2ð1 − cor 2 Þ, where I0 is the background intensity; A is a peak of the intensity of the ghost; and σx and σy areintensity variances along the x and y axis, respectively. For tracking of
M. mobile, the parameters σx, σy, and cor were required to achieve a proper
fitting, because single ghosts often appeared not as spherical but as ellipsoidal shapes. A was typically 800–2,000 under our capture conditions. In
speed
speed
Fig. 1F, V = Vmax
½ATP=ðKm + ½ATPÞ and V = Vmax
½ATPn =ð½ATP50 n + ½ATPn Þ
were used as formulae for Michaelis–Menten kinetics and the kinetics including the Hill coefficient, respectively. To estimate the size of the unitarystep, a PDF was independently adopted for each run showing stepwise
displacement. A histogram with 5-nm bin width is shown in Fig. 2B and was
fitted with the sum of the Gaussians with periodic intervals, Δ, as
P
2
2
2
2
i Ai exp½−ðx − iΔÞ =ð2σi Þ + A0 exp½−ðx − x0 Þ =ð2σ0 Þ. Note that, in the casethat stepwise displacements are always clean and pauses are stable, x0
should be 0 as indicated in the processive motor dynein (36). In contrast,
creeping motions were observed to some extent in our measurement and,thus, we set the parameter x0 in the fitting. Back steps, i = –1 or less, were notincluded in the fitting, because they were not observed in our measurement.

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8606 | www.pnas.org/cgi/doi/10.1073/pnas.1310355111

Step-Finding Algorithm. The step-finding algorithm was initially developedfor quantitative detection of the growth and shrinkage of microtubule ends
(17). The algorithm consists of three processes: (i) finding steps, (ii) evaluating the quality of the step fits, and (iii) finding the step distribution. (i)This process is the same as in the original method (17). Briefly, by fitting
a single large step to the data based on a calculation of the χ2 statistics, thefirst step is determined. Next, the subsequent steps are found by fitting newsteps to the dwells of the previous fitting, in the same way as for the firststep. (ii) The second process was modified as follows: The quality of thefitting was based on evaluation of the variance of the displacement in eachP
ðσ2i − Æσ2 æÞ2 . N is the number of steps.
dwell, which was defined as Q = 1= N+1i
σ2i is a variance of the displacement in the i-th dwell. Æσ2 æ is a mean of the^ = wQÞ was
variances in each dwell. In addition, Q weighed by w = 1=Æσ2 æ ðQ
used for the third process. In the case of under-fitting, the large values of^ In the caseðσ2i − Æσ2 æÞ2 and the weight (w) contribute to the low value of Q.
of overfitting, the values of ðσ2i − Æσ2 æÞ2 are small but N is large, so that the^ to be low. (iii) The highest Q^
summation of ðσ2i − Æσ2 æÞ2 allows the value of Q^
value basically represents the best fitting. In practical terms, a value of Q
that was not the highest value (usually the second highest value) toward theoverfitting, was often adopted as the best fitting, especially when some data
had nonuniformity of fluctuation of the displacement.
ACKNOWLEDGMENTS. We thank A. Upadhyaya for critical comments, M. P.
Sheetz for comments on an earlier draft, and M. Kaya and A. Tatsuguchi for
their technical advice and critical discussion. This study was supported in partby the Funding Program for Next Generation World-Leading Researchers
Grant LR033 (to T.N.) from the Japan Society for the Promotion of Science,by Grant-in-Aid for Scientific Research on Innovative Areas “Harmonized
Supramolecular Motility Machinery and Its Diversity (M.M. and T.N.) and“Fluctuation & Structure” (Grant 26103527 to T.N.), by Grant-in-Aid forYoung Scientists (Grant 25893230 to D.N.) from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan, by the Asahi GlassFoundation (D.N.), by the Institute for Fermentation (D.N.), and by the
Naito Foundation Natural Science Scholarship (D.N.).

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Bacteriocin Protein BacL1 of Enterococcus faecalis Targets Cell
Division Loci and Specifically Recognizes L-Ala2-Cross-Bridged
Peptidoglycan
Jun Kurushima,a Daisuke Nakane,b Takayuki Nishizaka,b Haruyoshi Tomitaa,cDepartment of Bacteriology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japana; Department of Physics, Faculty of Science, Gakushuin University,
Tokyo, Japanb; Laboratory of Bacterial Drug Resistance, Gunma University Graduate School of Medicine, Maebashi, Gunma, JapancEnterococcus faecalis is a commensal Gram-positive bacteriumin the intestinal tract of healthy humans or animals and is also
known to be an opportunistic pathogen causing various infectious
diseases, including urinary infectious disease, bacteremia, infective endocarditis, and others (1–3). The infection-derived E.
faecalis strains often produce various plasmid-encoded bacteriocins (4, 5).
Bacteriocins are bacterial peptides or proteins with antimicrobial activities (6). Heat- and acid-stable bacteriocin peptides produced by Gram-positive bacteria are divided into class I and class
II according to posttranslational modifications (7, 8). Class I bacteriocins are lantibiotics that contain nonproteinogenic amino acids generated by posttranslational modification (9). Only twoclass I bacteriocins have been identified in enterococci: ␤-hemolysin/bacteriocin (cytolysin) and enterocin W (10–14). In contrast, most enterococcal bacteriocins belong to class II and arenonmodified antimicrobial peptides, such as AS-48, enterocin A,
and others (7, 15, 16). We have found the enterococcal class IIbacteriocins, including Bac21, Bac31, Bac32, Bac43, and Bac51, inclinical strains of E. faecalis or Enterococcus faecium (17–21). Unlike the low-molecular-weight peptide-type class I and II bacteriocins, heat-labile antimicrobial proteins are referred to as bacteriolysins, previously named class III bacteriocins, and show
enzymatic bactericidal activity (22, 23). In enterococci, the bacteriolysins enterolysin A and bacteriocin 41 (Bac41) have been identified (24–26).
Bac41 was originally found expressed from the pheromoneresponsive plasmid pYI14 carried by the clinical strain E. faecalis
YI14 (26, 27). The Bac41-type bacteriocins were also found in theE. faecalis VanB-type vancomycin-resistant E. faecalis (VRE) outbreak strains (27). Bac41 is specifically active only against E. faeca-

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lis (26, 28). The determinant region of Bac41 contains six open
reading frames (ORFs), including bacL1, bacL2, bacA, and bacI
(Fig. 1A). The bactericidal activity of Bac41 is actually expressedby the two extracellular components, the bacL1- and bacA-encoded proteins BacL1 and BacA (26). BacL1 and BacA are secreted
proteins that coordinately exert bactericidal activity against E.
faecalis (26, 28). BacL2 positively regulates the transcripts of bacL1and bacL2 itself (unpublished data). BacI is the specific immunity
factor protecting a Bac41 producer from Bac41 activity (26).
We previously demonstrated that BacL1 is a peptidoglycan Disoglutamyl-L-lysine endopeptidase (28). BacL1 has 595 amino acids with a molecular mass of 64.5 kDa and consists of two distinct
peptidoglycan hydrolase homology domains and three repeats of
the SH3 domain (Fig. 1B). The two peptidoglycan hydrolase domains located in the regions from amino acid 3 to 140 and aminoacid 163 to 315 show homology to the bacteriophage-type pepti-

Received 11 August 2014 Accepted 27 October 2014
Accepted manuscript posted online 3 November 2014
Citation Kurushima J, Nakane D, Nishizaka T, Tomita H. 2015. Bacteriocin protein
BacL1 of Enterococcus faecalis targets cell division loci and specifically recognizes
L-Ala2-cross-bridged peptidoglycan. J Bacteriol 197:286 –295.
doi:10.1128/JB.02203-14.
Editor: P. J. Christie
Address correspondence to Haruyoshi Tomita, tomitaha@gunma-u.ac.jp.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.02203-14.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JB.02203-14

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Bacteriocin 41 (Bac41) is produced from clinical isolates of Enterococcus faecalis and consists of two extracellular proteins,
BacL1 and BacA. We previously reported that BacL1 protein (595 amino acids, 64.5 kDa) is a bacteriolytic peptidoglycan D-isoglutamyl-L-lysine endopeptidase that induces cell lysis of E. faecalis when an accessory factor, BacA, is copresent. However, thetarget of BacL1 remains unknown. In this study, we investigated the targeting specificity of BacL1. Fluorescence microscopy analysis using fluorescent dye-conjugated recombinant protein demonstrated that BacL1 specifically localized at the cell divisionassociated site, including the equatorial ring, division septum, and nascent cell wall, on the cell surface of target E. faecalis cells.
This specific targeting was dependent on the triple repeat of the SH3 domain located in the region from amino acid 329 to 590 of
BacL1. Repression of cell growth due to the stationary state of the growth phase or to treatment with bacteriostatic antibiotics
rescued bacteria from the bacteriolytic activity of BacL1 and BacA. The static growth state also abolished the binding and targeting of BacL1 to the cell division-associated site. Furthermore, the targeting of BacL1 was detectable among Gram-positive bacteria with an L-Ala-L-Ala-cross-bridging peptidoglycan, including E. faecalis, Streptococcus pyogenes, or Streptococcus pneumoniae, but not among bacteria with alternate peptidoglycan structures, such as Enterococcus faecium, Enterococcus hirae,
Staphylococcus aureus, or Listeria monocytogenes. These data suggest that BacL1 specifically targets the L-Ala-L-Ala-crossbridged peptidoglycan and potentially lyses the E. faecalis cells during cell division.

Bacteriocin Protein BacL1 Targets Cell Division Site

doglycan hydrolase and the NlpC/P60 family peptidoglycan hydrolase, respectively (26, 29, 30). The second peptidoglycanhydrolase homologue, with similarity to NlpC/P60, has D-isoglutamyl-L-lysine endopeptidase activity against the purified peptidoglycan component from E. faecalis (28). On the other hand, themolecular function of the first peptidoglycan hydrolase domain,with similarity to bacteriophage-type peptidoglycan hydrolase, remains to be elucidated but is still required for the bactericidal
activity against viable E. faecalis cells (28). The SH3 repeat domain
is located in the region from amino acid 329 to 590 and functions
as the binding domain to the peptidoglycan (28). However, BacL1
is not sufficient for bactericidal activity. BacA appears to be essen-

MATERIALS AND METHODS
Bacterial strains, plasmids, oligonucleotides, media, and antimicrobial
reagents. The bacterial strains and plasmids used in this study are shownin Table 1. A standard plasmid DNA methodology was used (34). Enterococcal strains were routinely grown in Todd-Hewitt broth (THB; Difco,
Detroit, MI) at 37°C (35), unless otherwise noted. Escherichia coli strains
were grown in Luria-Bertani medium (LB; Difco) at 37°C. Gram-positivebacterial species (other than Enterococcus) were grown in brain heart infusion (BHI) medium (Difco) at 37°C. The antibiotic concentrations forthe selection of E. coli were 100 mg liter⫺1 ampicillin and 30 mg liter⫺1
chloramphenicol. The concentration of chloramphenicol for the routine
selection of E. faecalis carrying plasmid pAM401 or its derivatives was 20
mg liter⫺1, unless otherwise noted. All antibiotics were obtained from
Sigma Co. (St. Louis, MO).
Recombinant proteins and antibodies. The histidine-tagged recombinant proteins of full-length BacL1, its truncated derivatives, and BacATABLE 1 Bacterial strains and plasmids used in this study
Strain or plasmid
Strains
E. faecalis OG1S
E. faecalis OG1X
E. faecalis OG1RF
E. faecalis FA2-2
E. faecium BM4105RF
E. hirae 9790
S. aureus F-182
S. pyogenes MGAS315
S. pneumoniae 262
L. monocytogenes EGD
E. coli DH5␣E. coli BL21

Plasmids
pAM401
pHT1100
pET22b(⫹)
pET::bacL1
pET::bacL1 ⌬1
pET::bacL1 ⌬2
pET::bacL1 ⌬1⌬2
pET::bacL1 ⌬3
pET::bacA

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Description

Source or reference

str, derivative of OG1
str, protease-negative derivative of OG1
rif fus, derivative of OG1
rif fus, derivative of JH2
rif fus, derivative of BM4105
Type strain
Clinical isolate, resistant to methicillin and oxacillin
Clinical isolate, serotype M3
Quality control strain, serotype 19F
Serovar 1/2a
endA1 recA1 gyrA96 thi-1 hsdR17 supE44 relA1 ⌬(argE-lacZYA)U169,
host for DNA cloning
ompT hsdSB(rB⫺ mB⫺) gal(␭cI 857 ind1 Sam7 nin5 lacUV5-T7gene1)
dcm(DE3), host for protein expression

35
35
35
60
61
ATCC 9790
ATCC 43300
ATCC BAA-595
ATCC 49619
ATCC BAA-679
Bethesda Research Laboratories

E. coli-E. faecalis shuttle plasmid; cat tet
pAM401 derivative containing wild-type Bac41 genes
Expression plasmid for His-tagged protein in E. coli
pET22b(⫹) derivative expressing BacL1
pET22b(⫹) derivative expressing BacL1⌬1
pET22b(⫹) derivative expressing BacL1⌬2
pET22b(⫹) derivative expressing BacL1⌬1⌬2
pET22b(⫹) derivative expressing BacL1⌬3
pET22b(⫹) derivative expressing BacA

62
26
Novagen
28
28
28
28
28
28

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FIG 1 Schematics of Bac41 gene organization and BacL1 structure. (A) Organization of Bac41-related genes (GI 169635857). (B) Molecular structure of
BacL1 (GI 169635864). Two domains with homology to distinct peptidoglycanhydrolases, bacteriophage-type hydrolase and NlpC/P60 family hydrolase, arepresent in the regions from amino acid (a.a.) 3 to 140 and amino acid 163 to
315, respectively. Three repeats of the bacterial SH3 domain are present in theregion from amino acid 329 to 590.

tial for bactericidal activity, together with BacL1, although its
function also remains to be determined (26, 28).
On the basis of cell morphology, enterococci are grouped in the
ovococci, whose cell shapes are elongated ellipsoids (31–33). In
ovococci, the model of the dividing cell wall assembly process isdistinct from that of other shaped bacteria, such as spherical cocci.
The cell division of ovococci is achieved by two distinct cell wallsynthesizing machineries that manage peripheral and septal cellwall growth. The peripheral cell wall growth is responsible for thelongitudinal cell elongation. On the other hand, the septal cell wall
growth occurs to allow splitting into separated daughter cells. In
this study, by using fluorescent dye-conjugated recombinant proteins, we demonstrated that BacL1 localized to the cell divisionrelated cell surface of target E. faecalis cells and that cell division
was required for susceptibility to the bactericidal activity expressed by BacL1 and BacA.

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RESULTSBacL1 targets the cell division-associated region on the E. faecalis surface via its cell wall binding domain. To investigate thelocalization of BacL1 on target E. faecalis cells, we coincubated E.
faecalis cells and the recombinant BacL1 labeled with red fluorescent dye in the absence or presence of BacA, followed by analysis
using fluorescence microscopy (Fig. 2A and B). A specific localization signal of BacL1 in the midcell was observed independentlyof BacA (Fig. 2A). Furthermore, the four characteristic localization patterns closely correlated with cell growth division were detected (31, 33, 39). First, the most typical localization signal of
BacL1 was detected in the midcell, which corresponds to the equatorial ring (Fig. 2B). The equatorial ring structure of the BacL1
localization in the midcell was clearly recognized by the reconstructed image of fluorescence microscopy analysis (see Movie S1
in the supplemental material). Second, the duplicated equatorial
ring structure was detected as the source of the localization signalof BacL1 in the cells initiating elongation prior to cell division.
Third, in the cells where the cell division process had progressed
further, to formation of the division septum, the localization signal of BacL1 was distributed in the area from the equatorial ring to
the division septum, where the cell wall is newly synthesized (nascent cell wall) (32). Furthermore, when cell division was completed, localization at the division septum between separated
daughter cells, as well as at the equatorial ring, was detected. In
addition, immunogold TEM analysis using anti-BacL1 antibodies
in E. faecalis cultures treated with BacL1 and BacA also showed theequatorial ring localization of BacL1 (Fig. 2C).
We previously reported that BacL1 binds to peptidoglycan of E.
faecalis via a C-terminal SH3 triple repeat domain localized in theregion from amino acid 329 to 590 (28). To investigate the domain
required for the specific targeting, domain deletion derivatives of
BacL1 were labeled with green fluorescent dye (Fig. 3A) and mixed
with E. faecalis cells, followed by analysis of their location signal byfluorescence microscopy (Fig. 3B). BacL1⌬3, the derivative withdeletion of the C-terminal SH3 repeat, failed to localize to theequatorial ring and did not show any detectable signal. In contrast,
BacL1⌬1, BacL1⌬2, and BacL1⌬1⌬2, derivatives with deletion of
the phage-type peptidoglycan hydrolase homology domain,
NlpC/P60 family peptidoglycan hydrolase homology domain, orboth domains, respectively, were targeted to the equatorial ringsimilarly to wild-type BacL1. These results indicate that the SH3
repeat was sufficient for the targeting to the equatorial ring on thecell surface of E. faecalis. Collectively, BacL1 appeared to target
the cell division-related cell surface, including the equatorial ring,the division septum, and the nascent cell wall, via its C-terminal
SH3 repeat domains.
Cell division is required for the septum targeting of BacL1
and for the cell lysis triggered by BacL1 and BacA. To analyze theinvolvement of cell division in Bac41 activity, we investigated therelationship of growth phase and susceptibility to Bac41-induced
lysis. E. faecalis was grown in fresh THB broth, and a mixture ofrecombinant BacL1 and BacA was added at different points in thegrowth phases (Fig. 4A). Adding BacL1 and BacA at the start ofincubation (time zero) completely inhibited the increase of thebacterial suspension’s turbidity (cell growth). When BacL1 andBacA were added at early or mid-exponential phase, bacterial turbidity was also dramatically decreased, indicating that cells werelysed. In contrast, treatment with BacL1 and BacA at the stationary

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were prepared by the Ni-nitrilotriacetic acid (NTA) system as previously
described (28). The green or red fluorescent dye-labeled recombinant
proteins were prepared with NH2-reactive fluorescein or NH2-reactive
HiLyte Fluor 555 (Dojindo, Kumamoto, Japan), respectively. By performing a soft-agar bacteriocin assay, we confirmed that fluorescent dye-conjugated BacL1 remains active (see Fig. S1 in the supplemental material).
Anti-BacL1 antibody was prepared by immunization of rabbits with recombinant BacL1-His protein as previously described (Operon Technologies, Alameda, CA) (28).
Fluorescence microscopy. Bacteria diluted with fresh medium weremixed with fluorescent recombinant protein as indicated and incubatedat 37°C for 1 h. The bacteria were collected by centrifugation at 5,800 ⫻ g
for 3 min and then fixed with 4% paraformaldehyde at room temperature(RT) for 15 min. The bacteria were rinsed and resuspended with distilledwater and mounted with Prolong gold antifade reagent with 4=,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA) on a glass
slide. The sample was analyzed by fluorescence microscopy (Axiovert 200;
Carl Zeiss, Oberkochen, Germany), and images were obtained with a
DP71 camera (Olympus, Tokyo, Japan).
Immunogold TEM. Bacteria in early exponential phase were treatedwith recombinant BacL1 and BacA as indicated and incubated at 37°C for
1 h. The bacteria were fixed with 3% paraformaldehyde– 0.1% glutaraldehyde for 10 min at RT and mounted on an electron microscopy (EM) grid.
After fixation, the sample grid was treated with 10-fold-diluted anti-BacL1
antibodies in phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA) at 37°C for 1 h, followed by a wash with PBS. Then,the grid was treated with 10-fold diluted colloidal gold (15 nm)-conjugated anti-rabbit IgG in PBS containing 2% BSA for 30 min at RT, washedwith PBS, and then negatively stained with 2% ammonium molybdate for
1 min at RT. The resulting samples were analyzed by transmission electron microscopy (TEM) (JEM-1010; JEOL Ltd., Tokyo, Japan).
Bacteriolytic assay. The soft-agar assay or liquid-phase assay for bacteriocin activity was performed as described previously (36). Briefly, thetest bacterial strain or 1 ␮l of the recombinant protein solution was inoculated onto THB soft agar (0.75%) containing the indicator strain and wasthen incubated at 37°C for 24 h. The formation of an inhibitory zone was
evaluated as susceptibility to the bacteriocin. For the agar-based bacteriolytic assays using Streptococcus pyogenes and Streptococcus pneumoniae, theindicator bacteria were spread on agar plates by swab instead of using thesoft agar. In this swab method, E. faecalis OG1S and E. faecium BM4105
RF were used for the positive control and the negative control, respectively. For the liquid-phase bactericidal assay, an overnight culture of theindicator strain was diluted with fresh medium, and then the recombinant
proteins were added and the sample was incubated at 37°C. Changes inturbidity were monitored by using a spectrometer (DU730; Beckman
Coulter, Fullerton, CA) or microplate reader (Thermo Scientific, Waltham, MA).
Cell wall degradation assay. For the cell wall degradation assay, a cellwall fraction was prepared as described previously, with slight modifications (28, 37). The bacterial culture was collected by centrifugation andrinsed with 1 M NaCl. The bacterial pellet was suspended in 4% SDS andheated at 95°C for 30 min. After rinsing with distilled water four times, thebacterial pellet was resuspended with distilled water and mechanicallydisrupted with 0.1-mm glass beads (As One, Osaka, Japan) using a FastPrep FP100A (Thermo Scientific, Waltham, MA). After unbroken cells
were removed by centrifugation at 1,000 rpm for 1 min, the cell wall
fraction in the supernatant was collected by centrifugation at 15,000 rpm
for 10 min and was then treated with 0.5 mg ml⫺1 trypsin (0.1 M Tris-HCl[pH 6.8], 20 mM CaCl2) at 37°C for 16 h. The sample was further washedwith distilled water four times and was resuspended in 10% trichloroacetic acid (TCA), followed by incubation at 4°C for 5 h, and then given
additional washes with distilled water four times (38). Finally, the cell wall
fraction was resuspended in PBS and quantified by measuring the turbidity for the cell wall degradation assay. Mutanolysin (Sigma) was used as apositive control for the cell wall degradation enzyme.

Bacteriocin Protein BacL1 Targets Cell Division Site

HiLyte Fluor 555 fluorescent dye-labeled (red) BacL1 (5 ␮g/ml) in the presence (bottom) or absence (middle) of BacA, followed by analysis using fluorescencemicroscopy. Bacteria grown without red fluorescent conjugate are also shown as a negative control (top). Phase contrast (Ph) is pseudocolored (green) in themerged image. (B) Extensive representation of the localization pattern of red fluorescent dye-labeled BacL1. The sample preparation was performed exactly as
described for panel A. DNA was visualized with DAPI (blue). The schematic on the right illustrates the four characteristic patterns of BacL1 localization and cell
division states. (C) E. faecalis treated with BacL1 and BacA (5 ␮g/ml each) was subjected to immunogold transmission electron microscopy using anti-BacL1
antibodies. The arrow indicates the septum localization of gold particles.

phase did not affect the bacterial turbidity, similar to the results
for the untreated culture. The bacterial viability test by colony
formation assay also indicated that the bactericidal activity of
BacL1 and BacA was effective only in early or exponential phase
but not stationary phase (Fig. 4B). In contrast, egg white lysozyme
was able to decrease the viability of bacteria even in stationary
phase (Fig. 4B). These observations indicated that E. faecalis instationary phase was not susceptible to the cell lysis induced by
BacL1 and BacA. Then, to test the growth phase dependence of the
septum targeting of BacL1, the red fluorescence-labeled BacL1 wasincubated with E. faecalis cells in early exponential or stationary
phase, and the BacL1 localization was analyzed by fluorescencemicroscopy (Fig. 4C). In the case of the bacteria in early exponential phase, BacL1 localized at the division septum. In contrast, theseptum localization of BacL1 was not observed in bacteria in stationary phase. BacL1 also failed to even bind to the cell surface instationary-phase bacteria (Fig. 4C). These results suggested that
BacL1 recognized the dividing cell surface. Furthermore, we investigated the susceptibility to bactericidal activity of BacL1 and BacA
when bacterial cell growth was artificially restricted with various
antibiotic reagents (Fig. 5). Treatment with bacteriostatic antibiotics, such as chloramphenicol and tetracycline, almost completely rescued the cells from the bacteriolytic activity of BacL1
and BacA (Fig. 5A and B). The localization of BacL1 to the equatorial ring was also abolished in the chloramphenicol- or tetracycline-treated bacteria (Fig. 5C). Treatment with vancomycin, abactericidal drug blocking cell wall synthesis, resulted in relief ofthe sensitivity of E. faecalis to lysis by BacL1 and BacA and abolished BacL1 targeting to the cell surface (Fig. 5A, B, and C). Interestingly, the bacteria treated with ampicillin, which has an elon-

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gating effect on bacterial cells by inhibiting the penicillin binding
protein (PBP) functions, appeared to be more susceptible to thebactericidal activity of BacL1 and BacA (Fig. 5A and B) and to theseptum targeting of BacL1 (Fig. 5C).
Specific recognition by BacL1 of L-Ala2-type peptidoglycan
cross-bridging structure. The composition and length of thecross-bridge peptide-linking stem peptides bound to N-acetylmuramic acid are diverse among bacterial species (Fig. 6A) (40,41). Lu et al. reported that the SH3 domain of ALE-1, a bacteriolytic peptidoglycan hydrolase of Staphylococcus aureus, specificallyrecognizes the pentaglycine cross bridge, which is a specific structure in the peptidoglycan of S. aureus (42). As shown by the resultsin Fig. 3, the SH3 domains appeared to be necessary for targetingthe cell division-related region. To investigate whether the SH3
domain of BacL1 also specifically recognizes the cross-bridging
structure in the peptidoglycan of E. faecalis, we analyzed the cell
division-associated targeting of BacL1 in various Gram-positivebacterial species, including E. faecalis OG1S, E. faecalis OG1X, E.
faecalis OG1RF, E. faecalis FA2-2, E. faecium BM4105RF, Enterococcus hirae 9790, S. pyogenes MGAS315, S. pneumoniae 262, S.
aureus F-182, and Listeria monocytogenes EGD (Fig. 6B and Table2). In bacteria with L-Ala-L-Ala-cross-bridging peptidoglycans,including E. faecalis strains OG1S, OG1X, OG1RF, and FA2-2 andS. pyogenes, BacL1 clearly localized in the equatorial ring (38, 43–45). In contrast, the BacL1 signal was not detected on E. faecium, E.
hirae, or S. aureus, which have L-Asp-, D-Asn-, or penta-Gly-crossbridging peptidoglycan, respectively (37, 46, 47). L. monocytogenes, which has direct bridging between stem peptides, was also
not bound with BacL1 (48, 49). In the case of S. pneumoniae, which
has a hetero-cross-bridging structure consisting of L-Ala-L-Ala

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FIG 2 Localization of BacL1 on the E. faecalis cell surface. (A) An overnight culture of E. faecalis OG1S diluted 5-fold with fresh THB broth was incubated with

Kurushima et al.

and L-Ala-L-Ser, the equatorial localization was not observed;
however, localization in the division septum was detected (50).
Collectively, these observations suggest that BacL1 specificallybinds to the L-Ala-L-Ala-cross-bridged peptidoglycan. On theother hand, the bactericidal phenotype of BacL1 and BacA wasobserved only in E. faecalis strains and not in the other bacterial
species in soft-agar bacteriocin assays (Table 2). It is notable that S.
pyogenes and S. pneumoniae were not susceptible to BacL1 andBacA despite the targeting of BacL1 to their cell surface (Table 2).
Immunity factor does not alter the BacL1 equatorial targeting. The BacL1- and BacA-producing E. faecalis has a self-resis-

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FIG 4 Growth phase dependence of the susceptibility to Bac41. (A) An overnight culture of E. faecalis OG1S diluted 100-fold with fresh THB broth was
incubated at 37°C. A mixture of recombinant BacL1 and BacA (5 ␮g/ml each)
was added at different growth phases corresponding to 0 h, 2 h, 3.5 h, or 5 h, as
indicated with arrows. An untreated culture served as the negative control. The
turbidity (optical density at 600 nm [OD600]) was monitored in each culture.
The data are presented as the mean results ⫾ standard deviations (SD) of three
independent experiments. (B) E. faecalis was treated with BacL1 and BacA at
different growth phases as described for panel A. After further incubation for 1
h from each time point of addition, the bacterial suspensions were seriallydiluted 10-fold with fresh THB broth and then spotted onto a THB agar plate,followed by incubation overnight. Colony formation was evaluated as a measure of bacterial viability. Lysozyme was used as a control. (C) E. faecalis wastreated with HiLyte Fluor 555-labeled (red) BacL1 (5 ␮g/ml) in the earlyexponential (2 h) or stationary (5 h) phase of growth. After further incubationfor 1 h from each time point of addition, the cells were fixed and analyzed byfluorescence microscopy. Phase contrast (Ph) is pseudocolored (green) in themerged images.

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FIG 3 Domain of BacL1 that is responsible for septum targeting. (A) Schematics of truncated BacL1 constructs. (B) Overnight culture of E. faecalis OG1S
diluted 5-fold with fresh THB broth was incubated with the fluorescein dyelabeled (green) truncated BacL1 proteins (5 ␮g/ml) depicted in panel A, followed by analysis using fluorescence microscopy. Phase contrast (Ph) is
pseudocolored (red) in merged images.

Bacteriocin Protein BacL1 Targets Cell Division Site

(Bac⫹) or without (Bac⫺) a mixture of recombinant BacL1 and BacA (5 ␮g/ml each) in the presence or absence of ampicillin (ABPC; 20 ␮g/ml), chloramphenicol (CP; 100 ␮g/ml), tetracycline (TC; 12.5 ␮g/ml), or vancomycin (VCM; 10 ␮g/ml). The turbidity at 600 nm was measured with a microplate reader during
the incubation period. The data are presented as the mean results ⫾ SD of three independent experiments. (B) E. faecalis was treated with (⫹) or without (⫺) a
mixture of BacL1 and BacA in the presence of antibiotics as described for panel A. After incubation for 6 h, the bacterial suspensions were serially diluted 10-foldwith fresh THB broth and then spotted onto a THB agar plate, followed by incubation overnight. Colony formation was evaluated as a measure of bacterial
viability. (C) An overnight culture of E. faecalis diluted 5-fold with fresh THB broth was treated with HiLyte Fluor 555-labeled (red) BacL1 (5 ␮g/ml) in thepresence of antibiotics as shown. After incubation for 1 h, the cells were fixed and analyzed by fluorescence microscopy. Phase contrast (Ph) is pseudocolored(green) in merged images.

tance factor, BacI, encoded in the vicinity of the bacA gene (Fig.
1A) (26). E. faecalis carrying the bacI gene is completely resistantto the bacteriolytic effect of BacL1 and BacA (26, 28). Therefore, byfluorescence microscopy, we investigated whether the immunity
factor bacI affects the BacL1 targeting. The equatorial localizationof BacL1 was observed in E. faecalis carrying pHT1100 (a plasmid
containing all Bac41 genes, including immunity factor bacI), as
well as in E. faecalis carrying pAM401 (a vector control without the
bacI gene) (Fig. 7A). Furthermore, the peptidoglycan purified
from E. faecalis carrying pHT1100 was still degraded by BacL1(Fig. 7B). These results suggest that the specific immunity factor,

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BacI, has no effect on the BacL1 activities of binding, targeting, anddegrading peptidoglycan.
DISCUSSION

In this study, we report that BacL1 targets the cell division-associated site, including the equatorial ring, division septum, and nascent synthesized cell wall (Fig. 2), to exert potential bactericidal
activity against E. faecalis cells in the dividing state (Fig. 4 and 5).
We also demonstrate that BacL1 specifically recognizes peptidoglycan structures cross-linked by L-Ala-L-Ala but not by otherpeptide linkers (Fig. 6). Although the entire cell wall in E. faecalis is

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FIG 5 Effects of antibiotics on the susceptibility to Bac41. (A) An overnight culture of E. faecalis OG1S diluted 5-fold with fresh THB broth was incubated with

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FIG 6 BacL1 localization in various Gram-positive bacterial species. (A) Peptidoglycan structure of E. faecalis, representing an example of the organizationof peptide chain-cross-linking by a dipeptide. The dotted-line frame indicatesthe cross-bridging peptide between stem peptides bound to N-acetylmuramic
acids. Arrows indicate the sites of cleavage by the endopeptidase activity of
BacL1. (B) Overnight cultures of Gram-positive bacteria, diluted 5-fold withfresh THB broth, were treated with HiLyte Fluor 555-labeled (red) BacL1 (5␮g/ml). After incubation for 1 h, the cells were fixed and analyzed by fluorescence microscopy. Phase contrast is pseudocolored (green) in the merged
images.

composed of L-Ala-LAla-cross-bridged peptidoglycan that is likely
to be recognized by BacL1, there must be an additional determinant(s) for the localized targeting of BacL1 to the cell divisionassociated sites. The equatorial ring is a characteristic structure
observed at the middle of ovococcus cells (32, 51, 52). This ring
structure marks the initiation site for the peripheral cell wall-synthesizing machinery to construct the new peptidoglycan during
cell elongation. Therefore, BacL1 might recognize the cell wallsynthesizing machinery complex that is formed at the equatorial
ring or division septum during cell division. Alternatively, therelatively extended distribution of BacL1, from equatorial ring todivision septum, raises the possibility that BacL1 preferentiallybinds to newly synthesized nascent cell wall. Martínez et al. demonstrated that a bacteriocin of Lactococcus lactis, lactococcin 972,
inhibits the septum formation to cause abnormal cell morphology
in sensitive target cells. Although they have not shown this, lactococcin 972 itself might be associated with the cell-dividing structure, like BacL1 (53). Understanding the determinant(s) restricting the targeting site of BacL1 to cell division-related areas requires
further analysis.
As shown by the results in Fig. 3, the SH3 repeat moiety of
BacL1 was required and sufficient for its localized targeting. These
repeats are present in the region from amino acid 329 to 590 of
BacL1 (see Fig. S2A in the supplemental material). These individual SH3 repeats are nearly identical to each other (see Fig. S2B).
The SH3 domain sequences of BacL1 also show significant homology to SH3 domains from other bactericidal proteins (see Fig.
S2C), such as ALE-1 from S. aureus (54). Crystal structure analysisof the SH3 domain in ALE-1 revealed that the N-terminal conserved motif YXXNKYGTXYXXESA is a recognition groove thatspecifically binds to penta-Gly-cross-bridging peptides in S. aureus peptidoglycan (42). The YXXNKYGTXYXXESA motif (seeFig. S2C, blue frames) is not present in the SH3 domain of BacL1.
Instead, extra conserved residues (see Fig. S2C, red frames) arepresent among the SH3 domains targeting bacteria with an L-AlaL-Ala-cross-bridged cell wall, including E. faecalis, Streptococcus
agalactiae, and S. pneumoniae. Furthermore, amino acids 15 and
14 in the N terminus and C terminus, respectively, are highly
conserved motifs (see Fig. S2C, magenta highlighting) among thethree SH3 domains of BacL1, suggesting that these conserved motifs in BacL1 may play a role in the specific recognition of the
L-Ala-L-Ala-cross-bridged peptidoglycan structure.
Lysostaphin, with activity specific against S. aureus, is able todistinguish the penta-Gly-cross-bridging structure in the peptidoglycan of S. aureus from the cross-bridging structures of otherpeptidoglycans (55). The lysostaphin-specific immunity factor
Lif, a FemABX-like protein, incorporates serine into the crossbridging peptides in peptidoglycan of S. aureus and converts itfrom the penta-Gly-type cross bridge (56). This conversion of thecross-bridging peptide in peptidoglycan results in resistance tolysostaphin. Zoocin A is a bacteriolytic endopeptidase against thecell wall of sensitive bacteria produced by Streptococcus equi subsp.
zooepidemicus strain 4881 (57). The cross bridge in peptidoglycanof S. equi is an L-Ala-L-Ala peptide and is susceptible to the peptidoglycan hydrolase activity of zoocin A (57). Zif, an immunity
factor of zoocin A, belongs to the FemABX-like protein family(58). It additively increases L-Ala residues in the cross bridges of
peptidoglycans and converts L-Ala-L-Ala into L-Ala-L-Ala-L-Ala,resulting in resistance to zoocin A activity. Meanwhile, BacI,Bacteriocin Protein BacL1 Targets Cell Division Site

TABLE 2 Summary of cross-bridge structure and phenotypes against Bac41 in various bacterial species
Presence of phenotypea
StrainCross-bridging peptide

Enterococcus faecalisEnterococcus faecalisEnterococcus faecalisEnterococcus faecalisEnterococcus faecium
Enterococcus hirae
Streptococcus pyogenes
Streptococcus pneumoniae
Staphylococcus aureus
Listeria monocytogenes

OG1S
OG1X
OG1RF
FA2-2
BM4105RF
9790
MGAS315
262
F-182
EGD

L-Ala-L-Ala
L-Ala-L-Ala
L-Ala-L-Ala
L-Ala-L-Ala
L-Asp
D-Asn
L-Ala-L-Ala
L-Ala-L-Ala/L-Ser
Gly5
NAd

Targetingof BacL1b

Susceptibility
to Bac41c

⫹
⫹
⫹
⫹
⫺
⫺
⫹
⫾
⫺
⫺

⫹
⫹
⫹
⫾
⫺
⫺
⫺
⫺
⫺
⫺

⫹, clear/positive; ⫾, obscure/weak; ⫺, negative.
Targeting of BacL1 was determined from the results shown in Fig. 6B.
cSusceptibility to Bac41 (BacL1 and BacA mixture) was determined by a soft-agar-based bacteriocin assay.
dNA, not applicable; L. monocytogenes has direct bridging between stem peptides.
ab

FIG 7 Involvement of Bac41 specific immunity factor BacI in the susceptibility of cell wall to BacL1. (A) An overnight culture of E. faecalis carrying
pAM401 (a vector control without the bacI gene) or pHT1100 (a pAM401
derivative containing all Bac41 genes, including immunity factor bacI) diluted
5-fold with fresh THB broth was treated with HiLyte Fluor 555-labeled (red)BacL1 (5 ␮g/ml). After incubation for 1 h, the cells were fixed and analyzed byfluorescence microscopy. Phase contrast (Ph) is pseudocolored (green) inmerged images. (B) A cell wall fraction prepared from E. faecalis carrying
pAM401 or pHT1100 in exponential phase was diluted with PBS. Recombinant BacL1 (5 ␮g/ml) or mutanolysin (1 ␮g/ml) was added to the cell wall
suspension, and the mixture incubated at 37°C. The turbidity at 600 nm wasquantified at the indicated times during incubation. The values presented are
the percentages of the initial turbidity of the respective samples. The PBStreated sample is presented in each graph as a negative control. The data arepresented as the mean results ⫾ SD of three independent experiments.

January 2015 Volume 197 Number 2

which is the cognate immunity factor against Bac41, did not affect
the BacL1 targeting (Fig. 7A). In addition, the cell wall fraction
prepared from E. faecalis that is resistant to Bac41 due to the presence of bacI was still susceptible to the peptidoglycan-degrading
activity of BacL1 (Fig. 7B), suggesting that BacI is not involved in
the activity of BacL1. This result suggests the possibility that BacI
confers immunity by acting on the function of BacA rather thanthat of BacL1 or that another factor(s) of target cells, such as molecules or receptors that are only present in the growing cells, isinvolved in the BacI-mediated resistance.
The bactericidal activity of Bac41 (BacL1 and BacA) is strictly
specific against E. faecalis, and Bac41 does not show any activity
against the other bacterial species tested (Table 2). The specificity
could be partially explained by the diversity of cross-bridging peptides of peptidoglycan among bacterial species. As demonstrated
by the results in Fig. 6B, BacL1 appears to discriminate target bacterial species from nontarget species by specific recognition of
L-Ala-L-Ala-cross-bridged peptidoglycan. Indeed, BacL1 is able totarget bacteria with L-Ala-L-Ala-cross-bridged peptidoglycan,such as S. pyogenes and S. pneumoniae, regardless of the bacterial
genus. In contrast, E. faecium and E. hirae, with peptidoglycans
cross bridged by L-Asp and D-Asn, respectively, were not recognized by BacL1 although they are phylogenetically classified in thesame genus as E. faecalis. These observations demonstrated thatthe activity of BacL1 is specific against bacteria with L-Ala-L-Alacross-bridged peptidoglycans. However, the bacteriolytic phenotype in the copresence of BacL1 and BacA appears to be more
complex (Table 2). The bactericidal effect of BacL1 and BacA
(Bac41) was observed only against E. faecalis even though otherbacteria are of the L-Ala-L-Ala-cross-bridge type. Interestingly, S.
pyogenes and S. pneumoniae were not susceptible to BacL1 andBacA although they were targeted with BacL1. One possibility isthat BacA is not able to access S. pyogenes and S. pneumoniae.
Furthermore, the susceptibility of E. faecalis FA2-2 to BacL1 andBacA was lower than that of E. faecalis OG1-derived strains, suchas OG1S, OG1X, and OG1RF. Thurlow et al. reported that enterococcal capsular polysaccharide is present in FA2-2 but not in OG1
strains (59). Thus, probably the capsule on the cell surface of strain
FA2-2 cells limits the access of BacA, resulting in the decreased
susceptibility to Bac41-induced lysis. To reveal the detailed mo-

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SpeciesKurushima et al.

ACKNOWLEDGMENTS
This work was supported by grants from the Japanese Ministry of Education, Culture, Sport, Science and Technology [Grant-in-Aid for Young
Scientists (B) 25870116, Gunma University Operation grants] and theJapanese Ministry of Health, Labor and Welfare (H24-Shinkou-Ippan010 and H24-Shokuhin-Ippan-008).

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