NAISTRESEARCHHIGHLIGHTS

http://w w w .naist.jp/en/

RE SE ARCH HIGHLIGHTS
30 YEARS OF HISTORY : 1991−2021

www.naist.jp/en

30
YEARS
OF
HISTORY:
1991−2021

About NAIST
Nara Institute of Science and Technology (NAIST) was founded in 1991 as a Japanese national university with the aim of
conducting advanced research and educating scientists and technologists to support the development of society. NAIST
consisted of graduate schools in the three areas of information science, biological sciences and materials science. In 2018,
NAIST underwent an organizational transformation to continue research in these areas while promoting interdisciplinary
research and education across traditional fields. At present, over 1,000 students – roughly 25% from overseas – are supervisedby roughly 200 faculty members.

Outgrow your limits

With its cutting-edge facilities and a 5 to 1 student-to-faculty ratio, NAIST’s world-leading research and education are adirect result of its rich, global environment and supportive infrastructure. The outstanding achievements of NAIST’s faculty
and students are shared worldwide through publications, patents, licenses and active exchange with overseas partners.

History

WebsiteTwitter

Promotional
Video

Facebook

www.naist.jp/enwww.twitter.com/
naist_main_en

www.naist.jp/en/about_naist/
facts_history/videos.html

www.facebook.com/
naist.jp.en

NAIST Research Highlights
ISSN 2189-714X

For further information on the research
published in NAIST Research Highlights
or to arrange an interview with a faculty,
please contact:

NAURA (NAIST URA Team)
Institute for Research Initiatives
Nara Institute of Science and Technology
8916-5 Takayama-cho, Ikoma, Nara 630-0192, JAPANE-mail: uramember@rsc.naist.jp©2022 NAIST All rights reserved.

Aims & Scope

NAURA

NAIST Research Highlights showcases
promising and important researchachievements at NAIST and presents its
current research and core technologies tothe public. The publication distills highly
technical research papers into short,easy-to-understand articles that appeal to a
global audience of both specialists andnon-specialists. NAIST Research Highlights
aims to inform readers of the latest
developments in NAIST ’ s pioneeringresearch and to stimulate new and existing
international collaborations.

NAURA (NAIST URA team) publishes
NAIST Research Highlights under the
auspices of the “Program for Promoting theEnhancement of Research Universities ”,
which is funded by Japan’ s Ministry of
Education, Culture, Sports, Science andT e c h n o l o g y ( M E X T ) . Th r o u g h t h i s
program, NAIST further supports its
cutting-edge research while expanding into
new interdisciplinary fields in science andtechnology toward becoming a globally
recognized education and research center.

1991

Nara Institute of Science and Technology established
Graduate School of Information Science established

1992

Graduate School of Biological Sciences established
Information Technology Center established

1993

Research and Education Center for
Genetic Information established

1996

Graduate School of Materials Scienceestablished

September 1992
May 1995

1998

Research and Education Center for
Materials Science established2004

Nara Institute of Science and Technology National
University Corporation established2017

Data Science Center established2018

Graduate School of Science and Technology
established (Graduate Schools of Information
Science, Biological Sciences and Materials Sciencemerged together)

2021

Center for Digital Green-innovation established

April 1999

NAIST Research Highlights

1About NAIST
Nara Institute of Science and Technology (NAIST) was founded in 1991 as a Japanese national university with the aim of
conducting advanced research and educating scientists and technologists to support the development of society. NAIST
consisted of graduate schools in the three areas of information science, biological sciences and materials science. In 2018,
NAIST underwent an organizational transformation to continue research in these areas while promoting interdisciplinary
research and education across traditional fields. At present, over 1,000 students – roughly 25% from overseas – are supervisedby roughly 200 faculty members.

Outgrow your limits

With its cutting-edge facilities and a 5 to 1 student-to-faculty ratio, NAIST’s world-leading research and education are adirect result of its rich, global environment and supportive infrastructure. The outstanding achievements of NAIST’s faculty
and students are shared worldwide through publications, patents, licenses and active exchange with overseas partners.

History

WebsiteTwitter

Promotional
Video

Facebook

www.naist.jp/enwww.twitter.com/
naist_main_en

www.naist.jp/en/about_naist/
facts_history/videos.html

www.facebook.com/
naist.jp.en

NAIST Research Highlights
ISSN 2189-714X

For further information on the research
published in NAIST Research Highlights
or to arrange an interview with a faculty,
please contact:

NAURA (NAIST URA Team)
Institute for Research Initiatives
Nara Institute of Science and Technology
8916-5 Takayama-cho, Ikoma, Nara 630-0192, JAPANE-mail: uramember@rsc.naist.jp©2022 NAIST All rights reserved.

Aims & Scope

NAURA

NAIST Research Highlights showcases
promising and important researchachievements at NAIST and presents its
current research and core technologies tothe public. The publication distills highly
technical research papers into short,easy-to-understand articles that appeal to a
global audience of both specialists andnon-specialists. NAIST Research Highlights
aims to inform readers of the latest
developments in NAIST ’ s pioneeringresearch and to stimulate new and existing
international collaborations.

NAURA (NAIST URA team) publishes
NAIST Research Highlights under the
auspices of the “Program for Promoting theEnhancement of Research Universities ”,
which is funded by Japan’ s Ministry of
Education, Culture, Sports, Science andT e c h n o l o g y ( M E X T ) . Th r o u g h t h i s
program, NAIST further supports its
cutting-edge research while expanding into
new interdisciplinary fields in science andtechnology toward becoming a globally
recognized education and research center.

1991

Nara Institute of Science and Technology established
Graduate School of Information Science established

1992

Graduate School of Biological Sciences established
Information Technology Center established

1993

Research and Education Center for
Genetic Information established

1996

Graduate School of Materials Scienceestablished

September 1992
May 1995

1998

Research and Education Center for
Materials Science established2004

Nara Institute of Science and Technology National
University Corporation established2017

Data Science Center established2018

Graduate School of Science and Technology
established (Graduate Schools of Information
Science, Biological Sciences and Materials Sciencemerged together)

2021

Center for Digital Green-innovation established

April 1999

NAIST Research Highlights

1

Contents
1 About NAIST

15 Molecular Biology – Yasuyuki Yamada

The same but different: Structures of a pair ofstereospecific tropinone reductases revealed

8 Developmental Biology – Toshihiko Ogura

16 Cell Biology – Jun-ya Kato

10 Cell Biology – Hiroshi Yoshikawa

17 Computer Vision – Naokazu Yokoya

The critical nature of T-box (Tbx) genes in avian
embryonic development
Determination of key proteins in cellularmechanisms for controlling the initiation of
DNA replication

12 Molecular Biology – Kunio Yasuda

A novel transcription factor for the precise
control of lens development

Releasing inhibitions: Jab1 counteracts inhibition of cell cycle progression by p27Kip1
Improving virtual reality: Design of a stereoscopic omnidirectional video capture system

18 Chemistry – Yukio ImanishiLight-responsive polymer serves as a reversible
on-off switch for enzyme solubility and activity
in organic solvents

14 Structural Biology – Toshio Hakoshima

Resolving the crystal structure of the bacterial
histidine phosphotransfer domain

The discovery of embryonic stem cell pluripotency and teratogenic factors: Groundbreaking
research that led to the development of induced
pluripotent stem cells and a Nobel Prize inPhysiology or Medicine

22 Plant Sciences – Seiji Takayama

How Brassica plants recognize their own pollen

24 Plant Sciences – Toshiharu Shikanai

Cyclic electron flow is critical for higher plant
photosynthesis

26 Plant Sciences – Ko Shimamoto

Blooming brilliant: Discovering the master
regulator of flowering timing in rice plants

28 Microbiology – Naotake Ogasawara

The minimal gene set required to sustain bacterial life

2

NAIST Research Highlights

40 Physics – Hiroshi Daimon

37 Chemistry – Jun-ichi Kikuchi

Paving the way through layer-by-layer assemblyof lipid bilayer vesicles

38 Chemistry – Yoshiaki Kobuke

A synthetic mimic of a biological light-harvesting molecule

44 Molecular Biology – Kenji Kohno

Endoplasmic reticulum stress: How the cellprevents misfolded proteins46 Developmental Biology – Yoshiko Takahashi

Identifying the key control center for sympathetic
nervous system development in early embryos
Adapt and thrive: How a group of proteins may
have helped plants to colonize land

50 Plant Sciences – Masaaki Umedas
30 Molecular Biology – Masahiro Shirakawa

Solving the protein structure of a key binding
domain sheds light on the neurodevelopmental
disorder Rett syndrome

51 Cell Biology – Naoyuki Inagaki

Shootin1 phosphorylation converts a chemical signal
into mechanical force to promote axon growth

52 Microbiology – Hiroshi Takagi

32 Molecular Biology – Akiho Yokota

53 Cell Biology – Kazuhiro Shiozaki

RuBisCO-like proteins may be the ancestral
enzymes of photosynthetic RuBisCO

33 Structural Biology – Toshio Hakoshima

How plant cells perceive and decode the gibberellin signal

34 Speech Recognition – Kiyohiro Shikano

A non-audible voice interface for enhanced
speech recognition

Atomic-layer resolution of the magnetism ofnanoscale nickel

41 Biochemistry – Mikio Kataoka

First evidence for low-barrier hydrogen bonds

42 Biochemistry – Shun Hirota

Trading helices leads to protein polymerization

56 Information Systems – Keiichi Yasumoto

Gamification of crowdsourced monitoring tasks

57 Software Engineering – Kenichi Matsumoto

Improving software defect detection withparameter optimization

58 Artificial Intelligence – Satoshi Nakamura

Text and speech waveform closed-loop deeplearning

59 Chemistry – Tsuyoshi Kawai

Shining a light on the structural basis for highly
sensitive photo-induced cyclization in organic
compounds

Forming giant cells in response to DNA damage

31 Plant Sciences – Takashi Hashimoto

The role of microtubules in the handedness ofhelical growth in plants

Rapid imaging of polycrystalline silicon solar cell
defects

2011 – 2021

48 Plant Sciences – Taku Demura

2001 – 2010
20 Cell Biology – Shinya Yamanaka

36 Robotics – Tsukasa Ogasawara

A robotic hand with vision-based feedback control

1991 – 2000
Right on target: Identification of the first known
targets of Rho

39 Applied Physics – Takashi Fuyuki

A genetic algorithm for optimizing multiple
destination itineraries

4 Editorial

6 Molecular Biology – Kozo Kaibuchi

35 Information Systems – Minoru Ito

Mpr1 is a yeast antioxidant with a uniquechemical structure
Original Sin1: Sin1 recruits substrates to TORC2
for phosphorylation

54 Plant Sciences – Toshiro Ito

Fine-tuning flowers: Plant genes work in balance
to get flower formation right

55 Bioinformatics – Shigehiko Kanaya

Integrated species–metabolite databases forplants distributed around the world

60 Biophysics – Yoichiroh Hosokawa

Femtosecond lasers: A tool for understandingcell–cell adhesion pathologies

61

Nanotechnology – Hisao Yanagi
Chemically modified gain medium developed
for organic lasers

62 Energy Material – Masakazu Nakamura

Weaving carbon nanotubes into fabric for
harvesting human body heat

63 Applied Physics – Jun Ohta

World’s smallest IR wireless energy receiverdeveloped for implantable biodevices and smart
sensors

NAIST Research Highlights

3

Contents
1 About NAIST

15 Molecular Biology – Yasuyuki Yamada

The same but different: Structures of a pair ofstereospecific tropinone reductases revealed

8 Developmental Biology – Toshihiko Ogura

16 Cell Biology – Jun-ya Kato

10 Cell Biology – Hiroshi Yoshikawa

17 Computer Vision – Naokazu Yokoya

The critical nature of T-box (Tbx) genes in avian
embryonic development
Determination of key proteins in cellularmechanisms for controlling the initiation of
DNA replication

12 Molecular Biology – Kunio Yasuda

A novel transcription factor for the precise
control of lens development

Releasing inhibitions: Jab1 counteracts inhibition of cell cycle progression by p27Kip1
Improving virtual reality: Design of a stereoscopic omnidirectional video capture system

18 Chemistry – Yukio ImanishiLight-responsive polymer serves as a reversible
on-off switch for enzyme solubility and activity
in organic solvents

14 Structural Biology – Toshio Hakoshima

Resolving the crystal structure of the bacterial
histidine phosphotransfer domain

The discovery of embryonic stem cell pluripotency and teratogenic factors: Groundbreaking
research that led to the development of induced
pluripotent stem cells and a Nobel Prize inPhysiology or Medicine

22 Plant Sciences – Seiji Takayama

How Brassica plants recognize their own pollen

24 Plant Sciences – Toshiharu Shikanai

Cyclic electron flow is critical for higher plant
photosynthesis

26 Plant Sciences – Ko Shimamoto

Blooming brilliant: Discovering the master
regulator of flowering timing in rice plants

28 Microbiology – Naotake Ogasawara

The minimal gene set required to sustain bacterial life

2

NAIST Research Highlights

40 Physics – Hiroshi Daimon

37 Chemistry – Jun-ichi Kikuchi

Paving the way through layer-by-layer assemblyof lipid bilayer vesicles

38 Chemistry – Yoshiaki Kobuke

A synthetic mimic of a biological light-harvesting molecule

44 Molecular Biology – Kenji Kohno

Endoplasmic reticulum stress: How the cellprevents misfolded proteins46 Developmental Biology – Yoshiko Takahashi

Identifying the key control center for sympathetic
nervous system development in early embryos
Adapt and thrive: How a group of proteins may
have helped plants to colonize land

50 Plant Sciences – Masaaki Umedas
30 Molecular Biology – Masahiro Shirakawa

Solving the protein structure of a key binding
domain sheds light on the neurodevelopmental
disorder Rett syndrome

51 Cell Biology – Naoyuki Inagaki

Shootin1 phosphorylation converts a chemical signal
into mechanical force to promote axon growth

52 Microbiology – Hiroshi Takagi

32 Molecular Biology – Akiho Yokota

53 Cell Biology – Kazuhiro Shiozaki

RuBisCO-like proteins may be the ancestral
enzymes of photosynthetic RuBisCO

33 Structural Biology – Toshio Hakoshima

How plant cells perceive and decode the gibberellin signal

34 Speech Recognition – Kiyohiro Shikano

A non-audible voice interface for enhanced
speech recognition

Atomic-layer resolution of the magnetism ofnanoscale nickel

41 Biochemistry – Mikio Kataoka

First evidence for low-barrier hydrogen bonds

42 Biochemistry – Shun Hirota

Trading helices leads to protein polymerization

56 Information Systems – Keiichi Yasumoto

Gamification of crowdsourced monitoring tasks

57 Software Engineering – Kenichi Matsumoto

Improving software defect detection withparameter optimization

58 Artificial Intelligence – Satoshi Nakamura

Text and speech waveform closed-loop deeplearning

59 Chemistry – Tsuyoshi Kawai

Shining a light on the structural basis for highly
sensitive photo-induced cyclization in organic
compounds

Forming giant cells in response to DNA damage

31 Plant Sciences – Takashi Hashimoto

The role of microtubules in the handedness ofhelical growth in plants

Rapid imaging of polycrystalline silicon solar cell
defects

2011 – 2021

48 Plant Sciences – Taku Demura

2001 – 2010
20 Cell Biology – Shinya Yamanaka

36 Robotics – Tsukasa Ogasawara

A robotic hand with vision-based feedback control

1991 – 2000
Right on target: Identification of the first known
targets of Rho

39 Applied Physics – Takashi Fuyuki

A genetic algorithm for optimizing multiple
destination itineraries

4 Editorial

6 Molecular Biology – Kozo Kaibuchi

35 Information Systems – Minoru Ito

Mpr1 is a yeast antioxidant with a uniquechemical structure
Original Sin1: Sin1 recruits substrates to TORC2
for phosphorylation

54 Plant Sciences – Toshiro Ito

Fine-tuning flowers: Plant genes work in balance
to get flower formation right

55 Bioinformatics – Shigehiko Kanaya

Integrated species–metabolite databases forplants distributed around the world

60 Biophysics – Yoichiroh Hosokawa

Femtosecond lasers: A tool for understandingcell–cell adhesion pathologies

61

Nanotechnology – Hisao Yanagi
Chemically modified gain medium developed
for organic lasers

62 Energy Material – Masakazu Nakamura

Weaving carbon nanotubes into fabric for
harvesting human body heat

63 Applied Physics – Jun Ohta

World’s smallest IR wireless energy receiverdeveloped for implantable biodevices and smart
sensors

NAIST Research Highlights

3

NARA INSTITUTE of SCIENCE and TECHNOLOGYRESEARCH HIGHLIGHTS

30 YEARS OF HISTORY:
1991 – 2021

Nurturing
discoveryW

elcome to NAIST RESEARCHHIGHLIGHTS – 30 YEARS OFHISTORY: 1991–2021. Thispublication provides an overview of highly
impactful work conducted by researchers at Nara
Institute of Science and Technology in the past 30
years since its establishment. In these pages we
present short features of 44 selected topics.
Of course, at the heart of such outcomes are ourresearchers. Each year NAIST attracts more leadersin scientific fields. We look forward to nurturing
their important work and the careers of all our
researchers in the atmosphere of cutting-edge
discovery that will continue to be fostered at
NAIST.

Biological Science

NAIST

On the cover

RESEARCHHIGHLIGHTSRE SE ARCH HIGHLIGHTS
30 YEARS OF HISTORY : 1991−2021//w w w .naist .j p/ e n/

30
Years
of
History

4

Information Science

NAIST Research Highlights

www.naist.jp/en

The main gate of the NAIST
campus. Three triangles represent the three mountains of
Yamato (Kagu-yama, Unebi-yama and Miminashi-yama)described in Manyoshu, theoldest anthology of poems inJapan. They also symbolize
NAIST's three research areas(information science, Biological
Science and materials science),
the mainstay of today's advanced
science and technology.

30 YEARS OF HISTORY

1991 – 2000
Materials Science

NARA INSTITUTE of SCIENCE and TECHNOLOGYRESEARCH HIGHLIGHTS

30 YEARS OF HISTORY:
1991 – 2021

Nurturing
discoveryW

elcome to NAIST RESEARCHHIGHLIGHTS – 30 YEARS OFHISTORY: 1991–2021. Thispublication provides an overview of highly
impactful work conducted by researchers at Nara
Institute of Science and Technology in the past 30
years since its establishment. In these pages we
present short features of 44 selected topics.
Of course, at the heart of such outcomes are ourresearchers. Each year NAIST attracts more leadersin scientific fields. We look forward to nurturing
their important work and the careers of all our
researchers in the atmosphere of cutting-edge
discovery that will continue to be fostered at
NAIST.

Biological Science

NAIST

On the cover

RESEARCHHIGHLIGHTSRE SE ARCH HIGHLIGHTS
30 YEARS OF HISTORY : 1991−2021//w w w .naist .j p/ e n/

30
Years
of
History

4

Information Science

NAIST Research Highlights

www.naist.jp/en

The main gate of the NAIST
campus. Three triangles represent the three mountains of
Yamato (Kagu-yama, Unebi-yama and Miminashi-yama)described in Manyoshu, theoldest anthology of poems inJapan. They also symbolize
NAIST's three research areas(information science, Biological
Science and materials science),
the mainstay of today's advanced
science and technology.

30 YEARS OF HISTORY

1991 – 2000
Materials ScienceMolecular Biology

30 YEARS OF HISTORY: 1991– 2000

Kozo Kaibuchi

Rho, a small guanosine triphosphate, binds to protein kinase N
and Rho-kinase, and activates Rho-kinase to enhance the formationof actin stress fibers and focal adhesions

S

ignaling cascades are crucial for transmitting messages
from the extracellular environment to the interior of
the cell, as well as regulating and modifying processes
within cells. However, these cascades are often very complex,
involving multiple key players and affecting a broad range oftargets. Thus, the discovery of an important signaling
molecule is often the first step in a journey to find interacting
partners for that molecule and understand what they do andhow they do it. One key intracellular signaling protein, theRas-like small guanosine triphosphate Rho, was known tohelp regulate processes as diverse as stress fiber formation,
cell morphology, cell aggregation and cytokinesis. However,despite the involvement of Rho in so many crucial cellular
activities, its downstream effectors remained unknown. In
1995 and 1996, a research team led by Kozo Kaibuchi at
NAIST published a series of papers in Science identifying two

6

NAIST Research Highlights

targets of Rho and demonstrating how one of these targets
mediates an important biological effect of Rho.
In a study published in Science in February of 1996, theresearch team sought to identify proteins that could mediate
RhoA’s effects using an affinity column approach, in which
different forms of RhoA were immobilized on a column andextracts from cow brain membranes were then run over thecolumns. All proteins that bound to the RhoA-saturated
column were then retrieved and analyzed. This strategy
yielded a protein that was determined to be protein kinase N(PKN), which had already been characterized in humans. The
researchers revealed that not only does PKN bind directly to
RhoA, but RhoA also induces PKN phosphorylation andstimulates PKN kinase activity, indicating that this interaction is functionally significant. Importantly, RhoA and PKN
also interacted directly in live cells, showing that this relation-©Adobe Stock/ Volha

Right on target:
Identification of the first known
targets of Rho

ship is not restricted to artificial laboratory conditions, butfunctions in real life.
A subsequent paper published in Science in July of 1996
reported the identification of additional targets of Rho that
could function in other signaling pathways. In this study, theresearch team focused specifically on the stimulation ofsmooth muscle contraction, which was known to involve
phosphorylation of a protein called MLC and was thought tobe regulated by a guanosine triphosphate (GTP)-binding
protein. Given that Rho is a GTP-binding protein that had
been implicated in smooth muscle contraction, it seemed
reasonable that Rho would help regulate this process throughan as-yet-unknown target protein. Using the same column
approach described earlier, the group identified the
myosin-binding subunit (MBS) of myosin phosphatase. The
tail end of MBS interacts directly with RhoA; intriguingly,

this part of the protein has a very similar structure to that of
PKN, suggesting that the specific shape of these proteins
mediates their binding to RhoA. Somewhat unexpectedly,
however, RhoA did not appear to modulate MBS activity,
suggesting that there may be an intermediate factor required
for this step of the signaling pathway. Given that a separate
study from the same research group had identified aserine-threonine kinase (that is, an enzyme that phosphorylates other proteins) known as Rho-kinase as an additionaltarget of RhoA, and that MBS thiophosphorylation is associated with its activity, the researchers suspected that
Rho-kinase was this intermediate factor. This indeed turned
out to be the case: Rho-kinase thiophosphorylated MBS, andthis process was enhanced by the presence of RhoA. Thus,
this study identified a novel Rho target and showed that thisinteraction is functionally important, because it helps
regulate smooth muscle contraction.
In a later paper published in Science in February of 1997,
the research team explored the role of Rho in the formationof stress fibers and focal adhesions, and specifically thefunction of Rho-kinase in this process. To address this, they
investigated the effect of different domains of the Rho-kinase
protein on phosphorylation of myosin light chain, which is acomponent of stress fibers. The catalytic domain of Rho-kinase
showed activity equivalent to that of the full protein, and thisactivity was inhibited by treatment with a protein kinase
inhibitor, suggesting that the catalytic domain is crucial formyosin light chain phosphorylation. Furthermore, injecting
live cells with the catalytic domain induced the formation ofboth stress fibers and focal adhesions, and treatment with thesame inhibitors prevented these effects. Thus, activation of
Rho leads to Rho-kinase–mediated regulation of stress fiberand focal adhesion formation.
Taken together, these three papers reported the identification of the first known targets of Rho, a central signaling
molecule involved in multiple cellular processes, and revealed
the mechanism by which Rho signals through one of thesetargets, Rho-kinase, to induce functionally important
structural changes within cells. This groundbreaking work
established the basis for much of what is known about Rho
signaling today and provided crucial insight into a fundamental signaling network that regulates a host of essential
cellular processes. The findings from these three studies
substantially changed the landscape of the cell signaling fieldand set the stage for much of the subsequent work that has
been done in the field since their publication 25 years ago.

References
Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K.,
Iwamatsu, A., Kaibuchi, K. 1996. Identification of a putative target for Rho as theserine-threonine kinase protein kinase N. Science, 271, 648–650.
Kimura, K., Ito, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M., Yamamori,B., Feng, J., Nakano, T., Okawa, K., Iwamatsu, A., Kaibuchi, K. 1996. Regulation of
myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science,
273, 245–248.
Amano, M., Chihara, K., Kimura, K., Fukata, Y., Nakamura, N., Matsuura, Y.,
Kaibuchi, K. 1997. Formation of actin stress fibers and focal adhesions enhanced
by Rho-kinase. Science, 275, 1308–1311.

NAIST Research Highlights

7

Molecular Biology

30 YEARS OF HISTORY: 1991– 2000

Kozo Kaibuchi

Rho, a small guanosine triphosphate, binds to protein kinase N
and Rho-kinase, and activates Rho-kinase to enhance the formationof actin stress fibers and focal adhesions

S

ignaling cascades are crucial for transmitting messages
from the extracellular environment to the interior of
the cell, as well as regulating and modifying processes
within cells. However, these cascades are often very complex,
involving multiple key players and affecting a broad range oftargets. Thus, the discovery of an important signaling
molecule is often the first step in a journey to find interacting
partners for that molecule and understand what they do andhow they do it. One key intracellular signaling protein, theRas-like small guanosine triphosphate Rho, was known tohelp regulate processes as diverse as stress fiber formation,
cell morphology, cell aggregation and cytokinesis. However,despite the involvement of Rho in so many crucial cellular
activities, its downstream effectors remained unknown. In
1995 and 1996, a research team led by Kozo Kaibuchi at
NAIST published a series of papers in Science identifying two

6

NAIST Research Highlights

targets of Rho and demonstrating how one of these targets
mediates an important biological effect of Rho.
In a study published in Science in February of 1996, theresearch team sought to identify proteins that could mediate
RhoA’s effects using an affinity column approach, in which
different forms of RhoA were immobilized on a column andextracts from cow brain membranes were then run over thecolumns. All proteins that bound to the RhoA-saturated
column were then retrieved and analyzed. This strategy
yielded a protein that was determined to be protein kinase N(PKN), which had already been characterized in humans. The
researchers revealed that not only does PKN bind directly to
RhoA, but RhoA also induces PKN phosphorylation andstimulates PKN kinase activity, indicating that this interaction is functionally significant. Importantly, RhoA and PKN
also interacted directly in live cells, showing that this relation-©Adobe Stock/ Volha

Right on target:
Identification of the first known
targets of Rho

ship is not restricted to artificial laboratory conditions, butfunctions in real life.
A subsequent paper published in Science in July of 1996
reported the identification of additional targets of Rho that
could function in other signaling pathways. In this study, theresearch team focused specifically on the stimulation ofsmooth muscle contraction, which was known to involve
phosphorylation of a protein called MLC and was thought tobe regulated by a guanosine triphosphate (GTP)-binding
protein. Given that Rho is a GTP-binding protein that had
been implicated in smooth muscle contraction, it seemed
reasonable that Rho would help regulate this process throughan as-yet-unknown target protein. Using the same column
approach described earlier, the group identified the
myosin-binding subunit (MBS) of myosin phosphatase. The
tail end of MBS interacts directly with RhoA; intriguingly,

this part of the protein has a very similar structure to that of
PKN, suggesting that the specific shape of these proteins
mediates their binding to RhoA. Somewhat unexpectedly,
however, RhoA did not appear to modulate MBS activity,
suggesting that there may be an intermediate factor required
for this step of the signaling pathway. Given that a separate
study from the same research group had identified aserine-threonine kinase (that is, an enzyme that phosphorylates other proteins) known as Rho-kinase as an additionaltarget of RhoA, and that MBS thiophosphorylation is associated with its activity, the researchers suspected that
Rho-kinase was this intermediate factor. This indeed turned
out to be the case: Rho-kinase thiophosphorylated MBS, andthis process was enhanced by the presence of RhoA. Thus,
this study identified a novel Rho target and showed that thisinteraction is functionally important, because it helps
regulate smooth muscle contraction.
In a later paper published in Science in February of 1997,
the research team explored the role of Rho in the formationof stress fibers and focal adhesions, and specifically thefunction of Rho-kinase in this process. To address this, they
investigated the effect of different domains of the Rho-kinase
protein on phosphorylation of myosin light chain, which is acomponent of stress fibers. The catalytic domain of Rho-kinase
showed activity equivalent to that of the full protein, and thisactivity was inhibited by treatment with a protein kinase
inhibitor, suggesting that the catalytic domain is crucial formyosin light chain phosphorylation. Furthermore, injecting
live cells with the catalytic domain induced the formation ofboth stress fibers and focal adhesions, and treatment with thesame inhibitors prevented these effects. Thus, activation of
Rho leads to Rho-kinase–mediated regulation of stress fiberand focal adhesion formation.
Taken together, these three papers reported the identification of the first known targets of Rho, a central signaling
molecule involved in multiple cellular processes, and revealed
the mechanism by which Rho signals through one of thesetargets, Rho-kinase, to induce functionally important
structural changes within cells. This groundbreaking work
established the basis for much of what is known about Rho
signaling today and provided crucial insight into a fundamental signaling network that regulates a host of essential
cellular processes. The findings from these three studies
substantially changed the landscape of the cell signaling fieldand set the stage for much of the subsequent work that has
been done in the field since their publication 25 years ago.

References
Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K.,
Iwamatsu, A., Kaibuchi, K. 1996. Identification of a putative target for Rho as theserine-threonine kinase protein kinase N. Science, 271, 648–650.
Kimura, K., Ito, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M., Yamamori,B., Feng, J., Nakano, T., Okawa, K., Iwamatsu, A., Kaibuchi, K. 1996. Regulation of
myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science,
273, 245–248.
Amano, M., Chihara, K., Kimura, K., Fukata, Y., Nakamura, N., Matsuura, Y.,
Kaibuchi, K. 1997. Formation of actin stress fibers and focal adhesions enhanced
by Rho-kinase. Science, 275, 1308–1311.

NAIST Research Highlights

7

Developmental Biology

30 YEARS OF HISTORY: 1991– 2000

The critical natureof T-box (Tbx) genes
in avian embryonic development
Tbx4/5 expression is required for proper determination of limb identity
and Tbx5 is critical for eye shape developmentEmbryonic development is a detailed and precise processthat is facilitated by specific changes in gene expression
in different parts of the embryo. This allows for theformation of various organs and structures at defined times,
but also helps in the determination of anatomical directionality and identity. This is of particular importance for limb
development, where the fore- and hindlimbs begin to form
similarly but ultimately become different structures. Thisphenomenon can be seen in many species, such as arms andlegs in humans and wings and legs in birds. Understanding
these processes helps provide insight into the genetic components of development to better appreciate how and why
abnormalities can occur.
One relevant family of genes is the T-box (Tbx) genes,
which code for transcription factors. Previous researchdemonstrated that Tbx4 is expressed in the leg buds and Tbx5
is expressed in the wing buds of developing chickens. In
addition, the Tbx genes are part of the same family of transcription factors as the Drosophila optomoter blind gene, which is
involved in Drosophila limb and optic lobe development.

8

NAIST Research Highlights

Combined with the expression pattern of Tbx4/5, thissuggested that these genes are involved in limb identity inchickens, but it was not clear how significant their roles werein development.
In a study published in Nature in 1999, a research team led
by Toshihiko Ogura at NAIST used a system called in ovo
electroporation where they injected a retroviral plasmid into
chick embryonic cells to “misexpress” Tbx4 in the wing bud
and Tbx5 in the leg bud. Interestingly, expressing Tbx5 in theleg resulted in partial wing skeletal morphology development
and the growth of feathers where scales should be present.
Tbx4 expression in the wing showed opposite effects, including leg-like formation with clawed digits and scale growth.
Expression of control genes, such as green fluorescent protein,
did not cause any of these changes, suggesting they
are Tbx-specific.
Mechanistically, the researchers showed that these Tbx
genes can regulate leg and wing developmental identity by
inducing the expression of limb-specific genes, including
Hoxc9 in the leg and Hoxd9 in the wing. Plasmid-driven© iStock/ SteveMcsweeny

Toshihiko Ogura

expression of Tbx4 induced Hoxc9 and suppressed Hoxd9,
while the opposite was observed with Tbx5. Additionally, theyfound that Tbx4 or Tbx5 could be specifically selected given
certain genetic factors. Expression of a gene called Pitx1 could
induce Tbx4, while Tbx5 expression could inhibit Tbx4. This
allows for specific gene expression repertoires in both the wing
and leg, facilitating limb determination.
Because of its importance in the limbs, the research teamthen became interested in examining the developmental roleof Tbx5 in other parts of the body. They investigated thisgene’s function in the chick eye, publishing their findings inScience in 2000. Previous research showed that Tbx5 is
expressed in the dorsal side of the eye during development,
leading to the hypothesis that this gene is critical for
dorsal-ventral axis determination.
Tbx5 expression was observed in the dorsal retina during early
developmental stages. Similar to their approach in chick limbs,
the team used in ovo electroporation to misexpress Tbx5 in theventral eye. After doing this in stage 8 embryos, oval-shaped eyes
developed, suggesting the eye cup was elongated in both

directions. Expression levels of ventral-specific genes like Pax2and Vax were repressed. However, expression of EphrinB1/2,
which is typically restricted to the dorsal side, was observed in
the ventral retina following Tbx5 expression.
Furthermore, the researchers examined the axons of theretinal ganglion cells, because these normally enter the optic
tectum. When Tbx5 was misexpressed in the ventral retina,some but not all of these axons showed abnormal trajectories
into the tectum. They also examined the role of bone morphogenetic proteins (BMPs), as they have been implicated in
dorsal-ventral axis determination. Interestingly, misexpression of mouse BMP4 in the ventral eye cups resulted in
upregulated Tbx5 and lower levels of Pax2 and Vax. Overall,these findings suggest that formation of the eye dorsal-ventral
axis involves a pathway where BMP4 can induce Tbx5, whichthen induces or represses specific downstream genes.
These two highly impactful publications from the researchteam demonstrated key findings in chicken limb and eye
development. They both centered on Tbx5, a critical gene forwing formation and eye dorsal-ventral axis determination.
Their study also identified specific downstream genes
involved in signaling cascades that mediate these phenotypic
effects. Importantly, these two articles collectively showedthat the same gene is involved in two crucial, yet very different, developmental processes. Their findings therefore
highlight the indispensable nature of Tbx5 in chicken developmental biology. Additionally, these mechanistic results arealso useful for understanding the genetic components ofabnormal development, possibly opening the door for therapeutic intervention or tissue regeneration and transplantationin humans.

Interactions regulating the wing/leg identity of limb buds
(Takeuchi et al., 1999, Nature).

References
Takeuchi, J.K., Koshiba-Takeuchi, K., Matsumoto, K., Vogel-Höpker, A.,
Naitoh-Matsuo, M., Ogura, K., Takahashi, N., Yasuda, K., Ogura, T. 1999. Tbx5
and Tbx4 genes determine the wing/leg identity of limb buds. Nature, 398, 810-814.
Koshiba-Takeuchi, K., Takeuchi, J.K., Matsumoto, K., Momose, T., Uno, K., Hoepker,
V., Ogura, K., Takahashi, N., Nakamura, H., Yasuda, K., Ogura, T. 2000. Tbx5 andthe retinotectum projection. Science, 287, 134-137.

NAIST Research Highlights

9Developmental Biology

30 YEARS OF HISTORY: 1991– 2000

The critical natureof T-box (Tbx) genes
in avian embryonic development
Tbx4/5 expression is required for proper determination of limb identity
and Tbx5 is critical for eye shape developmentEmbryonic development is a detailed and precise processthat is facilitated by specific changes in gene expression
in different parts of the embryo. This allows for theformation of various organs and structures at defined times,
but also helps in the determination of anatomical directionality and identity. This is of particular importance for limb
development, where the fore- and hindlimbs begin to form
similarly but ultimately become different structures. Thisphenomenon can be seen in many species, such as arms andlegs in humans and wings and legs in birds. Understanding
these processes helps provide insight into the genetic components of development to better appreciate how and why
abnormalities can occur.
One relevant family of genes is the T-box (Tbx) genes,
which code for transcription factors. Previous researchdemonstrated that Tbx4 is expressed in the leg buds and Tbx5
is expressed in the wing buds of developing chickens. In
addition, the Tbx genes are part of the same family of transcription factors as the Drosophila optomoter blind gene, which is
involved in Drosophila limb and optic lobe development.

8

NAIST Research Highlights

Combined with the expression pattern of Tbx4/5, thissuggested that these genes are involved in limb identity inchickens, but it was not clear how significant their roles werein development.
In a study published in Nature in 1999, a research team led
by Toshihiko Ogura at NAIST used a system called in ovo
electroporation where they injected a retroviral plasmid into
chick embryonic cells to “misexpress” Tbx4 in the wing bud
and Tbx5 in the leg bud. Interestingly, expressing Tbx5 in theleg resulted in partial wing skeletal morphology development
and the growth of feathers where scales should be present.
Tbx4 expression in the wing showed opposite effects, including leg-like formation with clawed digits and scale growth.
Expression of control genes, such as green fluorescent protein,
did not cause any of these changes, suggesting they
are Tbx-specific.
Mechanistically, the researchers showed that these Tbx
genes can regulate leg and wing developmental identity by
inducing the expression of limb-specific genes, including
Hoxc9 in the leg and Hoxd9 in the wing. Plasmid-driven© iStock/ SteveMcsweeny

Toshihiko Ogura

expression of Tbx4 induced Hoxc9 and suppressed Hoxd9,
while the opposite was observed with Tbx5. Additionally, theyfound that Tbx4 or Tbx5 could be specifically selected given
certain genetic factors. Expression of a gene called Pitx1 could
induce Tbx4, while Tbx5 expression could inhibit Tbx4. This
allows for specific gene expression repertoires in both the wing
and leg, facilitating limb determination.
Because of its importance in the limbs, the research teamthen became interested in examining the developmental roleof Tbx5 in other parts of the body. They investigated thisgene’s function in the chick eye, publishing their findings inScience in 2000. Previous research showed that Tbx5 is
expressed in the dorsal side of the eye during development,
leading to the hypothesis that this gene is critical for
dorsal-ventral axis determination.
Tbx5 expression was observed in the dorsal retina during early
developmental stages. Similar to their approach in chick limbs,
the team used in ovo electroporation to misexpress Tbx5 in theventral eye. After doing this in stage 8 embryos, oval-shaped eyes
developed, suggesting the eye cup was elongated in both

directions. Expression levels of ventral-specific genes like Pax2and Vax were repressed. However, expression of EphrinB1/2,
which is typically restricted to the dorsal side, was observed in
the ventral retina following Tbx5 expression.
Furthermore, the researchers examined the axons of theretinal ganglion cells, because these normally enter the optic
tectum. When Tbx5 was misexpressed in the ventral retina,some but not all of these axons showed abnormal trajectories
into the tectum. They also examined the role of bone morphogenetic proteins (BMPs), as they have been implicated in
dorsal-ventral axis determination. Interestingly, misexpression of mouse BMP4 in the ventral eye cups resulted in
upregulated Tbx5 and lower levels of Pax2 and Vax. Overall,these findings suggest that formation of the eye dorsal-ventral
axis involves a pathway where BMP4 can induce Tbx5, whichthen induces or represses specific downstream genes.
These two highly impactful publications from the researchteam demonstrated key findings in chicken limb and eye
development. They both centered on Tbx5, a critical gene forwing formation and eye dorsal-ventral axis determination.
Their study also identified specific downstream genes
involved in signaling cascades that mediate these phenotypic
effects. Importantly, these two articles collectively showedthat the same gene is involved in two crucial, yet very different, developmental processes. Their findings therefore
highlight the indispensable nature of Tbx5 in chicken developmental biology. Additionally, these mechanistic results arealso useful for understanding the genetic components ofabnormal development, possibly opening the door for therapeutic intervention or tissue regeneration and transplantationin humans.

Interactions regulating the wing/leg identity of limb buds
(Takeuchi et al., 1999, Nature).

References
Takeuchi, J.K., Koshiba-Takeuchi, K., Matsumoto, K., Vogel-Höpker, A.,
Naitoh-Matsuo, M., Ogura, K., Takahashi, N., Yasuda, K., Ogura, T. 1999. Tbx5
and Tbx4 genes determine the wing/leg identity of limb buds. Nature, 398, 810-814.
Koshiba-Takeuchi, K., Takeuchi, J.K., Matsumoto, K., Momose, T., Uno, K., Hoepker,
V., Ogura, K., Takahashi, N., Nakamura, H., Yasuda, K., Ogura, T. 2000. Tbx5 andthe retinotectum projection. Science, 287, 134-137.

NAIST Research Highlights

9Cell Biology

30 YEARS OF HISTORY: 1991– 2000

Hiroshi Yoshikawa© Adobe Stock/ sabelskaya , BillionPhotos.com

Determination of key proteinsin cellular mechanisms for controllingthe initiation of DNA replication
Two proteins are shown to be critical for regulating progression through the cell cycle after
DNA replication and effecting the cell’s response to DNA damage

DNA replication is a fundamental biological process thatis key for cell division and growth. It takes place during
the ‘S phase’ of the cell cycle, and is tightly regulated topreserve the fidelity of the genetic information. Cell-cycle
dysregulation is thought to occur in a range of illnesses such as
inflammation, neurodegenerative disorders, heart disease andcancer; therefore, research into the molecules controlling DNA
replication have wide potential clinical application. The mechanisms by which the cell regulates the timing of the various stepsof the process remained shrouded in mystery until 1998, whena research group led by Hiroshi Yoshikawa at NAIST obtained
new insights into this process. Using the model organism
Saccharomyces cerevisiae, known commonly as budding yeast, they
identified the role of two specific checkpoint proteins, Rad53 andOrc2, in the timing and surveillance of DNA replication.
Chromosome VI of budding yeast contains multiple places
at which DNA replication begins, known as the Origin of
Replication (ORI). In wild-type cells these can be seen to fire atdifferent times in a defined sequence throughout S phase. To
investigate the temporal control of the various ORIs of chromosome VI, the group used an agent that damages DNA, called
MMS (methyl methane sulphonate). MMS halts the cell cycle
at the S phase because detection of DNA damage is a key cellular

10

NAIST Research Highlights

checkpoint that prevents the cell cycle from progressing until
the damage is resolved. The protein Rad53 was thought to be
involved in the mechanism by which the cell monitors progression through S phase, but the detailed mechanism was
unknown. Another important protein in this process is Orc2.
This protein is part of the complex that recognizes and binds thereplication origins, which is crucial for replication initiation.
When wild-type yeast were treated with MMS, they stalledin the S phase and the initiation of DNA replication from thelate-initiating ORIs was blocked. The S phase surveillancemechanism was still in effect, because the cells were still viable.
In contrast, when rad53 and orc2 mutant yeast were treatedwith MMS, they progressed through S phase into the next
phase of the cell cycle, known as G2 phase, albeit with aconcomitant loss of viability. Because entry into the S phasenaturally occurs at different times within a population of cells,
the researchers confirmed these results using cells with cycles
that were artificially synchronized to enter the S phase at thesame time. When MMS was present, wild-type yeast showed aclear reduction in the numbers of cells that progressed to S
phase. However, yeast lacking Rad53 exhibited a slower thanusual progression through the S phase and were still able toreach the G2 phase.

The researchers then went on to look at the sequential firingof the ORIs identified on the right arm of chromosome VI.
When treated with MMS, wild-type cells showed replication
initiation at the early ORI, but late and weak ORIs showed no
replication initiation. Cells lacking Rad53 or Orc2, however,exhibited no such block of the initiation of replication at thelate or weak ORIs. Under normal conditions, in the absence of
MMS, cells lacking Rad53 or Orc2 initiated replication at thelate ORIs more frequently than wild-type cells did.
To determine how general these results were, the researchers then studied another late ORI located on chromosome V
and observed the same results for this origin: MMS blocked
initiation in wild-type cells but not in cells lacking either Rad53
or Orc2. This confirmed that both Rad53 and Orc2 are involvedin blocking initiation from late and weak ORIs as a response to
DNA damage.
Further investigation using Rad53 and Orc2 mutants
revealed the related but distinct roles of these checkpoint
proteins at the different ORIs. The researchers found that
Rad53 mutation affected both the timing of initiation at late
ORIs and the movement of the replication fork at the end of thechromosome, whereas mutation of Orc2 affected the timing of
initiation of both late and early ORIs but did not affect the

movement of the replication fork. Under normal conditions,in the absence of MMS, Rad53 and Orc2 mutation increased
the efficiency of replication initiation at the late ORI and both
early and late ORIs, respectively. They also saw that Rad53
mutation accelerated the timing of initiation of late ORIs.
Taken together, these results suggested that Rad53 acts bysuppressing activation of late ORIs to regulate the efficiency
and timing of replication initiation from them, whereas Orc2
could regulate the activation of early ORIs as well as suppressing late ones.
The cellular response to DNA damage and proper management of the cell cycle is essential for the maintenance ofgenomic stability and prevention of cell death. In higher organisms, dysregulation of these processes can lead to the
propagation of damaged DNA and precursor cell mechanisms
in the development of cancer. The results of this study may be
useful for greater understanding of conditions thought to berelated to cell-cycle dysregulation, such as neurodegenerative
disorders, inflammation and cardiovascular disease.
Reference
Shirahige, K., Hori, Y., Shiraishi, K., Yamashita, M., Takahashi, K., Obuse, C.,
Tsurimoto, T., Yoshikawa, H. 1998. Regulation of DNA-replication origins duringcell-cycle progression. Nature, 395, 618–621.

NAIST Research Highlights

11

Cell Biology

30 YEARS OF HISTORY: 1991– 2000

Hiroshi Yoshikawa© Adobe Stock/ sabelskaya , BillionPhotos.com

Determination of key proteinsin cellular mechanisms for controllingthe initiation of DNA replication
Two proteins are shown to be critical for regulating progression through the cell cycle after
DNA replication and effecting the cell’s response to DNA damage

DNA replication is a fundamental biological process thatis key for cell division and growth. It takes place during
the ‘S phase’ of the cell cycle, and is tightly regulated topreserve the fidelity of the genetic information. Cell-cycle
dysregulation is thought to occur in a range of illnesses such as
inflammation, neurodegenerative disorders, heart disease andcancer; therefore, research into the molecules controlling DNA
replication have wide potential clinical application. The mechanisms by which the cell regulates the timing of the various stepsof the process remained shrouded in mystery until 1998, whena research group led by Hiroshi Yoshikawa at NAIST obtained
new insights into this process. Using the model organism
Saccharomyces cerevisiae, known commonly as budding yeast, they
identified the role of two specific checkpoint proteins, Rad53 andOrc2, in the timing and surveillance of DNA replication.
Chromosome VI of budding yeast contains multiple places
at which DNA replication begins, known as the Origin of
Replication (ORI). In wild-type cells these can be seen to fire atdifferent times in a defined sequence throughout S phase. To
investigate the temporal control of the various ORIs of chromosome VI, the group used an agent that damages DNA, called
MMS (methyl methane sulphonate). MMS halts the cell cycle
at the S phase because detection of DNA damage is a key cellular

10

NAIST Research Highlights

checkpoint that prevents the cell cycle from progressing until
the damage is resolved. The protein Rad53 was thought to be
involved in the mechanism by which the cell monitors progression through S phase, but the detailed mechanism was
unknown. Another important protein in this process is Orc2.
This protein is part of the complex that recognizes and binds thereplication origins, which is crucial for replication initiation.
When wild-type yeast were treated with MMS, they stalledin the S phase and the initiation of DNA replication from thelate-initiating ORIs was blocked. The S phase surveillancemechanism was still in effect, because the cells were still viable.
In contrast, when rad53 and orc2 mutant yeast were treatedwith MMS, they progressed through S phase into the next
phase of the cell cycle, known as G2 phase, albeit with aconcomitant loss of viability. Because entry into the S phasenaturally occurs at different times within a population of cells,
the researchers confirmed these results using cells with cycles
that were artificially synchronized to enter the S phase at thesame time. When MMS was present, wild-type yeast showed aclear reduction in the numbers of cells that progressed to S
phase. However, yeast lacking Rad53 exhibited a slower thanusual progression through the S phase and were still able toreach the G2 phase.

The researchers then went on to look at the sequential firingof the ORIs identified on the right arm of chromosome VI.
When treated with MMS, wild-type cells showed replication
initiation at the early ORI, but late and weak ORIs showed no
replication initiation. Cells lacking Rad53 or Orc2, however,exhibited no such block of the initiation of replication at thelate or weak ORIs. Under normal conditions, in the absence of
MMS, cells lacking Rad53 or Orc2 initiated replication at thelate ORIs more frequently than wild-type cells did.
To determine how general these results were, the researchers then studied another late ORI located on chromosome V
and observed the same results for this origin: MMS blocked
initiation in wild-type cells but not in cells lacking either Rad53
or Orc2. This confirmed that both Rad53 and Orc2 are involvedin blocking initiation from late and weak ORIs as a response to
DNA damage.
Further investigation using Rad53 and Orc2 mutants
revealed the related but distinct roles of these checkpoint
proteins at the different ORIs. The researchers found that
Rad53 mutation affected both the timing of initiation at late
ORIs and the movement of the replication fork at the end of thechromosome, whereas mutation of Orc2 affected the timing of
initiation of both late and early ORIs but did not affect the

movement of the replication fork. Under normal conditions,in the absence of MMS, Rad53 and Orc2 mutation increased
the efficiency of replication initiation at the late ORI and both
early and late ORIs, respectively. They also saw that Rad53
mutation accelerated the timing of initiation of late ORIs.
Taken together, these results suggested that Rad53 acts bysuppressing activation of late ORIs to regulate the efficiency
and timing of replication initiation from them, whereas Orc2
could regulate the activation of early ORIs as well as suppressing late ones.
The cellular response to DNA damage and proper management of the cell cycle is essential for the maintenance ofgenomic stability and prevention of cell death. In higher organisms, dysregulation of these processes can lead to the
propagation of damaged DNA and precursor cell mechanisms
in the development of cancer. The results of this study may be
useful for greater understanding of conditions thought to berelated to cell-cycle dysregulation, such as neurodegenerative
disorders, inflammation and cardiovascular disease.
Reference
Shirahige, K., Hori, Y., Shiraishi, K., Yamashita, M., Takahashi, K., Obuse, C.,
Tsurimoto, T., Yoshikawa, H. 1998. Regulation of DNA-replication origins duringcell-cycle progression. Nature, 395, 618–621.

NAIST Research Highlights

11

Molecular Biology

30 YEARS OF HISTORY: 1991– 2000

Kunio Yasuda

A novel transcription factor for the
precise control of lens developmentT

he process by which a single cell develops into a fully
formed organism is one of the enduring mysteries ofscience. It is posited that the complex interplay between
gene expression, external signals and cell differentiation must
play a crucial role. By studying the emergence of specificstructures, such as the lens of the eye, scientists can start to form

12

NAIST Research Highlights

a better understanding of the intricacies of this process. The
development of the lens is an intriguing model system for thispurpose because it contains specialized crystallin proteins thatprovide transparency and are not found in other parts of the
body. In addition, the cellular differentiation required fordevelopment in vertebrates is known to be precisely controlled© Adobe Stock/ Andrés García

The L-Maf protein discovered in vertebrates regulates genes that control
cell differentiation in the lens, which may provide insights
for the development of regenerative medicine technologies

by intercellular signaling and transcription factors. An improved understandingof the molecular mechanisms governing
this process could be applied more widelyto the development of artificial organs andtissues for use in transplantation or regenerative medicine. As a result, this area of
research has the potential to provide
crucial insights that could revolutionize
human health and longevity.
A series of studies in chickens
demonstrated that the gene expression of
αA-crystallin is controlled by a lens-specific enhancer element, named αCE2,which is a portion of DNA surrounding
Ectopic expression of L-Maf induces lens differentiation in chick embryos
the gene. This enhancer is located about(Ogino & Yasuda, 1998, Science).
100 base pairs upstream of the gene’s
transcription start site, and is conservedin other crystallin genes. These findings indicate that thisThis retina-to-lens cell conversion was not seen with cells
sequence plays an important regulatory role, and a researchwithout L-Maf transfection.
group led by Kunio Yasuda at NAIST worked to identify
Finally, a retrovirus expressing L-Maf was generated, andprotein factors that could bind to αCE2 to better understandallowed to infect retinal cell cultures. Compared with
their role in lens formation.
uninfected cells, the L-Maf retrovirus could induce expresThe researchers first screened an embryonic lens cDNA
sion of αA-, βB1- and δ1-crystallin expression at both thelibrary with an αCE2 oligonucleotide probe and mapped geneprotein and RNA levels. The researchers also found that theexpression in the lens tissues of chicken embryos. They foundRNA expression of filensin, a marker of terminally differentithat an important event in development occurred when cells fromated lens fiber cells, was significantly higher in the L-Maf
the head ectoderm tissue layer made contact with the optic
retrovirus-infected cells.
vesicle. The mRNA expression remained confined to the lensOverall, this study demonstrated that L-Maf, a previously
throughout subsequent stages of development, and αA-crystallin
unidentified transcription factor, has the ability to activate
genetic transcripts, a newly detected mRNA type, were seen evengenes by binding to the αCE2 element in their promoter
before δ1-crystallin mRNA, an early marker of lens developregions. Moreover, these genes were shown to be specific toment.When these transcripts were further characterized, they
the lens, and activating their transcription could induce otherwere found to encode a protein with 286 amino acids, predicted
cell types to differentiate into lens cells. While eye developto be a transcription factor similar to those of the maf family. Thisment is very complex and many other genes are also involved,
novel factor was named “lens-specific Maf ” (L-Maf).
such as Pax-6 and Six3, L-Maf is the gene most strictly
The researchers next performed transfection experiments
localized to the developing lens. This leads to the conclusion
to examine the effects of specific DNA sequences by addingthat L-Maf is likely directly targeted by signals coming fromsynthetic versions directly to cells. Using a bioluminescent
the optic vesicle, especially because of its specificity to the lensdetection system based on the enzyme luciferase, the αA-crysand rapid expression.
tallin promoter was found to produce significantly more light
Obtaining an enhanced understanding of the genetic
when placed into chicken lens primary cells with a plasmid
cascade involved in lens development is a significant
expressing L-Maf, compared with other sequences. These
achievement for the overarching objective of being able toobservations suggest that L-Maf can transactivate αA-crystallin
predict or even control organ development. For example,
expression via the αCE2 sequence in its promoter.
replacement lenses might be grown for patients based on
To further test the role of L-Maf in lens development, thetheir own cells. Similarly, while the need for other organs
researchers conducted gel mobility shift DNA binding assays.
like hearts and kidneys today far outstrips the number
Overexpression of L-Maf could transcriptionally activate any
available for transplant, in the future they may also be generof the crystallin elements tested, including several from chickated by exposing adult stem cells to the correct sequence ofens and two from mice. However, activation was not observed
signaling factors. This would represent a breakthrough forin a gene without a crystallin element, nor in the chicken αAtissue engineering and regenerative medicine.
reporter with a mutated αCE2 sequence.
The researchers then examined the role of L-Maf in lens
cell differentiation by transiently transfecting the L-Maf
plasmid into chicken embryo neural retina cells. These cells
Reference
were found to express both αA- and δ1-crystallin. FurtherOgino, H., Yasuda, K. 1998. Induction of lens differentiation by activation of a bZIP
transcription factor, L-Maf. Science, 280, 115–118.
more, the cells displayed morphology similar to lens fiber cells.

NAIST Research Highlights

13

Molecular Biology

30 YEARS OF HISTORY: 1991– 2000

Kunio Yasuda

A novel transcription factor for the
precise control of lens developmentT

he process by which a single cell develops into a fully
formed organism is one of the enduring mysteries ofscience. It is posited that the complex interplay between
gene expression, external signals and cell differentiation must
play a crucial role. By studying the emergence of specificstructures, such as the lens of the eye, scientists can start to form

12

NAIST Research Highlights

a better understanding of the intricacies of this process. The
development of the lens is an intriguing model system for thispurpose because it contains specialized crystallin proteins thatprovide transparency and are not found in other parts of the
body. In addition, the cellular differentiation required fordevelopment in vertebrates is known to be precisely controlled© Adobe Stock/ Andrés García

The L-Maf protein discovered in vertebrates regulates genes that control
cell differentiation in the lens, which may provide insights
for the development of regenerative medicine technologies

by intercellular signaling and transcription factors. An improved understandingof the molecular mechanisms governing
this process could be applied more widelyto the development of artificial organs andtissues for use in transplantation or regenerative medicine. As a result, this area of
research has the potential to provide
crucial insights that could revolutionize
human health and longevity.
A series of studies in chickens
demonstrated that the gene expression of
αA-crystallin is controlled by a lens-specific enhancer element, named αCE2,which is a portion of DNA surrounding
Ectopic expression of L-Maf induces lens differentiation in chick embryos
the gene. This enhancer is located about(Ogino & Yasuda, 1998, Science).
100 base pairs upstream of the gene’s
transcription start site, and is conservedin other crystallin genes. These findings indicate that thisThis retina-to-lens cell conversion was not seen with cells
sequence plays an important regulatory role, and a researchwithout L-Maf transfection.
group led by Kunio Yasuda at NAIST worked to identify
Finally, a retrovirus expressing L-Maf was generated, andprotein factors that could bind to αCE2 to better understandallowed to infect retinal cell cultures. Compared with
their role in lens formation.
uninfected cells, the L-Maf retrovirus could induce expresThe researchers first screened an embryonic lens cDNA
sion of αA-, βB1- and δ1-crystallin expression at both thelibrary with an αCE2 oligonucleotide probe and mapped geneprotein and RNA levels. The researchers also found that theexpression in the lens tissues of chicken embryos. They foundRNA expression of filensin, a marker of terminally differentithat an important event in development occurred when cells fromated lens fiber cells, was significantly higher in the L-Maf
the head ectoderm tissue layer made contact with the optic
retrovirus-infected cells.
vesicle. The mRNA expression remained confined to the lensOverall, this study demonstrated that L-Maf, a previously
throughout subsequent stages of development, and αA-crystallin
unidentified transcription factor, has the ability to activate
genetic transcripts, a newly detected mRNA type, were seen evengenes by binding to the αCE2 element in their promoter
before δ1-crystallin mRNA, an early marker of lens developregions. Moreover, these genes were shown to be specific toment.When these transcripts were further characterized, they
the lens, and activating their transcription could induce otherwere found to encode a protein with 286 amino acids, predicted
cell types to differentiate into lens cells. While eye developto be a transcription factor similar to those of the maf family. Thisment is very complex and many other genes are also involved,
novel factor was named “lens-specific Maf ” (L-Maf).
such as Pax-6 and Six3, L-Maf is the gene most strictly
The researchers next performed transfection experiments
localized to the developing lens. This leads to the conclusion
to examine the effects of specific DNA sequences by addingthat L-Maf is likely directly targeted by signals coming fromsynthetic versions directly to cells. Using a bioluminescent
the optic vesicle, especially because of its specificity to the lensdetection system based on the enzyme luciferase, the αA-crysand rapid expression.
tallin promoter was found to produce significantly more light
Obtaining an enhanced understanding of the genetic
when placed into chicken lens primary cells with a plasmid
cascade involved in lens development is a significant
expressing L-Maf, compared with other sequences. These
achievement for the overarching objective of being able toobservations suggest that L-Maf can transactivate αA-crystallin
predict or even control organ development. For example,
expression via the αCE2 sequence in its promoter.
replacement lenses might be grown for patients based on
To further test the role of L-Maf in lens development, thetheir own cells. Similarly, while the need for other organs
researchers conducted gel mobility shift DNA binding assays.
like hearts and kidneys today far outstrips the number
Overexpression of L-Maf could transcriptionally activate any
available for transplant, in the future they may also be generof the crystallin elements tested, including several from chickated by exposing adult stem cells to the correct sequence ofens and two from mice. However, activation was not observed
signaling factors. This would represent a breakthrough forin a gene without a crystallin element, nor in the chicken αAtissue engineering and regenerative medicine.
reporter with a mutated αCE2 sequence.
The researchers then examined the role of L-Maf in lens
cell differentiation by transiently transfecting the L-Maf
plasmid into chicken embryo neural retina cells. These cells
Reference
were found to express both αA- and δ1-crystallin. FurtherOgino, H., Yasuda, K. 1998. Induction of lens differentiation by activation of a bZIP
transcription factor, L-Maf. Science, 280, 115–118.
more, the cells displayed morphology similar to lens fiber cells.

NAIST Research Highlights

13

Molecular Biology

30 YEARS OF HISTORY: 1991– 2000

Toshio Hakoshima

Yasuyuki Yamada

Resolving the crystal structureof the bacterial histidine
phosphotransfer domain

The same but different:
Structures of a pair of stereospecifictropinone reductases revealed

X-ray diffraction analysis reveals the 3D structure of the histidine phosphotransfer
domain of anaerobic sensor kinase ArcB, providing insights into an importantsignaling system for environmental responses in bacteria

The crystal structures of two tropinone reductases are solved to determine the featuresthat influence the stereochemistry of medicinally important tropane alkaloid productsT

he ability to detect and respond to external stimuli areessential features of living systems. Many organisms
have evolved highly specialized signaling circuits thatare able to transmit information regarding the outside environment from where it is sensed, at the exterior of the cell, to otherlocations where it can be used to modulate activity. In particular, some cells use a specific two-component regulatory system
in which a membrane-bound histidine kinase protein can betriggered by external conditions to transfer a phosphoryl group
to an internal response-regulating domain. By employing thissystem, changes in the environmental oxygen level or osmotic
concentration, for example, can trigger alterations in behavior.
However, while these two-component regulatory systems are
known to be very common in bacteria, the extent of structuralsimilarity between the signaling proteins used by prokaryotes
and eukaryotes was not well established.
To shed light on this question, a research team led by Toshio
Hakoshima at NAIST used X-ray crystallography to determine
the structure of the histidine phosphotransfer (HPt) domainof the bacterial signaling protein ArcB. This segment of theprotein is made up of six alpha helices that combine to form atightly packed core. The scientists focused on the phosphorelay
system, in which an active histidine amino acid acts as part ofthe chain that sends a phosphoryl group to an aspartate on areceiver protein. These "hybrid" histidine kinases are more
complex compared with other signaling systems, because theHPt histidine is just the final part of a longer relay that receives
the phosphoryl group before transferring it to the response
regulator protein.
Even though HPt domains vary significantly between
bacterial species – so much so that the genetic sequences share
only about 20% similarity with each other – the researchers
found conserved structural features linked to the phosphorelay
function. This indicated that these features were essential forthe correct operation of the system, which was especially true
for the His-717 active site, a common aspect of all protein
domain instances studied. Moreover, similarities in structural
motif were seen with eukaryotic sensor kinases, including
those used by yeasts.
Understanding which structural features of signaling
domains are conserved across species can be very useful when
designing new antibiotic drugs. This is because certain HPt

14

30 YEARS OF HISTORY: 1991– 2000

NAIST Research Highlights

domains are known to be involved in the process of activating
the virulence of certain bacteria. Developing inhibitory drugs
that target these conserved regions would be useful across awider range of pathogens, and it would be much harder forthese critical domains to mutate to confer resistance.

Structure of the histidine phosphotransfer domain of ArcB
(Kato et al., 1997, Cell).

Reference
Kato, M., Mizuno, T., Shimizu, T., Hakoshima, T. 1997. Insights into multistep
phosphorelay from the crystal structure of the C-terminal HPt domain of ArcB.
Cell, 88, 717–723.

N

atural products often exist as stereoisomers – molecules with the same structural
formula, but different orientations in 3D
space. The reactions that lead to these different
products are catalyzed by pairs of enzymes thateach give a specific isomer. Identifying thedifferences within pairs of enzymes that lead totheir specificity, contributes to the continued
understanding of how complex molecules
within the cells of living beings are produced
during biosynthesis.
Tropinone reductases (TRs) catalyze thereduction of tropinones to give tropane alkaloids.
However, the structural differences between
TR-I, which produces the tropine stereoisomer,
and TR-II, which produces the pseudotropine
stereoisomer, were not understood until a
Structures of TR dimers (Nakajima et al., 1998, PNAS).
research group led by Yasuyuki Yamada at
NAIST published their crystal structures in 1998.
binding sites, and the positions of the active site residues were
Tropane alkaloids are natural molecules known to elicit
almost the same in both cases. And the hydrophobic natureeffects as stimulants and anticholinergics, with cocaine andof many of the residues at the active site was common toatropine being widely known examples. The reduction ofboth enzymes.
tropinones is therefore an important reaction in the research
However, differences in the charged residues between theand development of new physiologically active compounds.
two TRs resulted in variations in the electrostatic environTwo pairs of TRs have been identified from the species
Datura stramonium and Hyoscyamus niger. And bothments. Modeling of these differences showed that they
TR-I/TR-II pairs are known to share 64% of their amino acidinfluenced the orientation of the tropinone binding to the
enzyme, which ultimately led to opposing stereochemistry inresidues, indicating that they diverged from a common protein
relatively recently. The researchers therefore focused on
the reaction products.
Not all stereospecific enzyme pairs are as structurally
determining the structural features that result in the diastereosimilar as the TRs pairs were found to be, with some enzymes
meric configurations of the reaction products.
differing almost entirely except for at the active sites, which
The crystal structures of the two D. stramonium derived
have evolved to be mirror images of each other. Stereospecificenzymes were determined with 2.4- and 2.3-Å resolution andenzymes are crucial for the manufacture of stereospecificthe 120-amino acid sequence at the C-terminal of the enzymes
chemicals and compounds such as pharmaceuticals, flavorings– which represents most of the substrate binding site – wasand fragrances. Gaining a full understanding of the structuralinvestigated to determine how structural differences in that
region influence the reaction products.
similarities of TRs provided an important piece of the overall
53 residues in the sequence were found to differ between
picture of stereospecific enzyme evolution.
TR-I and TR-II. Determining whether these variations result
Referencein differences in folding, or whether only a small number of the
Nakajima, K., Yamashita, A., Akama, H., Nakatsu, T., Kato, H., Hashimoto, T., Oda, J.,differences influence the activities, was an important focus of
Yamada, Y. 1998. Crystal structures of two tropinone reductases: Different reaction
the work. The overall folding of the proteins was found to
stereospecificities in the same protein fold. Proceedings of the National Academy of
Sciences of the Unites States of America, 95, 4876–4881.
be almost identical for both enzymes. In addition, the cofactor© 1998 National Academy of Sciences

Structural Biology

NAIST Research Highlights

15

Molecular Biology

30 YEARS OF HISTORY: 1991– 2000

Toshio Hakoshima

Yasuyuki Yamada

Resolving the crystal structureof the bacterial histidine
phosphotransfer domain

The same but different:
Structures of a pair of stereospecifictropinone reductases revealed

X-ray diffraction analysis reveals the 3D structure of the histidine phosphotransfer
domain of anaerobic sensor kinase ArcB, providing insights into an importantsignaling system for environmental responses in bacteria

The crystal structures of two tropinone reductases are solved to determine the featuresthat influence the stereochemistry of medicinally important tropane alkaloid productsT

he ability to detect and respond to external stimuli areessential features of living systems. Many organisms
have evolved highly specialized signaling circuits thatare able to transmit information regarding the outside environment from where it is sensed, at the exterior of the cell, to otherlocations where it can be used to modulate activity. In particular, some cells use a specific two-component regulatory system
in which a membrane-bound histidine kinase protein can betriggered by external conditions to transfer a phosphoryl group
to an internal response-regulating domain. By employing thissystem, changes in the environmental oxygen level or osmotic
concentration, for example, can trigger alterations in behavior.
However, while these two-component regulatory systems are
known to be very common in bacteria, the extent of structuralsimilarity between the signaling proteins used by prokaryotes
and eukaryotes was not well established.
To shed light on this question, a research team led by Toshio
Hakoshima at NAIST used X-ray crystallography to determine
the structure of the histidine phosphotransfer (HPt) domainof the bacterial signaling protein ArcB. This segment of theprotein is made up of six alpha helices that combine to form atightly packed core. The scientists focused on the phosphorelay
system, in which an active histidine amino acid acts as part ofthe chain that sends a phosphoryl group to an aspartate on areceiver protein. These "hybrid" histidine kinases are more
complex compared with other signaling systems, because theHPt histidine is just the final part of a longer relay that receives
the phosphoryl group before transferring it to the response
regulator protein.
Even though HPt domains vary significantly between
bacterial species – so much so that the genetic sequences share
only about 20% similarity with each other – the researchers
found conserved structural features linked to the phosphorelay
function. This indicated that these features were essential forthe correct operation of the system, which was especially true
for the His-717 active site, a common aspect of all protein
domain instances studied. Moreover, similarities in structural
motif were seen with eukaryotic sensor kinases, including
those used by yeasts.
Understanding which structural features of signaling
domains are conserved across species can be very useful when
designing new antibiotic drugs. This is because certain HPt

14

30 YEARS OF HISTORY: 1991– 2000

NAIST Research Highlights

domains are known to be involved in the process of activating
the virulence of certain bacteria. Developing inhibitory drugs
that target these conserved regions would be useful across awider range of pathogens, and it would be much harder forthese critical domains to mutate to confer resistance.

Structure of the histidine phosphotransfer domain of ArcB
(Kato et al., 1997, Cell).

Reference
Kato, M., Mizuno, T., Shimizu, T., Hakoshima, T. 1997. Insights into multistep
phosphorelay from the crystal structure of the C-terminal HPt domain of ArcB.
Cell, 88, 717–723.

N

atural products often exist as stereoisomers – molecules with the same structural
formula, but different orientations in 3D
space. The reactions that lead to these different
products are catalyzed by pairs of enzymes thateach give a specific isomer. Identifying thedifferences within pairs of enzymes that lead totheir specificity, contributes to the continued
understanding of how complex molecules
within the cells of living beings are produced
during biosynthesis.
Tropinone reductases (TRs) catalyze thereduction of tropinones to give tropane alkaloids.
However, the structural differences between
TR-I, which produces the tropine stereoisomer,
and TR-II, which produces the pseudotropine
stereoisomer, were not understood until a
Structures of TR dimers (Nakajima et al., 1998, PNAS).
research group led by Yasuyuki Yamada at
NAIST published their crystal structures in 1998.
binding sites, and the positions of the active site residues were
Tropane alkaloids are natural molecules known to elicit
almost the same in both cases. And the hydrophobic natureeffects as stimulants and anticholinergics, with cocaine andof many of the residues at the active site was common toatropine being widely known examples. The reduction ofboth enzymes.
tropinones is therefore an important reaction in the research
However, differences in the charged residues between theand development of new physiologically active compounds.
two TRs resulted in variations in the electrostatic environTwo pairs of TRs have been identified from the species
Datura stramonium and Hyoscyamus niger. And bothments. Modeling of these differences showed that they
TR-I/TR-II pairs are known to share 64% of their amino acidinfluenced the orientation of the tropinone binding to the
enzyme, which ultimately led to opposing stereochemistry inresidues, indicating that they diverged from a common protein
relatively recently. The researchers therefore focused on
the reaction products.
Not all stereospecific enzyme pairs are as structurally
determining the structural features that result in the diastereosimilar as the TRs pairs were found to be, with some enzymes
meric configurations of the reaction products.
differing almost entirely except for at the active sites, which
The crystal structures of the two D. stramonium derived
have evolved to be mirror images of each other. Stereospecificenzymes were determined with 2.4- and 2.3-Å resolution andenzymes are crucial for the manufacture of stereospecificthe 120-amino acid sequence at the C-terminal of the enzymes
chemicals and compounds such as pharmaceuticals, flavorings– which represents most of the substrate binding site – wasand fragrances. Gaining a full understanding of the structuralinvestigated to determine how structural differences in that
region influence the reaction products.
similarities of TRs provided an important piece of the overall
53 residues in the sequence were found to differ between
picture of stereospecific enzyme evolution.
TR-I and TR-II. Determining whether these variations result
Referencein differences in folding, or whether only a small number of the
Nakajima, K., Yamashita, A., Akama, H., Nakatsu, T., Kato, H., Hashimoto, T., Oda, J.,differences influence the activities, was an important focus of
Yamada, Y. 1998. Crystal structures of two tropinone reductases: Different reaction
the work. The overall folding of the proteins was found to
stereospecificities in the same protein fold. Proceedings of the National Academy of
Sciences of the Unites States of America, 95, 4876–4881.
be almost identical for both enzymes. In addition, the cofactor© 1998 National Academy of Sciences

Structural Biology

NAIST Research Highlights

15

Cell Biology

Computer Vision

30 YEARS OF HISTORY: 1991– 2000

Jun-ya Kato

Naokazu Yokoya

Releasing inhibitions:
Jab1 counteracts inhibition ofcell cycle progression by p27Kip1

Improving virtual reality:
Design of a stereoscopic
omnidirectional video
capture system

Jab1 translocates the cyclin-dependent-kinase inhibitor p27Kip1 from the nucleus to the cytoplasm,
where its proteasome-mediated degradation enables cell cycle progression
not result in its degradation, suggesting that Jab1 may accelerate p27Kip1 degradation by specifically bringing it into proximitywith degradation machinery in the cytoplasm. Importantly, ata functional level, expressing p27Kip1 in mouse fibroblasts inhibited cell cycle progression, and coexpression of Jab1 rescued
this effect.
Overall, the findings from this study showed that Jab1
negatively regulates p27Kip1 by causing it to translocate from thenucleus to the cytoplasm and promoting its degradation by theproteasome. This process results in decreased levels of p27Kip1,
thereby releasing inhibition of cell cycle progression and allowing cells to exit the G1 phase and enter the S phase of the cell
cycle. Given the importance of p27Kip1 abundance in predicting
survival in breast and colon cancer patients, the findings fromthis study provide important insight into how regulation of
p27Kip1 levels may be involved in cancer progression. More
broadly, the interaction between Jab1 and p27Kip1 sheds newlight on cell cycle regulation in general.

T

Reference
Tomoda, K., Kubota, Y., Kato, J. 1999. Degradation of the cyclin-dependent-kinase
inhibitor p27Kip1 is instigated by Jab1. Nature, 398, 160–164.

echnological advances in computing
power and graphics rendering capabilities have made virtual reality (in which
a user interacts with an immersive simulated
environment) and mixed reality (in which
computer-generated graphics are overlaid onlive video) more practical than ever before.
However, conventional video capture methods lack the high resolution and dynamic
wide-angle scope to create believable scenes.
In addition, panoramic approaches often
create extreme image distortion, as with afisheye lens or paraboloid mirror. As a result,an omnidirectional video system that cansimultaneously capture images at video-rate
in all directions would represent a significant
advance for this area.
To address this need, a research team led by
Naokazu Yokoya at NAIST developed animage capture system that produces stereoscopic, omnidirectional images from a single
viewpoint using two sets of six cameras.
Precisely aligned mirrors in the form ofequilateral hexagonal pyramids help combine
the camera shots resulting in real-time video
in all directions simultaneously. Each camera
must have a wide viewing angle to enable anomnidirectional image to be obtained from asingle viewpoint; however, the wide viewing
angle led to undesirable distortion in therecorded images.
To overcome the problem of radial image
distortion, a correction process known as
Tsai's calibration method was implemented.
Tsai’s method for camera calibration has often
been utilized with stereoscopic systems todetermine the interior and exterior orientation parameters in the form of a power seriesof distortion coefficients. Combined with animage scaling factor, the recorded image
coordinates can be mapped to known calibration points. This process can be done quickly
with open-source image software and acalibration grid of black dots. The raw images
also have to be transformed to compensate forthe hexagonal shape of the mirrors. This isdone by digitally projecting the data onto avirtual cylindrical surface with parametersthat are found heuristically based on continuity of the image and consistency between thetwo sensor arrays. The data are recorded as 12
still images for each “frame” of video, which
can then be recombined into an omnidirectional stereo movie.
The system the researchers developed
is among the first to achieve all fourof the requirements for complete scene
capture – namely, it is omnidirectional, stereoscopic, high-resolution, and records witha video-speed frame rate. On the basis of theseresults, the use of mirrored camera arrays canbe applied more broadly to other situations
that require real-time panoramic imaging.
Future versions of this imaging system could
be mounted on vehicles for dynamic video
capture of wide areas or used to achieve
seamless integration of real and virtual
elements in simulated environments for urban
planning of smart cities.

Reference
Kawanishi, T., Yamazawa, K., Iwasa, H., Takemura, H., Yokoya,
N. 1998. Generation of high-resolution stereo panoramic
images by omnidirectional imaging sensor using hexagonal
pyramidal mirrors. Fourteenth International Conference on
Pattern Recognition, 485–489.

An omnidirectional stereo panoramic image (Kawanishi et al., 1998, ICPR).

NAIST Research Highlights

A 12-camera omnidirectional video capture system is created thatcan produce high-resolution video with stereoscopic perspective,
which may allow for greater immersive, panoramic experiences© iStock /BeholdingEyeT

he process of mammalian cell proliferation is carefully
regulated to ensure that cells do not grow out of control.
Multiple cell cycle checkpoints and other regulatory
mechanisms exist to prevent proliferation of damaged cells andappropriately time the proliferation of healthy cells. Disruptionof these systems is a common feature of cancers, which typicallyinvolve unregulated or inappropriate cell proliferation. One
component of the cell cycle regulatory system is the cyclin-dependent-kinase inhibitory protein p27Kip1. Reduced levels of
p27Kip1 expression are associated with poor survival in patientswith breast or colon cancer, and p27Kip1 abundance is regulated
by a variety of mechanisms. A research group led by Jun-ya
Kato at NAIST found a novel cellular mechanism that decreases
p27Kip1 levels.
To identify factors involved in the regulation of p27Kip1
abundance, the researchers performed a yeast two-hybrid
screen designed to detect proteins that interact directly with
p27Kip1. One of the candidates retrieved from this screen was aprotein called Jab1 that was originally identified as a co-activator of c-Jun and JunD. Different portions of Jab1 were thenfused to a variety of tags and reporters to determine how itinteracts with p27Kip1 and where it is active within the cell.
The results showed that Jab1 binds to amino acids 97 to 151
of p27Kip1, and that phosphorylation of a threonine residue
at position 187 of p27 Kip1
may reduce the stabilityof this interaction. Overexpressing Jab1 in mammaliancells significantly decreased
the half-life of p27 Kip1 ina p ro t e a s o m e - d e p e n d e n t
m a n n e r, su g ge st i ng t h at
it promotes p27Kip1 proteasomemediated degradation.
Indeed, further investigation
showed that, while p27Kip1
was primarily located in the
nucleus, coexpression of Jab1
resulted in a shift of both proteinsto the cytoplasm. Simply moving
p27Kip1 to the cytoplasm by fusing
it to a nuclear export signal did

16

30 YEARS OF HISTORY: 1991– 2000

NAIST Research Highlights

17

Cell Biology

Computer Vision

30 YEARS OF HISTORY: 1991– 2000

Jun-ya Kato

Naokazu Yokoya

Releasing inhibitions:
Jab1 counteracts inhibition ofcell cycle progression by p27Kip1

Improving virtual reality:
Design of a stereoscopic
omnidirectional video
capture system

Jab1 translocates the cyclin-dependent-kinase inhibitor p27Kip1 from the nucleus to the cytoplasm,
where its proteasome-mediated degradation enables cell cycle progression
not result in its degradation, suggesting that Jab1 may accelerate p27Kip1 degradation by specifically bringing it into proximitywith degradation machinery in the cytoplasm. Importantly, ata functional level, expressing p27Kip1 in mouse fibroblasts inhibited cell cycle progression, and coexpression of Jab1 rescued
this effect.
Overall, the findings from this study showed that Jab1
negatively regulates p27Kip1 by causing it to translocate from thenucleus to the cytoplasm and promoting its degradation by theproteasome. This process results in decreased levels of p27Kip1,
thereby releasing inhibition of cell cycle progression and allowing cells to exit the G1 phase and enter the S phase of the cell
cycle. Given the importance of p27Kip1 abundance in predicting
survival in breast and colon cancer patients, the findings fromthis study provide important insight into how regulation of
p27Kip1 levels may be involved in cancer progression. More
broadly, the interaction between Jab1 and p27Kip1 sheds newlight on cell cycle regulation in general.

T

Reference
Tomoda, K., Kubota, Y., Kato, J. 1999. Degradation of the cyclin-dependent-kinase
inhibitor p27Kip1 is instigated by Jab1. Nature, 398, 160–164.

echnological advances in computing
power and graphics rendering capabilities have made virtual reality (in which
a user interacts with an immersive simulated
environment) and mixed reality (in which
computer-generated graphics are overlaid onlive video) more practical than ever before.
However, conventional video capture methods lack the high resolution and dynamic
wide-angle scope to create believable scenes.
In addition, panoramic approaches often
create extreme image distortion, as with afisheye lens or paraboloid mirror. As a result,an omnidirectional video system that cansimultaneously capture images at video-rate
in all directions would represent a significant
advance for this area.
To address this need, a research team led by
Naokazu Yokoya at NAIST developed animage capture system that produces stereoscopic, omnidirectional images from a single
viewpoint using two sets of six cameras.
Precisely aligned mirrors in the form ofequilateral hexagonal pyramids help combine
the camera shots resulting in real-time video
in all directions simultaneously. Each camera
must have a wide viewing angle to enable anomnidirectional image to be obtained from asingle viewpoint; however, the wide viewing
angle led to undesirable distortion in therecorded images.
To overcome the problem of radial image
distortion, a correction process known as
Tsai's calibration method was implemented.
Tsai’s method for camera calibration has often
been utilized with stereoscopic systems todetermine the interior and exterior orientation parameters in the form of a power seriesof distortion coefficients. Combined with animage scaling factor, the recorded image
coordinates can be mapped to known calibration points. This process can be done quickly
with open-source image software and acalibration grid of black dots. The raw images
also have to be transformed to compensate forthe hexagonal shape of the mirrors. This isdone by digitally projecting the data onto avirtual cylindrical surface with parametersthat are found heuristically based on continuity of the image and consistency between thetwo sensor arrays. The data are recorded as 12
still images for each “frame” of video, which
can then be recombined into an omnidirectional stereo movie.
The system the researchers developed
is among the first to achieve all fourof the requirements for complete scene
capture – namely, it is omnidirectional, stereoscopic, high-resolution, and records witha video-speed frame rate. On the basis of theseresults, the use of mirrored camera arrays canbe applied more broadly to other situations
that require real-time panoramic imaging.
Future versions of this imaging system could
be mounted on vehicles for dynamic video
capture of wide areas or used to achieve
seamless integration of real and virtual
elements in simulated environments for urban
planning of smart cities.

Reference
Kawanishi, T., Yamazawa, K., Iwasa, H., Takemura, H., Yokoya,
N. 1998. Generation of high-resolution stereo panoramic
images by omnidirectional imaging sensor using hexagonal
pyramidal mirrors. Fourteenth International Conference on
Pattern Recognition, 485–489.

An omnidirectional stereo panoramic image (Kawanishi et al., 1998, ICPR).

NAIST Research Highlights

A 12-camera omnidirectional video capture system is created thatcan produce high-resolution video with stereoscopic perspective,
which may allow for greater immersive, panoramic experiences© iStock /BeholdingEyeT

he process of mammalian cell proliferation is carefully
regulated to ensure that cells do not grow out of control.
Multiple cell cycle checkpoints and other regulatory
mechanisms exist to prevent proliferation of damaged cells andappropriately time the proliferation of healthy cells. Disruptionof these systems is a common feature of cancers, which typicallyinvolve unregulated or inappropriate cell proliferation. One
component of the cell cycle regulatory system is the cyclin-dependent-kinase inhibitory protein p27Kip1. Reduced levels of
p27Kip1 expression are associated with poor survival in patientswith breast or colon cancer, and p27Kip1 abundance is regulated
by a variety of mechanisms. A research group led by Jun-ya
Kato at NAIST found a novel cellular mechanism that decreases
p27Kip1 levels.
To identify factors involved in the regulation of p27Kip1
abundance, the researchers performed a yeast two-hybrid
screen designed to detect proteins that interact directly with
p27Kip1. One of the candidates retrieved from this screen was aprotein called Jab1 that was originally identified as a co-activator of c-Jun and JunD. Different portions of Jab1 were thenfused to a variety of tags and reporters to determine how itinteracts with p27Kip1 and where it is active within the cell.
The results showed that Jab1 binds to amino acids 97 to 151
of p27Kip1, and that phosphorylation of a threonine residue
at position 187 of p27 Kip1
may reduce the stabilityof this interaction. Overexpressing Jab1 in mammaliancells significantly decreased
the half-life of p27 Kip1 ina p ro t e a s o m e - d e p e n d e n t
m a n n e r, su g ge st i ng t h at
it promotes p27Kip1 proteasomemediated degradation.
Indeed, further investigation
showed that, while p27Kip1
was primarily located in the
nucleus, coexpression of Jab1
resulted in a shift of both proteinsto the cytoplasm. Simply moving
p27Kip1 to the cytoplasm by fusing
it to a nuclear export signal did

16

30 YEARS OF HISTORY: 1991– 2000

NAIST Research Highlights

17

Chemistry

30 YEARS OF HISTORY: 1991– 2000

Yukio ImanishiLight-responsive polymer serves
as a reversible on-off switch for enzyme
solubility and activity in organic solvents
Modifying an enzyme with a polymer that undergoes a photo-reversible structuralchange allows for control over its solubility in organic media, and thus its catalytic
activity, potentially enabling improved biological catalyst recovery and efficiencyE

resulting suspension was irradiated with visible light, the PSP
nzymes are known to catalyze many importantbiochemical reactions which, harnessed and controlled
was converted back to its nonpolar form, and all of the precipitated enzyme was resolubilized. The UV/visible
appropriately, could be extremely valuable for industrial
irradiation-induced solubility changes could be repeated multiapplications involving chemical synthesis. However, enzymes
ple times, and, at any point, precipitation via UV irradiation(which are polar) are largely insoluble in the nonpolar organic
solvents that are usually used in industrial synthesis. Thus,
could lead to complete recovery of the coupled PSP-Sub.
solubility in nonpolar organic media is highly beneficial
To confirm that the enzyme’s solubility in organic media
because this would increase the enzyme’s efficiency by removenhanced its activity, the catalytic transesterification reactivity
ing the barrier of diffusion, thereby facilitating the substrate’sof the native Sub was compared with that of the PSP-Sub, inaccess to the catalytic active site.
terms of the initial reaction rate in toluene. Remarkably, theTo modulate their solubility in nonpolar solvents, enzymes
catalytic activity of PSP-Sub (soluble) was more than 100 times
that of native Sub (insoluble), and the modified enzyme system
are often chemically modified, but it is difficult or costly toretained this high level of reactivity even after several precipitarecover or reuse the modified enzymes after the reactions.
tion/solubilization cycles. As a proof-of-concept, the PSP-Sub
In 1999, a research team led by Yukio Imanishi at NAIST
also exhibited photo-reversible solubility in chloroform, as welldemonstrated that attaching a photo-responsive polymer to an
as enhanced catalytic activity relative to native Sub (insoluble
enzyme enabled control over its solubility in organic media via
ultraviolet (UV) and visible light treatment; furthermore,in chloroform), although not as high as in toluene.
the solubilized enzyme showed enhanced reactivity relative
This work highlights an effective approach for expanding
to the native enzyme suspended in nonpolar organic
the use of enzymes in organic synthesis by controlling their
media (toluene).
solubility in organic medial through coupling with photo-reSubtilisin (Sub) is an enzyme that catalyzes industriallysponsive polymers. This solubility control could enhance theapplicable transesterification reactions, which involve exchangindustrial application of biological enzymes in the production
ing the organic R-groups between an ester and an alcohol. The
of chemicals with increased efficiency and sustainability, owing
to improved catalyst recovery.
photo-sensitive polymer (PSP) used in this study contained
spiropyran, which is an organic group that undergoes a nonpolar-to-polar structural change under UV irradiation.
Reference
Importantly, this photo-induced reaction is reversible underIto, Y., Sugimura, N., Kwon, O.H., Imanishi, Y. 1999. Enzyme modification by polymers
visible light irradiation. The researchers successfully coupledwith solubilities that change in response to photoirradiation in organic media. Naturethese two components to generate PSP-Sub, and chromatoBiotechnology, 17, 73–75.
graphic characterization confirmedthat the product contained several PSP
chains per Sub enzyme. This indicatedthe potential for reversibly controllingthe modified enzyme’s solubility.
Importantly, although the native
Sub is insoluble in toluene, the enzyme
modified with the nonpolar PSP was
highly soluble. After irradiation with
UV light, the modified enzyme precipitated from the organic solution because
the photo-induced structural change in
the PSP rendered it polar. When the
Photoisomerization of spiropyran in response to UV light (Ito et al., 1999, Nat Biotechnol).

18

NAIST Research Highlights

30 YEARS OF HISTORY

2001 – 2010

Chemistry

30 YEARS OF HISTORY: 1991– 2000

Yukio ImanishiLight-responsive polymer serves
as a reversible on-off switch for enzyme
solubility and activity in organic solvents
Modifying an enzyme with a polymer that undergoes a photo-reversible structuralchange allows for control over its solubility in organic media, and thus its catalytic
activity, potentially enabling improved biological catalyst recovery and efficiencyE

resulting suspension was irradiated with visible light, the PSP
nzymes are known to catalyze many importantbiochemical reactions which, harnessed and controlled
was converted back to its nonpolar form, and all of the precipitated enzyme was resolubilized. The UV/visible
appropriately, could be extremely valuable for industrial
irradiation-induced solubility changes could be repeated multiapplications involving chemical synthesis. However, enzymes
ple times, and, at any point, precipitation via UV irradiation(which are polar) are largely insoluble in the nonpolar organic
solvents that are usually used in industrial synthesis. Thus,
could lead to complete recovery of the coupled PSP-Sub.
solubility in nonpolar organic media is highly beneficial
To confirm that the enzyme’s solubility in organic media
because this would increase the enzyme’s efficiency by removenhanced its activity, the catalytic transesterification reactivity
ing the barrier of diffusion, thereby facilitating the substrate’sof the native Sub was compared with that of the PSP-Sub, inaccess to the catalytic active site.
terms of the initial reaction rate in toluene. Remarkably, theTo modulate their solubility in nonpolar solvents, enzymes
catalytic activity of PSP-Sub (soluble) was more than 100 times
that of native Sub (insoluble), and the modified enzyme system
are often chemically modified, but it is difficult or costly toretained this high level of reactivity even after several precipitarecover or reuse the modified enzymes after the reactions.
tion/solubilization cycles. As a proof-of-concept, the PSP-Sub
In 1999, a research team led by Yukio Imanishi at NAIST
also exhibited photo-reversible solubility in chloroform, as welldemonstrated that attaching a photo-responsive polymer to an
as enhanced catalytic activity relative to native Sub (insoluble
enzyme enabled control over its solubility in organic media via
ultraviolet (UV) and visible light treatment; furthermore,in chloroform), although not as high as in toluene.
the solubilized enzyme showed enhanced reactivity relative
This work highlights an effective approach for expanding
to the native enzyme suspended in nonpolar organic
the use of enzymes in organic synthesis by controlling their
media (toluene).
solubility in organic medial through coupling with photo-reSubtilisin (Sub) is an enzyme that catalyzes industriallysponsive polymers. This solubility control could enhance theapplicable transesterification reactions, which involve exchangindustrial application of biological enzymes in the production
ing the organic R-groups between an ester and an alcohol. The
of chemicals with increased efficiency and sustainability, owing
to improved catalyst recovery.
photo-sensitive polymer (PSP) used in this study contained
spiropyran, which is an organic group that undergoes a nonpolar-to-polar structural change under UV irradiation.
Reference
Importantly, this photo-induced reaction is reversible underIto, Y., Sugimura, N., Kwon, O.H., Imanishi, Y. 1999. Enzyme modification by polymers
visible light irradiation. The researchers successfully coupledwith solubilities that change in response to photoirradiation in organic media. Naturethese two components to generate PSP-Sub, and chromatoBiotechnology, 17, 73–75.
graphic characterization confirmedthat the product contained several PSP
chains per Sub enzyme. This indicatedthe potential for reversibly controllingthe modified enzyme’s solubility.
Importantly, although the native
Sub is insoluble in toluene, the enzyme
modified with the nonpolar PSP was
highly soluble. After irradiation with
UV light, the modified enzyme precipitated from the organic solution because
the photo-induced structural change in
the PSP rendered it polar. When the
Photoisomerization of spiropyran in response to UV light (Ito et al., 1999, Nat Biotechnol).

18

NAIST Research Highlights

30 YEARS OF HISTORY

2001 – 2010

Cell Biology

30 YEARS OF HISTORY: 2001– 2010

Shinya Yamanaka

The discovery of embryonic stem cell
pluripotency and teratogenic factors:
Groundbreaking research that led
to the development of induced
pluripotent stem cells and a Nobel
Prize in Physiology or MedicineEmbryonic stem cells are a special cell type in earlymammalian embryos that have a unique feature called
pluripotency, which means that they are capable ofdifferentiating into every cell type that will comprise the adultorganism. The field of stem cell medicine is based on the ideathat pluripotent embryonic stem cells could be used to regrow
mature cell populations, thereby restoring the function lostwith the loss of the original cells. However, this approach faces

20

NAIST Research Highlights

some challenges. First, embryonic stem cells can only beisolated from embryos, which raises substantial ethicalconcerns. Second, implanted embryonic stem cells can produce
tumors. In 2003, two seminal papers were published by aresearch group led by Shinya Yamanaka at NAIST, recipient of
the Nobel Prize in Physiology or Medicine 2012, that exploredthe molecular basis of embryonic stem cell pluripotency andteratogenic behavior to discover solutions to these challenges.

© Adobe Stock/ fusebulb, Alexandr Mitiuc, Artur© shutterstock/ Jose Luis Calvo, K.K.T Madhusanka

The discovery of Nanog, the fundamental factor underlying embryonicstem cell pluripotency, and ERas, an oncogene that is important for
the tumor-forming behavior of embryonic stem cells, paved the way
for the later development of induced pluripotent cells, an invaluable
tool for stem cell medicine and medical researchPrior to these investigations of embryonic stem cell pluripotency, it was known that leukemia inhibitory factor (LIF)
signaling through Stat3 could maintain symmetrical self-renewal – the process by which cells divide into two identicaldaughter cells – of mouse embryonic stem cells. However,
LIF/Stat3 signaling is not required for maintenance of the innercell mass of developing embryos, where these cells are located,
nor is it necessary for human embryonic stem cell self-renewal.
Therefore, the aim was to identify the factor, or factors, that are
crucial for pluripotency by detecting genes that are expressed
specifically in embryonic stem cells.
The researchers initially identified 20 candidate genes, someof which had already been experimentally identified as embryonic stem cell–specific markers. Nine uncharacterized genes
were expressed specifically in embryonic stem cells, but only
one, when expressed constitutively in embryonic stem cells
without LIF, resulted in retention of their stem cell morphology.
This gene was named nanog, for Tir Na Nog, which in Irish
mythology is “the land of the ever young”; nanog was expressedin four independent embryonic stem cell lines, but not in twelve
somatic organs, confirming that it is embryonic stem cell–specific. In addition, when nanog was deleted, the cells grew
more slowly, changed shape, and expressed markers ofendodermal cell lineages, suggesting that they had lost
pluripotency and were committed to an endoderm cell fate.
Furthermore, nanog-deficient mouse embryos exhibited a
disrupted tissue structure, and cells retrieved from the innercell mass of these embryos differentiated into endoderm-likecells, again reflecting a loss of pluripotency.
The groundbreaking work reported in this paper published
in Cell showed for the first time that nanog is the fundamental
factor underlying pluripotency in the inner cell mass and inembryonic stem cells. The identification of this critical molecular regulator of cell fate provided crucial insight into early
embryogenesis, and broke new ground in stem cell medicine
by delineating one way in which cells could be induced to adopt
and/or maintain a pluripotent state for differentiation into
clinically relevant cell types for therapeutic applications.
In the second study published in Nature, Yamanaka’s
research group sought to identify the factor(s) responsible forthe tumor-like properties of embryonic stem cells. They identified a gene expressed specifically in embryonic stem cells inmice that exhibited a high level of homology, or similarity, toknown Ras genes, and was therefore named ERas. Comparison
with human genes showed considerable similarities between
ERas and HRasp, which was previously thought to be a
pseudogene but in fact is the human ortholog of ERas, and wastherefore renamed human ERas. Importantly, both mouse andhuman ERas transformed cells in vitro and induced tumor
formation in nude mice, confirming that these are teratogenic
factors. When ERas was deleted from mouse embryonic stem
cells, the mice grew and developed normally, although the cells
themselves grew slowly and produced smaller tumors thanwild-type cells in nude mice, suggesting that ERas is required
for the tumor-like properties of embryonic stem cells, but notfor embryonic stem cell pluripotency.
The findings from this study show that ERas is a transform-ing oncogene that is at least partly responsible for thetumor-like behavior of embryonic stem cells. This has important implications for stem cell therapeutics, because it could help
engineer cells that are pluripotent but not tumor-forming. The
identification of ERas therefore provided a crucial new avenue
for adapting embryonic stem cells for clinical use.
Taken together, these two papers from Yamanaka’slaboratory represented significant advances in our understanding of stem cell biology. Crucially, this groundbreaking work
later led to the development of induced pluripotent stem cells,
which are mature, adult cells that have been induced todedifferentiate into an embryonic stem cell-like state by theapplication of only a few factors, one of which is Nanog. The
ability to create these cells from adult cells circumvents the
ethical dilemmas associated with using stem cells fromembryos, and they are thus an invaluable tool for stem cell
medicine. Yamanaka’s seminal contributions to the development of induced pluripotent stem cells led to him being
awarded the Nobel Prize in Physiology or Medicine in 2012.

Morphology of wild type RF8 cells grown on STO
feeder cells (top) and nanog null cells grown withor without STO feeder cells (middle or bottom,
respectively) (Mitsui et al., 2003, Cell ).

References
Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., Maruyama,M., Maeda, M., Yamanaka, S. 2003. The homeoprotein Nanog is required formaintenance of pluripotency in mouse epiblast and ES cells. Cell, 113, 631–642.
Takahashi, K., Mitsui, K., Yamanaka, S. 2003. Role of ERas in promoting tumour-like
properties in mouse embryonic stem cells. Nature, 423, 541–545.

NAIST Research Highlights

21

Cell Biology

30 YEARS OF HISTORY: 2001– 2010

Shinya Yamanaka

The discovery of embryonic stem cell
pluripotency and teratogenic factors:
Groundbreaking research that led
to the development of induced
pluripotent stem cells and a Nobel
Prize in Physiology or MedicineEmbryonic stem cells are a special cell type in earlymammalian embryos that have a unique feature called
pluripotency, which means that they are capable ofdifferentiating into every cell type that will comprise the adultorganism. The field of stem cell medicine is based on the ideathat pluripotent embryonic stem cells could be used to regrow
mature cell populations, thereby restoring the function lostwith the loss of the original cells. However, this approach faces

20

NAIST Research Highlights

some challenges. First, embryonic stem cells can only beisolated from embryos, which raises substantial ethicalconcerns. Second, implanted embryonic stem cells can produce
tumors. In 2003, two seminal papers were published by aresearch group led by Shinya Yamanaka at NAIST, recipient of
the Nobel Prize in Physiology or Medicine 2012, that exploredthe molecular basis of embryonic stem cell pluripotency andteratogenic behavior to discover solutions to these challenges.

© Adobe Stock/ fusebulb, Alexandr Mitiuc, Artur© shutterstock/ Jose Luis Calvo, K.K.T Madhusanka

The discovery of Nanog, the fundamental factor underlying embryonicstem cell pluripotency, and ERas, an oncogene that is important for
the tumor-forming behavior of embryonic stem cells, paved the way
for the later development of induced pluripotent cells, an invaluable
tool for stem cell medicine and medical researchPrior to these investigations of embryonic stem cell pluripotency, it was known that leukemia inhibitory factor (LIF)
signaling through Stat3 could maintain symmetrical self-renewal – the process by which cells divide into two identicaldaughter cells – of mouse embryonic stem cells. However,
LIF/Stat3 signaling is not required for maintenance of the innercell mass of developing embryos, where these cells are located,
nor is it necessary for human embryonic stem cell self-renewal.
Therefore, the aim was to identify the factor, or factors, that are
crucial for pluripotency by detecting genes that are expressed
specifically in embryonic stem cells.
The researchers initially identified 20 candidate genes, someof which had already been experimentally identified as embryonic stem cell–specific markers. Nine uncharacterized genes
were expressed specifically in embryonic stem cells, but only
one, when expressed constitutively in embryonic stem cells
without LIF, resulted in retention of their stem cell morphology.
This gene was named nanog, for Tir Na Nog, which in Irish
mythology is “the land of the ever young”; nanog was expressedin four independent embryonic stem cell lines, but not in twelve
somatic organs, confirming that it is embryonic stem cell–specific. In addition, when nanog was deleted, the cells grew
more slowly, changed shape, and expressed markers ofendodermal cell lineages, suggesting that they had lost
pluripotency and were committed to an endoderm cell fate.
Furthermore, nanog-deficient mouse embryos exhibited a
disrupted tissue structure, and cells retrieved from the innercell mass of these embryos differentiated into endoderm-likecells, again reflecting a loss of pluripotency.
The groundbreaking work reported in this paper published
in Cell showed for the first time that nanog is the fundamental
factor underlying pluripotency in the inner cell mass and inembryonic stem cells. The identification of this critical molecular regulator of cell fate provided crucial insight into early
embryogenesis, and broke new ground in stem cell medicine
by delineating one way in which cells could be induced to adopt
and/or maintain a pluripotent state for differentiation into
clinically relevant cell types for therapeutic applications.
In the second study published in Nature, Yamanaka’s
research group sought to identify the factor(s) responsible forthe tumor-like properties of embryonic stem cells. They identified a gene expressed specifically in embryonic stem cells inmice that exhibited a high level of homology, or similarity, toknown Ras genes, and was therefore named ERas. Comparison
with human genes showed considerable similarities between
ERas and HRasp, which was previously thought to be a
pseudogene but in fact is the human ortholog of ERas, and wastherefore renamed human ERas. Importantly, both mouse andhuman ERas transformed cells in vitro and induced tumor
formation in nude mice, confirming that these are teratogenic
factors. When ERas was deleted from mouse embryonic stem
cells, the mice grew and developed normally, although the cells
themselves grew slowly and produced smaller tumors thanwild-type cells in nude mice, suggesting that ERas is required
for the tumor-like properties of embryonic stem cells, but notfor embryonic stem cell pluripotency.
The findings from this study show that ERas is a transform-ing oncogene that is at least partly responsible for thetumor-like behavior of embryonic stem cells. This has important implications for stem cell therapeutics, because it could help
engineer cells that are pluripotent but not tumor-forming. The
identification of ERas therefore provided a crucial new avenue
for adapting embryonic stem cells for clinical use.
Taken together, these two papers from Yamanaka’slaboratory represented significant advances in our understanding of stem cell biology. Crucially, this groundbreaking work
later led to the development of induced pluripotent stem cells,
which are mature, adult cells that have been induced todedifferentiate into an embryonic stem cell-like state by theapplication of only a few factors, one of which is Nanog. The
ability to create these cells from adult cells circumvents the
ethical dilemmas associated with using stem cells fromembryos, and they are thus an invaluable tool for stem cell
medicine. Yamanaka’s seminal contributions to the development of induced pluripotent stem cells led to him being
awarded the Nobel Prize in Physiology or Medicine in 2012.

Morphology of wild type RF8 cells grown on STO
feeder cells (top) and nanog null cells grown withor without STO feeder cells (middle or bottom,
respectively) (Mitsui et al., 2003, Cell ).

References
Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., Maruyama,M., Maeda, M., Yamanaka, S. 2003. The homeoprotein Nanog is required formaintenance of pluripotency in mouse epiblast and ES cells. Cell, 113, 631–642.
Takahashi, K., Mitsui, K., Yamanaka, S. 2003. Role of ERas in promoting tumour-like
properties in mouse embryonic stem cells. Nature, 423, 541–545.

NAIST Research Highlights

21

Plant Sciences

30 YEARS OF HISTORY: 2001– 2010

Seiji Takayama© Adobe Stock/ KYOTO

How Brassica plants
recognize their own pollen
The identification of a novel signaling protein and small non-coding RNA
revolutionizes our understanding of the self-incompatibility response in plants

M

any flowering plants have evolved to develop a self-incompatibility (SI) response, avoiding self-fertilizationfrom its own pollen and the potentially negative genetic
consequences resulting from this. This is the case in Brassica
plants, which have SI regulated by a series of genes called S
haplotypes. Together, the pistil determinant gene and pollen
determinant gene from the S haplotype help initiate the molecular signaling cascade associated with the SI response. A
thorough investigation of the specific details of this cascade wastherefore necessary to fully understand this complicated but
crucial plant process.
Previous work in the field demonstrated that the S locus
contains three genes vital for SI in Brassica: S-receptor kinase(SRK), S-locus protein 11 (SP11) and S-locus glycoprotein(SLG). The SRK protein is a membrane-bound kinase that
determines the S-haplotype specificity of the stigma, theportion of the pistil that receives pollen, while the SP11 protein
is the pollen S-haplotype determinant and is part of the pollencoat. SP11 can be secreted by the pollen and bind to the respective SRK, initiating the SI response in the stigmatic papilla cells.
However, the role of SLG in this process was a point of contention. This gap in the knowledge interested Seiji Takayama at
NAIST, who then spent years researching the nuances of the SI
signaling cascade in Brassica and ultimately made significant
contributions to the field.
In a study published in Nature in 2001, a research team led
by Takayama investigated previous observations that SLG – aprotein secreted by the stigma and very similar to a portion of
SRK – could enhance the SI response. Here, they studied the S8
haplotype. They first isolated and purified the S8-SP11 proteinfrom the pollen, and then based on mass spectrometry analysis,
they showed that its structure is stabilized by four intramolecular disulfide bonds. Interestingly,
after performing crosslinking
experiments to identify proteinsthat S8-SP11 can interact with,they found that it binds to both
S8-SRK and S8-SLG to form alarger complex. Formation of thiscomplex resulted in autophosphorylation of S8-SRK and thus
the start of the SI cascade. This did
not occur with S8-SP11, emphasizing the S-haplotype specificity.
Though it was clear that asignalingcascade is initiatedBrassica mm plants (right) show defective self-incompatibility response with penetrated pollen tubes (arrow)
when self-fertilization occurs, the(Murase et al., 2004, Science).

22

NAIST Research Highlights

molecule(s) and effects downstream of autophosphorylated
SRK were unknown.The research team therefore continued
their characterization of this pathway with a study published in
Science in 2004. The M locus became of interest, because plants
with a recessive mutation in the modifier (m) gene had complete
absence of the SI response in the stigma with normal activity inthe pollen. In fact, the protein encoded by the M gene was fully
undetermined. The team identified this as a 404-amino acidprotein kinase, which they called M locus protein kinase(MLPK). They showed that MLPK expression was mainly inthe stigma and levels increased immediately prior to flowering,similar to the expression pattern of SRK. The researchers thenpinpointed a single nucleotide difference in the mutant MLPK
gene (m) that resulted in an amino acid substitution preventing
the kinase’s autophosphorylation activity. Moreover, the MLPK
protein was undetectable in mutant plants, suggesting thesubstitution affected the protein’s stability. Transiently expressing wild-type MLPK in mutant papilla cells restored the SI
response, demonstrating that functional MLPK is indispensable for proper rejection of self-pollen in these plants.
The researchers provided crucial data that revolutionized
our understanding of the molecular signaling governing the SI
response in Brassica. However, they remained interested in the
upstream S-haplotype genes and how their dominant/recessive
relationship is regulated in these plants. In particular, SP11
genes are expressed in a monoallelic pattern, meaning theversion of the gene from one parent is expressed (dominant
allele), while the version from the other parent is not (recessive
allele). The molecular details of how this is controlled remained
elusive to the team, leading to a set of experiments that were
published in Nature in 2010. The group previously found that

promoter methylation could turn off expression of the recessive
allele. Intriguingly, they observed inverted genomic sequencesin regions surrounding dominant SP11 alleles that were similar
to sequences in recessive SP11 promoters. Expression of thissequence led to the production of a 24-nucleotide small
non-coding RNA that they named SP11 methylation inducer(Smi). This RNA can work in trans to activate methylation of
the recessive allele promoter, repressing transcription. This was
the first report of a small non-coding RNA working in thismanner to affect a dominant/recessive allele relationship.
Through a series of high-impact publications spanning overa decade, the research team revealed significant molecular
information regarding the SI response process in Brassica
plants. This included from S locus haplotype gene expression
mechanisms to the SI signaling cascade. The team identified
two novel factors: MLPK as a crucial SI signaling mediator andSmi as a trans-acting non-coding RNA in marking the recessive
SP11 allele. Furthermore, they found that the SLG protein ispart of a larger SI-associated complex and the mutant MLPK
gene erases the SI response in the stigma. These results weregroundbreaking for a greater understanding of self-recognitionin plants.
References
Takayama, S., Shimosato, H., Shiba, H., Funato, M., Che, F.S., Watanabe, M., Iwano,
M., Isogai, A. 2001. Direct ligand–receptor complex interaction controls Brassica
self-incompatibility. Nature, 413, 534–538.
Murase, K., Shiba, H., Iwano, M., Che, F.S., Watanabe, M., Isogai, A., Takayama, S.
2004. A membrane-anchored protein kinase involved in Brassica self-incompatibility signaling. Science, 303, 1516–1519.
Tarutani, Y., Shiba, H., Iwano, M., Kakizaki, T., Suzuki, G., Watanabe, M., Isogai, A.,
Takayama, S. 2010. Trans-acting small RNA determines dominance relationships
in Brassica self-incompatibility. Nature, 466, 983–986.

NAIST Research Highlights

23

Plant Sciences

30 YEARS OF HISTORY: 2001– 2010

Seiji Takayama© Adobe Stock/ KYOTO

How Brassica plants
recognize their own pollen
The identification of a novel signaling protein and small non-coding RNA
revolutionizes our understanding of the self-incompatibility response in plants

M

any flowering plants have evolved to develop a self-incompatibility (SI) response, avoiding self-fertilizationfrom its own pollen and the potentially negative genetic
consequences resulting from this. This is the case in Brassica
plants, which have SI regulated by a series of genes called S
haplotypes. Together, the pistil determinant gene and pollen
determinant gene from the S haplotype help initiate the molecular signaling cascade associated with the SI response. A
thorough investigation of the specific details of this cascade wastherefore necessary to fully understand this complicated but
crucial plant process.
Previous work in the field demonstrated that the S locus
contains three genes vital for SI in Brassica: S-receptor kinase(SRK), S-locus protein 11 (SP11) and S-locus glycoprotein(SLG). The SRK protein is a membrane-bound kinase that
determines the S-haplotype specificity of the stigma, theportion of the pistil that receives pollen, while the SP11 protein
is the pollen S-haplotype determinant and is part of the pollencoat. SP11 can be secreted by the pollen and bind to the respective SRK, initiating the SI response in the stigmatic papilla cells.
However, the role of SLG in this process was a point of contention. This gap in the knowledge interested Seiji Takayama at
NAIST, who then spent years researching the nuances of the SI
signaling cascade in Brassica and ultimately made significant
contributions to the field.
In a study published in Nature in 2001, a research team led
by Takayama investigated previous observations that SLG – aprotein secreted by the stigma and very similar to a portion of
SRK – could enhance the SI response. Here, they studied the S8
haplotype. They first isolated and purified the S8-SP11 proteinfrom the pollen, and then based on mass spectrometry analysis,
they showed that its structure is stabilized by four intramolecular disulfide bonds. Interestingly,
after performing crosslinking
experiments to identify proteinsthat S8-SP11 can interact with,they found that it binds to both
S8-SRK and S8-SLG to form alarger complex. Formation of thiscomplex resulted in autophosphorylation of S8-SRK and thus
the start of the SI cascade. This did
not occur with S8-SP11, emphasizing the S-haplotype specificity.
Though it was clear that asignalingcascade is initiatedBrassica mm plants (right) show defective self-incompatibility response with penetrated pollen tubes (arrow)
when self-fertilization occurs, the(Murase et al., 2004, Science).

22

NAIST Research Highlights

molecule(s) and effects downstream of autophosphorylated
SRK were unknown.The research team therefore continued
their characterization of this pathway with a study published in
Science in 2004. The M locus became of interest, because plants
with a recessive mutation in the modifier (m) gene had complete
absence of the SI response in the stigma with normal activity inthe pollen. In fact, the protein encoded by the M gene was fully
undetermined. The team identified this as a 404-amino acidprotein kinase, which they called M locus protein kinase(MLPK). They showed that MLPK expression was mainly inthe stigma and levels increased immediately prior to flowering,similar to the expression pattern of SRK. The researchers thenpinpointed a single nucleotide difference in the mutant MLPK
gene (m) that resulted in an amino acid substitution preventing
the kinase’s autophosphorylation activity. Moreover, the MLPK
protein was undetectable in mutant plants, suggesting thesubstitution affected the protein’s stability. Transiently expressing wild-type MLPK in mutant papilla cells restored the SI
response, demonstrating that functional MLPK is indispensable for proper rejection of self-pollen in these plants.
The researchers provided crucial data that revolutionized
our understanding of the molecular signaling governing the SI
response in Brassica. However, they remained interested in the
upstream S-haplotype genes and how their dominant/recessive
relationship is regulated in these plants. In particular, SP11
genes are expressed in a monoallelic pattern, meaning theversion of the gene from one parent is expressed (dominant
allele), while the version from the other parent is not (recessive
allele). The molecular details of how this is controlled remained
elusive to the team, leading to a set of experiments that were
published in Nature in 2010. The group previously found that

promoter methylation could turn off expression of the recessive
allele. Intriguingly, they observed inverted genomic sequencesin regions surrounding dominant SP11 alleles that were similar
to sequences in recessive SP11 promoters. Expression of thissequence led to the production of a 24-nucleotide small
non-coding RNA that they named SP11 methylation inducer(Smi). This RNA can work in trans to activate methylation of
the recessive allele promoter, repressing transcription. This was
the first report of a small non-coding RNA working in thismanner to affect a dominant/recessive allele relationship.
Through a series of high-impact publications spanning overa decade, the research team revealed significant molecular
information regarding the SI response process in Brassica
plants. This included from S locus haplotype gene expression
mechanisms to the SI signaling cascade. The team identified
two novel factors: MLPK as a crucial SI signaling mediator andSmi as a trans-acting non-coding RNA in marking the recessive
SP11 allele. Furthermore, they found that the SLG protein ispart of a larger SI-associated complex and the mutant MLPK
gene erases the SI response in the stigma. These results weregroundbreaking for a greater understanding of self-recognitionin plants.
References
Takayama, S., Shimosato, H., Shiba, H., Funato, M., Che, F.S., Watanabe, M., Iwano,
M., Isogai, A. 2001. Direct ligand–receptor complex interaction controls Brassica
self-incompatibility. Nature, 413, 534–538.
Murase, K., Shiba, H., Iwano, M., Che, F.S., Watanabe, M., Isogai, A., Takayama, S.
2004. A membrane-anchored protein kinase involved in Brassica self-incompatibility signaling. Science, 303, 1516–1519.
Tarutani, Y., Shiba, H., Iwano, M., Kakizaki, T., Suzuki, G., Watanabe, M., Isogai, A.,
Takayama, S. 2010. Trans-acting small RNA determines dominance relationships
in Brassica self-incompatibility. Nature, 466, 983–986.

NAIST Research Highlights

23

Plant Sciences

30 YEARS OF HISTORY: 2001– 2010

Toshiharu Shikanai© Adobe Stock/ candy1812

Cyclic electron flow is criticalfor higher plant photosynthesis
Biochemical and genetic studies on cyclic electron flow in photosynthesis
uncover crucial proteins involved in this processP

hotosynthesis is the process by which plants convert
light energy to chemical energy that can be used as fuelfor its metabolic reactions. This is performed by photosystem I (PSI) and photosystem II (PSII), protein complexes
that operate in the thylakoid membrane of chloroplasts.
Elucidating the different molecular mechanisms that govern
photosynthesis, particularly under conditions of stress such as
extreme light, low temperature and drought, is important tobetter understand plant biology. This will also lend a greaterappreciation of the differences between plant species at themolecular level.
During photosynthesis, light energy is used to generate a
proton gradient (ΔpH) across the thylakoid membrane, whichis then used to synthesize ATP. This gradient can be generated
via at least two routes: linear electron flow and cyclic electron
flow. Linear electron flow involves both PSI and PSII, where
electrons produced by the splitting of water are used to form
reduced ferredoxin or NADPH in the chloroplast stroma. In
contrast, cyclic electron flow is driven solely by PSI – electrons
are recycled around PSI to plastoquinone from either reduced
ferredoxin or NADPH.
Cyclic electron flow and the generation of ΔpH are central
to energy production in plants. However, the specific details ofthese processes and how they are maintained under various
stress conditions were poorly understood. A series of studies
led by Toshiharu Shikanai at NAIST provided key insights into
the mechanisms of cyclic electron flow in photosystems.
Under excessive light conditions, PSII is downregulated todissipate heat to avoid production of dangerous byproducts.
However, electron transport is often limited when the plant isunder stress, bringing into question how ΔpH is maintained inthese cases. In a study published in Cell in 2002, the research

24

NAIST Research Highlights

team hypothesized that cyclic electron flow is induced tofacilitate this heat dissipation. They examined the role of the
proton gradient regulation (PGR5) gene in cyclic electron flow
around PSI by isolating an Arabidopsis thaliana mutant, pgr5,
that exhibited impairment of PSII photochemistry downregulation in response to intense light. pgr5 mutants are therefore
defective in cyclic flow. The team revealed that PGR5 encodes
a previously unidentified thylakoid membrane protein thatplays a role in electron transfer from ferredoxin to plastoquinone. These results suggested that the PGR5 pathway
contributes to the generation of a ΔpH that triggers heat loss
when Calvin cycle activity is reduced and also prevents PSI
photoinhibition by limiting overreduction of its acceptor side.
By identifying PGR5, these findings helped characterize themolecular nature of the alternative electron transfer pathway
for the first time.
Following this increased understanding of cyclic flow, theteam aimed to investigate its effects on photosynthesis
efficiency. Their results were published in Nature in 2004.
Double mutants of A. thaliana were constructed in which both
PSI cyclic pathways were impaired by crossing pgr5 mutant
plants with crr plants (impaired NA(P)DH dehydrogenase(NDH) activity). Severe phenotypes were observed in these
double mutants, including slowed growth and reduced chlorophyll content. These results suggested that the significance of
cyclic flow had been underestimated, and that linear flow
cannot sustain the required ratio of ATP/NADPH productionin the absence of cyclic flow. This ultimately results in excessive
accumulation of NADPH in the stroma, and consequently its
overreduction. These findings showed that cyclic flow is essential for the prevention of stroma overreduction, and therefore
key to efficient photosynthesis. These data also further

expanded upon their earlier demonstration of the importanceof PGR5 in this process.
After appreciating the importance of cyclic electron flow inphotosynthesis, the researchers examined the genetic components that govern this machinery and how they are regulated,which was published in Nature in 2005. The chloroplast
genome of higher plants encodes 11 subunits (ndhA–ndhK)of the NDH complex. Interestingly, when examining the A.
thaliana crr4 mutants (impaired NDH activity) they investigated previously, the team identified crr4 transcripts with
deficient RNA editing. RNA editing is a process of RNA
maturation involved in nucleotide modification, deletion or
insertion. In higher plant organelle transcripts, editing often

leads to restoration of conserved amino acid residues, an essential process for protein function in plastids. Further
experiments indicated that a pentatricopeptide repeat (PPR)
protein acts as a factor for recognizing the target RNA in theseplants and enables the access of a general factor that contains
cytidine deaminase activity. The genetic approach used by theteam was not previously done in higher plants. Their reported
discovery of PPR as crucial to RNA editing in chloroplasts
provided significant insight into the machinery controlling thisprocess in these plants.
The results of the extensive work done by the researchteam demonstrated key details about cyclic electron flow inphotosynthetic pathways in certain plants, and provided
molecular characterization of the genes and factors involved
in regulating these processes. The team identified PGR5 as thethylakoid membrane protein involved with cyclic electron
flow in plants that helps dissipate heat. They also showed that
this cyclic electron flow is essential for efficient photosynthesis
and prevention of chloroplast stroma overreduction. Furthermore, the team discovered PPR as a core part of the RNA
editing machinery in these plants’ chloroplasts.These findings
revolutionized our molecular understanding of how thephotosynthesis process responds to excess light.
References

Visible phenotype of Arabidopsis thaliana mutants(Munekage et al., 2004, Nature).

Munekage, Y., Hojo, M., Meurer, J., Endo, T., Tasaka, M., Shikanai, T. 2002. PGR5 isinvolved in cyclic electron flow around photosystem I and is essential forphotoprotection in Arabidopsis. Cell, 110, 361–371.
Munekage, Y., Hashimoto, M., Miyake, C., Tomizawa, K., Endo, T., Tasaka, M.,
Shikanai, T. 2004. Cyclic electron flow around photosystem I is essential forphotosynthesis. Nature, 429, 579–582.
Kotera, E., Tasaka, M., Shikanai, T. 2005. A pentatricopeptide repeat protein isessential for RNA editing in chloroplasts. Nature, 433, 326–330.

NAIST Research Highlights

25

Plant Sciences

30 YEARS OF HISTORY: 2001– 2010

Toshiharu Shikanai© Adobe Stock/ candy1812

Cyclic electron flow is criticalfor higher plant photosynthesis
Biochemical and genetic studies on cyclic electron flow in photosynthesis
uncover crucial proteins involved in this processP

hotosynthesis is the process by which plants convert
light energy to chemical energy that can be used as fuelfor its metabolic reactions. This is performed by photosystem I (PSI) and photosystem II (PSII), protein complexes
that operate in the thylakoid membrane of chloroplasts.
Elucidating the different molecular mechanisms that govern
photosynthesis, particularly under conditions of stress such as
extreme light, low temperature and drought, is important tobetter understand plant biology. This will also lend a greaterappreciation of the differences between plant species at themolecular level.
During photosynthesis, light energy is used to generate a
proton gradient (ΔpH) across the thylakoid membrane, whichis then used to synthesize ATP. This gradient can be generated
via at least two routes: linear electron flow and cyclic electron
flow. Linear electron flow involves both PSI and PSII, where
electrons produced by the splitting of water are used to form
reduced ferredoxin or NADPH in the chloroplast stroma. In
contrast, cyclic electron flow is driven solely by PSI – electrons
are recycled around PSI to plastoquinone from either reduced
ferredoxin or NADPH.
Cyclic electron flow and the generation of ΔpH are central
to energy production in plants. However, the specific details ofthese processes and how they are maintained under various
stress conditions were poorly understood. A series of studies
led by Toshiharu Shikanai at NAIST provided key insights into
the mechanisms of cyclic electron flow in photosystems.
Under excessive light conditions, PSII is downregulated todissipate heat to avoid production of dangerous byproducts.
However, electron transport is often limited when the plant isunder stress, bringing into question how ΔpH is maintained inthese cases. In a study published in Cell in 2002, the research

24

NAIST Research Highlights

team hypothesized that cyclic electron flow is induced tofacilitate this heat dissipation. They examined the role of the
proton gradient regulation (PGR5) gene in cyclic electron flow
around PSI by isolating an Arabidopsis thaliana mutant, pgr5,
that exhibited impairment of PSII photochemistry downregulation in response to intense light. pgr5 mutants are therefore
defective in cyclic flow. The team revealed that PGR5 encodes
a previously unidentified thylakoid membrane protein thatplays a role in electron transfer from ferredoxin to plastoquinone. These results suggested that the PGR5 pathway
contributes to the generation of a ΔpH that triggers heat loss
when Calvin cycle activity is reduced and also prevents PSI
photoinhibition by limiting overreduction of its acceptor side.
By identifying PGR5, these findings helped characterize themolecular nature of the alternative electron transfer pathway
for the first time.
Following this increased understanding of cyclic flow, theteam aimed to investigate its effects on photosynthesis
efficiency. Their results were published in Nature in 2004.
Double mutants of A. thaliana were constructed in which both
PSI cyclic pathways were impaired by crossing pgr5 mutant
plants with crr plants (impaired NA(P)DH dehydrogenase(NDH) activity). Severe phenotypes were observed in these
double mutants, including slowed growth and reduced chlorophyll content. These results suggested that the significance of
cyclic flow had been underestimated, and that linear flow
cannot sustain the required ratio of ATP/NADPH productionin the absence of cyclic flow. This ultimately results in excessive
accumulation of NADPH in the stroma, and consequently its
overreduction. These findings showed that cyclic flow is essential for the prevention of stroma overreduction, and therefore
key to efficient photosynthesis. These data also further

expanded upon their earlier demonstration of the importanceof PGR5 in this process.
After appreciating the importance of cyclic electron flow inphotosynthesis, the researchers examined the genetic components that govern this machinery and how they are regulated,which was published in Nature in 2005. The chloroplast
genome of higher plants encodes 11 subunits (ndhA–ndhK)of the NDH complex. Interestingly, when examining the A.
thaliana crr4 mutants (impaired NDH activity) they investigated previously, the team identified crr4 transcripts with
deficient RNA editing. RNA editing is a process of RNA
maturation involved in nucleotide modification, deletion or
insertion. In higher plant organelle transcripts, editing often

leads to restoration of conserved amino acid residues, an essential process for protein function in plastids. Further
experiments indicated that a pentatricopeptide repeat (PPR)
protein acts as a factor for recognizing the target RNA in theseplants and enables the access of a general factor that contains
cytidine deaminase activity. The genetic approach used by theteam was not previously done in higher plants. Their reported
discovery of PPR as crucial to RNA editing in chloroplasts
provided significant insight into the machinery controlling thisprocess in these plants.
The results of the extensive work done by the researchteam demonstrated key details about cyclic electron flow inphotosynthetic pathways in certain plants, and provided
molecular characterization of the genes and factors involved
in regulating these processes. The team identified PGR5 as thethylakoid membrane protein involved with cyclic electron
flow in plants that helps dissipate heat. They also showed that
this cyclic electron flow is essential for efficient photosynthesis
and prevention of chloroplast stroma overreduction. Furthermore, the team discovered PPR as a core part of the RNA
editing machinery in these plants’ chloroplasts.These findings
revolutionized our molecular understanding of how thephotosynthesis process responds to excess light.
References

Visible phenotype of Arabidopsis thaliana mutants(Munekage et al., 2004, Nature).

Munekage, Y., Hojo, M., Meurer, J., Endo, T., Tasaka, M., Shikanai, T. 2002. PGR5 isinvolved in cyclic electron flow around photosystem I and is essential forphotoprotection in Arabidopsis. Cell, 110, 361–371.
Munekage, Y., Hashimoto, M., Miyake, C., Tomizawa, K., Endo, T., Tasaka, M.,
Shikanai, T. 2004. Cyclic electron flow around photosystem I is essential forphotosynthesis. Nature, 429, 579–582.
Kotera, E., Tasaka, M., Shikanai, T. 2005. A pentatricopeptide repeat protein isessential for RNA editing in chloroplasts. Nature, 433, 326–330.

NAIST Research Highlights

25

Plant Sciences

30 YEARS OF HISTORY: 2001– 2010

Ko Shimamoto

Blooming brilliant:Discovering the master regulatorof flowering timing in rice plants

P

lants have a complex lifecycle that is highly regulated bymany environmental factors, such as the availability ofwater, nutrients and sunlight. In addition to daily
variations in these conditions, plants are also subject toseasonal changes that affect their growth and development. A
key moment in a plant’s life that is central to its reproduction,and therefore survival, is when it flowers, and the timing of

26

NAIST Research Highlights

flowering is very carefully regulated at a genetic level in
response to internal and external cues. Day length in particular
is a crucial determinant of plant flowering, and all plants can
be classified as long-day plants or short-day plants based on
how day length promotes their flowering. Arabidopsis, which
is a common plant model, is a long-day plant, and the genetic
regulation of its flowering cycle had been well-characterized.

© shutterstock/ NOPPHARAT7824

Hd3a is a mobile signal in rice plants that travels from the leaves to the shoots,
where it forms a functional protein complex that induces flowering

However, the flowering process in short-day plants was considmechanism by which this factor functions to effectively
erably less well understood.
promote flowering still remained unknown. Therefore, theTo address this, a research team led by Ko Shimamoto atresearchers next sought to identify the molecular mechanism
NAIST explored the regulation of flowering in rice, an importby which Hd3a induces flowering in rice, publishing theirant short-day plant. The Arabidopsis GIGANTEA gene wasfindings in Nature in 2011. The team identified proteins thatknown to promote flowering, so the research team began bybound directly to Hd3a and found that one (GF14c) was aoverexpressing the rice equivalent of this gene, OsGI, in rice,
known 14-3-3 protein, while the others contained a SAP motif,
publishing their findings in Nature in 2003. Unexpectedly, thiswhich is similar to the 14-3-3 recognition motif. Further analyresulted in later flowering, indicating that OsGI has thesis showed that Hd3a interacts with GF14c in the shoot apex
opposite role of GIGANTEA and suppresses flowering in rice.
of rice plants, and that the active, stable complex also includes
Overexpression of OsGI also led to decreased expression ofa protein called OsFD1. This three-protein complex was named
Hd1(Se1) and Hd3a, rice equivalents
the florigen activation complex (FAC)
of Arabidopsis genes that are also
and was shown to directly bind the
involved in the regulation of floweringpromoter region of AP1, a gene thattime. Interestingly, in wild-type riceleads to flowering. Analysis of whereplants both OsGI and Hd1(Se1)these proteins are located at different
showed similar expression patterns
points suggested a model in which
when the plants were exposed to
Hd3a travels from the leaves to theshort-day or long-day conditions,shoot apex, where it binds to 14-3-3
while Hd3a was strongly suppressed
proteins in the cytoplasm; thisunder long-day conditions but
two-protein complex then enters theshowed a daily pattern of expression
nucleus, where it binds to OsFD1 tochanges under short-day conditions.
create the complete FAC that activates
Taken together, these findings
transcription of AP1, thereby inducsuggested that, similar to Arabidopsis,ing flowering.
in rice OsGI activates Hd1(Se1) toTaken together, this groundbreakpromote flowering, except undering series of studies identified Hd3a aslong-day conditions, when Hd1(Se1)a key regulator of flowering time insuppresses Hd3a expression, thereby
rice, a short-day plant, that behavessuppressing flowering. This study notin an opposite manner to its equivaonly highlighted the conservation oflent protein in Arabidopsis, a long-day
an important regulatory network
plant. Importantly, Hd3a was thenamong plants, but also underlined theconfirmed to be the factor previously
central role that Hd3a plays in regulatknown as florigen, a mobile floweringing the timing of flowering in rice.
signal that travels from the leaves to the
In a study published in Science inshoot apex to induce flowering.
2007, the research group explored theCharacterization of the FAC provided
possibility that Hd3a could be thecrucial insight into how this signal
elusive molecule referred to as
functions on a molecular basis to‘florigen,’ a mobile flowering signal
regulate the central process of flower14-3-3 interaction is required for OsMADS15 activationby Hd3a and OsFD1 (Taoka et al., 2011, Nature).
that was known to move from planting during the rice plant’s lifecycle.
leaves to the shoot apex to induce
This series of studies not only
flowering, but whose identify remained unknown for many
answered longstanding questions in the field of plant biology,
years. Earlier studies had suggested that the FLOWERING
but also represents a substantial advance in our understanding
LOCUS T (FT) gene in Arabidopsis could encode the factorof the conservation and function of central pathways in plant
known as florigen, as the FT protein is only expressed in leaves
cell biology, as well as providing new tools and conceptual
but interacts with a transcription factor that is only expressedframeworks for future studies.
in the shoot apex. Therefore, the research team investigated theexpression of rice Hd3a, the FT equivalent, in different tissues
and found that this gene too is expressed almost exclusively in
References
the leaves. Importantly, Hd3a protein was detected in the shootHayama, R., Yokoi, S., Tamaki, S., Yano, M., Shimamoto, K. 2003. Adaptation of
apex, suggesting that it migrates through the plant like FT tophotoperiodic control pathways produces short-day flowering in rice. Nature,422, 719–722.
reach the area where it can actively induce flowering. This
Tamaki, S., Matsuo, S., Wong, H.L., Yokoi, S., Shimamoto, K. 2007. Hd3a protein is agroundbreaking finding strongly suggested that Hd3a ismobile flowering signal in rice. Science, 316, 1033–1036.
Taoka, K., Ohki, I., Tsuji, H., Furuita, K., Hayashi, K., Yanase, T., Yamaguchi, M.,
therefore florigen, the mobile flowering signal that had
Nakashima, C., Purwestri, Y.A., Tamaki, S., Ogaki, Y., Shimada, C., Nakagawa, A.,remained unidentified for so long.
Kojima, C., Shimamoto, K. 2011. 14-3-3 proteins act as intracellular receptors forrice Hd3a florigen. Nature, 476, 332–335.
While this study clearly revealed the identity of florigen, the

NAIST Research Highlights

27

Plant Sciences

30 YEARS OF HISTORY: 2001– 2010

Ko Shimamoto

Blooming brilliant:Discovering the master regulatorof flowering timing in rice plants

P

lants have a complex lifecycle that is highly regulated bymany environmental factors, such as the availability ofwater, nutrients and sunlight. In addition to daily
variations in these conditions, plants are also subject toseasonal changes that affect their growth and development. A
key moment in a plant’s life that is central to its reproduction,and therefore survival, is when it flowers, and the timing of

26

NAIST Research Highlights

flowering is very carefully regulated at a genetic level in
response to internal and external cues. Day length in particular
is a crucial determinant of plant flowering, and all plants can
be classified as long-day plants or short-day plants based on
how day length promotes their flowering. Arabidopsis, which
is a common plant model, is a long-day plant, and the genetic
regulation of its flowering cycle had been well-characterized.

© shutterstock/ NOPPHARAT7824

Hd3a is a mobile signal in rice plants that travels from the leaves to the shoots,
where it forms a functional protein complex that induces flowering

However, the flowering process in short-day plants was considmechanism by which this factor functions to effectively
erably less well understood.
promote flowering still remained unknown. Therefore, theTo address this, a research team led by Ko Shimamoto atresearchers next sought to identify the molecular mechanism
NAIST explored the regulation of flowering in rice, an importby which Hd3a induces flowering in rice, publishing theirant short-day plant. The Arabidopsis GIGANTEA gene wasfindings in Nature in 2011. The team identified proteins thatknown to promote flowering, so the research team began bybound directly to Hd3a and found that one (GF14c) was aoverexpressing the rice equivalent of this gene, OsGI, in rice,
known 14-3-3 protein, while the others contained a SAP motif,
publishing their findings in Nature in 2003. Unexpectedly, thiswhich is similar to the 14-3-3 recognition motif. Further analyresulted in later flowering, indicating that OsGI has thesis showed that Hd3a interacts with GF14c in the shoot apex
opposite role of GIGANTEA and suppresses flowering in rice.
of rice plants, and that the active, stable complex also includes
Overexpression of OsGI also led to decreased expression ofa protein called OsFD1. This three-protein complex was named
Hd1(Se1) and Hd3a, rice equivalents
the florigen activation complex (FAC)
of Arabidopsis genes that are also
and was shown to directly bind the
involved in the regulation of floweringpromoter region of AP1, a gene thattime. Interestingly, in wild-type riceleads to flowering. Analysis of whereplants both OsGI and Hd1(Se1)these proteins are located at different
showed similar expression patterns
points suggested a model in which
when the plants were exposed to
Hd3a travels from the leaves to theshort-day or long-day conditions,shoot apex, where it binds to 14-3-3
while Hd3a was strongly suppressed
proteins in the cytoplasm; thisunder long-day conditions but
two-protein complex then enters theshowed a daily pattern of expression
nucleus, where it binds to OsFD1 tochanges under short-day conditions.
create the complete FAC that activates
Taken together, these findings
transcription of AP1, thereby inducsuggested that, similar to Arabidopsis,ing flowering.
in rice OsGI activates Hd1(Se1) toTaken together, this groundbreakpromote flowering, except undering series of studies identified Hd3a aslong-day conditions, when Hd1(Se1)a key regulator of flowering time insuppresses Hd3a expression, thereby
rice, a short-day plant, that behavessuppressing flowering. This study notin an opposite manner to its equivaonly highlighted the conservation oflent protein in Arabidopsis, a long-day
an important regulatory network
plant. Importantly, Hd3a was thenamong plants, but also underlined theconfirmed to be the factor previously
central role that Hd3a plays in regulatknown as florigen, a mobile floweringing the timing of flowering in rice.
signal that travels from the leaves to the
In a study published in Science inshoot apex to induce flowering.
2007, the research group explored theCharacterization of the FAC provided
possibility that Hd3a could be thecrucial insight into how this signal
elusive molecule referred to as
functions on a molecular basis to‘florigen,’ a mobile flowering signal
regulate the central process of flower14-3-3 interaction is required for OsMADS15 activationby Hd3a and OsFD1 (Taoka et al., 2011, Nature).
that was known to move from planting during the rice plant’s lifecycle.
leaves to the shoot apex to induce
This series of studies not only
flowering, but whose identify remained unknown for many
answered longstanding questions in the field of plant biology,
years. Earlier studies had suggested that the FLOWERING
but also represents a substantial advance in our understanding
LOCUS T (FT) gene in Arabidopsis could encode the factorof the conservation and function of central pathways in plant
known as florigen, as the FT protein is only expressed in leaves
cell biology, as well as providing new tools and conceptual
but interacts with a transcription factor that is only expressedframeworks for future studies.
in the shoot apex. Therefore, the research team investigated theexpression of rice Hd3a, the FT equivalent, in different tissues
and found that this gene too is expressed almost exclusively in
References
the leaves. Importantly, Hd3a protein was detected in the shootHayama, R., Yokoi, S., Tamaki, S., Yano, M., Shimamoto, K. 2003. Adaptation of
apex, suggesting that it migrates through the plant like FT tophotoperiodic control pathways produces short-day flowering in rice. Nature,422, 719–722.
reach the area where it can actively induce flowering. This
Tamaki, S., Matsuo, S., Wong, H.L., Yokoi, S., Shimamoto, K. 2007. Hd3a protein is agroundbreaking finding strongly suggested that Hd3a ismobile flowering signal in rice. Science, 316, 1033–1036.
Taoka, K., Ohki, I., Tsuji, H., Furuita, K., Hayashi, K., Yanase, T., Yamaguchi, M.,
therefore florigen, the mobile flowering signal that had
Nakashima, C., Purwestri, Y.A., Tamaki, S., Ogaki, Y., Shimada, C., Nakagawa, A.,remained unidentified for so long.
Kojima, C., Shimamoto, K. 2011. 14-3-3 proteins act as intracellular receptors forrice Hd3a florigen. Nature, 476, 332–335.
While this study clearly revealed the identity of florigen, the

NAIST Research Highlights27

Microbiology

30 YEARS OF HISTORY: 2001– 2010

clues as to what they do. However, the remaining four of these
unknown essential genes remain completely uncharacterized,
despite their apparently pivotal role in B. subtilis survival.
The identification of essential genes in this study provided
new insight into the processes that sustain bacterial life. By fully
understanding the essential elements required for free living,new possibilities arise for bacterial genomic engineering andsynthetic biology approaches with potential therapeutic orindustrial applications. Newly identified essential genes that,at present, have no known function will undoubtedly reveal
their purpose in future studies that will follow this important
study. The benefits of this new knowledge will thus be revealedin the fullness of time.

Naotake OgasawaraT

he single thread that ties all organisms together, from thelargest predators to the smallest microbes, is that they arealive. Given the incredibly variety on the theme of ‘life,’a fundamental question in biology is what elements are actuallyrequired to sustain life. This question is important, because its
answer may illuminate the most fundamental functions
required for life. Certain processes, such as the taking in of
nutrients and turning them into energy, are clearly common to
all living organisms. But without the systematic analyses ofresearch, it is difficult to say whether these obvious functions
are all that is really needed to be a living organism; there may beother, less evident, processes that are equally important andshared among all living things. In addition, defining theminimal requirements for life has important implications forbiotechnology, in that it could help optimize and streamline
laboratory-based manufacturing processes designed to generateclinically and industrially important products using bacteria asliving ‘factories.’ Therefore, an outstanding question in the life
sciences is what is the smallest amount of genetic informationneeded for an organism to be alive.
To explore this question, a research team led by Naotake
Ogasawara at NAIST focused on Bacillus subtilis, a modelorganism that is one of the best understood prokaryotes interms of molecular and cellular biology, and one that is also of

28

NAIST Research Highlights

biotechnological significance. To obtain a reliable estimate of
the minimal protein-encoding gene set required for bacterial
life, the researchers systematically inactivated each gene in theB. subtilis genome by inserting a nonreplicating plasmid into
the target gene. This resulted in complete elimination of expression of that gene and allowed the group to classify each gene as
essential or nonessential. A gene was considered essential if it
was unable to be inactivated by insertion, because this meantthat the bacterium did not survive without it.
Intriguingly, even though the B. subtilis genome comprises
approximately 4,100 genes, only 271 genes were identified as
being indispensable for growth when inactivated one at a time.
Of the essential genes that were identified, most were conserved
across a wide range of Bacteria, and almost 70% were also foundin Archaea and Eucarya, indicating genetic conservation acrosskingdoms. Many organisms therefore appear to rely on asimilar set of essential functions. The researchers further foundthat the number of B. subtilis essential gene homologues
present in an organism depended on two main factors: evolutionary proximity to B. subtilis and the size of the genome. Over
80% of essential B. subtilis gene homologues were found to bepresent in all bacteria with genomes over 3 Mb, and evenbacteria with the smallest genomes (such as mycoplasma)retained 57% of these gene homologues. This prompted arather minimalist view of a bacterial cell, consisting of acompartment (or cell), enclosed by a membrane and a wall,that houses the machinery necessary to synthesize proteins that
perform some basic essential functions: (1) duplication andinheritance of genetic information; (2) cell division; and (3)energy production.
The team’s study also revealed some unexpected essential
genes under the experimental conditions used, including eightgenes involved in the EMP glycolytic pathway. The ubiquitous
EMP glycolytic pathway is usually necessary for energy generation; however, it was not expected to perform an essentialfunction during growth in nutrient-rich medium (used tocreate optimal growth conditions in this study) because themedium itself contains various compounds capable of providing the energy and building blocks required for cellularsurvival. It is therefore possible that the enzymes within the
EMP glycolytic pathway that were identified as essential for lifein this study may have novel, as yet unidentified roles within
the cell.
Intriguingly, the researchers also identified 11 essential
genes that at present do not have any known function. Basedon comparison with known gene sequences, physiological roles
could be suggested for two of these genes, and another fivecontain recognizable domains or motifs that could provide© 2003 National Academy of Sciences

Systematic inactivation of Bacillus subtilis genes reveals
the minimal set of genes that are indispensable for bacterial life

© Adobe Stock/ Alex

The minimal gene set required
to sustain bacterial life

Phylogenetic profiling of the 271 essential genes(Kobayashi et al., 2003, PNAS).

Reference
Kobayashi, K., Ehrlich, S.D., Albertini, A., Amati, G., et al. 2003. Essential Bacillus
subtilis genes. Proceedings of the National Academy of Sciences of the United Statesof America, 100, 4678–4683.

NAIST Research Highlights29

Microbiology

30 YEARS OF HISTORY: 2001– 2010

clues as to what they do. However, the remaining four of these
unknown essential genes remain completely uncharacterized,
despite their apparently pivotal role in B. subtilis survival.
The identification of essential genes in this study provided
new insight into the processes that sustain bacterial life. By fully
understanding the essential elements required for free living,new possibilities arise for bacterial genomic engineering andsynthetic biology approaches with potential therapeutic orindustrial applications. Newly identified essential genes that,at present, have no known function will undoubtedly reveal
their purpose in future studies that will follow this important
study. The benefits of this new knowledge will thus be revealedin the fullness of time.

Naotake OgasawaraT

he single thread that ties all organisms together, from thelargest predators to the smallest microbes, is that they arealive. Given the incredibly variety on the theme of ‘life,’a fundamental question in biology is what elements are actuallyrequired to sustain life. This question is important, because its
answer may illuminate the most fundamental functions
required for life. Certain processes, such as the taking in of
nutrients and turning them into energy, are clearly common to
all living organisms. But without the systematic analyses ofresearch, it is difficult to say whether these obvious functions
are all that is really needed to be a living organism; there may beother, less evident, processes that are equally important andshared among all living things. In addition, defining theminimal requirements for life has important implications forbiotechnology, in that it could help optimize and streamline
laboratory-based manufacturing processes designed to generateclinically and industrially important products using bacteria asliving ‘factories.’ Therefore, an outstanding question in the life
sciences is what is the smallest amount of genetic informationneeded for an organism to be alive.
To explore this question, a research team led by Naotake
Ogasawara at NAIST focused on Bacillus subtilis, a modelorganism that is one of the best understood prokaryotes interms of molecular and cellular biology, and one that is also of

28

NAIST Research Highlights

biotechnological significance. To obtain a reliable estimate of
the minimal protein-encoding gene set required for bacterial
life, the researchers systematically inactivated each gene in theB. subtilis genome by inserting a nonreplicating plasmid into
the target gene. This resulted in complete elimination of expression of that gene and allowed the group to classify each gene as
essential or nonessential. A gene was considered essential if it
was unable to be inactivated by insertion, because this meantthat the bacterium did not survive without it.
Intriguingly, even though the B. subtilis genome comprises
approximately 4,100 genes, only 271 genes were identified as
being indispensable for growth when inactivated one at a time.
Of the essential genes that were identified, most were conserved
across a wide range of Bacteria, and almost 70% were also foundin Archaea and Eucarya, indicating genetic conservation acrosskingdoms. Many organisms therefore appear to rely on asimilar set of essential functions. The researchers further foundthat the number of B. subtilis essential gene homologues
present in an organism depended on two main factors: evolutionary proximity to B. subtilis and the size of the genome. Over
80% of essential B. subtilis gene homologues were found to bepresent in all bacteria with genomes over 3 Mb, and evenbacteria with the smallest genomes (such as mycoplasma)retained 57% of these gene homologues. This prompted arather minimalist view of a bacterial cell, consisting of acompartment (or cell), enclosed by a membrane and a wall,that houses the machinery necessary to synthesize proteins that
perform some basic essential functions: (1) duplication andinheritance of genetic information; (2) cell division; and (3)energy production.
The team’s study also revealed some unexpected essential
genes under the experimental conditions used, including eightgenes involved in the EMP glycolytic pathway. The ubiquitous
EMP glycolytic pathway is usually necessary for energy generation; however, it was not expected to perform an essentialfunction during growth in nutrient-rich medium (used tocreate optimal growth conditions in this study) because themedium itself contains various compounds capable of providing the energy and building blocks required for cellularsurvival. It is therefore possible that the enzymes within the
EMP glycolytic pathway that were identified as essential for lifein this study may have novel, as yet unidentified roles within
the cell.
Intriguingly, the researchers also identified 11 essential
genes that at present do not have any known function. Basedon comparison with known gene sequences, physiological roles
could be suggested for two of these genes, and another fivecontain recognizable domains or motifs that could provide© 2003 National Academy of Sciences

Systematic inactivation of Bacillus subtilis genes reveals
the minimal set of genes that are indispensable for bacterial life

© Adobe Stock/ Alex

The minimal gene set required
to sustain bacterial life

Phylogenetic profiling of the 271 essential genes(Kobayashi et al., 2003, PNAS).

Reference
Kobayashi, K., Ehrlich, S.D., Albertini, A., Amati, G., et al. 2003. Essential Bacillus
subtilis genes. Proceedings of the National Academy of Sciences of the United Statesof America, 100, 4678–4683.

NAIST Research Highlights

29

Molecular Biology

30 YEARS OF HISTORY: 2001– 2010

30 YEARS OF HISTORY: 2001– 2010

Masahiro Shirakawa

Takashi Hashimoto

Solving the protein structureof a key binding domain sheds
light on the neurodevelopmental
disorder Rett syndrome

The role of microtubulesin the handedness of helical
growth in plants

Structural analysis of the methyl-CpG binding domain reveals how proteins containing
this domain can interact with methylated DNA, which enables the interpretationof disease-causing mutations in Rett syndrome

D

NA methylation is an important biological mechanism
for the regulation of gene expression and overall genomic
stability. The proper functioning of this process isessential for normal development in mammals. Throughmethylation, the activity of DNA can be modified via additionof a methyl group, without changing the DNA sequence itself.
Erroneous methylation can result in the inappropriate regulation of genes, leading to various diseases such as cancer or
cardiovascular disease.
The consequences of DNA methylation are often mediatedby proteins that contain methyl-CpG binding domains (MBDs).
The structure of the MBD was unknown until 2001, when aresearch group led by Masahiro Shirakawa at NAIST reported
the structure of the MBD in a human protein, MBD1.
The MBD is highly conserved, meaning that its structure isshared with other MBD-containing proteins in several species.
There are five MBD protein family members characterized in
mammals, one of which is MeCP2. Seventeen different
mutations in MeCP2 have been found to cause Rett syndrome
which affects brain development leading to physical andintellectual disability in girls. Eleven of these mutations sit
within the MBD, indicating the importance of this domain.
The team overexpressed human MBD1 in E. coli and labeledand purified it for nuclear magnetic resonance analysis. Thisrevealed that when MBD binds DNA, structural rearrangements occur to form a loop in the protein structure, causing it
to fold into a new DNA binding interface.
They found that five residues contact the major groove of themethylated DNA to identify the two methyl groups that form
the methylated CpG group. These five residues form a hydrophobic patch. The lack of symmetry of this region indicates thateach of the two methyl groups is recognized distinctly.
A notable feature of the interaction between the MBD and
DNA is the small size of the contact area. This small contact
area, which sits only on the major groove of the DNA, allows
the MBD to access methyl-CpG sites on nucleosome coreswithout any steric interference from the chromatin structure.
As a result of the findings of this study, residues with known
mutations in Rett syndrome could be analyzed. Some of the30

Plant Sciences

NAIST Research Highlights

The MBD-DNA contacts lie exclusively within the major groove of DNA (Ohkiet al., 2001, Cell ).

mutations in MeCP2 may result in failure of the MBD to contact
the DNA bases. One reported mutation, in which an alanine is
replaced by a valine, likely interferes with the DNA binding
affinity, because valine has a bulky side chain. Mutations toother residues probably interfere with MeCP2’s activity by
causing global structural changes.
This study definitively outlined the protein structure of
MBDs, which demonstrated the involvement of several physicalinteractions between the DNA strand and protein side chains,
revealing for the first time how the methyl-CpG sequence canbe recognized by the MBD-containing family of proteins. Thesefindings facilitate a greater understanding of the causes of Rett
syndrome and may assist research into other disease pathologies
in which DNA methylation has been implicated, including
vascular tissue changes in atherosclerosis and carcinogenesis.

Two Arabidopsis thaliana mutants, lefty1 and lefty2, are found to exhibit left-handed
helical growth and clockwise twisting in elongating organs, and have cortical
microtubules with increased sensitivity to microtubule-specific drugs

P

lants usually grow in a linear or circumferential way, butmany plants exhibit some type of helical growth in theform of spirals, twists, or coils. Plant parts that show
helical growth include the stems or tendrils of twining plants,
fan-blade shaped flower petals, coiling roots, and spiral seed
pods. Helical growth is relevant to agriculture and horticulture
because it underpins plant forms that may be desirable, such as
aesthetically pleasing flowers or plant shapes, or plants that are
free to explore new environments, like climbing vines.
Conversely, this type of growth may be undesirable if it resultsin plant products that cannot be mechanically processed (e.g.,
misshapen root vegetables). Understanding the molecularmechanisms that produce helical growth can contribute to thedevelopment of new ways of improving this trait to better utilize
crops in horticulture and agriculture.
The left–right asymmetry, or handedness, of helical plant
forms is often fixed within species, and this phenomenon isfound in a broad range of taxa. This indicates that only a few
genes underlie such asymmetry. Wild-type forms of the model
plant Arabidopsis thaliana are symmetrical; however, thissymmetry can be disrupted by mutations, including theright-hand twisting mutants, spiral1 (spr1) and spr2. In an
attempt to discover suppressor mutants of the right-handed
helical growth mutant spr1, a research group led by Takashi
Hashimoto at NAIST identified two independent mutant loci,lefty1 and lefty2.
It was determined that the lefty1 and lefty2 mutations were
the result of single nucleotide exchanges in the α-tubulin genes

TUA6 and TUA4, respectively. It was shown that these dominantnegative mutations result in left-handed helical growth andclockwise twisting in A. thaliana’s elongating organs. The study
demonstrated that the mutant tubulins integrate into microtubule polymers, yielding right-handed cortical arrays in the cellsof the root epidermis. The mutants’ cortical microtubules had aheightened sensitivity to microtubule-specific drugs; theseresults indicated that diminished microtubule stability can resultin left-handed helical growth in plants.
A number of other helical growth mutants with fixed
handedness had been reported in Arabidopsis prior to this study;
however, the cellular and molecular causes of their asymmetrical
growth were not known. The results of this study indicated thatoblique orientation of cortical microtubules offered a possible
mechanism for producing the left–right asymmetry in elongating plant organs. They also presented a potential molecularexplanation for the establishment of handedness in Arabidopsis
twisting mutants, as well as the natural asymmetry of vines,
tendrils, and other twisting plant organs. In addition to contributing to a broadened understanding of helical growth in plants,
the results were also relevant to herbicide sensitivity (e.g.,dinitroaniline-like herbicides) in plants, as demonstrated by theincreased sensitivity to microtubule-specific drugs shown by the
mutants’ cortical microtubules.
Reference
Thitamadee, S., Tuchihara, K., Hashimoto, T. 2002. Microtubule basis for left-handed
helical growth in Arabidopsis. Nature, 417, 193–196.

Reference
Ohki, I., Shimotake, N., Fujita, N., Jee, J., Ikegami, T., Nakao, M., Shirakawa, M. 2001.
Solution structure of the methyl-CpG binding domain of human MBD1 in complex
with methylated DNA. Cell, 105, 487–497.

Flowers of a wild type (left), a lefty1 mutant (middle) and a lefty2 mutant (right) of Arabidopsis thaliana (Thitamadee etal., 2002, Nature).

NAIST Research Highlights

31

Molecular Biology

30 YEARS OF HISTORY: 2001– 2010

30 YEARS OF HISTORY: 2001– 2010

Masahiro Shirakawa

Takashi Hashimoto

Solving the protein structureof a key binding domain sheds
light on the neurodevelopmental
disorder Rett syndrome

The role of microtubulesin the handedness of helical
growth in plants

Structural analysis of the methyl-CpG binding domain reveals how proteins containing
this domain can interact with methylated DNA, which enables the interpretationof disease-causing mutations in Rett syndrome

D

NA methylation is an important biological mechanism
for the regulation of gene expression and overall genomic
stability. The proper functioning of this process isessential for normal development in mammals. Throughmethylation, the activity of DNA can be modified via additionof a methyl group, without changing the DNA sequence itself.
Erroneous methylation can result in the inappropriate regulation of genes, leading to various diseases such as cancer or
cardiovascular disease.
The consequences of DNA methylation are often mediatedby proteins that contain methyl-CpG binding domains (MBDs).
The structure of the MBD was unknown until 2001, when aresearch group led by Masahiro Shirakawa at NAIST reported
the structure of the MBD in a human protein, MBD1.
The MBD is highly conserved, meaning that its structure isshared with other MBD-containing proteins in several species.
There are five MBD protein family members characterized in
mammals, one of which is MeCP2. Seventeen different
mutations in MeCP2 have been found to cause Rett syndrome
which affects brain development leading to physical andintellectual disability in girls. Eleven of these mutations sit
within the MBD, indicating the importance of this domain.
The team overexpressed human MBD1 in E. coli and labeledand purified it for nuclear magnetic resonance analysis. Thisrevealed that when MBD binds DNA, structural rearrangements occur to form a loop in the protein structure, causing it
to fold into a new DNA binding interface.
They found that five residues contact the major groove of themethylated DNA to identify the two methyl groups that form
the methylated CpG group. These five residues form a hydrophobic patch. The lack of symmetry of this region indicates thateach of the two methyl groups is recognized distinctly.
A notable feature of the interaction between the MBD and
DNA is the small size of the contact area. This small contact
area, which sits only on the major groove of the DNA, allows
the MBD to access methyl-CpG sites on nucleosome coreswithout any steric interference from the chromatin structure.
As a result of the findings of this study, residues with known
mutations in Rett syndrome could be analyzed. Some of the30

Plant Sciences

NAIST Research Highlights

The MBD-DNA contacts lie exclusively within the major groove of DNA (Ohkiet al., 2001, Cell ).

mutations in MeCP2 may result in failure of the MBD to contact
the DNA bases. One reported mutation, in which an alanine is
replaced by a valine, likely interferes with the DNA binding
affinity, because valine has a bulky side chain. Mutations toother residues probably interfere with MeCP2’s activity by
causing global structural changes.
This study definitively outlined the protein structure of
MBDs, which demonstrated the involvement of several physicalinteractions between the DNA strand and protein side chains,
revealing for the first time how the methyl-CpG sequence canbe recognized by the MBD-containing family of proteins. Thesefindings facilitate a greater understanding of the causes of Rett
syndrome and may assist research into other disease pathologies
in which DNA methylation has been implicated, including
vascular tissue changes in atherosclerosis and carcinogenesis.

Two Arabidopsis thaliana mutants, lefty1 and lefty2, are found to exhibit left-handed
helical growth and clockwise twisting in elongating organs, and have cortical
microtubules with increased sensitivity to microtubule-specific drugs

P

lants usually grow in a linear or circumferential way, butmany plants exhibit some type of helical growth in theform of spirals, twists, or coils. Plant parts that show
helical growth include the stems or tendrils of twining plants,
fan-blade shaped flower petals, coiling roots, and spiral seed
pods. Helical growth is relevant to agriculture and horticulture
because it underpins plant forms that may be desirable, such as
aesthetically pleasing flowers or plant shapes, or plants that are
free to explore new environments, like climbing vines.
Conversely, this type of growth may be undesirable if it resultsin plant products that cannot be mechanically processed (e.g.,
misshapen root vegetables). Understanding the molecularmechanisms that produce helical growth can contribute to thedevelopment of new ways of improving this trait to better utilize
crops in horticulture and agriculture.
The left–right asymmetry, or handedness, of helical plant
forms is often fixed within species, and this phenomenon isfound in a broad range of taxa. This indicates that only a few
genes underlie such asymmetry. Wild-type forms of the model
plant Arabidopsis thaliana are symmetrical; however, thissymmetry can be disrupted by mutations, including theright-hand twisting mutants, spiral1 (spr1) and spr2. In an
attempt to discover suppressor mutants of the right-handed
helical growth mutant spr1, a research group led by Takashi
Hashimoto at NAIST identified two independent mutant loci,lefty1 and lefty2.
It was determined that the lefty1 and lefty2 mutations were
the result of single nucleotide exchanges in the α-tubulin genes

TUA6 and TUA4, respectively. It was shown that these dominantnegative mutations result in left-handed helical growth andclockwise twisting in A. thaliana’s elongating organs. The study
demonstrated that the mutant tubulins integrate into microtubule polymers, yielding right-handed cortical arrays in the cellsof the root epidermis. The mutants’ cortical microtubules had aheightened sensitivity to microtubule-specific drugs; theseresults indicated that diminished microtubule stability can resultin left-handed helical growth in plants.
A number of other helical growth mutants with fixed
handedness had been reported in Arabidopsis prior to this study;
however, the cellular and molecular causes of their asymmetrical
growth were not known. The results of this study indicated thatoblique orientation of cortical microtubules offered a possible
mechanism for producing the left–right asymmetry in elongating plant organs. They also presented a potential molecularexplanation for the establishment of handedness in Arabidopsis
twisting mutants, as well as the natural asymmetry of vines,
tendrils, and other twisting plant organs. In addition to contributing to a broadened understanding of helical growth in plants,
the results were also relevant to herbicide sensitivity (e.g.,dinitroaniline-like herbicides) in plants, as demonstrated by theincreased sensitivity to microtubule-specific drugs shown by the
mutants’ cortical microtubules.
Reference
Thitamadee, S., Tuchihara, K., Hashimoto, T. 2002. Microtubule basis for left-handed
helical growth in Arabidopsis. Nature, 417, 193–196.

Reference
Ohki, I., Shimotake, N., Fujita, N., Jee, J., Ikegami, T., Nakao, M., Shirakawa, M. 2001.
Solution structure of the methyl-CpG binding domain of human MBD1 in complex
with methylated DNA. Cell, 105, 487–497.

Flowers of a wild type (left), a lefty1 mutant (middle) and a lefty2 mutant (right) of Arabidopsis thaliana (Thitamadee etal., 2002, Nature).

NAIST Research Highlights

31

Molecular Biology

30 YEARS OF HISTORY: 2001– 2010

Akiho Yokota

Structural Biology

30 YEARS OF HISTORY: 2001– 2010

Toshio Hakoshima

RuBisCO-like proteins may be
the ancestral enzymes ofphotosynthetic RuBisCO
Carbon-fixing proteins found in Bacillus and involved in photosynthesis share
a common function that recycles sulfur, indicating a potential ancestral relationshipP

hotosynthesis, the metabolic process responsible forwith RuBisCO enzymes or other RLPs with RuBP-carboxylase
fixing atmospheric carbon into organic molecules, dates
activity. Furthermore, the codon usage and GC content of theback to the origins of Earth’s biological life. Ribulose
RLP gene were typical of the organism. On the basis of thesebisphosphate carboxylase/oxygenase (RuBisCO) is thefindings and previous literature, the group proposed that the
predominant CO2-fixing enzyme in the biosphere. It has bothgenes for RLP and photosynthetic RuBisCo did not originate
from lateral transfer of a gene encoding an RuBP-carboxylating
carboxylase and oxygenase activity, and it is the carboxylation
enzyme from another unrelated organism, such as an archaeonand oxygenation of ribulose 1,5-bisphosphate (RuBP) that areor photosynthetic bacterium. Instead, the gene for RLP, which
the primary events in photosynthesis and photorespiration,
in B. subtilis is part of the methionine salvage pathway, and therespectively. At a high CO2 concentration, RuBisCO functions
only as a carboxylase and participates in the Calvin cycle,
gene for photosynthetic RuBisCO originated from a common
whereas, at a high O2 concentration and a low CO2 concentraancestral gene.
However, as the group highlighted, the evolutionary origins
tion, RuBisCO functions as an oxygenase, as manifested byof bacteria and Archaea that possess RLPs far pre-date thephotorespiration. Photorespiration emerged later than photosynthesis in evolutionary history, along with the increase in theemergence of the Calvin cycle in photosynthetic bacteria. This
oxygen content of the atmosphere.
led to the conclusion that RLPs may be the ancestral enzymes
All living organisms possess RuBisCO itself or a RuBisof photosynthetic RuBisCO. An improved understanding of
CO-like protein (RLP) from the same superfamily. However,
the origin of early RLP-encoding genes may offer insight into
not all of these proteins possess carboxylase activity. The
how these fundamentally important CO2-fixing enzymes havefunctional and evolutionary relationship of RLPs to photosynovercome changes in global environments throughout history.
thetic RuBisCO remained unknown until 2003, when aresearch team led by Akiho Yokota at NAIST described theReference
functional relationship between a RuBisCO-like protein of
Ashida, H., Saito, Y., Kojima, C., Kobayashi, K., Ogasawara, N., Yokota, A. 2003. A
Bacillus subtilis and photosynthetic RuBisCO.
functional link between RuBisCO-like protein of Bacillus and photosynthetic
RuBisCO. Science, 302, 286–290.
The research group found that the RLP of B. subtilis
functions as a 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P) enolase in
the methionine salvage pathway – theprocess by which sulfur is recycled following certain biosytheses – but possesses no
RuBP-carboxylation activity. Interestingly,
the growth defect resulting from mutationof this RLP-encoding gene in B. subtilis was
rescued by the RuBisCO gene from thephotosynthetic bacterium Rhodospirillum
rubrum, as demonstrated by in vivo experiments. This finding was significant because
it proved that the photosynthetic RuBisCO
from R. rubrum retained the ability tofunction in the methionine salvage pathway
in B. subtilis, potentially indicating common
ancestry.
Phylogenetically, the researchers foundPhylogenetic relationships of the large subunits of RuBisCO (forms I and II) and RLPs (forms III and IV)from various organisms (Ashida et al., 2003, Science).
that the RLP from B. subtilis did not cluster

32

NAIST Research Highlights

Structure of the GA3–GID1A–DELLA complex (Murase et al., 2008, Nature).

How plant cells perceive anddecode the gibberellin signal
A structural model of a plant hormone receptor is identified that differs from the previously
known underlying mechanism of hormone sensing and effector recognition in auxin receptors

G

ibberellins (GAs) are a large, important group of plant
hormones that are involved in growth and development
in higher plants, including shoot elongation, seedgermination, and the development of flowers and fruit. These
hormones have a broad range of uses in agriculture, from theproduction of seedless fruit to the promotion of flowering. GAs
regulate gene expression; however, the exact mechanism bywhich the GA signal is recognized and decoded by plant cells
was a key question in plant biology that had remainedunanswered for years.
A research group led by Toshio Hakoshima at NAIST
addressed this question using the model plant Arabidopsis
thaliana. GAs regulate gene expression by binding to the receptor GIBBERELLIN INSENSITIVE DWARF1 (GID1), andenabling degradation of DELLA proteins, which negatively
regulate GA signaling, including GIBBERELLIN INSENSITIVE (GAI). Because bioactive GAs control various
developmental processes during a plant’s life cycle, the teamspecifically examined how bioactive GAs are sensed by andactivate GID1 for binding to DELLA proteins. The team set out
to determine the structure of GA receptors to understand thedetailed relationships between structure and activity regardingthese molecules.
Crystallographic and biochemical methods were used to
examine how the GA receptor GID1A perceives GA, and toinvestigate GA-induced binding of GID1A to GAI. The result
was the presentation of a crystal structure of a ternary complex
comprising GID1A, a bioactive gibberellin, and the amino-ter-minal DELLA domain of GAI. In this complex, GID1A traps
GA in a deep binding pocket, which is covered by its N-terminal region. This region then interacts with the DELLA domainthat contains DELLA, VHYNP and LExLE motifs, which are
involved in GID1 binding.
The results of the study offered a model for the structure ofa plant hormone receptor that differs from the hormone
sensing and effector recognition mechanism of the known
auxin receptors. Just prior to this study, auxin was revealed toact as the ‘molecular glue’ that brings a substrate protein
together with the receptor, which in the case of the auxin
pathway is an F-box protein. Conversely, the GA receptor is atrue receptor protein that is activated by GA to serve asa ‘ubiquitylation chaperon’ that triggers recognition of thesubstrate by the SCF complex, which is involved in protein
degradation. The structural investigation in this study
furthered the understanding of cellular function regulationthat is dependent on degradation signals, and the results
revealed a structural mechanism underpinning the perceptionof GA and DELLA protein recognition by GID1. The findings
also suggested an alternative way forward regarding development of GA derivatives for regulating the growth of ornamental
plants and crops.
Reference
Murase, K., Hirano, Y., Sun, T-p., Hakoshima, T. 2008. Gibberellin-induced DELLA
recognition by the gibberellin receptor GID1. Nature, 456, 459–463.

NAIST Research Highlights

33

Molecular Biology

30 YEARS OF HISTORY: 2001– 2010

Akiho Yokota

Structural Biology

30 YEARS OF HISTORY: 2001– 2010

Toshio Hakoshima

RuBisCO-like proteins may be
the ancestral enzymes ofphotosynthetic RuBisCO
Carbon-fixing proteins found in Bacillus and involved in photosynthesis share
a common function that recycles sulfur, indicating a potential ancestral relationshipP

hotosynthesis, the metabolic process responsible forwith RuBisCO enzymes or other RLPs with RuBP-carboxylase
fixing atmospheric carbon into organic molecules, dates
activity. Furthermore, the codon usage and GC content of theback to the origins of Earth’s biological life. Ribulose
RLP gene were typical of the organism. On the basis of thesebisphosphate carboxylase/oxygenase (RuBisCO) is thefindings and previous literature, the group proposed that the
predominant CO2-fixing enzyme in the biosphere. It has bothgenes for RLP and photosynthetic RuBisCo did not originate
from lateral transfer of a gene encoding an RuBP-carboxylating
carboxylase and oxygenase activity, and it is the carboxylation
enzyme from another unrelated organism, such as an archaeonand oxygenation of ribulose 1,5-bisphosphate (RuBP) that areor photosynthetic bacterium. Instead, the gene for RLP, which
the primary events in photosynthesis and photorespiration,
in B. subtilis is part of the methionine salvage pathway, and therespectively. At a high CO2 concentration, RuBisCO functions
only as a carboxylase and participates in the Calvin cycle,
gene for photosynthetic RuBisCO originated from a common
whereas, at a high O2 concentration and a low CO2 concentraancestral gene.
However, as the group highlighted, the evolutionary origins
tion, RuBisCO functions as an oxygenase, as manifested byof bacteria and Archaea that possess RLPs far pre-date thephotorespiration. Photorespiration emerged later than photosynthesis in evolutionary history, along with the increase in theemergence of the Calvin cycle in photosynthetic bacteria. This
oxygen content of the atmosphere.
led to the conclusion that RLPs may be the ancestral enzymes
All living organisms possess RuBisCO itself or a RuBisof photosynthetic RuBisCO. An improved understanding of
CO-like protein (RLP) from the same superfamily. However,
the origin of early RLP-encoding genes may offer insight into
not all of these proteins possess carboxylase activity. The
how these fundamentally important CO2-fixing enzymes havefunctional and evolutionary relationship of RLPs to photosynovercome changes in global environments throughout history.
thetic RuBisCO remained unknown until 2003, when aresearch team led by Akiho Yokota at NAIST described theReference
functional relationship between a RuBisCO-like protein of
Ashida, H., Saito, Y., Kojima, C., Kobayashi, K., Ogasawara, N., Yokota, A. 2003. A
Bacillus subtilis and photosynthetic RuBisCO.
functional link between RuBisCO-like protein of Bacillus and photosynthetic
RuBisCO. Science, 302, 286–290.
The research group found that the RLP of B. subtilis
functions as a 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P) enolase in
the methionine salvage pathway – theprocess by which sulfur is recycled following certain biosytheses – but possesses no
RuBP-carboxylation activity. Interestingly,
the growth defect resulting from mutationof this RLP-encoding gene in B. subtilis was
rescued by the RuBisCO gene from thephotosynthetic bacterium Rhodospirillum
rubrum, as demonstrated by in vivo experiments. This finding was significant because
it proved that the photosynthetic RuBisCO
from R. rubrum retained the ability tofunction in the methionine salvage pathway
in B. subtilis, potentially indicating common
ancestry.
Phylogenetically, the researchers foundPhylogenetic relationships of the large subunits of RuBisCO (forms I and II) and RLPs (forms III and IV)from various organisms (Ashida et al., 2003, Science).
that the RLP from B. subtilis did not cluster

32

NAIST Research Highlights

Structure of the GA3–GID1A–DELLA complex (Murase et al., 2008, Nature).

How plant cells perceive anddecode the gibberellin signal
A structural model of a plant hormone receptor is identified that differs from the previously
known underlying mechanism of hormone sensing and effector recognition in auxin receptors

G

ibberellins (GAs) are a large, important group of plant
hormones that are involved in growth and development
in higher plants, including shoot elongation, seedgermination, and the development of flowers and fruit. These
hormones have a broad range of uses in agriculture, from theproduction of seedless fruit to the promotion of flowering. GAs
regulate gene expression; however, the exact mechanism bywhich the GA signal is recognized and decoded by plant cells
was a key question in plant biology that had remainedunanswered for years.
A research group led by Toshio Hakoshima at NAIST
addressed this question using the model plant Arabidopsis
thaliana. GAs regulate gene expression by binding to the receptor GIBBERELLIN INSENSITIVE DWARF1 (GID1), andenabling degradation of DELLA proteins, which negatively
regulate GA signaling, including GIBBERELLIN INSENSITIVE (GAI). Because bioactive GAs control various
developmental processes during a plant’s life cycle, the teamspecifically examined how bioactive GAs are sensed by andactivate GID1 for binding to DELLA proteins. The team set out
to determine the structure of GA receptors to understand thedetailed relationships between structure and activity regardingthese molecules.
Crystallographic and biochemical methods were used to
examine how the GA receptor GID1A perceives GA, and toinvestigate GA-induced binding of GID1A to GAI. The result
was the presentation of a crystal structure of a ternary complex
comprising GID1A, a bioactive gibberellin, and the amino-ter-minal DELLA domain of GAI. In this complex, GID1A traps
GA in a deep binding pocket, which is covered by its N-terminal region. This region then interacts with the DELLA domainthat contains DELLA, VHYNP and LExLE motifs, which are
involved in GID1 binding.
The results of the study offered a model for the structure ofa plant hormone receptor that differs from the hormone
sensing and effector recognition mechanism of the known
auxin receptors. Just prior to this study, auxin was revealed toact as the ‘molecular glue’ that brings a substrate protein
together with the receptor, which in the case of the auxin
pathway is an F-box protein. Conversely, the GA receptor is atrue receptor protein that is activated by GA to serve asa ‘ubiquitylation chaperon’ that triggers recognition of thesubstrate by the SCF complex, which is involved in protein
degradation. The structural investigation in this study
furthered the understanding of cellular function regulationthat is dependent on degradation signals, and the results
revealed a structural mechanism underpinning the perceptionof GA and DELLA protein recognition by GID1. The findings
also suggested an alternative way forward regarding development of GA derivatives for regulating the growth of ornamental
plants and crops.
Reference
Murase, K., Hirano, Y., Sun, T-p., Hakoshima, T. 2008. Gibberellin-induced DELLA
recognition by the gibberellin receptor GID1. Nature, 456, 459–463.

NAIST Research Highlights

33

Speech Recognition

30 YEARS OF HISTORY: 2001– 2010

Kiyohiro Shikano

Information Systems

30 YEARS OF HISTORY: 2001– 2010

Minoru Ito

A genetic algorithm for optimizing
multiple destination itineraries
A personal navigation system that utilizes a genetic algorithm to find optimized routes for trips
with multiple destinations, which may assist both tourists and commercial delivery services

M

A voice-recognition interface with a stethoscopic microphone attached behind the ear (Nakajima et al., 2003, ICASSP).

A non-audible voice interface forenhanced speech recognition
A microphone to be attached directly to a user’s skin is designed to register subaudible
sounds for voice-recognition software, which may lead to more accurate computer interfacesAs smart devices become increasingly portable, and evermore reliant on voice commands instead of keyboards,
robust interfaces that work under a wide range ofsituations with varying levels of background noise are required.
Additionally, as these electronics reach nearly universal
adoption, the voice commands themselves begin to add to theinterference between users in the same room, potentiallylimiting use outdoors, in public spaces or inside vehicles. The
development of new devices that can overcome these issues
contributes greatly to continued technological progress in thisfield, as well as being more user friendly and secure.
A research team led by Kiyohiro Shikano at NAIST
developed a voice-recognition interface that operates by
capturing non-audible murmur (NAM) vocalizations via astethoscopic microphone that can be attached directly behindthe ear. Because acoustic vibrations travel more easily throughbone and human tissue compared with air, this microphone
can decern sounds with intensities much lower than canconventional systems. Normally, once sound energy from thevocal cords leaves a person’s mouth, it will propagate in all
directions in an unfocused fashion. This requires the user tospeak louder, which in turn adds to the room’s ambient noise.
By contrast, a vocalization with an intensity below the usual
threshold of audibility can be directed by the human anatomy
to produce vibrations at other locations on the head, to bepicked up by the stethoscopic microphone; hence, this system

34

NAIST Research Highlights

can recognize commands that are too quiet to be heard in the
air. This can lead to quieter working environments andpromote data privacy. Moreover, this approach is much lesssusceptible to noise interference and may be suitable for people
who are unable to speak loudly owing to health reasons.
The researchers tested the system using the Japanese Dictation Toolkit, which includes voice-recognition software alongwith acoustic and language modeling capabilities. Accuracy
ratings around 90% were achieved, even with backgroundsounds present. The team studied common types of ambient
noise, such as a Bach concerto and a television news broadcast.
The results are even more impressive given the limitations ofvoice-recognition software available at the time. The fidelity is
expected to be even higher when implemented using contemporary applications.
The results of this work may substantially improve the stateof voice-recognition artificial intelligence by providing moreaccurate commands. In particular, this research may makewearable computers considerably more attractive by providing
a reliable, cost-effective hands-free user interface that will workin loud environments.
Reference
Nakajima, Y., Kashioka, H., Shikano, K., Campbell, N. 2003. Non-audible murmur
recognition input interface using stethoscopic microphone attached to the skin.
IEEE International Conference on Acoustics, Speech, and Signal Processing, 127–130.

anaging travel itineraries with multiple destinations
map-based test, selecting up to 13 destinations with specificand limited time has been a challenging task fortime restrictions associated with four destinations. Because
centuries. One example is the “traveling salesman”
the route search algorithm was to be executed on a remoteproblem, which requires finding the shortest path between
server, it could be computed more efficiently compared withlist of cities, visiting each exactly once. Despite the relative
being located on the user’s device. Future extensions of thealgorithm can be made to include multiple transport modalilack of constraints, this problem is known to be “NP-hard”,
ties, such as riding on trains, buses, or walking.
meaning that no algorithm can efficiently find the absolute
The approach demonstrated by the research can also be
best solution unless the number of cities is small. Tourists inapplied to more general sets of travel constraints experienced
the real world face even more practical requirements, such as
throughout the transportation industry. This may be of signifbusiness hours, reservation times, or changes in plans. The
icant value specifically for commercial delivery services that
potential benefits of a personal navigation system (PNS) thatwould save on fuel and labor costs with shortened routes.
could map out reasonable itineraries was recognized for all
Additionally, as factories and warehouses increase in size andkinds of travelers.
automation, the need for indoor itinerary optimization is
To help achieve this goal – in a study that predates both
expected to increase in the future.
Google Maps and the iPhone – a research group led byMinoru Ito at NAIST created a genetic algorithm thatcomputes “semi-optimal solutions.” While previous methods
were limited to finding the best way to travel between two
given points, the algorithm they made can handle trips with
Reference
multiple destinations. Genetic algorithms are different from
Maruyama, A., Shibata, N., Murata, Y., Yasumoto, K., Ito, M. 2004. A personal tourism
other search optimization methods in that they use a populanavigation system to support traveling multiple destinations with time restrictions.
tion of candidate solutions. Then, using a process similar toInternational Conference on Advanced Information Networking and Applications, 18–21.
the natural selection of organisms, the solutions that arefound to have the highest“fitness” for solving the problem
are chosen to be retained. These
high-fitness candidate solutions
undergo “crossover” and “mutation” operations to produce new
possible itineraries.
In the complete program, thewell-known A* search algorithm
is first used to determine theshortest route between each pairof desired locations. Then, thenovel genetic algorithm is used tofind the best possible routes. One
benefit of this approach is thatmultiple candidate solutions are
considered during each step, so
an approximate solution is always
available. In addition, the user
can be provided a list of possible
routes from which to choose.
The researchers conducted a
The entire route of six destinations obtained by the genetic algorithm (Maruyama et al., 2004, AINA).

NAIST Research Highlights

35

Speech Recognition

30 YEARS OF HISTORY: 2001– 2010

Kiyohiro Shikano

Information Systems

30 YEARS OF HISTORY: 2001– 2010

Minoru Ito

A genetic algorithm for optimizing
multiple destination itineraries
A personal navigation system that utilizes a genetic algorithm to find optimized routes for trips
with multiple destinations, which may assist both tourists and commercial delivery services

M

A voice-recognition interface with a stethoscopic microphone attached behind the ear (Nakajima et al., 2003, ICASSP).

A non-audible voice interface forenhanced speech recognition
A microphone to be attached directly to a user’s skin is designed to register subaudible
sounds for voice-recognition software, which may lead to more accurate computer interfacesAs smart devices become increasingly portable, and evermore reliant on voice commands instead of keyboards,
robust interfaces that work under a wide range ofsituations with varying levels of background noise are required.
Additionally, as these electronics reach nearly universal
adoption, the voice commands themselves begin to add to theinterference between users in the same room, potentiallylimiting use outdoors, in public spaces or inside vehicles. The
development of new devices that can overcome these issues
contributes greatly to continued technological progress in thisfield, as well as being more user friendly and secure.
A research team led by Kiyohiro Shikano at NAIST
developed a voice-recognition interface that operates by
capturing non-audible murmur (NAM) vocalizations via astethoscopic microphone that can be attached directly behindthe ear. Because acoustic vibrations travel more easily throughbone and human tissue compared with air, this microphone
can decern sounds with intensities much lower than canconventional systems. Normally, once sound energy from thevocal cords leaves a person’s mouth, it will propagate in all
directions in an unfocused fashion. This requires the user tospeak louder, which in turn adds to the room’s ambient noise.
By contrast, a vocalization with an intensity below the usual
threshold of audibility can be directed by the human anatomy
to produce vibrations at other locations on the head, to bepicked up by the stethoscopic microphone; hence, this system

34

NAIST Research Highlights

can recognize commands that are too quiet to be heard in the
air. This can lead to quieter working environments andpromote data privacy. Moreover, this approach is much lesssusceptible to noise interference and may be suitable for people
who are unable to speak loudly owing to health reasons.
The researchers tested the system using the Japanese Dictation Toolkit, which includes voice-recognition software alongwith acoustic and language modeling capabilities. Accuracy
ratings around 90% were achieved, even with backgroundsounds present. The team studied common types of ambient
noise, such as a Bach concerto and a television news broadcast.
The results are even more impressive given the limitations ofvoice-recognition software available at the time. The fidelity is
expected to be even higher when implemented using contemporary applications.
The results of this work may substantially improve the stateof voice-recognition artificial intelligence by providing moreaccurate commands. In particular, this research may makewearable computers considerably more attractive by providing
a reliable, cost-effective hands-free user interface that will workin loud environments.
Reference
Nakajima, Y., Kashioka, H., Shikano, K., Campbell, N. 2003. Non-audible murmur
recognition input interface using stethoscopic microphone attached to the skin.
IEEE International Conference on Acoustics, Speech, and Signal Processing, 127–130.

anaging travel itineraries with multiple destinations
map-based test, selecting up to 13 destinations with specificand limited time has been a challenging task fortime restrictions associated with four destinations. Because
centuries. One example is the “traveling salesman”
the route search algorithm was to be executed on a remoteproblem, which requires finding the shortest path between
server, it could be computed more efficiently compared withlist of cities, visiting each exactly once. Despite the relative
being located on the user’s device. Future extensions of thealgorithm can be made to include multiple transport modalilack of constraints, this problem is known to be “NP-hard”,
ties, such as riding on trains, buses, or walking.
meaning that no algorithm can efficiently find the absolute
The approach demonstrated by the research can also be
best solution unless the number of cities is small. Tourists inapplied to more general sets of travel constraints experienced
the real world face even more practical requirements, such as
throughout the transportation industry. This may be of signifbusiness hours, reservation times, or changes in plans. The
icant value specifically for commercial delivery services that
potential benefits of a personal navigation system (PNS) thatwould save on fuel and labor costs with shortened routes.
could map out reasonable itineraries was recognized for all
Additionally, as factories and warehouses increase in size andkinds of travelers.
automation, the need for indoor itinerary optimization is
To help achieve this goal – in a study that predates both
expected to increase in the future.
Google Maps and the iPhone – a research group led byMinoru Ito at NAIST created a genetic algorithm thatcomputes “semi-optimal solutions.” While previous methods
were limited to finding the best way to travel between two
given points, the algorithm they made can handle trips with
Reference
multiple destinations. Genetic algorithms are different from
Maruyama, A., Shibata, N., Murata, Y., Yasumoto, K., Ito, M. 2004. A personal tourism
other search optimization methods in that they use a populanavigation system to support traveling multiple destinations with time restrictions.
tion of candidate solutions. Then, using a process similar toInternational Conference on Advanced Information Networking and Applications, 18–21.
the natural selection of organisms, the solutions that arefound to have the highest“fitness” for solving the problem
are chosen to be retained. These
high-fitness candidate solutions
undergo “crossover” and “mutation” operations to produce new
possible itineraries.
In the complete program, thewell-known A* search algorithm
is first used to determine theshortest route between each pairof desired locations. Then, thenovel genetic algorithm is used tofind the best possible routes. One
benefit of this approach is thatmultiple candidate solutions are
considered during each step, so
an approximate solution is always
available. In addition, the user
can be provided a list of possible
routes from which to choose.
The researchers conducted a
The entire route of six destinations obtained by the genetic algorithm (Maruyama et al., 2004, AINA).

NAIST Research Highlights

3530 YEARS OF HISTORY: 2001– 2010

30 YEARS OF HISTORY: 2001– 2010

Tsukasa Ogasawara

Jun-ichi Kikuchi

A robotic hand with vision-based
feedback control

Paving the way through layer-by-layer
assembly of lipid bilayer vesicles

A robotic hand that mimics human physiology and uses fingertip cameras
to measure contact forces leads to prosthetic devices with increased functionality,
as well as more robust automated object manipulation

Layer-by-layer assembly of cationic and anionic Cerasomes gives
three-dimensional hierarchical bilayer structures on solid substrates

R

feedback loop, the team used a mathematical model in which
obotic arms are employed in many automated tasks,
the radius of the contact area is proportional to the normalsuch as automotive manufacturing. However, theseforce raised to the ⅓ power. When an incipient slip was
appendages have typically lacked the dexterity of humandetected, the traction force could be adjusted before the object
hands, and they have been unable to reliably pick up andmoved too much.
manipulate objects. In particular, robots have a difficult time
The advances presented by the NAIST Hand provided an
calculating and applying the correct amount of force to grasp
important foundation for subsequent improvements in robotic
something without crushing it. Being able to construct a
hand technology. Future versions of the robotic hand may be
robotic hand that can mimic human motion is an important
controlled using a motion-capture glove. Operators would begoal for both prosthetics and industrial applications.
able to move heavy equipment without risk of injury. AdditionA research team led by Tsukasa Ogasawara at NAIST
ally, patients requiring a prosthetic hand may be able todeveloped a robotic hand that uses a vision-based tactile sensor
experience an improved quality of life by being able to graspin each fingertip. The hand has four fingers, and each finger isfragile objects, like paper cups, more easily.
capable of three degrees of freedom. The team used a speciallydesigned gear mechanism that placed all three actuators in thepalm section, thus avoiding space limitations, as well as theneed for a wire-control system. Rotation along each of the three
Reference
axes is controlled independently by a separate driving mechaUeda, J., Ishida, Y., Kondo, M., Ogasawara, T. 2005. Development of the NAIST-Handnism. One of the joints in each finger is capable ofwith vision-based tactile fingertip sensor. IEEE International Conference on Robotics
and Automation, 2332–2337.
adduction/abduction as well as flexion/extension, while asecond joint has flexion/extension, similar tohuman physiology.
To carefully monitor the force being
applied by each fingertip, the researchers
attached a hemispherical transparent gelwith LED light. An embedded camera
underneath looked for signs of distortion
caused by the compression of the gel and was
coupled with a force detector. By measuring
the eccentricity of the gel’s image, thedisplacement could be calculated. When thiswas combined with the radius of the contact
area, the “slip margin” could be estimated.
While some previous approaches to slip
detection used accelerometers, these often
have been found to experience too muchnoise to be practical. Similarly, measuring
the strain distribution required a large
number of sensors to obtain the necessary
spatial resolution. Using a camera-based
method had many advantages, including
improved accuracy of contact area measurements. In addition, a stable grip could beobtained by using real-time feedback of theslip margin, even when the coefficient offriction was not known. To control the
Motion transfer to the NAIST Hand (Ueda et al., 2005, ICRA).

36

Chemistry

NAIST Research HighlightsT

he lipid bilayer membrane thatsurrounds cells is so effective at
its job that it is used as inspiration for synthetic products. For
example, vesicles made of a lipid
membrane protecting an aqueous
environment have been used as
carriers for drugs and as artificial cell
membranes. The lipid used for thebilayer of these synthetic systems canbe purposefully selected, meaning
features such as particle size can becontrolled. It should therefore followthat different lipids could be combinedto achieve multiple functions within
one system. However, this advancerelies on assembling vesicles that arestable enough to maintain their
Schematic drawing of the three-dimensional assembled structure of the anionic and cationic Cerasomes
integrity in a variety of conditions. This
(Katagiri et al., 2002, J Am Chem Soc).
was not achieved until a research teamled by Jun-ichi Kikuchi at NAIST
Cerasome layer, which was anticipated owing to their larger
reported vesicles assembled from organic-inorganic hybrid
particle diameter. When compared with layer-by-layer
proamphiphiles in 2002.
exposure to traditional cationic and anionic liposomes, theThe research team used two different organo-alkoxysilanes
Cerasomes resulted in much greater mass increases, showing
to form Cerasomes – organic-inorganic hybrids with a liposothat stable buildup was achieved, unlike for the liposomes.
mal membrane and a ceramic surface. The ceramic outer layer
Atomic force microscopy images were acquired to confirm
was expected to act as a protective barrier against the collapse orthat the integrity of the Cerasomes in each layer was
fusion of the vesicles. Two different types of Cerasome weremaintained. The images showed the distinct sizes of the vesicles
assembled: one that was cationic at pH 9.0 and one that wasin each layer, nicely demonstrating the stability of the vesicle
anionic. Transmission electron microscopy showed that themorphologies. The packing of the layered structure was likened
vesicles formed were 70–300 nm and 20–100 nm in diameter,
to a pavement, with the larger anionic Cerasomes acting as
for the anionic and cationic Cerasomes, respectively.
paving stones that the smaller cationic structures packed
In addition, the Cerasomes had properties that would beclosely around like cement.
expected for bilayer vesicles; for example, they showed phase
The three-dimensional structure reported in this study wastransition behavior from the gel to the liquid crystalline state.
the first packed hierarchical structure assembled on a solid
The phase transition temperatures were found to be
substrate using the layer-by-layer technique. The stability of the10.5 and 25.7 °C for the anionic and cationic Cerasomes,
different vesicle types demonstrated the exciting potential torespectively, showing that they responded differently toinclude different functions within the same system, laying thetemperature triggers.
foundations for many functional systems, such as responsive
The researchers showed that the opposing charges of the
drug delivery systems that release therapeutics at specificCerasomes could be used to build up layers of vesicles thatlocations in the body and complex artificial cells.
maintained their discreet morphologies without collapsing, or
the membranes fusing. Quartz crystal microbalance measureReference
ment showed that alternately exposing a substrate to aqueous
Katagiri, K., Hamasaki, R., Ariga, K., Kikuchi, J. 2002. Layered paving of vesicularsolutions of each Cerasome led to sequential increases in mass
nanoparticles formed with cerasome as a bioinspired organic-inorganic hybrid.
Journal of the American Chemical Society, 124, 7892–7893.
with each layer. The mass increases were greater for the anionic© 2002 American Chemical Society

Robotics

NAIST Research Highlights

37

30 YEARS OF HISTORY: 2001– 2010

30 YEARS OF HISTORY: 2001– 2010

Tsukasa Ogasawara

Jun-ichi Kikuchi

A robotic hand with vision-based
feedback control

Paving the way through layer-by-layer
assembly of lipid bilayer vesicles

A robotic hand that mimics human physiology and uses fingertip cameras
to measure contact forces leads to prosthetic devices with increased functionality,
as well as more robust automated object manipulation

Layer-by-layer assembly of cationic and anionic Cerasomes gives
three-dimensional hierarchical bilayer structures on solid substrates

R

feedback loop, the team used a mathematical model in which
obotic arms are employed in many automated tasks,
the radius of the contact area is proportional to the normalsuch as automotive manufacturing. However, theseforce raised to the ⅓ power. When an incipient slip was
appendages have typically lacked the dexterity of humandetected, the traction force could be adjusted before the object
hands, and they have been unable to reliably pick up andmoved too much.
manipulate objects. In particular, robots have a difficult time
The advances presented by the NAIST Hand provided an
calculating and applying the correct amount of force to grasp
important foundation for subsequent improvements in robotic
something without crushing it. Being able to construct a
hand technology. Future versions of the robotic hand may be
robotic hand that can mimic human motion is an important
controlled using a motion-capture glove. Operators would begoal for both prosthetics and industrial applications.
able to move heavy equipment without risk of injury. AdditionA research team led by Tsukasa Ogasawara at NAIST
ally, patients requiring a prosthetic hand may be able todeveloped a robotic hand that uses a vision-based tactile sensor
experience an improved quality of life by being able to graspin each fingertip. The hand has four fingers, and each finger isfragile objects, like paper cups, more easily.
capable of three degrees of freedom. The team used a speciallydesigned gear mechanism that placed all three actuators in thepalm section, thus avoiding space limitations, as well as theneed for a wire-control system. Rotation along each of the three
Reference
axes is controlled independently by a separate driving mechaUeda, J., Ishida, Y., Kondo, M., Ogasawara, T. 2005. Development of the NAIST-Handnism. One of the joints in each finger is capable ofwith vision-based tactile fingertip sensor. IEEE International Conference on Robotics
and Automation, 2332–2337.
adduction/abduction as well as flexion/extension, while asecond joint has flexion/extension, similar tohuman physiology.
To carefully monitor the force being
applied by each fingertip, the researchers
attached a hemispherical transparent gelwith LED light. An embedded camera
underneath looked for signs of distortion
caused by the compression of the gel and was
coupled with a force detector. By measuring
the eccentricity of the gel’s image, thedisplacement could be calculated. When thiswas combined with the radius of the contact
area, the “slip margin” could be estimated.
While some previous approaches to slip
detection used accelerometers, these often
have been found to experience too muchnoise to be practical. Similarly, measuring
the strain distribution required a large
number of sensors to obtain the necessary
spatial resolution. Using a camera-based
method had many advantages, including
improved accuracy of contact area measurements. In addition, a stable grip could beobtained by using real-time feedback of theslip margin, even when the coefficient offriction was not known. To control the
Motion transfer to the NAIST Hand (Ueda et al., 2005, ICRA).

36

Chemistry

NAIST Research HighlightsT

he lipid bilayer membrane thatsurrounds cells is so effective at
its job that it is used as inspiration for synthetic products. For
example, vesicles made of a lipid
membrane protecting an aqueous
environment have been used as
carriers for drugs and as artificial cell
membranes. The lipid used for thebilayer of these synthetic systems canbe purposefully selected, meaning
features such as particle size can becontrolled. It should therefore followthat different lipids could be combinedto achieve multiple functions within
one system. However, this advancerelies on assembling vesicles that arestable enough to maintain their
Schematic drawing of the three-dimensional assembled structure of the anionic and cationic Cerasomes
integrity in a variety of conditions. This
(Katagiri et al., 2002, J Am Chem Soc).
was not achieved until a research teamled by Jun-ichi Kikuchi at NAIST
Cerasome layer, which was anticipated owing to their larger
reported vesicles assembled from organic-inorganic hybrid
particle diameter. When compared with layer-by-layer
proamphiphiles in 2002.
exposure to traditional cationic and anionic liposomes, theThe research team used two different organo-alkoxysilanes
Cerasomes resulted in much greater mass increases, showing
to form Cerasomes – organic-inorganic hybrids with a liposothat stable buildup was achieved, unlike for the liposomes.
mal membrane and a ceramic surface. The ceramic outer layer
Atomic force microscopy images were acquired to confirm
was expected to act as a protective barrier against the collapse orthat the integrity of the Cerasomes in each layer was
fusion of the vesicles. Two different types of Cerasome weremaintained. The images showed the distinct sizes of the vesicles
assembled: one that was cationic at pH 9.0 and one that wasin each layer, nicely demonstrating the stability of the vesicle
anionic. Transmission electron microscopy showed that themorphologies. The packing of the layered structure was likened
vesicles formed were 70–300 nm and 20–100 nm in diameter,
to a pavement, with the larger anionic Cerasomes acting as
for the anionic and cationic Cerasomes, respectively.
paving stones that the smaller cationic structures packed
In addition, the Cerasomes had properties that would beclosely around like cement.
expected for bilayer vesicles; for example, they showed phase
The three-dimensional structure reported in this study wastransition behavior from the gel to the liquid crystalline state.
the first packed hierarchical structure assembled on a solid
The phase transition temperatures were found to be
substrate using the layer-by-layer technique. The stability of the10.5 and 25.7 °C for the anionic and cationic Cerasomes,
different vesicle types demonstrated the exciting potential torespectively, showing that they responded differently toinclude different functions within the same system, laying thetemperature triggers.
foundations for many functional systems, such as responsive
The researchers showed that the opposing charges of the
drug delivery systems that release therapeutics at specificCerasomes could be used to build up layers of vesicles thatlocations in the body and complex artificial cells.
maintained their discreet morphologies without collapsing, or
the membranes fusing. Quartz crystal microbalance measureReference
ment showed that alternately exposing a substrate to aqueous
Katagiri, K., Hamasaki, R., Ariga, K., Kikuchi, J. 2002. Layered paving of vesicularsolutions of each Cerasome led to sequential increases in mass
nanoparticles formed with cerasome as a bioinspired organic-inorganic hybrid.
Journal of the American Chemical Society, 124, 7892–7893.
with each layer. The mass increases were greater for the anionic© 2002 American Chemical Society

Robotics

NAIST Research Highlights

37

Chemistry

30 YEARS OF HISTORY: 2001– 2010

Takashi Fuyuki

A synthetic mimic of a biologicallight-harvesting molecule

Rapid imaging of polycrystalline
silicon solar cell defects

A self-assembled, porphyrin-based barrel structure is synthesized, enabling
structure–function insights into light harvesting by photosynthetic organisms

Quantitating the main parameter that indicates the efficiency of solar cells with routine
instrumentation enables performance evaluations during mass production

P

P

NAIST Research Highlights© 2003 American Chemical Society

Yoshiaki Kobuke

hotosynthetic organisms use intricate
macromolecular assemblies to absorb
solar energy for subsequent conversion
into the chemical energy necessary to perform
life functions. However, researchers have been
unable to fabricate close synthetic mimics ofthese assemblies, a necessary step to understandhow the assemblies work and how to apply themto modern technology. Thus, it can be difficult
to optimize light harvesting in artificial
constructs, such as in solar cells that may exceed
the efficiency of even the most advanced
silicon-based designs in use today.
The first step of photosynthesis is absorptionof solar energy by light-harvesting complexes
that are arranged in a barrel architecture. Artificial analogs of such complexes, based on
porphyrin rings, have been synthesized.
However, the foundational porphyrin macroring architecture in biology – based on dimers
arranged in a slipped-cofacial geometry – has
not been synthesized. A research team led by
Yoshiaki Kobuke at NAIST published work on
the self-assembly of a supramolecule which has
intramolecular orientations and distances thatreflect the architecture of biological light-harA structural model of the target cyclic hexamer (Takahashi & Kobuke, 2003, J Am Chem Soc).
vesting complexes, as confirmed by rigorous
experimental characterization.
copy to quantitate its light absorption. Bands characteristic of
The macroring design was based on two principles. First,
the expected porphyrin absorption were present. Furthermore,
the geometry of the zinc-containing porphyrin-based dimer
the fluorescence quantum yield of the macroring relative
facilitates light absorption and minimizes energy waste after
to that of the porphyrin dimer subunits was 0.51, which islight absorption. Second, the geometry of the phenylene spacer
consistent with analogous observations in bacterial
that links each dimer enables ring closure into a barrel architeclight-harvesting complexes.
ture which has center-to-center distances of 6.1 and 11.0
These results provide a means of experimentally testing
angstroms. This closely mimics the architecture of light-harlight-harvesting structure–function relationships that are
vesting complexes found in photosynthetic purple bacteria.
otherwise intractable except by theoretical calculations. OngoGel permeation chromatography indicated that theing questions on the molecular-level mechanism of lightintended macroring was synthesized, rather than a linear
harvesting can be addressed systematically and quantitatively
polymer. Atomic force microscopy indicated that the complex
through this research. Such knowledge could help researchersconsisted of round particles ~1.5 nanometers in height, consisoptimize light harvesting in technology that does not depend
tent with the intended barrel shape. Small-angle X-rayon biology, such as organic solar cells.
scattering indicated a particle radius of gyration of ~41
angstroms, consistent with the outer diameter predicted by
Referencemolecular mechanics simulations.
Takahashi, R., Kobuke, Y. 2003. Hexameric macroring of gable-porphyrins as a
To demonstrate that the macroring was capable of light
light-harvesting antenna mimic. Journal of the American Chemical Society, 125,
2372–2373.
harvesting, the researchers used ultraviolet-visible spectros-

38

Applied Physics

30 YEARS OF HISTORY: 2001– 2010

5 compared with the light-beam-induced current method,
olycrystalline silicon is a major raw material for themeaning that smaller defects in the cell could be identified
global solar cell industry and is a workhorse thatand resolved.
converts sunlight into electricity. It is imperative tocheck solar cell performance during the manufacturing process
To further verify these results, the researchers quantitatedfor rapid quality control, and this is doubly important
the relationship between the measured electroluminescenceand the calibrated MCDL. The results demonstrated that awith the increasing demand for photovoltaic systems
straightforward photographic survey of electroluminescenceas the world begins to take the threat of climate change
is a reliable approach for determining the MCDL in silicon
seriously. However, commonly used simple checks – such as
solar cells, though not with absolute precision.
current/voltage output under simulated light – do not provide
These results provide a means of rigorously evaluating
sufficiently thorough metrics to correct manufacturing
problems. Thus, it can be difficult to correct solar cell
the quality of polycrystalline solar cells by using common
fabrication errors in real time, which increases costs for
instrumentation. The solar cell industry can thus identify
defects in the production line in real time without investing in
the consumer.
A fundamental parameter for rigorously evaluating thespecialized instrumentation or adding significant time to theperformance of solar cells is the minority carrier diffusion
manufacturing process. This development provided a solution
to industry that will ultimately lower the cost of solar cells
length (MCDL). This parameter is pertinent to the efficiency
to global consumers and increase the economic feasibility ofof a cell's energy production. However, the laboratory-grade
solar energy.
instrumentation that works for quantita ting the MCDL isn’t
suitable for the production line. A research group led by
Takashi Fuyuki at NAIST explored the use of a routine imaging
technology – a charge-coupled device (CCD) camera – toReferencequantitate the electroluminescence and thus estimate the
Fuyuki, T., Kondo, H., Yamazaki, T., Takahashi, Y., Uraoka, Y. 2005. Photographic
MCDL of polycrystalline silicon solar cells. The benefit ofsurveying of minority carrier diffusion length in polycrystalline silicon solar cells
by electroluminescence. Applied Physics Letters, 86, 262108.
having this MCDL estimation is that it enables rapid detectionof deteriorated areas in the cells.
The imaging design was based onelectroluminescence: applying a voltage toa solar cell and measuring the resulting
emitted infrared light with a CCD camera.
The intensity of the emitted light can beused to estimate the MCDL. The electroluminescence results were then comparedwith a standard research method –light-beam-induced current – where theyused four wavelengths of light from thered to near-infrared range to exactly
quantitate the MCDL. The distribution of
the intensity of the electroluminescenceemitted by the solar cell as measured by
the CCD camera corresponded to themapping of the MCDL. Specifically,
regions in the cell that appeared dark byelectroluminescence were areas of short
MCDL. Furthermore, the spatial resolution of the electroluminescence method
Distribution of the minority carrier diffusion length in polycrystalline Si cell(Fuyuki et al., 2005, Appl Phys Lett).
was improved by an approximate factor of

NAIST Research Highlights

39

Chemistry

30 YEARS OF HISTORY: 2001– 2010

Takashi Fuyuki

A synthetic mimic of a biologicallight-harvesting molecule

Rapid imaging of polycrystalline
silicon solar cell defects

A self-assembled, porphyrin-based barrel structure is synthesized, enabling
structure–function insights into light harvesting by photosynthetic organisms

Quantitating the main parameter that indicates the efficiency of solar cells with routine
instrumentation enables performance evaluations during mass production

P

P

NAIST Research Highlights© 2003 American Chemical Society

Yoshiaki Kobuke

hotosynthetic organisms use intricate
macromolecular assemblies to absorb
solar energy for subsequent conversion
into the chemical energy necessary to perform
life functions. However, researchers have been
unable to fabricate close synthetic mimics ofthese assemblies, a necessary step to understandhow the assemblies work and how to apply themto modern technology. Thus, it can be difficult
to optimize light harvesting in artificial
constructs, such as in solar cells that may exceed
the efficiency of even the most advanced
silicon-based designs in use today.
The first step of photosynthesis is absorptionof solar energy by light-harvesting complexes
that are arranged in a barrel architecture. Artificial analogs of such complexes, based on
porphyrin rings, have been synthesized.
However, the foundational porphyrin macroring architecture in biology – based on dimers
arranged in a slipped-cofacial geometry – has
not been synthesized. A research team led by
Yoshiaki Kobuke at NAIST published work on
the self-assembly of a supramolecule which has
intramolecular orientations and distances thatreflect the architecture of biological light-harA structural model of the target cyclic hexamer (Takahashi & Kobuke, 2003, J Am Chem Soc).
vesting complexes, as confirmed by rigorous
experimental characterization.
copy to quantitate its light absorption. Bands characteristic of
The macroring design was based on two principles. First,
the expected porphyrin absorption were present. Furthermore,
the geometry of the zinc-containing porphyrin-based dimer
the fluorescence quantum yield of the macroring relative
facilitates light absorption and minimizes energy waste after
to that of the porphyrin dimer subunits was 0.51, which islight absorption. Second, the geometry of the phenylene spacer
consistent with analogous observations in bacterial
that links each dimer enables ring closure into a barrel architeclight-harvesting complexes.
ture which has center-to-center distances of 6.1 and 11.0
These results provide a means of experimentally testing
angstroms. This closely mimics the architecture of light-harlight-harvesting structure–function relationships that are
vesting complexes found in photosynthetic purple bacteria.
otherwise intractable except by theoretical calculations. OngoGel permeation chromatography indicated that theing questions on the molecular-level mechanism of lightintended macroring was synthesized, rather than a linear
harvesting can be addressed systematically and quantitatively
polymer. Atomic force microscopy indicated that the complex
through this research. Such knowledge could help researchersconsisted of round particles ~1.5 nanometers in height, consisoptimize light harvesting in technology that does not depend
tent with the intended barrel shape. Small-angle X-rayon biology, such as organic solar cells.
scattering indicated a particle radius of gyration of ~41
angstroms, consistent with the outer diameter predicted by
Referencemolecular mechanics simulations.
Takahashi, R., Kobuke, Y. 2003. Hexameric macroring of gable-porphyrins as a
To demonstrate that the macroring was capable of light
light-harvesting antenna mimic. Journal of the American Chemical Society, 125,
2372–2373.
harvesting, the researchers used ultraviolet-visible spectros-

38

Applied Physics

30 YEARS OF HISTORY: 2001– 2010

5 compared with the light-beam-induced current method,
olycrystalline silicon is a major raw material for themeaning that smaller defects in the cell could be identified
global solar cell industry and is a workhorse thatand resolved.
converts sunlight into electricity. It is imperative tocheck solar cell performance during the manufacturing process
To further verify these results, the researchers quantitatedfor rapid quality control, and this is doubly important
the relationship between the measured electroluminescenceand the calibrated MCDL. The results demonstrated that awith the increasing demand for photovoltaic systems
straightforward photographic survey of electroluminescenceas the world begins to take the threat of climate change
is a reliable approach for determining the MCDL in silicon
seriously. However, commonly used simple checks – such as
solar cells, though not with absolute precision.
current/voltage output under simulated light – do not provide
These results provide a means of rigorously evaluating
sufficiently thorough metrics to correct manufacturing
problems. Thus, it can be difficult to correct solar cell
the quality of polycrystalline solar cells by using common
fabrication errors in real time, which increases costs for
instrumentation. The solar cell industry can thus identify
defects in the production line in real time without investing in
the consumer.
A fundamental parameter for rigorously evaluating thespecialized instrumentation or adding significant time to theperformance of solar cells is the minority carrier diffusion
manufacturing process. This development provided a solution
to industry that will ultimately lower the cost of solar cells
length (MCDL). This parameter is pertinent to the efficiency
to global consumers and increase the economic feasibility ofof a cell's energy production. However, the laboratory-grade
solar energy.
instrumentation that works for quantita ting the MCDL isn’t
suitable for the production line. A research group led by
Takashi Fuyuki at NAIST explored the use of a routine imaging
technology – a charge-coupled device (CCD) camera – toReferencequantitate the electroluminescence and thus estimate the
Fuyuki, T., Kondo, H., Yamazaki, T., Takahashi, Y., Uraoka, Y. 2005. Photographic
MCDL of polycrystalline silicon solar cells. The benefit ofsurveying of minority carrier diffusion length in polycrystalline silicon solar cells
by electroluminescence. Applied Physics Letters, 86, 262108.
having this MCDL estimation is that it enables rapid detectionof deteriorated areas in the cells.
The imaging design was based onelectroluminescence: applying a voltage toa solar cell and measuring the resulting
emitted infrared light with a CCD camera.
The intensity of the emitted light can beused to estimate the MCDL. The electroluminescence results were then comparedwith a standard research method –light-beam-induced current – where theyused four wavelengths of light from thered to near-infrared range to exactly
quantitate the MCDL. The distribution of
the intensity of the electroluminescenceemitted by the solar cell as measured by
the CCD camera corresponded to themapping of the MCDL. Specifically,
regions in the cell that appeared dark byelectroluminescence were areas of short
MCDL. Furthermore, the spatial resolution of the electroluminescence method
Distribution of the minority carrier diffusion length in polycrystalline Si cell(Fuyuki et al., 2005, Appl Phys Lett).
was improved by an approximate factor of

NAIST Research Highlights

39

Physics

30 YEARS OF HISTORY: 2001– 2010

Hiroshi Daimon

Biochemistry

30 YEARS OF HISTORY: 2001– 2010

Mikio Kataoka

First evidence for low-barrierhydrogen bonds
A combination of neutron and X-ray crystallography provides evidence for low-barrier hydrogenbonds in photoactive yellow protein, which may be important for fundamental insights
into intermolecular bonding

Atomic-layer resolution of
the magnetism of nanoscale nickel
Diffraction spectroscopy is developed for quantitating the magnetism of ultrathin nickel films, yielding
quantitative data that can be compared with theoretical studies of advanced data storage mediaMaximizing the density of a device's data storage capacity increases the processing speed and lowers the costof computer technology. However, there are ongoing
demands for a continually increasing data storage density;
diverse technical approaches are necessary to meet thesedemands and thus the increased memory needs of future
devices. Magnetism based on nanostructured nickel would
advance the state-of-the-art, but experiments have not beenable to confirm the outstanding potential suggested by theoretical work.
The layer-by-layer, atomic-scale architecture of ultrathin
films of nickel on copper is challenging to experimentallymeasure. There are means of interrogating, for example, themagnetic structure of the buried nickel–copper interface.
However, direct measurements at atomic-layer resolution are
required to experimentally test theoretical predictions ofmagnetism enabled by nanoscale architectures. A researchteam led by Hiroshi Daimon at NAIST aimed to address thislack of experimental testing by devising diffraction spectroscopy for direct, layer-by-layer measurements of the electronic
configuration of energetic atoms.
A new spectroscopy method was first used to measure theenergies and two-dimensional angular distributions ofelectrons emitted from ultrathin coatings of nickel on a copper(001) surface. Data that were consistent with nickel atoms inthe second or third layer of atoms were obtained from one- or
two-monolayer-thick coatings of nickel. This was direct
evidence that some of the nickel atoms mixed with the underlying copper.
X-ray absorption near-edge structure measurements werepossible up to a depth of 10 monolayers of nickel atoms. The

40

NAIST Research Highlights

results of these measurements differed depending on the depthof the atomic layer. One of the interpretations of theselayer-by-layer studies was that from the standpoint ofelectronic properties, the surface of the nickel consisted of only
three atomic layers.
To demonstrate unprecedented experimental interrogationof the advanced magnetic properties of nanoscale nickel, X-ray
magnetic circular dichroism measurements were taken. There
was in-plane magnetism for thin films consisting of eightmonolayers of nickel atoms, and perpendicular magnetism
when the film consisted of 15 monolayers. Thus, the spin
magnetic moment of the film could be altered by adjusting the
thickness of the film. This finding is consistent with theoreticalpredictions and is central to maximizing the efficiency ofcomputer memory technology.
These results provided a means of experimentally testing
theories of nanoscale magnetism. Ongoing questions of how
such magnetism depends on atomic-level organization within
ultrathin films of nickel can be addressed quantitatively
through this field of research inquiry. Such knowledge could
enable the high-density data storage – at a reasonable cost –that is essential for future high-speed computer technologies.

Reference
Matsui, F., Matsushita, T., Kato, Y., Hashimoto, M., Inaji, K., Guo, F.Z., Daimon, H. 2008.
Atomic-layer resolved magnetic and electronic structure analysis of Ni thin film ona Cu(001) surface by diffraction spectroscopy. Physical Review Letters, 100, 207201.

P

roteins and enzymes are important cogs in the machinwere able determine the locations of the hydrogen atoms
ery of our bodies, and investigating their molecularparticipating in the bonds of interest. They found that the SHB
structures – how their atoms bond and interact –between pCA and amino acid E46 is in fact a LBHB, while thefacilitates our understanding of their function and how they
other investigated SHB between pCA and amino acid Y42 is
not and could be considered a short ionic hydrogenwork. Armed with this knowledge, we can better counteract
bond (SIHB).
the proteins and enzymes involved in diseases, which is crucial
The study was the first definitive example of transient
for the design of new and improved drugs and therapies.
LBHBs being formed and provided important insight into the
Low-barrier hydrogen bonds (LBHBs) were first reported
to contribute to enzymatic processes and proton transfer in the
additional roles played by hydrogen bonds in protein function
1990s, with transient LBHBs being thought to stabilize– for example, influencing a protein’s ability to stabilize anintermediates during reactions. However, in the absence ofisolated charge and allow rapid proton transfer.
hard experimental evidence for the positions of hydrogen
The implications of the findings continue to be far-reachingfor the understanding of natural processes in our bodies andatoms, they proved a contentious concept until a research
the world around us, which in turn provides starting points forgroup led by Mikio Kataoka at NAIST demonstrated their
existence in photoactive yellow protein (PYP) in 2009.
developing vital therapeutics and treatments.
In contrast to standard hydrogen bonds where the dipolesof a donor and an acceptor atom interact, in LBHBs the hydroReference
gen atom is shared. As a result, LBHBs have a shorter bondYamaguchi, S., Kamikubo, H., Kurihara, K., Kuroki, R., Niimura, N., Shimizu, N.,
length than a standard hydrogen bond. Identifying the
Yamazaki, Y., Kataoka, M. 2009. Low-barrier hydrogen bond in photoactive yellow
bond therefore required determining the positions of theprotein. Proceedings of the National Academy of Sciences of the United States ofhydrogen atom and the acceptor and donor atoms. However,America, 106, 440–444.
because hydrogen atoms are so small, it is very difficult
to detect them with crystallography techniques, which
is why the subject was open to debate. To finally provide
the experimental evidence that was lacking, theresearchers investigated PYP using neutron and X-ray
crystallography together.
PYP is a light sensor protein found in certain
bacteria. It has a photoactive center that contains thesmall molecule p-coumaric acid (pCA) acid in a hydrophobic pocket. When light excites the pCA it
rearranges into a different structure and the thermal
reactions that follow cause reorganization of the hydrogen bonding structure.
Two short hydrogen bonds (SHBs) in PYP that arenear the reactive center are known to be present evenin the ground state. These SHBs were therefore selected
as the focus of the investigation. Neutron and X-ray
crystallography of PYP were achieved with resolutionsof 1.5- and 2.5-Å, respectively.
By refining both sets of data, the researchers wereable to achieve nuclear density maps for 87% of thehydrogen and deuterium atoms in the protein. And
based on the deuterium atoms – which have larger
Structure of PYP including hydrogen atom positions (Yamaguchi et al., 2009, PNAS).
nuclei making them easier to detect – the researchers© 2009 National Academy of Sciences

Ni L3-edge peak shift from bulk for each atomic layer in various Ni film thicknesses (Matsui et al., 2008, Phys Rev Lett).

NAIST Research Highlights

41

Physics

30 YEARS OF HISTORY: 2001– 2010

Hiroshi Daimon

Biochemistry

30 YEARS OF HISTORY: 2001– 2010

Mikio Kataoka

First evidence for low-barrierhydrogen bonds
A combination of neutron and X-ray crystallography provides evidence for low-barrier hydrogenbonds in photoactive yellow protein, which may be important for fundamental insights
into intermolecular bonding

Atomic-layer resolution of
the magnetism of nanoscale nickel
Diffraction spectroscopy is developed for quantitating the magnetism of ultrathin nickel films, yielding
quantitative data that can be compared with theoretical studies of advanced data storage mediaMaximizing the density of a device's data storage capacity increases the processing speed and lowers the costof computer technology. However, there are ongoing
demands for a continually increasing data storage density;
diverse technical approaches are necessary to meet thesedemands and thus the increased memory needs of future
devices. Magnetism based on nanostructured nickel would
advance the state-of-the-art, but experiments have not beenable to confirm the outstanding potential suggested by theoretical work.
The layer-by-layer, atomic-scale architecture of ultrathin
films of nickel on copper is challenging to experimentallymeasure. There are means of interrogating, for example, themagnetic structure of the buried nickel–copper interface.
However, direct measurements at atomic-layer resolution are
required to experimentally test theoretical predictions ofmagnetism enabled by nanoscale architectures. A researchteam led by Hiroshi Daimon at NAIST aimed to address thislack of experimental testing by devising diffraction spectroscopy for direct, layer-by-layer measurements of the electronic
configuration of energetic atoms.
A new spectroscopy method was first used to measure theenergies and two-dimensional angular distributions ofelectrons emitted from ultrathin coatings of nickel on a copper(001) surface. Data that were consistent with nickel atoms inthe second or third layer of atoms were obtained from one- or
two-monolayer-thick coatings of nickel. This was direct
evidence that some of the nickel atoms mixed with the underlying copper.
X-ray absorption near-edge structure measurements werepossible up to a depth of 10 monolayers of nickel atoms. The

40

NAIST Research Highlights

results of these measurements differed depending on the depthof the atomic layer. One of the interpretations of theselayer-by-layer studies was that from the standpoint ofelectronic properties, the surface of the nickel consisted of only
three atomic layers.
To demonstrate unprecedented experimental interrogationof the advanced magnetic properties of nanoscale nickel, X-ray
magnetic circular dichroism measurements were taken. There
was in-plane magnetism for thin films consisting of eightmonolayers of nickel atoms, and perpendicular magnetism
when the film consisted of 15 monolayers. Thus, the spin
magnetic moment of the film could be altered by adjusting the
thickness of the film. This finding is consistent with theoreticalpredictions and is central to maximizing the efficiency ofcomputer memory technology.
These results provided a means of experimentally testing
theories of nanoscale magnetism. Ongoing questions of how
such magnetism depends on atomic-level organization within
ultrathin films of nickel can be addressed quantitatively
through this field of research inquiry. Such knowledge could
enable the high-density data storage – at a reasonable cost –that is essential for future high-speed computer technologies.

Reference
Matsui, F., Matsushita, T., Kato, Y., Hashimoto, M., Inaji, K., Guo, F.Z., Daimon, H. 2008.
Atomic-layer resolved magnetic and electronic structure analysis of Ni thin film ona Cu(001) surface by diffraction spectroscopy. Physical Review Letters, 100, 207201.

P

roteins and enzymes are important cogs in the machinwere able determine the locations of the hydrogen atoms
ery of our bodies, and investigating their molecularparticipating in the bonds of interest. They found that the SHB
structures – how their atoms bond and interact –between pCA and amino acid E46 is in fact a LBHB, while thefacilitates our understanding of their function and how they
other investigated SHB between pCA and amino acid Y42 is
not and could be considered a short ionic hydrogenwork. Armed with this knowledge, we can better counteract
bond (SIHB).
the proteins and enzymes involved in diseases, which is crucial
The study was the first definitive example of transient
for the design of new and improved drugs and therapies.
LBHBs being formed and provided important insight into the
Low-barrier hydrogen bonds (LBHBs) were first reported
to contribute to enzymatic processes and proton transfer in the
additional roles played by hydrogen bonds in protein function
1990s, with transient LBHBs being thought to stabilize– for example, influencing a protein’s ability to stabilize anintermediates during reactions. However, in the absence ofisolated charge and allow rapid proton transfer.
hard experimental evidence for the positions of hydrogen
The implications of the findings continue to be far-reachingfor the understanding of natural processes in our bodies andatoms, they proved a contentious concept until a research
the world around us, which in turn provides starting points forgroup led by Mikio Kataoka at NAIST demonstrated their
existence in photoactive yellow protein (PYP) in 2009.
developing vital therapeutics and treatments.
In contrast to standard hydrogen bonds where the dipolesof a donor and an acceptor atom interact, in LBHBs the hydroReference
gen atom is shared. As a result, LBHBs have a shorter bondYamaguchi, S., Kamikubo, H., Kurihara, K., Kuroki, R., Niimura, N., Shimizu, N.,
length than a standard hydrogen bond. Identifying the
Yamazaki, Y., Kataoka, M. 2009. Low-barrier hydrogen bond in photoactive yellow
bond therefore required determining the positions of theprotein. Proceedings of the National Academy of Sciences of the United States ofhydrogen atom and the acceptor and donor atoms. However,America, 106, 440–444.
because hydrogen atoms are so small, it is very difficult
to detect them with crystallography techniques, which
is why the subject was open to debate. To finally provide
the experimental evidence that was lacking, theresearchers investigated PYP using neutron and X-ray
crystallography together.
PYP is a light sensor protein found in certain
bacteria. It has a photoactive center that contains thesmall molecule p-coumaric acid (pCA) acid in a hydrophobic pocket. When light excites the pCA it
rearranges into a different structure and the thermal
reactions that follow cause reorganization of the hydrogen bonding structure.
Two short hydrogen bonds (SHBs) in PYP that arenear the reactive center are known to be present evenin the ground state. These SHBs were therefore selected
as the focus of the investigation. Neutron and X-ray
crystallography of PYP were achieved with resolutionsof 1.5- and 2.5-Å, respectively.
By refining both sets of data, the researchers wereable to achieve nuclear density maps for 87% of thehydrogen and deuterium atoms in the protein. And
based on the deuterium atoms – which have larger
Structure of PYP including hydrogen atom positions (Yamaguchi et al., 2009, PNAS).
nuclei making them easier to detect – the researchers© 2009 National Academy of Sciences

Ni L3-edge peak shift from bulk for each atomic layer in various Ni film thicknesses (Matsui et al., 2008, Phys Rev Lett).

NAIST Research Highlights

41

Biochemistry

30 YEARS OF HISTORY: 2001– 2010

Shun Hirota

Trading helices leads toprotein polymerization
Structural studies reveal a protein polymerization mechanism where a helix fromone monomer dissociates and plugs into another in a successive chain-linking reaction,
thus providing broad insights into disease-causing protein fibril formation

C© 2010 National Academy of Sciences

ytochrome c (cyt c) is a heme-containing protein that plays a key role inmitochondrial respiration, although it
is also involved in the steps preceding
apoptosis (cell death). This protein naturally
exists and functions as a monomer (single
protein unit) comprising three α-helices, but
it is also known to form polymers or
oligomers (comprising many or several
protein units, respectively), albeit through anundetermined mechanism. In fact, when cyt
c is heated, monomers aggregate to form
amyloid fibrils similar to those associatedwith neurodegenerative disorders, such as
Parkinson’s disease. Therefore, understanding
the structural changes and thermodynamics
Schematic summary of cyt c polymerization (Hirota et al., 2010, PNAS).
that govern cyt c oligomerization could
provide critical insights into other protein
because the ethanol solvent can stabilize the lone helix to allow
polymerization processes and hence, numerous otherpathologies and potential therapies.
for successive domain-swapping processes. Indeed, cyt cTo this end, a research group led by Shun Hirota at NAIST
polymers with up to 50 protein units were identified withconfigurations similar to those of the smaller oligomers.
treated monomeric cyt c with ethanol and isolated its dimer,
trimer and tetramer forms. The X-ray crystal structure of
The oligomerization was also monitored according to thethe dimer revealed that the C-terminal helices of the twoheme-iron environment in cyt c, specifically, the coordination
monomers had swapped places; that is, a helix from one monoof an axial methionine (Met) residue. This aspect was particumer now occupied the analogous helix’s place in the otherlarly crucial because Met dissociation from the iron allows formonomer’s folded structure, and vice versa, thus linking thehydroxide coordination, which triggers peroxidase activity –two protein units into a “closed-end mutually swapped” dimer.
an initial step of apoptosis. Disruption of the Met-iron coordiIn the trimer’s structure, the C-terminal helix displaced fromnation during cyt c oligomerization enabled the C-terminal
the second monomer was instead inserted into a third
helix swap, but the original heme coordination environment
could be recovered upon heating to obtain the monomers.
monomer’s structure whose displaced helix was fit into
Moreover, the Met-iron de-coordination contributed 90% of
the first monomer’s vacant site, thus forming a “cyclic”
trimer structure. These results confirmed the so-called
the thermodynamic driving force for oligomer dissociation.
domain-swapping phenomenon.
The characterization of cyt c oligomers confirmed a succesThe oligomers were all stable at low temperature but could
sive C-terminal domain-swapping mechanism, thus offering
be converted back to the monomer form by heating or addition
key insights into protein polymerization processes, includingof surfactant. Certain other additives could convert the less-stathose linked to disease states. This knowledge presented a step
ble linear configurations (where the final C-terminal helix does
forward in understanding the pathogenesis of such diseases,
not occupy the vacant region of another monomer but insteadand may assist in the development of therapeutic interventions.
dangles in an open-ended fashion) to the mutually swapped
dimer or cyclic trimer structures. Together, these results
Referencesuggested a certain equilibrium between the monomer and
Hirota, S., Hattori, Y., Nagao, S., Taketa, M., Komori, H., Kamikubo, H., Wang, Z.,oligomer forms, and between the linear (open) and closed
Takahashi, I., Negi, S., Sugiura, Y., Kataoka, M., Higuchi, Y. 2010. Cytochrome cstructures. The linear forms with “free” helix protrusions
polymerization by successive domain swapping at the C-terminal helix. Proceedings
highlight the potential for higher-order polymeric structuresof the National Academy of Sciences of the United States of America, 107, 12854–12859.

42

NAIST Research Highlights

30 YEARS OF HISTORY

2011 – 2021

Biochemistry

30 YEARS OF HISTORY: 2001– 2010

Shun Hirota

Trading helices leads toprotein polymerization
Structural studies reveal a protein polymerization mechanism where a helix fromone monomer dissociates and plugs into another in a successive chain-linking reaction,
thus providing broad insights into disease-causing protein fibril formation

C© 2010 National Academy of Sciences

ytochrome c (cyt c) is a heme-containing protein that plays a key role inmitochondrial respiration, although it
is also involved in the steps preceding
apoptosis (cell death). This protein naturally
exists and functions as a monomer (single
protein unit) comprising three α-helices, but
it is also known to form polymers or
oligomers (comprising many or several
protein units, respectively), albeit through anundetermined mechanism. In fact, when cyt
c is heated, monomers aggregate to form
amyloid fibrils similar to those associatedwith neurodegenerative disorders, such as
Parkinson’s disease. Therefore, understanding
the structural changes and thermodynamics
Schematic summary of cyt c polymerization (Hirota et al., 2010, PNAS).
that govern cyt c oligomerization could
provide critical insights into other protein
because the ethanol solvent can stabilize the lone helix to allow
polymerization processes and hence, numerous otherpathologies and potential therapies.
for successive domain-swapping processes. Indeed, cyt cTo this end, a research group led by Shun Hirota at NAIST
polymers with up to 50 protein units were identified withconfigurations similar to those of the smaller oligomers.
treated monomeric cyt c with ethanol and isolated its dimer,
trimer and tetramer forms. The X-ray crystal structure of
The oligomerization was also monitored according to thethe dimer revealed that the C-terminal helices of the twoheme-iron environment in cyt c, specifically, the coordination
monomers had swapped places; that is, a helix from one monoof an axial methionine (Met) residue. This aspect was particumer now occupied the analogous helix’s place in the otherlarly crucial because Met dissociation from the iron allows formonomer’s folded structure, and vice versa, thus linking thehydroxide coordination, which triggers peroxidase activity –two protein units into a “closed-end mutually swapped” dimer.
an initial step of apoptosis. Disruption of the Met-iron coordiIn the trimer’s structure, the C-terminal helix displaced fromnation during cyt c oligomerization enabled the C-terminal
the second monomer was instead inserted into a third
helix swap, but the original heme coordination environment
could be recovered upon heating to obtain the monomers.
monomer’s structure whose displaced helix was fit into
Moreover, the Met-iron de-coordination contributed 90% of
the first monomer’s vacant site, thus forming a “cyclic”
trimer structure. These results confirmed the so-called
the thermodynamic driving force for oligomer dissociation.
domain-swapping phenomenon.
The characterization of cyt c oligomers confirmed a succesThe oligomers were all stable at low temperature but could
sive C-terminal domain-swapping mechanism, thus offering
be converted back to the monomer form by heating or addition
key insights into protein polymerization processes, includingof surfactant. Certain other additives could convert the less-stathose linked to disease states. This knowledge presented a step
ble linear configurations (where the final C-terminal helix does
forward in understanding the pathogenesis of such diseases,
not occupy the vacant region of another monomer but insteadand may assist in the development of therapeutic interventions.
dangles in an open-ended fashion) to the mutually swapped
dimer or cyclic trimer structures. Together, these results
Referencesuggested a certain equilibrium between the monomer and
Hirota, S., Hattori, Y., Nagao, S., Taketa, M., Komori, H., Kamikubo, H., Wang, Z.,oligomer forms, and between the linear (open) and closed
Takahashi, I., Negi, S., Sugiura, Y., Kataoka, M., Higuchi, Y. 2010. Cytochrome cstructures. The linear forms with “free” helix protrusions
polymerization by successive domain swapping at the C-terminal helix. Proceedings
highlight the potential for higher-order polymeric structuresof the National Academy of Sciences of the United States of America, 107, 12854–12859.

42

NAIST Research Highlights

30 YEARS OF HISTORY

2011 – 2021

Molecular Biology

30 YEARS OF HISTORY: 2011– 2021

Kenji Kohno© iStock/ artur Pavliuk

Endoplasmic reticulum stress:
How the cell prevents
misfolded proteinsmRNA encodes the transcription factor that controls the ER-stress regulation
pathway, facilitating its instant reaction to the build-up of ER stressT

he endoplasmic reticulum (ER) serves as a site forprotein synthesis and folding in cells and thus involves
a major interplay of many important factors. For certain
proteins, translation begins at ribosomes throughout thecytosol, but association with the ER and completion of translation require the signal recognition particle (SRP). This critical
function means that the ER is susceptible to effects of cellularstress conditions, such as a viral infection. ER stress occurs
when there is an imbalance between new proteins entering the
ER and its folding capacity, and the ER cannot keep up with its
expected workload. Understanding the mechanisms
controlling this is important, because ER stress can lead to
misfolded proteins and is associated with the pathogenesis ofsome degenerative disorders like Alzheimer’s disease.
The inositol requiring enzyme 1α (IRE1α)-X-box-binding
protein 1 (XBP1) pathway is the main eukaryotic mechanism
for addressing ER stress. IRE1α is an ER membrane protein thatis activated upon ER stress, which allows its RNase domain tocleave two sites of XBP1 precursor mRNA (XBP1u). This
spliced mRNA (XBP1s) encodes the XBP1s transcription
factor, which promotes the transcription of ER stress-alleviating genes.
Because of the importance of ER stress in human disease,this pathway became a primary focus of a research group led
by Kenji Kohno at NAIST. The group previously showed that
the XBP1u-encoded protein, known as the mRNA-ribosome-nascent chain (R-RNC), can recruit the XBP1umRNA-ribosome complex to the ER membrane through its
hydrophobic region 2 (HR2) as it is being translated, serving
to increase splicing efficiency during ER stress. In a 2011 paper
published in Science, the group used in vitro translation experiments to demonstrate that XBP1u mRNA contains a signal thatcauses translational pausing. Serial truncation of a luciferase-XBP1u fusion protein indicated that the translationalpausing is mediated by a C-terminal
26 amino acid region of XBP1u.
Interestingly, this region is evolutionarily conserved, indicating thatits function is important and not
limited to certain species.
The in vitro observations werealso seen in mammalian celllines, where it was determinedthat L246A and W256A mutations
could prevent the translationalpausing, while the S255A mutation
could increase it. Additionalin vitro translation experiments
using pausing-defective L246A andA model for nascent chain-mediated membrane-targeting of the XBP1u mRNA (Yanagitani et al., 2011, Science).

44

NAIST Research Highlights

W256A mutants resulted in XBP1u peptide accumulation in
the soluble fraction rather than the membrane fraction, implying that translational pausing is necessary for the XBP1uprotein to be targeted to the ER membrane. Similar findings
were seen in vivo in HEK293T cells. Furthermore, the L246A
and W256A mutants had significantly reduced splicing
efficiency of XBP1u mRNA, suggesting that translationalpausing is also important for splicing.
The exact details and mechanism governing the recruitment
of XBP1u-RNC to the ER were not fully clarified in the paper
published in 2011. The team continued investigating thispathway and published a follow-up article in PNAS in 2016.
Using immunofluorescence, they observed XBP1u protein
localized in the ER and absent from the mitochondria and
Golgi, suggesting it is specific to the ER membrane. A
protease-treated microsome showed significantly lower affinity
to XBP1u, implying that an ER membrane-based protein
component is needed for this protein to be targeted there.
The research group investigated this protein component
further through coimmunoprecipitation and mass spectrometry approaches. Proteins associated with SRP and the Sec61
translocon were identified and confirmed to interact with
XBP1u. Because the Sec61 translocon is a membrane protein
complex involved with transport to the ER, the researchers
hypothesized that SRP recognizes the XBP1u-RNC andrecruits it to the Sec61 translocon. However, a mechanism
controlling this required further study. The association with
SRP was confirmed to be in an HR2-dependent manner.
Following membrane-flotation assays, it became evident thatthe presence of SRP is indispensable for XBP1u-RNC to betargeted to the ER. This was also seen in cells, where knockdown of SRP54 protein resulted in inhibited ER targeting of

XBP1u mRNA, as well as a modest reduction of XBP1u splicing
efficiency under ER stress.
Furthermore, observed interactions of XBP1u-RNC with
SRP and the translocon were weakened with the W256A
mutant and strengthened with the S255A mutant, suggestingthat the translational pausing is critical for these associations.
It was also shown that XBP1u does not enter into the ER lumen
but stays on the membrane, implying that a noncanonicalmechanism is at play.
These groundbreaking findings by Kohno and colleagues
identify a novel mechanism where HR2 in XBP1u-RNC is
recognized by SRP but does not result in translocon pore
opening and entrance to the ER lumen. Furthermore, their data
highlight the critical need for translational pausing for this toproceed properly and the cell to efficiently respond to ER stress.
This work may lead to the identification of other mRNAs thatare targeted and spliced in this manner. Importantly, a better
understanding of how the cell responds to ER stress can aid in
the investigation behind the mechanisms of conditions such as
Alzheimer’s disease which are associated with misfolded
proteins. Gaining clarity over the innate cellular mechanisms
responsible for preventing protein misfolding may strengthen
efforts to develop therapies for treating diseases associated with
ER stress.
References
Yanagitani, K., Kimata, Y., Kadokura, H., Kohno, K. 2011. Translational pausing
ensures membrane targeting and cytoplasmic splicing of XBP1u mRNA. Science,
331, 586–589.
Kanda, S., Yanagitani, K., Yokota, Y., Esaki, Y., Kohno, K. 2016. Autonomoustranslational pausing is required for XBP1u mRNA recruitment to the ER via the
SRP pathway. Proceedings of the National Academy of Sciences of the United States ofAmerica, 113, E5886–E5895.

NAIST Research Highlights

45

Molecular Biology

30 YEARS OF HISTORY: 2011– 2021

Kenji Kohno© iStock/ artur Pavliuk

Endoplasmic reticulum stress:
How the cell prevents
misfolded proteinsmRNA encodes the transcription factor that controls the ER-stress regulation
pathway, facilitating its instant reaction to the build-up of ER stressT

he endoplasmic reticulum (ER) serves as a site forprotein synthesis and folding in cells and thus involves
a major interplay of many important factors. For certain
proteins, translation begins at ribosomes throughout thecytosol, but association with the ER and completion of translation require the signal recognition particle (SRP). This critical
function means that the ER is susceptible to effects of cellularstress conditions, such as a viral infection. ER stress occurs
when there is an imbalance between new proteins entering the
ER and its folding capacity, and the ER cannot keep up with its
expected workload. Understanding the mechanisms
controlling this is important, because ER stress can lead to
misfolded proteins and is associated with the pathogenesis ofsome degenerative disorders like Alzheimer’s disease.
The inositol requiring enzyme 1α (IRE1α)-X-box-binding
protein 1 (XBP1) pathway is the main eukaryotic mechanism
for addressing ER stress. IRE1α is an ER membrane protein thatis activated upon ER stress, which allows its RNase domain tocleave two sites of XBP1 precursor mRNA (XBP1u). This
spliced mRNA (XBP1s) encodes the XBP1s transcription
factor, which promotes the transcription of ER stress-alleviating genes.
Because of the importance of ER stress in human disease,this pathway became a primary focus of a research group led
by Kenji Kohno at NAIST. The group previously showed that
the XBP1u-encoded protein, known as the mRNA-ribosome-nascent chain (R-RNC), can recruit the XBP1umRNA-ribosome complex to the ER membrane through its
hydrophobic region 2 (HR2) as it is being translated, serving
to increase splicing efficiency during ER stress. In a 2011 paper
published in Science, the group used in vitro translation experiments to demonstrate that XBP1u mRNA contains a signal thatcauses translational pausing. Serial truncation of a luciferase-XBP1u fusion protein indicated that the translationalpausing is mediated by a C-terminal
26 amino acid region of XBP1u.
Interestingly, this region is evolutionarily conserved, indicating thatits function is important and not
limited to certain species.
The in vitro observations werealso seen in mammalian celllines, where it was determinedthat L246A and W256A mutations
could prevent the translationalpausing, while the S255A mutation
could increase it. Additionalin vitro translation experiments
using pausing-defective L246A andA model for nascent chain-mediated membrane-targeting of the XBP1u mRNA (Yanagitani et al., 2011, Science).

44

NAIST Research Highlights

W256A mutants resulted in XBP1u peptide accumulation in
the soluble fraction rather than the membrane fraction, implying that translational pausing is necessary for the XBP1uprotein to be targeted to the ER membrane. Similar findings
were seen in vivo in HEK293T cells. Furthermore, the L246A
and W256A mutants had significantly reduced splicing
efficiency of XBP1u mRNA, suggesting that translationalpausing is also important for splicing.
The exact details and mechanism governing the recruitment
of XBP1u-RNC to the ER were not fully clarified in the paper
published in 2011. The team continued investigating thispathway and published a follow-up article in PNAS in 2016.
Using immunofluorescence, they observed XBP1u protein
localized in the ER and absent from the mitochondria and
Golgi, suggesting it is specific to the ER membrane. A
protease-treated microsome showed significantly lower affinity
to XBP1u, implying that an ER membrane-based protein
component is needed for this protein to be targeted there.
The research group investigated this protein component
further through coimmunoprecipitation and mass spectrometry approaches. Proteins associated with SRP and the Sec61
translocon were identified and confirmed to interact with
XBP1u. Because the Sec61 translocon is a membrane protein
complex involved with transport to the ER, the researchers
hypothesized that SRP recognizes the XBP1u-RNC andrecruits it to the Sec61 translocon. However, a mechanism
controlling this required further study. The association with
SRP was confirmed to be in an HR2-dependent manner.
Following membrane-flotation assays, it became evident thatthe presence of SRP is indispensable for XBP1u-RNC to betargeted to the ER. This was also seen in cells, where knockdown of SRP54 protein resulted in inhibited ER targeting of

XBP1u mRNA, as well as a modest reduction of XBP1u splicing
efficiency under ER stress.
Furthermore, observed interactions of XBP1u-RNC with
SRP and the translocon were weakened with the W256A
mutant and strengthened with the S255A mutant, suggestingthat the translational pausing is critical for these associations.
It was also shown that XBP1u does not enter into the ER lumen
but stays on the membrane, implying that a noncanonicalmechanism is at play.
These groundbreaking findings by Kohno and colleagues
identify a novel mechanism where HR2 in XBP1u-RNC is
recognized by SRP but does not result in translocon pore
opening and entrance to the ER lumen. Furthermore, their data
highlight the critical need for translational pausing for this toproceed properly and the cell to efficiently respond to ER stress.
This work may lead to the identification of other mRNAs thatare targeted and spliced in this manner. Importantly, a better
understanding of how the cell responds to ER stress can aid in
the investigation behind the mechanisms of conditions such as
Alzheimer’s disease which are associated with misfolded
proteins. Gaining clarity over the innate cellular mechanisms
responsible for preventing protein misfolding may strengthen
efforts to develop therapies for treating diseases associated with
ER stress.
References
Yanagitani, K., Kimata, Y., Kadokura, H., Kohno, K. 2011. Translational pausing
ensures membrane targeting and cytoplasmic splicing of XBP1u mRNA. Science,
331, 586–589.
Kanda, S., Yanagitani, K., Yokota, Y., Esaki, Y., Kohno, K. 2016. Autonomoustranslational pausing is required for XBP1u mRNA recruitment to the ER via the
SRP pathway. Proceedings of the National Academy of Sciences of the United States ofAmerica, 113, E5886–E5895.

NAIST Research Highlights

45

Developmental Biology

30 YEARS OF HISTORY: 2011– 2021

Identifying the key control center for
sympathetic nervous systemdevelopment in early embryos
The diversification of the sympathetic nervous system is coordinated by signaling from the dorsal aorta,enhancing our fundamental understanding of a key step in embryonic developmentThe autonomic nervous system (ANS) plays a key role invarious involuntary physiological functions including
stress defenses (fight-or-flight response) and homeostasis. ANS dysfunction can occur by itself or in diseases such as
diabetes and autoimmune disorders, and usually results in an
inability to properly regulate biological processes such as bloodpressure or heart rate. Proper functioning of the ANS requires
multiple cooperating cell types; however, how the different celllineages of the ANS develop in the early embryo remainedlargely unknown.
The ANS comprises a variety of cell types that are responsible for specific processes. These include the adrenal medullathat secrete hormones such as adrenaline in response to stress,
the sympathetic neurons that control the body’s rapid responseto stressful stimuli, and the parasympathetic neurons that
control involuntary functions of the body at rest, such as salivation and digestion. The sympathetic (Sg) and medullary (Am)lineages both develop from common progenitor cells, known
as SA progenitors, that are formed from neural crest cells

46

NAIST Research Highlights

during embryogenesis.
Previous research had shown that the SA progenitors firstmigrate towards the dorsal aorta, the first blood vessel that
forms in a developing embryo. Then, in the region around theaorta, they begin to differentiate into the separate Sg and Am
lineages. This indicated that signaling from the dorsal aorta
may play a role in this differentiation process. However, theexact mechanism of action had proved difficult to uncover,because it was very challenging to manipulate both the dorsal
aorta and the cells of the SA lineage separately yet at the sametime in a single embryo.
In 2012, a research team led by Yoshiko Takahashi at NAIST
identified the dorsal aorta as a key relay point that coordinatedthe diversification process using a combination of mouse
genetics and avian gene manipulation to show that the dorsal
aorta acts as a critical developmental relay point, known as amorphogenetic center, for SA progenitor cell migration andthe subsequent separation of Sg and Am cells. Bone morphogenetic proteins (BMPs), a class of signaling molecules, were also

© Adobe Stock/ solvod

Yoshiko Takahashi

shown to play a key role in this process.
Using cell lines in which the expression of certain genes had
been reduced or removed, alongside antibody staining todetermine the precise locations of cells that were expressing
key proteins, the team showed that BMPs from the aorta
induced the expression of two other cytokines in the region
around the aorta. These cytokines are known as SDF1 (stromalcell-derived factor 1) and NRG1 (neuregulin 1). The expressionof SDF1 and NRG1 went on to directly regulate the differentiation of the SA progenitor cells.
In vivo tissue transplantation experiments and in vitro
time-lapse imaging were then used to show that SDF1 and
NRG1 were responsible for the migration of the SA progenitor
cells towards the dorsal aorta by acting as chemical “attractants.” They also showed that inhibition of the receptors of
SDF1 and NRG1 interfered with the accumulation of SA
progenitor cells around the dorsal aorta. Transplanting a pieceof dorsal aorta or an aggregate of cells that were producing
SDF1 and NRG1 elsewhere in the embryo caused the SA

progenitor cells to migrate towards the implanted tissue andform an ectopic accumulation.
Upon further investigation into the segregation of the SA
progenitor cells into the Sg and Am lineages, BMP signaling
was found to be essential. The dorsal aorta is the most likely
source of the BMP signaling molecules BMP4 and BMP7.
These molecules caused segregation of the Am cells from theSg cells, and also set up a localized pattern of the cytokine
NRG1. NRG1 then attracted the Am cells, causing a physicaldisplacement of the different cell types.
This study outlined the mechanisms, both molecular andcellular, that lead to the establishment of both the sympathetic
nervous system and the adrenal medulla. The dorsal aorta is a
critical control point for all three processes: the migration of
the SA progenitor cells, the segregation of the Sg and Am cells,
and the subsequent movement of the Am cells. This process
involves multiple factors that are regulated not only temporally
throughout the process but also spatially within the embryo.
The cells alter how responsive they are to the various signaling
factors that they encounter. This allows the various events tooccur in the correct sequence to result in the different cell types
being established in the appropriate regions of the embryo.
This work provided significant insight into a complex
cascade of signaling which controls one of the earliest interactions between neurons and blood vessels guiding thedevelopment of embryos. The outcomes of this study support
the identification of new therapeutic targets for the treatment
of developmental disorders of the autonomic nervous system.

SDF1 and NRG1 act directly on migrating SA
progenitors (NT, neural tube; n, notochord; DA, dorsal
aorta) (Saito et al., 2012, Science).

Reference
Saito, D., Takase, Y., Murai, H., Takahashi, Y. 2012. The dorsal aorta initiates amolecular cascade that instructs sympatho-adrenal specification. Science, 336,
1578–1581.

NAIST Research Highlights

47

Developmental Biology

30 YEARS OF HISTORY: 2011– 2021

Identifying the key control center for
sympathetic nervous systemdevelopment in early embryos
The diversification of the sympathetic nervous system is coordinated by signaling from the dorsal aorta,enhancing our fundamental understanding of a key step in embryonic developmentThe autonomic nervous system (ANS) plays a key role invarious involuntary physiological functions including
stress defenses (fight-or-flight response) and homeostasis. ANS dysfunction can occur by itself or in diseases such as
diabetes and autoimmune disorders, and usually results in an
inability to properly regulate biological processes such as bloodpressure or heart rate. Proper functioning of the ANS requires
multiple cooperating cell types; however, how the different celllineages of the ANS develop in the early embryo remainedlargely unknown.
The ANS comprises a variety of cell types that are responsible for specific processes. These include the adrenal medullathat secrete hormones such as adrenaline in response to stress,
the sympathetic neurons that control the body’s rapid responseto stressful stimuli, and the parasympathetic neurons that
control involuntary functions of the body at rest, such as salivation and digestion. The sympathetic (Sg) and medullary (Am)lineages both develop from common progenitor cells, known
as SA progenitors, that are formed from neural crest cells

46

NAIST Research Highlights

during embryogenesis.
Previous research had shown that the SA progenitors firstmigrate towards the dorsal aorta, the first blood vessel that
forms in a developing embryo. Then, in the region around theaorta, they begin to differentiate into the separate Sg and Am
lineages. This indicated that signaling from the dorsal aorta
may play a role in this differentiation process. However, theexact mechanism of action had proved difficult to uncover,because it was very challenging to manipulate both the dorsal
aorta and the cells of the SA lineage separately yet at the sametime in a single embryo.
In 2012, a research team led by Yoshiko Takahashi at NAIST
identified the dorsal aorta as a key relay point that coordinatedthe diversification process using a combination of mouse
genetics and avian gene manipulation to show that the dorsal
aorta acts as a critical developmental relay point, known as amorphogenetic center, for SA progenitor cell migration andthe subsequent separation of Sg and Am cells. Bone morphogenetic proteins (BMPs), a class of signaling molecules, were also

© Adobe Stock/ solvod

Yoshiko Takahashi

shown to play a key role in this process.
Using cell lines in which the expression of certain genes had
been reduced or removed, alongside antibody staining todetermine the precise locations of cells that were expressing
key proteins, the team showed that BMPs from the aorta
induced the expression of two other cytokines in the region
around the aorta. These cytokines are known as SDF1 (stromalcell-derived factor 1) and NRG1 (neuregulin 1). The expressionof SDF1 and NRG1 went on to directly regulate the differentiation of the SA progenitor cells.
In vivo tissue transplantation experiments and in vitro
time-lapse imaging were then used to show that SDF1 and
NRG1 were responsible for the migration of the SA progenitor
cells towards the dorsal aorta by acting as chemical “attractants.” They also showed that inhibition of the receptors of
SDF1 and NRG1 interfered with the accumulation of SA
progenitor cells around the dorsal aorta. Transplanting a pieceof dorsal aorta or an aggregate of cells that were producing
SDF1 and NRG1 elsewhere in the embryo caused the SA

progenitor cells to migrate towards the implanted tissue andform an ectopic accumulation.
Upon further investigation into the segregation of the SA
progenitor cells into the Sg and Am lineages, BMP signaling
was found to be essential. The dorsal aorta is the most likely
source of the BMP signaling molecules BMP4 and BMP7.
These molecules caused segregation of the Am cells from theSg cells, and also set up a localized pattern of the cytokine
NRG1. NRG1 then attracted the Am cells, causing a physicaldisplacement of the different cell types.
This study outlined the mechanisms, both molecular andcellular, that lead to the establishment of both the sympathetic
nervous system and the adrenal medulla. The dorsal aorta is a
critical control point for all three processes: the migration of
the SA progenitor cells, the segregation of the Sg and Am cells,
and the subsequent movement of the Am cells. This process
involves multiple factors that are regulated not only temporally
throughout the process but also spatially within the embryo.
The cells alter how responsive they are to the various signaling
factors that they encounter. This allows the various events tooccur in the correct sequence to result in the different cell types
being established in the appropriate regions of the embryo.
This work provided significant insight into a complex
cascade of signaling which controls one of the earliest interactions between neurons and blood vessels guiding thedevelopment of embryos. The outcomes of this study support
the identification of new therapeutic targets for the treatment
of developmental disorders of the autonomic nervous system.

SDF1 and NRG1 act directly on migrating SA
progenitors (NT, neural tube; n, notochord; DA, dorsal
aorta) (Saito et al., 2012, Science).

Reference
Saito, D., Takase, Y., Murai, H., Takahashi, Y. 2012. The dorsal aorta initiates amolecular cascade that instructs sympatho-adrenal specification. Science, 336,
1578–1581.

NAIST Research Highlights

47

Plant Sciences

30 YEARS OF HISTORY: 2011– 2021

Taku Demura© iStock/ Liam Preece

Adapt and thrive:
How a group of proteins may havehelped plants to colonize land
NAC transcription factors, which regulate the differentiation of water-conducting
and support cells in vascular plants, may have helped them to adapt to living on landT

he water-to-land transition was a key step in plant
evolution, facilitated by the emergence of specialized
cells that conduct water and provide mechanicalsupport. Xylem vessel elements perform these functions invascular plants, enabling them to colonize the terrestrial
environment. In this plant group, NAC transcription factors
are the proteins that control the differentiation of these cells,
as well as regulating the development of fiber cells, such as
those found in the supporting sclerenchyma tissue of stems
and bark. This suggests that there is an evolutionary link
between fiber cells and vessels, and that the system that
regulates transcription has been evolutionarily conserved,
providing potential clues to the evolutionary origins of how
land plants developed the vascular systems that enabled themto invade land. However, exactly how plants with xylem vessel
elements evolved from nonvascular plants was not previously
well-understood.
To investigate the novel features needed by plants to adapt
to land, a research team led by Taku Demura at NAIST studied
genes that encode NAC proteins in the moss Physcomitrella
patens. In P. patens, these genes regulate the development ofstereids and hydroids, which are specialized water-conducting and structural support cells. Prior to this study, it was

48

NAIST Research Highlights

unclear whether there was an evolutionary relationshipbetween hydroids and stereids and xylem vessels and fiber
cells, respectively. There are eight loci in the P. patens genome
similar to the VND/NST/SND transcription factors seen invascular plants, and the team sequenced complementary
DNA from these genes, naming the gene family PpVNS[VND-, NST/SND-, SMB (SOMBRERO)-related protein]. The
team searched publicly available transcriptomic and genomic
sequence data for phylogenetic analysis; the results suggested
that VNS genes underwent an early expansion in prevascularplants.
The tissue-specific expression patterns of PpVNS in tissues
from protonemata and gametophytes were then examined. In
P. patens, after haploid spore germination in the gametophytic
generation, filamentous cells called protonemata are formed,
and leafy gametophores develop from these. The results
suggested that PpVNS genes are involved in midrib development, as well as that of other tissues. Consequently, theresearchers investigated three of the PpVNS genes that wereprominently expressed in the midrib. Water transport inleaves from three loss-of-function mutant lines was examined
and, compared with the wild type, was revealed to be
deficient, which may have resulted from malformation ofhydroid cells. The mutants also exhibited abnormalities intheir stereids and sporophyte cells, indicating that PpVNS
genes play crucial roles in the development of these specialized cell types in P. patens.
The research group then conducted overexpression of the
PpVNS genes in P. patens and A. thaliana, and the results
suggested that the PpVNS family is involved in programmed
cell death in P. patens. Further analysis indicated evolutionary
conservation of VNS downstream genes. Building on existing
research, the team concluded that the expression of genes that
support the formation of water-conducting cells is promotedby the overexpression of PpVNS genes, and in P. patens andA. thaliana, those developmental mechanisms lead to ectopic
thickening of cell walls and programmed cell death.
The results of this study demonstrated that hydroid celldifferentiation is regulated by PpVNS proteins via induced
cell death, and that these proteins regulate the differentiationof stereid cells by enabling the thickening of cell walls and theclearance of cellular contents. In mosses and vascular plants,
the same gene families are regulated by PpVNS, indicating anevolutionary conservation of the genetic basis for the formation of both supporting and water-conducting tissues among
these groups. In mosses, stereids and hydroids are developed

during the gametophytic generation; in vascular plants, thevascular system is established in the sporophytic generation.
Therefore, it appears that in the gametophyte and sporophyte
generations, the VNS-based gene regulatory networks arehomologous, and during vascular plant evolution wereexpanded in the sporophyte generation. Additionally, water
is conducted much more readily via dead cells than by theliving cells of the parenchyma. Therefore, water conduction
may have been facilitated by the evolution of the cell death
program. Similarly, the production of large, complex plantbodies requires supporting elements. During the transitionof plants to land, the NAC transcription factors may haveplayed a role in the evolution of both supporting andwater-conducting cells. This research enhances our fundamental understanding of the genetic changes in the evolutionof land plants and may contribute to future developments inagricultural biotechnology.
Reference
Xu, B., Ohtani, M., Yamaguchi, M., Toyooka, K., Wakazaki, M., Sato, M., Kubo, M.,
Nakano, Y., Sano, R., Hiwatashi, Y., Murata, T., Kurata, T., Yoneda, A., Kato, K.,
Hasebe, M. Demura, T. 2014. Contribution of NAC transcription factors to plant
adaptation to land. Science, 343, 1505–1508.

NAIST Research Highlights

49

Plant Sciences

30 YEARS OF HISTORY: 2011– 2021

Taku Demura© iStock/ Liam Preece

Adapt and thrive:
How a group of proteins may havehelped plants to colonize land
NAC transcription factors, which regulate the differentiation of water-conducting
and support cells in vascular plants, may have helped them to adapt to living on landT

he water-to-land transition was a key step in plant
evolution, facilitated by the emergence of specialized
cells that conduct water and provide mechanicalsupport. Xylem vessel elements perform these functions invascular plants, enabling them to colonize the terrestrial
environment. In this plant group, NAC transcription factors
are the proteins that control the differentiation of these cells,
as well as regulating the development of fiber cells, such as
those found in the supporting sclerenchyma tissue of stems
and bark. This suggests that there is an evolutionary link
between fiber cells and vessels, and that the system that
regulates transcription has been evolutionarily conserved,
providing potential clues to the evolutionary origins of how
land plants developed the vascular systems that enabled themto invade land. However, exactly how plants with xylem vessel
elements evolved from nonvascular plants was not previously
well-understood.
To investigate the novel features needed by plants to adapt
to land, a research team led by Taku Demura at NAIST studied
genes that encode NAC proteins in the moss Physcomitrella
patens. In P. patens, these genes regulate the development ofstereids and hydroids, which are specialized water-conducting and structural support cells. Prior to this study, it was

48

NAIST Research Highlights

unclear whether there was an evolutionary relationshipbetween hydroids and stereids and xylem vessels and fiber
cells, respectively. There are eight loci in the P. patens genome
similar to the VND/NST/SND transcription factors seen invascular plants, and the team sequenced complementary
DNA from these genes, naming the gene family PpVNS[VND-, NST/SND-, SMB (SOMBRERO)-related protein]. The
team searched publicly available transcriptomic and genomic
sequence data for phylogenetic analysis; the results suggested
that VNS genes underwent an early expansion in prevascularplants.
The tissue-specific expression patterns of PpVNS in tissues
from protonemata and gametophytes were then examined. In
P. patens, after haploid spore germination in the gametophytic
generation, filamentous cells called protonemata are formed,
and leafy gametophores develop from these. The results
suggested that PpVNS genes are involved in midrib development, as well as that of other tissues. Consequently, theresearchers investigated three of the PpVNS genes that wereprominently expressed in the midrib. Water transport inleaves from three loss-of-function mutant lines was examined
and, compared with the wild type, was revealed to be
deficient, which may have resulted from malformation ofhydroid cells. The mutants also exhibited abnormalities intheir stereids and sporophyte cells, indicating that PpVNS
genes play crucial roles in the development of these specialized cell types in P. patens.
The research group then conducted overexpression of the
PpVNS genes in P. patens and A. thaliana, and the results
suggested that the PpVNS family is involved in programmed
cell death in P. patens. Further analysis indicated evolutionary
conservation of VNS downstream genes. Building on existing
research, the team concluded that the expression of genes that
support the formation of water-conducting cells is promotedby the overexpression of PpVNS genes, and in P. patens andA. thaliana, those developmental mechanisms lead to ectopic
thickening of cell walls and programmed cell death.
The results of this study demonstrated that hydroid celldifferentiation is regulated by PpVNS proteins via induced
cell death, and that these proteins regulate the differentiationof stereid cells by enabling the thickening of cell walls and theclearance of cellular contents. In mosses and vascular plants,
the same gene families are regulated by PpVNS, indicating anevolutionary conservation of the genetic basis for the formation of both supporting and water-conducting tissues among
these groups. In mosses, stereids and hydroids are developed

during the gametophytic generation; in vascular plants, thevascular system is established in the sporophytic generation.
Therefore, it appears that in the gametophyte and sporophyte
generations, the VNS-based gene regulatory networks arehomologous, and during vascular plant evolution wereexpanded in the sporophyte generation. Additionally, water
is conducted much more readily via dead cells than by theliving cells of the parenchyma. Therefore, water conduction
may have been facilitated by the evolution of the cell death
program. Similarly, the production of large, complex plantbodies requires supporting elements. During the transitionof plants to land, the NAC transcription factors may haveplayed a role in the evolution of both supporting andwater-conducting cells. This research enhances our fundamental understanding of the genetic changes in the evolutionof land plants and may contribute to future developments inagricultural biotechnology.
Reference
Xu, B., Ohtani, M., Yamaguchi, M., Toyooka, K., Wakazaki, M., Sato, M., Kubo, M.,
Nakano, Y., Sano, R., Hiwatashi, Y., Murata, T., Kurata, T., Yoneda, A., Kato, K.,
Hasebe, M. Demura, T. 2014. Contribution of NAC transcription factors to plant
adaptation to land. Science, 343, 1505–1508.

NAIST Research Highlights

49

Plant Sciences

Cell Biology

30 YEARS OF HISTORY: 2011– 2021

Masaaki Umeda

Naoyuki Inagaki

Forming giant cells in response
to DNA damage

Shootin1 phosphorylation convertsa chemical signal into mechanicalforce to promote axon growth

In response to double-strand DNA breaks induced by radiation, plant cells replicate
the nuclear genome to help survive the effects of genotoxic stress and avoid cell death

Phosphorylation of shootin1 by Pak1 enhances its interactions with actin filaments,
enabling them to couple more strongly with extracellular substrates and create
traction force to promote axon outgrowth

induce a programmed response of endoreduplication in ArabiNA can be damaged by many toxic stimuli, such as
dopsis. This strategy represents a third option for responding
radiation and chemicals. If this damage goes undetected,
to the damage induced by ionizing radiation, in addition to theit can lead to serious consequences, such as cancer. Thus,
classical options of delaying cell division or inducing cell death.
identifying and repairing areas of DNA damage is a crucial
Although SOG1 was involved in this signaling process, its
cellular process that is built into the cell cycle in the form ofspecific role appeared to vary depending on whether it wascheckpoints, which give the cell a chance to detect damage andactive in the ATM- or ATR-dependent pathway, opening up
decided what to do with it before replicating and potentially
new avenues for research into activity of this transcription
passing on a damaged genome to its daughter cells. In metazofactor in Arabidopsis. In addition to revealing an importantans, double-strand breaks halt cell cycle progression at the
aspect of double-strand DNA break repair in plants, theDNA damage checkpoint. This checkpoint is vital in ensuringfindings from this study provide crucial insight into a universal
genome integrity by giving the cell time to either repair theprocess that is key to maintaining genome integrity in all
break or induce apoptosis. While there is some conservation
living organisms.
of DNA damage checkpoint and double-strand break repair
system components across species, plants seem to lack manyof the factors known to participate in these processes inReference
animals. Therefore, a research group led by Masaaki Umeda
Adachi, S., Minamisawa, K., Okushima, Y., Inagaki, S., Yoshiyama, K., Kondou, Y.,
Kaminuma, E., Kawashima, M., Toyoda, T., Matsui, M., Kurihara, D., Matsunaga,
at NAIST sought to investigate the factors involved in theS., Umeda, M. 2011. Programmed induction of endoreduplication by DNA
cellular response to double-strand breaks in Arabidopsis, adouble-strand breaks in Arabidopsis. Proceedings of the National Academy of Sciencesof the United States of America, 108, 10004–10009.
common plant model, to better understand how cells manage
what could otherwise be a catastrophic cellular event.
To do this, the researchers treated Arabidopsis roots with zeocin, an agent that mimics
radiation damage, and found that the plants
responded by forming giant cells, indicating
the occurrence of endoreduplication, which is
replication of the entire genome without cell
division. Gamma irradiation, another agent
that causes double-strand breaks, had a similareffect on Arabidopsis cells, suggesting that
this type of DNA damage specificallyinduces endoreduplication.
Further investigation of the signaling
pathways involved in this process showed that
ATM and ATR, which are orthologs ofmetazoan proteins that are known to play key
roles in the DNA damage response, as well as
SOG1, a plant-specific transcription factor, allhelped mediate endoreduplication. In addition,
cells that demonstrated endoreduplication after
treatment with zeocin exhibited significant
changes in the expression of cell cycle–related
genes and induced degradation of CDKB2, an
important cyclin-dependent kinase.
Taken together, the findings from this study
demonstrated that double-strand DNA breaks
Cell enlargement (right) in root tips (Adachi et al., 2011, PNAS).
affect the expression of cell cycle regulators to

NAIST Research Highlights

C© 2011 National Academy of Sciences

1991

2000

D

50

30 YEARS OF HISTORY: 2011– 2021

ell movement and growth can be directed by soluble
guidance cues that modulate adhesion andcytoskeletal dynamics. The combination of actin
filament (F-actin) polymerization at the leading edgeof motile cells and myosin II activity causes F-actins,
which couple with extracellular substrates, to flow
backwards within cells, resulting in movement or growth.
One example of this phenomenon is the growth ofthe long neuronal cell extensions called axons, which
occurs at the tip of axons from a region known as
the growth cone. Retrograde flow of F-actins, coupled witha variety of extracellular molecules known collectively as
substrates, is thought to generate traction force that enables
outgrowth of the axon. However, it was unclear how
neurons regulate this process, and specifically how
cell signaling affected the efficiency of coupling
between F-actins and these substrates to create traction
and facilitate growth.
To address this, a research group led by Naoyuki Inagaki
at NAIST tested the effects of netrin-1, which was known
to promote axonal outgrowth, on axonal growth cones.
They utilized traction force microscopy to detect filopodium extension and fluorescent speckle microscopy tovisualize F-actin flow. They also investigated the expression andphosphorylation of downstream effector molecules using
standard techniques, including in vitro kinase assays, immunoblotting, and Immunocytochemical analysis, to gain a moredetailed understanding of the cellular signaling systems
regulating these processes.
The results showed that netrin-1 promoted traction forcesat growth cones. This process involved phosphorylation ofshootin1, a linker molecule known to promote axon outgrowthby coupling F-actin flow with extracellular substrates, by Pak1,
which had also been shown to play a role in axon outgrowth.
Shootin1 was phosphorylated at two sites, Ser101 and Ser249,
and this modification increased when Pak1 was expressed
constitutively and when the cells were stimulated with netrin-1.
Furthermore, phosphorylated shootin1 was expressed at highlevels in areas of the axon growth cones where retrograde
F-actin flow and traction forces were also observed, suggested
an enhanced association between shootin1 and F-actin flow at

Fluorescence image of an axonal growth cone (Toriyama et al., 2013, Curr Biol).

growth cones. This resulted in greater coupling with substrates,
thereby generating traction force that “pulled” the growing
axon forward.
Taken together, these findings suggested that the dynamic
interactions between shootin1 and F-actin at axon growthcones are regulated by Pak1-mediated phosphorylation ofshootin1. Importantly, this discovery provides clear evidence
for the conversion of a chemical signal into traction force,
which in this case functions to promote axon outgrowth. This
molecular level of insight into cells regulating the signaling
process to control growth and movement could potentiallyhave important clinical implications – for example, in the fieldsof regenerative medicine and neurology, where directing nerve
regrowth after injury would have a crucial impact on recovery
from catastrophic spinal damage.
Reference
Toriyama, M., Kozawa, S., Sakumura, Y., Inagaki, N. 2013. Conversion of a signal into
forces for axon outgrowth through Pak1-mediated shootin1 phosphorylation.
Current Biology, 23, 529–534.

NAIST Research Highlights

51

Plant Sciences

Cell Biology

30 YEARS OF HISTORY: 2011– 2021

Masaaki Umeda

Naoyuki Inagaki

Forming giant cells in response
to DNA damage

Shootin1 phosphorylation convertsa chemical signal into mechanicalforce to promote axon growth

In response to double-strand DNA breaks induced by radiation, plant cells replicate
the nuclear genome to help survive the effects of genotoxic stress and avoid cell death

Phosphorylation of shootin1 by Pak1 enhances its interactions with actin filaments,
enabling them to couple more strongly with extracellular substrates and create
traction force to promote axon outgrowth

induce a programmed response of endoreduplication in ArabiNA can be damaged by many toxic stimuli, such as
dopsis. This strategy represents a third option for responding
radiation and chemicals. If this damage goes undetected,
to the damage induced by ionizing radiation, in addition to theit can lead to serious consequences, such as cancer. Thus,
classical options of delaying cell division or inducing cell death.
identifying and repairing areas of DNA damage is a crucial
Although SOG1 was involved in this signaling process, its
cellular process that is built into the cell cycle in the form ofspecific role appeared to vary depending on whether it wascheckpoints, which give the cell a chance to detect damage andactive in the ATM- or ATR-dependent pathway, opening up
decided what to do with it before replicating and potentially
new avenues for research into activity of this transcription
passing on a damaged genome to its daughter cells. In metazofactor in Arabidopsis. In addition to revealing an importantans, double-strand breaks halt cell cycle progression at the
aspect of double-strand DNA break repair in plants, theDNA damage checkpoint. This checkpoint is vital in ensuringfindings from this study provide crucial insight into a universal
genome integrity by giving the cell time to either repair theprocess that is key to maintaining genome integrity in all
break or induce apoptosis. While there is some conservation
living organisms.
of DNA damage checkpoint and double-strand break repair
system components across species, plants seem to lack manyof the factors known to participate in these processes inReference
animals. Therefore, a research group led by Masaaki Umeda
Adachi, S., Minamisawa, K., Okushima, Y., Inagaki, S., Yoshiyama, K., Kondou, Y.,
Kaminuma, E., Kawashima, M., Toyoda, T., Matsui, M., Kurihara, D., Matsunaga,
at NAIST sought to investigate the factors involved in theS., Umeda, M. 2011. Programmed induction of endoreduplication by DNA
cellular response to double-strand breaks in Arabidopsis, adouble-strand breaks in Arabidopsis. Proceedings of the National Academy of Sciencesof the United States of America, 108, 10004–10009.
common plant model, to better understand how cells manage
what could otherwise be a catastrophic cellular event.
To do this, the researchers treated Arabidopsis roots with zeocin, an agent that mimics
radiation damage, and found that the plants
responded by forming giant cells, indicating
the occurrence of endoreduplication, which is
replication of the entire genome without cell
division. Gamma irradiation, another agent
that causes double-strand breaks, had a similareffect on Arabidopsis cells, suggesting that
this type of DNA damage specificallyinduces endoreduplication.
Further investigation of the signaling
pathways involved in this process showed that
ATM and ATR, which are orthologs ofmetazoan proteins that are known to play key
roles in the DNA damage response, as well as
SOG1, a plant-specific transcription factor, allhelped mediate endoreduplication. In addition,
cells that demonstrated endoreduplication after
treatment with zeocin exhibited significant
changes in the expression of cell cycle–related
genes and induced degradation of CDKB2, an
important cyclin-dependent kinase.
Taken together, the findings from this study
demonstrated that double-strand DNA breaks
Cell enlargement (right) in root tips (Adachi et al., 2011, PNAS).
affect the expression of cell cycle regulators to

NAIST Research Highlights

C© 2011 National Academy of Sciences

1991

2000

D

50

30 YEARS OF HISTORY: 2011– 2021

ell movement and growth can be directed by soluble
guidance cues that modulate adhesion andcytoskeletal dynamics. The combination of actin
filament (F-actin) polymerization at the leading edgeof motile cells and myosin II activity causes F-actins,
which couple with extracellular substrates, to flow
backwards within cells, resulting in movement or growth.
One example of this phenomenon is the growth ofthe long neuronal cell extensions called axons, which
occurs at the tip of axons from a region known as
the growth cone. Retrograde flow of F-actins, coupled witha variety of extracellular molecules known collectively as
substrates, is thought to generate traction force that enables
outgrowth of the axon. However, it was unclear how
neurons regulate this process, and specifically how
cell signaling affected the efficiency of coupling
between F-actins and these substrates to create traction
and facilitate growth.
To address this, a research group led by Naoyuki Inagaki
at NAIST tested the effects of netrin-1, which was known
to promote axonal outgrowth, on axonal growth cones.
They utilized traction force microscopy to detect filopodium extension and fluorescent speckle microscopy tovisualize F-actin flow. They also investigated the expression andphosphorylation of downstream effector molecules using
standard techniques, including in vitro kinase assays, immunoblotting, and Immunocytochemical analysis, to gain a moredetailed understanding of the cellular signaling systems
regulating these processes.
The results showed that netrin-1 promoted traction forcesat growth cones. This process involved phosphorylation ofshootin1, a linker molecule known to promote axon outgrowthby coupling F-actin flow with extracellular substrates, by Pak1,
which had also been shown to play a role in axon outgrowth.
Shootin1 was phosphorylated at two sites, Ser101 and Ser249,
and this modification increased when Pak1 was expressed
constitutively and when the cells were stimulated with netrin-1.
Furthermore, phosphorylated shootin1 was expressed at highlevels in areas of the axon growth cones where retrograde
F-actin flow and traction forces were also observed, suggested
an enhanced association between shootin1 and F-actin flow at

Fluorescence image of an axonal growth cone (Toriyama et al., 2013, Curr Biol).

growth cones. This resulted in greater coupling with substrates,
thereby generating traction force that “pulled” the growing
axon forward.
Taken together, these findings suggested that the dynamic
interactions between shootin1 and F-actin at axon growthcones are regulated by Pak1-mediated phosphorylation ofshootin1. Importantly, this discovery provides clear evidence
for the conversion of a chemical signal into traction force,
which in this case functions to promote axon outgrowth. This
molecular level of insight into cells regulating the signaling
process to control growth and movement could potentiallyhave important clinical implications – for example, in the fieldsof regenerative medicine and neurology, where directing nerve
regrowth after injury would have a crucial impact on recovery
from catastrophic spinal damage.
Reference
Toriyama, M., Kozawa, S., Sakumura, Y., Inagaki, N. 2013. Conversion of a signal into
forces for axon outgrowth through Pak1-mediated shootin1 phosphorylation.
Current Biology, 23, 529–534.

NAIST Research Highlights

51

Microbiology

30 YEARS OF HISTORY: 2011– 2021

30 YEARS OF HISTORY: 2011– 2021

Kazuhiro Shiozaki

Mpr1 is a yeast antioxidant
with a unique chemical structure

Original Sin1: Sin1 recruits substrates
to TORC2 for phosphorylation

The crystal structure of the antioxidant Mpr1 reveals key differences from other membersof the same enzyme superfamily that account for its unique substrate specificity

A central ubiquitin-fold domain in yeast Sin1 is required for recruitment of substrates
to the TORC2 complex, where they are phosphorylated and activatedO© 2013 National Academy of Sciences

engineered a stable form of the protein and subjected it tosingle-wavelength anomalous dispersion. They also analyzed
the structure of Mpr1 bound to one of its known substrates toclarify the interactions within this complex. Analysis of X-ray
diffraction data resulted in high-resolution three-dimensional
structures of both unbound and bound Mpr1.
The results showed that, even though Mpr1 exhibits poorsequence homology with other GNAT enzymes, it has a similar
structure, with eight-stranded mixed β-sheets and six α-helices.
In addition, the substrate was bound at the expected site.
Interestingly, however, Mpr1 lacks a β-bulge structure near theactive site that is common to several other GNAT proteins.
Mutational analysis of Mpr1 based on the crystal structure
showed that residues Asn135 and Asn178 are important for
substrate recognition and catalysis, respectively. Furthermore,mutation of either of these two residues reduced the ability ofMpr1 in yeast to acetylate P5C, which normally inhibits
mitochondrial respiration and leads to the generation ofreactive oxygen species, thus demonstrating an important
physiological role for Mpr1 in protecting yeast fromoxidative damage.
Taken together, the findings from this study show that Mpr1
has a similar structure to other GNAT superfamily member
proteins, but that key differences account for its distinctive
substrate specificity, highlighting the diversity of this enzyme
superfamily. Given that expression of Mpr1 increases theresistance of industrial yeast strains to oxidative stress such as
ethanol and freezing, engineering a form ofMpr1 based on the structural findings fromthis study could have applications in improved
industrial fermentation for the production of
biofuel or as new chemical catalysts for thesynthesis of natural products. Mpr1 is only
found in yeasts and fungi; therefore, understanding the mechanism of Mpr1 may also
have important clinical application regarding
the development of antifungal drugs andcombating antifungal drug resistance.

The structure of Mpr1, superimposed with those of other GNAT proteins shown in green,orange and gray (Nasuno et al., 2013, PNAS).

NAIST Research Highlights

Reference
Nasuno, R., Hirano, Y., Itoh, T., Hakoshima, T., Hibi, T., Takagi,
H. 2013. Structural and functional analysis of the yeast
N-acetyltransferase Mpr1 involved in oxidative stress
tolerance via proline metabolism. Proceedings of the National
Academy of Sciences of the United States of America, 110,11821–11826.

Licensed under CC BY 4.0

Hiroshi Takagi

xidative stress occurs when there is an imbalance in free
radicals and antioxidants within an organism. Free
radicals, also known as reactive oxygen species, candamage DNA, fats and proteins, and in humans this cancontribute to the development of serious conditions such as
diabetes, hypertension, and neurodegenerative disease.
Antioxidants, whether generated by the cell or acquired fromthe environment, play an important role in neutralizing
reactive oxygen species and thereby reducing the amount ofdamage they can cause.
Mpr1 is a protein and antioxidant enzyme found in yeastthat provides protection against oxidative stresses, such as
those that occur under extreme temperature. In contrast toother members of the Gcn5-related N-acetyltransferase(GNAT) superfamily of enzymes, which act on primaryamines, the two known substrates of Mpr1 are cyclic secondaryamines. Furthermore, Mpr1 has little homology with other
GNAT enzymes, suggesting that it may have a unique mechanism by which it recognizes substrates and catalyzes reactions.
However, the structural features conferring these uniqueproperties were previously poorly understood. A researchgroup led by Hiroshi Takagi at NAIST showed the X-ray crystal
structure of Mpr1, providing important insights into thestructure-function properties of this specific enzyme and wider
evolutional relationships among the biologically importantN-acetyltransferases.
To determine the structure of Mpr1, the researchers

52

Cell Biology

The molecular surface of SpSin1CRIM colored according to the electrostatic potential ranging from positive (blue) to negative(red) charge (Tatebe et al., 2017, eLife).

S

everal key molecules and signaling pathways are
implicated in more than one cancer, and as such makeattractive targets for designing new treatments that
could be effective against a variety of tumors. One of thesecentral players in cancer development is mammalian target of
rapamycin complex 2 (mTORC2), a multi-subunit complex
that mediates phosphorylation of several important cellulartargets, notably AKT. However, even though the function ofmTORC2 has been relatively well characterized, how it recognizes and targets specific substrates for phosphorylation
remained unknown.
To address this, a research team led by Kazuhiro Shiozaki
used the yeast equivalent of mTORC2, known as TORC2, anda yeast target protein with a high degree of similarity to human
AKT, known as Gad8, to determine the mechanism by which
this complex targets specific proteins for modification. Because
previous studies had shown that a subunit of the TORC2
complex called Sin1 plays a critical role in this process, theresearchers chose to focus on exploring the function of Sin1 inthis context.
The results showed that Sin1 is not required for assembly of
the TORC2 complex, as the complex formed normally evenwhen the gene encoding Sin1 was deleted. Because it was notimportant for TORC2 structure, the researchers next sought
to determine whether it was required for TORC2 function bydeleting domains that had been previously associated with Sin1
function. Unexpectedly, removal of both known functional
domain in Sin1 had no effect on Gad8 phosphorylation.
Instead, the conserved region in the middle (CRIM) was
required for Sin1 function, although deletion of this domain

had no effect on complex formation. Importantly, deletion of
the CRIM in human Sin1 made it unable to phosphorylate its
target proteins. The researchers then performed a structural
analysis of the Sin1CRIM and found that it forms a ubiquitin
fold, which is common to proteins that interact with a selectgroup of target proteins. In addition, the Sin1CRIM exhibiteda protruding loop structure that was essential for recruitment
and phosphorylation of its target proteins. Finally, Gad8 that
was artificially brought into proximity with the TORC2
complex lacking Sin1 was phosphorylated, indicating that the
primary role of the Sin1CRIM is to recruit target proteins tothe complex.
The findings from this study suggest that Sin1 is thesubstrate-recruiting element of the TORC2 complex, and that
its CRIM domain, and specifically the protruding loop part of
the ubiquitin fold, is responsible for this activity. Importantly,
these findings in yeast appeared to hold true in humans as well,
as phosphorylation of AKT by the human SIN1 protein also
required this region.
Given that over-activation of the mTORC2 complex is acentral event in the development of a variety of cancers, inhibition of this complex could be an effective means of controlling
tumor growth. The functional and structural insights provided
by this study could therefore be valuable in designing
small-molecule inhibitors that disrupt the interaction between
mTORC2 and its target proteins.
Reference
Tatebe, H., Murayama, S., Yonekura, T., Hatano, T., Richter, D., Furuya, T., Kataoka, S.,
Furuita, K., Kojima, C., Shiozaki, K. 2017. Substrate specificity of TOR complex 2 isdetermined by a ubiquitin-fold domain of the Sin1 subunit. eLife, 6, e19594.

NAIST Research Highlights

53

Microbiology

30 YEARS OF HISTORY: 2011– 2021

30 YEARS OF HISTORY: 2011– 2021

Kazuhiro Shiozaki

Mpr1 is a yeast antioxidant
with a unique chemical structure

Original Sin1: Sin1 recruits substrates
to TORC2 for phosphorylation

The crystal structure of the antioxidant Mpr1 reveals key differences from other membersof the same enzyme superfamily that account for its unique substrate specificity

A central ubiquitin-fold domain in yeast Sin1 is required for recruitment of substrates
to the TORC2 complex, where they are phosphorylated and activatedO© 2013 National Academy of Sciences

engineered a stable form of the protein and subjected it tosingle-wavelength anomalous dispersion. They also analyzed
the structure of Mpr1 bound to one of its known substrates toclarify the interactions within this complex. Analysis of X-ray
diffraction data resulted in high-resolution three-dimensional
structures of both unbound and bound Mpr1.
The results showed that, even though Mpr1 exhibits poorsequence homology with other GNAT enzymes, it has a similar
structure, with eight-stranded mixed β-sheets and six α-helices.
In addition, the substrate was bound at the expected site.
Interestingly, however, Mpr1 lacks a β-bulge structure near theactive site that is common to several other GNAT proteins.
Mutational analysis of Mpr1 based on the crystal structure
showed that residues Asn135 and Asn178 are important for
substrate recognition and catalysis, respectively. Furthermore,mutation of either of these two residues reduced the ability ofMpr1 in yeast to acetylate P5C, which normally inhibits
mitochondrial respiration and leads to the generation ofreactive oxygen species, thus demonstrating an important
physiological role for Mpr1 in protecting yeast fromoxidative damage.
Taken together, the findings from this study show that Mpr1
has a similar structure to other GNAT superfamily member
proteins, but that key differences account for its distinctive
substrate specificity, highlighting the diversity of this enzyme
superfamily. Given that expression of Mpr1 increases theresistance of industrial yeast strains to oxidative stress such as
ethanol and freezing, engineering a form ofMpr1 based on the structural findings fromthis study could have applications in improved
industrial fermentation for the production of
biofuel or as new chemical catalysts for thesynthesis of natural products. Mpr1 is only
found in yeasts and fungi; therefore, understanding the mechanism of Mpr1 may also
have important clinical application regarding
the development of antifungal drugs andcombating antifungal drug resistance.

The structure of Mpr1, superimposed with those of other GNAT proteins shown in green,orange and gray (Nasuno et al., 2013, PNAS).

NAIST Research Highlights

Reference
Nasuno, R., Hirano, Y., Itoh, T., Hakoshima, T., Hibi, T., Takagi,
H. 2013. Structural and functional analysis of the yeast
N-acetyltransferase Mpr1 involved in oxidative stress
tolerance via proline metabolism. Proceedings of the National
Academy of Sciences of the United States of America, 110,11821–11826.

Licensed under CC BY 4.0

Hiroshi Takagi

xidative stress occurs when there is an imbalance in free
radicals and antioxidants within an organism. Free
radicals, also known as reactive oxygen species, candamage DNA, fats and proteins, and in humans this cancontribute to the development of serious conditions such as
diabetes, hypertension, and neurodegenerative disease.
Antioxidants, whether generated by the cell or acquired fromthe environment, play an important role in neutralizing
reactive oxygen species and thereby reducing the amount ofdamage they can cause.
Mpr1 is a protein and antioxidant enzyme found in yeastthat provides protection against oxidative stresses, such as
those that occur under extreme temperature. In contrast toother members of the Gcn5-related N-acetyltransferase(GNAT) superfamily of enzymes, which act on primaryamines, the two known substrates of Mpr1 are cyclic secondaryamines. Furthermore, Mpr1 has little homology with other
GNAT enzymes, suggesting that it may have a unique mechanism by which it recognizes substrates and catalyzes reactions.
However, the structural features conferring these uniqueproperties were previously poorly understood. A researchgroup led by Hiroshi Takagi at NAIST showed the X-ray crystal
structure of Mpr1, providing important insights into thestructure-function properties of this specific enzyme and wider
evolutional relationships among the biologically importantN-acetyltransferases.
To determine the structure of Mpr1, the researchers

52

Cell Biology

The molecular surface of SpSin1CRIM colored according to the electrostatic potential ranging from positive (blue) to negative(red) charge (Tatebe et al., 2017, eLife).

S

everal key molecules and signaling pathways are
implicated in more than one cancer, and as such makeattractive targets for designing new treatments that
could be effective against a variety of tumors. One of thesecentral players in cancer development is mammalian target of
rapamycin complex 2 (mTORC2), a multi-subunit complex
that mediates phosphorylation of several important cellulartargets, notably AKT. However, even though the function ofmTORC2 has been relatively well characterized, how it recognizes and targets specific substrates for phosphorylation
remained unknown.
To address this, a research team led by Kazuhiro Shiozaki
used the yeast equivalent of mTORC2, known as TORC2, anda yeast target protein with a high degree of similarity to human
AKT, known as Gad8, to determine the mechanism by which
this complex targets specific proteins for modification. Because
previous studies had shown that a subunit of the TORC2
complex called Sin1 plays a critical role in this process, theresearchers chose to focus on exploring the function of Sin1 inthis context.
The results showed that Sin1 is not required for assembly of
the TORC2 complex, as the complex formed normally evenwhen the gene encoding Sin1 was deleted. Because it was notimportant for TORC2 structure, the researchers next sought
to determine whether it was required for TORC2 function bydeleting domains that had been previously associated with Sin1
function. Unexpectedly, removal of both known functional
domain in Sin1 had no effect on Gad8 phosphorylation.
Instead, the conserved region in the middle (CRIM) was
required for Sin1 function, although deletion of this domain

had no effect on complex formation. Importantly, deletion of
the CRIM in human Sin1 made it unable to phosphorylate its
target proteins. The researchers then performed a structural
analysis of the Sin1CRIM and found that it forms a ubiquitin
fold, which is common to proteins that interact with a selectgroup of target proteins. In addition, the Sin1CRIM exhibiteda protruding loop structure that was essential for recruitment
and phosphorylation of its target proteins. Finally, Gad8 that
was artificially brought into proximity with the TORC2
complex lacking Sin1 was phosphorylated, indicating that the
primary role of the Sin1CRIM is to recruit target proteins tothe complex.
The findings from this study suggest that Sin1 is thesubstrate-recruiting element of the TORC2 complex, and that
its CRIM domain, and specifically the protruding loop part of
the ubiquitin fold, is responsible for this activity. Importantly,
these findings in yeast appeared to hold true in humans as well,
as phosphorylation of AKT by the human SIN1 protein also
required this region.
Given that over-activation of the mTORC2 complex is acentral event in the development of a variety of cancers, inhibition of this complex could be an effective means of controlling
tumor growth. The functional and structural insights provided
by this study could therefore be valuable in designing
small-molecule inhibitors that disrupt the interaction between
mTORC2 and its target proteins.
Reference
Tatebe, H., Murayama, S., Yonekura, T., Hatano, T., Richter, D., Furuya, T., Kataoka, S.,
Furuita, K., Kojima, C., Shiozaki, K. 2017. Substrate specificity of TOR complex 2 isdetermined by a ubiquitin-fold domain of the Sin1 subunit. eLife, 6, e19594.

NAIST Research Highlights

53

Plant Sciences

Bioinformatics

30 YEARS OF HISTORY: 2011– 2021

Toshiro Ito

30 YEARS OF HISTORY: 2011– 2021

Shigehiko Kanaya

Fine-tuning flowers:
Plant genes work in balance
to get flower formation right
The plant gene CRABS CLAW regulates auxin homeostasis, which controls
the transition from floral meristem termination to gynoecium development

I

54

NAIST Research Highlights

Main window of the KNApSAcK family.

Integrated species–metabolite databases
for plants distributed around the world
A family of databases to facilitate research on plant metabolites, with a focus on medicinal plants

P© Adobe Stock /2Dvisualizen plants, all development after the embryonic stage isshown that TRN2 regulates auxin homeostasis, demonstrating
underpinned by stem cells. Because of this, the balancea relationship between gynoecium development andbetween the differentiation and proliferation of plant
floral meristem termination via the fine-tuning by CRC oforgans must be carefully established. For flowering plants to
auxin homeostasis.
reproduce successfully, the control of floral stem cell terminaThe results of the study revealed the molecular framework
for AG-mediated floral meristem termination via auxin, whichtion must be coordinated with the start of female reproductive
organ development, both in time and space. The source of theseis needed for the subsequent development of the gynoecium.
stem cells, floral meristem, is where flowers are formed. Floral
The researchers found that in the developing gynoecium, CRC
meristem is of particular significance to humans, because thiscontrols auxin homeostasis via negative regulation of TRN2,
gives rise to flowers and fruits, from which most agricultural
therefore avoiding floral meristem overgrowth when KNU is
and horticultural products are derived. A detailed understandmutated. CRC and KNU work together to regulate the terminaing of mechanisms behind developmental processes in flowers
tion of floral meristem, and mutation of these genes, which act
is beneficial, for example in applications for crop improvement.
downstream of AG, disrupts determinacy of the floral
meristem. The verification of the remarkable, synergistic
In the model plant Arabidopsis thaliana, the transcription
regulation pathway for floral meristem termination had beenmutant phenotype seen in crc knu highlights the key roles
documented. However, prior to this study conducted by aplayed by these genes.
research team led by Toshiro Ito at NAIST, the mechanisms
The mechanisms revealed in this study provided an importunderlying the coordination of these stages were unclear. In
ant addition to the knowledge of flowering plant reproduction.
Arabidopsis, the gene AGAMOUS (AG) is pivotal to the processThis improved understanding of floral development hasof floral meristem termination and establishment of theimplications for the study of plant biology overall, andidentity of reproductive organs. Floral meristem termination
especially to agriculture and horticulture.
is dependent on activation of the genes CRABS CLAW (CRC)
Referenceand KNUCKLES (KNU), which are direct targets of AG.
Yamaguchi, N., Huang, J., Xu, Y., Tanoi, K., Ito, T. 2017. Fine-tuning of auxin homeostaPrevious studies had revealed that auxin homeostasis plays asis governs the transition from floral stem cell maintenance to gynoecium formation.
Nature Communications, 8,1125.
role in generating auxin maxima that are key to development.
Although it was known that auxin plays a crucialrole in gynoecium development, its potentialfunction during floral meristem termination
was unknown.
Previously, a crc knu double mutant had been
demonstrated to exhibit strong indeterminacy
in its floral meristem, and therefore, the crc
mutant phenotype could be partly rescued with
an auxin transport inhibitor. Because of this, the
NAIST researchers used A. thaliana mutants toinvestigate the role of auxin at the terminationof floral meristem. They found that CRC andKNU coordinate to regulate the repression of the
WUSCHEL (WUS) gene, which is essential formeristem maintenance. Additionally, CRC wasdemonstrated to repress expression of the
TORNADO2 (TRN2) gene and control thedeterminacy of floral meristem. It was further
Arabidopsis thaliana

lants produce a broad variety of metabolites, which
include an array of medicinal compounds. Studies on theeffects of these plant metabolites in humans provide
invaluable insights, particularly in the field of healthcare andmedicinal plants. Owing to their great importance, many
scientists have investigated and characterized plant metabolites.
Such studies have generally employed mass spectrometry, which
can accurately measure the masses of thousands of compounds
within a single sample. From various studies, metabolite data
have been collected for numerous organisms. However, systematic evaluations of the relationships between metabolites andtheir originating plants have been hindered by the vast numberof plant metabolites in existence. In a major step towards
connecting widely dispersed data and plant use knowledge, a
research team led by Shigehiko Kanaya at NAIST developed an
integrated family of species–metabolite databases for plants,
called the KNApSAcK Family Databases.
By collecting information available in the scientific literature,
the research team developed a family of plant metabolite
databases that can facilitate systematic research on metabolites.
Several databases were developed as part of this initiative. As a
primary database within this family, the KNApSAcK Core
Database contains information for more than 100,000
species–metabolite relationships based on over 20,000 species
and over 50,000 metabolites. For this database, the investigatorsdeveloped a search engine, enabling researchers to search formetabolites based on various characteristics, such as metabolite
mass, name, spectra, or molecular formula.

Other databases include a world map function that provides
information regarding medicinal and edible plants for different
geographic zones. A biological activity database provides
information on the biological activity of medicinal and edible
plants. Two additional databases provide the formulas of medicinal plants based on Kampo (Japanese medicine) and Jamu
(Indonesian medicine). These databases are highly extensive: the15,240 medicinal plants in the database correspond to 72% of all
medicinal plants listed by the World Health Organization.
By statistically analyzing the metabolites in the database, theresearch team predicted that approximately one million plant
metabolites exist on Earth. This large number highlights theneed for computational tools, such as the KNApSAcK
Database, to allow for timely and comprehensive studiesof plant metabolomics.
The researchers have made these databases, along with an
instruction manual, freely available online. By providing freeaccess to this resource, scientists worldwide are able to systematically study plant metabolites, particularly metabolites that may
be of medicinal value. In addition to accelerating discoveries in
the use of medicinal plants, this family of databases will contribute to our holistic understanding of plant function andhealthcare, bolstering efforts to create sustainable societies.
Reference
Afendi, F.M., Okada, T., Yamazaki, M., Hirai-Morita, A., Nakamura, Y., Nakamura, K.,
Ikeda, S., Takahashi, H., Altaf-Ul-Amin, M., Darusman, L.K., Saito, K., Kanaya, S.
2012. KNApSAcK family databases: Integrated metabolite–plant species databases
for multifaceted plant research. Plant and Cell Physiology, 53, e1.

NAIST Research Highlights

55

Plant Sciences

Bioinformatics

30 YEARS OF HISTORY: 2011– 2021

Toshiro Ito

30 YEARS OF HISTORY: 2011– 2021

Shigehiko Kanaya

Fine-tuning flowers:
Plant genes work in balance
to get flower formation right
The plant gene CRABS CLAW regulates auxin homeostasis, which controls
the transition from floral meristem termination to gynoecium development

I

54

NAIST Research Highlights

Main window of the KNApSAcK family.

Integrated species–metabolite databases
for plants distributed around the world
A family of databases to facilitate research on plant metabolites, with a focus on medicinal plants

P© Adobe Stock /2Dvisualizen plants, all development after the embryonic stage isshown that TRN2 regulates auxin homeostasis, demonstrating
underpinned by stem cells. Because of this, the balancea relationship between gynoecium development andbetween the differentiation and proliferation of plant
floral meristem termination via the fine-tuning by CRC oforgans must be carefully established. For flowering plants to
auxin homeostasis.
reproduce successfully, the control of floral stem cell terminaThe results of the study revealed the molecular framework
for AG-mediated floral meristem termination via auxin, whichtion must be coordinated with the start of female reproductive
organ development, both in time and space. The source of theseis needed for the subsequent development of the gynoecium.
stem cells, floral meristem, is where flowers are formed. Floral
The researchers found that in the developing gynoecium, CRC
meristem is of particular significance to humans, because thiscontrols auxin homeostasis via negative regulation of TRN2,
gives rise to flowers and fruits, from which most agricultural
therefore avoiding floral meristem overgrowth when KNU is
and horticultural products are derived. A detailed understandmutated. CRC and KNU work together to regulate the terminaing of mechanisms behind developmental processes in flowers
tion of floral meristem, and mutation of these genes, which act
is beneficial, for example in applications for crop improvement.
downstream of AG, disrupts determinacy of the floral
meristem. The verification of the remarkable, synergistic
In the model plant Arabidopsis thaliana, the transcription
regulation pathway for floral meristem termination had beenmutant phenotype seen in crc knu highlights the key roles
documented. However, prior to this study conducted by aplayed by these genes.
research team led by Toshiro Ito at NAIST, the mechanisms
The mechanisms revealed in this study provided an importunderlying the coordination of these stages were unclear. In
ant addition to the knowledge of flowering plant reproduction.
Arabidopsis, the gene AGAMOUS (AG) is pivotal to the processThis improved understanding of floral development hasof floral meristem termination and establishment of theimplications for the study of plant biology overall, andidentity of reproductive organs. Floral meristem termination
especially to agriculture and horticulture.
is dependent on activation of the genes CRABS CLAW (CRC)
Referenceand KNUCKLES (KNU), which are direct targets of AG.
Yamaguchi, N., Huang, J., Xu, Y., Tanoi, K., Ito, T. 2017. Fine-tuning of auxin homeostaPrevious studies had revealed that auxin homeostasis plays asis governs the transition from floral stem cell maintenance to gynoecium formation.
Nature Communications, 8,1125.
role in generating auxin maxima that are key to development.
Although it was known that auxin plays a crucialrole in gynoecium development, its potentialfunction during floral meristem termination
was unknown.
Previously, a crc knu double mutant had been
demonstrated to exhibit strong indeterminacy
in its floral meristem, and therefore, the crc
mutant phenotype could be partly rescued with
an auxin transport inhibitor. Because of this, the
NAIST researchers used A. thaliana mutants toinvestigate the role of auxin at the terminationof floral meristem. They found that CRC andKNU coordinate to regulate the repression of the
WUSCHEL (WUS) gene, which is essential formeristem maintenance. Additionally, CRC wasdemonstrated to repress expression of the
TORNADO2 (TRN2) gene and control thedeterminacy of floral meristem. It was further
Arabidopsis thaliana

lants produce a broad variety of metabolites, which
include an array of medicinal compounds. Studies on theeffects of these plant metabolites in humans provide
invaluable insights, particularly in the field of healthcare andmedicinal plants. Owing to their great importance, many
scientists have investigated and characterized plant metabolites.
Such studies have generally employed mass spectrometry, which
can accurately measure the masses of thousands of compounds
within a single sample. From various studies, metabolite data
have been collected for numerous organisms. However, systematic evaluations of the relationships between metabolites andtheir originating plants have been hindered by the vast numberof plant metabolites in existence. In a major step towards
connecting widely dispersed data and plant use knowledge, a
research team led by Shigehiko Kanaya at NAIST developed an
integrated family of species–metabolite databases for plants,
called the KNApSAcK Family Databases.
By collecting information available in the scientific literature,
the research team developed a family of plant metabolite
databases that can facilitate systematic research on metabolites.
Several databases were developed as part of this initiative. As a
primary database within this family, the KNApSAcK Core
Database contains information for more than 100,000
species–metabolite relationships based on over 20,000 species
and over 50,000 metabolites. For this database, the investigatorsdeveloped a search engine, enabling researchers to search formetabolites based on various characteristics, such as metabolite
mass, name, spectra, or molecular formula.

Other databases include a world map function that provides
information regarding medicinal and edible plants for different
geographic zones. A biological activity database provides
information on the biological activity of medicinal and edible
plants. Two additional databases provide the formulas of medicinal plants based on Kampo (Japanese medicine) and Jamu
(Indonesian medicine). These databases are highly extensive: the15,240 medicinal plants in the database correspond to 72% of all
medicinal plants listed by the World Health Organization.
By statistically analyzing the metabolites in the database, theresearch team predicted that approximately one million plant
metabolites exist on Earth. This large number highlights theneed for computational tools, such as the KNApSAcK
Database, to allow for timely and comprehensive studiesof plant metabolomics.
The researchers have made these databases, along with an
instruction manual, freely available online. By providing freeaccess to this resource, scientists worldwide are able to systematically study plant metabolites, particularly metabolites that may
be of medicinal value. In addition to accelerating discoveries in
the use of medicinal plants, this family of databases will contribute to our holistic understanding of plant function andhealthcare, bolstering efforts to create sustainable societies.
Reference
Afendi, F.M., Okada, T., Yamazaki, M., Hirai-Morita, A., Nakamura, Y., Nakamura, K.,
Ikeda, S., Takahashi, H., Altaf-Ul-Amin, M., Darusman, L.K., Saito, K., Kanaya, S.
2012. KNApSAcK family databases: Integrated metabolite–plant species databases
for multifaceted plant research. Plant and Cell Physiology, 53, e1.

NAIST Research Highlights

55

Information Systems

30 YEARS OF HISTORY: 2011– 2021

30 YEARS OF HISTORY: 2011– 2021

Keiichi Yasumoto

Kenichi Matsumoto

Gamification of crowdsourced
monitoring tasks

Improving software defect detection
with parameter optimization

Adding game-like elements encourages participation in a mobile phone app basedon feelings of accomplishment and increased social status

Optimizing the model parameters of machine-learning algorithms used for
identifying software bugs may lead to more accurate artificial intelligenceOnline monitoring services, such as those that engage inthe real-time recording of store crowd sizes, sometimes
enlist people in the area to detect and relay accurate
information to a central server via an app. Participation in this“crowdsourcing” of sensing tasks can be substantially enhanced
if the monitoring service offers incentives, financial or otherwise, to users. To minimize the cost of paying these benefits,
status-based rewards, such as badges or ranking lists, can also
be included. While many companies now implement non-financial inducements to encourage increased user engagement,
systematic studies of apps that can promote real-world sensing
tasks have been lacking.
To help address this need, a research team led by Keiichi
Yasumoto at NAIST has proposed an algorithm that combines
both a financial incentive, such as airline miles, with statusrewards. This idea was based on the psychological concept of“gamification,” in which users are much more likely to completedifficult or onerous tasks when presented in the form of a gamewith points to be scored. The hypothesis is that the satisfaction
users receive from the sense of accomplishment will compensate for reduced financial incentives.
A heuristic algorithm was created to determine which setof users should receive each request, and the appropriate
number of reward points to offer as an incentive. In addition,a status scheme was added that classifies users into ranks basedon points accrued. Users with higher status were offered
increased reward points for additional sensing tasks. Rankings
were made public to all users with leader boards. Finally, digitalbadges could be earned, along with bonus points, for accomplishing conditions set by the system.

The application was then built on the Foursquare platform,
which is a popular smartphone app in which users “check in”
at various locations. During the experiment, the new algorithm
decided to whom requests should be sent by sorting users
based on their expected performance per cost, which was
defined as the ratio of probability of participation divided by
the reward points offered for the task. These requests came in
the form of an email that contains the content of the sensing
task related to the location where the user checked in, thereward points, and the time limit. For this test, the tasks all
involved uploading a photograph within 15 minutes. For example, the user might be asked to submit an image of a restaurant’smenu or cars in a parking garage to gauge usage rate. To findthe impact of gamification, users were randomly offered eithera monetary incentive only, or a monetary incentive coupledwith a gamification-based reward.
When they trialed this approach with 18 users over 30 days,
the researchers found that their gamification algorithm
increased participation probability to 73%, up from 53%
without gamification. Possible applications of this research arevery broad, including the real-time monitoring of weather
condition or highway congestion at various locations. Optimizing the gamification aspects of crowdsourcing apps could
greatly increase participation rates while not incurring
excessive payment costs.
Reference
Ueyama, Y., Tamai, M., Arakawa, Y., Yasumoto, K. 2014. Gamification-based incentive
mechanism for participatory sensing. Pervasive Computing and Communications
Workshops, 98–103.

The overview of the participatory sensing system (Ueyama et al., 2014, PERCOM).

56

Software Engineering

NAIST Research Highlights

An overview of the approach employed (Tantithamthavorn et al., 2016, ICSE).

The complexity of modern software has rapidly
outstripped the comprehension of any single coder. The
length of the source code that lies behind a major
computer program or website can easily run to tens of millionsof lines. It has become standard practice for organizations totest each module for bugs that could potentially lead to failure
or suboptimal performance under certain future conditions.
As part of this effort, the software quality assurance processusually employs specialized automated machine-learning
prediction models trained to identify software that may bevulnerable to defects. These tools are based on statistical ormachine-learning classification approaches that use predefined
search parameters, for example, the number of decision trees
to use in a random forest classifier. However, the optimal
settings for these parameters are not known ahead of time, andthere is reason to believe that defect prediction models may
suffer from reduced accuracy if they are trained under suboptimal parameter settings. Because the number of possible
parameter permutations is far too large to try exhaustively, amore systematic approach is needed.
To study this situation, a research team led by Kenichi
Matsumoto at NAIST used the automated parameter optimization tool called Caret using examples from 18 datasets,
including both proprietary and open-source software. They
found that changing parameter settings for the defect-detection
models can lead to significant improvement in performance.

Of the 26 machine-learning methods considered, Caret could
be used for 16 of them, including commonly used approaches
like random Forest, support vector machines, and artificial
neural networks.
Caret works by evaluating potential combinations of thecandidate parameter settings, up to a preset limit. The researchers used 100 repetitions to estimate the performance ofclassifiers that are trained using each of the candidate parameter settings. They found that using Caret improved the area
under the curve (the primary measure of model accuracy) of
defect prediction models by as much as 40 percentage points.
This approach also increased the chance that a top-performingclassifier would be identified by up to 83%. To ensure that theconclusions regarding the models were robust, out-of-sample
bootstrap validation was applied.
The results of this work can be applied throughout the fieldof machine learning. In particular, the accuracy gains due toparameter optimization can be beneficial for almost any kindof classification task. This project can help guide developers
determine how many resources to invest in this process basedon the prospective increase in performance.
Reference
Tantithamthavorn, C., McIntosh, S., Hassan, A.E., Matsumoto, K. 2016. Automatedparameter optimization of classification techniques for defect prediction models.
International Conference on Software Engineering, 321–332.

NAIST Research Highlights

57

Information Systems

30 YEARS OF HISTORY: 2011– 2021

30 YEARS OF HISTORY: 2011– 2021

Keiichi Yasumoto

Kenichi Matsumoto

Gamification of crowdsourced
monitoring tasks

Improving software defect detection
with parameter optimization

Adding game-like elements encourages participation in a mobile phone app basedon feelings of accomplishment and increased social status

Optimizing the model parameters of machine-learning algorithms used for
identifying software bugs may lead to more accurate artificial intelligenceOnline monitoring services, such as those that engage inthe real-time recording of store crowd sizes, sometimes
enlist people in the area to detect and relay accurate
information to a central server via an app. Participation in this“crowdsourcing” of sensing tasks can be substantially enhanced
if the monitoring service offers incentives, financial or otherwise, to users. To minimize the cost of paying these benefits,
status-based rewards, such as badges or ranking lists, can also
be included. While many companies now implement non-financial inducements to encourage increased user engagement,
systematic studies of apps that can promote real-world sensing
tasks have been lacking.
To help address this need, a research team led by Keiichi
Yasumoto at NAIST has proposed an algorithm that combines
both a financial incentive, such as airline miles, with statusrewards. This idea was based on the psychological concept of“gamification,” in which users are much more likely to completedifficult or onerous tasks when presented in the form of a gamewith points to be scored. The hypothesis is that the satisfaction
users receive from the sense of accomplishment will compensate for reduced financial incentives.
A heuristic algorithm was created to determine which setof users should receive each request, and the appropriate
number of reward points to offer as an incentive. In addition,a status scheme was added that classifies users into ranks basedon points accrued. Users with higher status were offered
increased reward points for additional sensing tasks. Rankings
were made public to all users with leader boards. Finally, digitalbadges could be earned, along with bonus points, for accomplishing conditions set by the system.

The application was then built on the Foursquare platform,
which is a popular smartphone app in which users “check in”
at various locations. During the experiment, the new algorithm
decided to whom requests should be sent by sorting users
based on their expected performance per cost, which was
defined as the ratio of probability of participation divided by
the reward points offered for the task. These requests came in
the form of an email that contains the content of the sensing
task related to the location where the user checked in, thereward points, and the time limit. For this test, the tasks all
involved uploading a photograph within 15 minutes. For example, the user might be asked to submit an image of a restaurant’smenu or cars in a parking garage to gauge usage rate. To findthe impact of gamification, users were randomly offered eithera monetary incentive only, or a monetary incentive coupledwith a gamification-based reward.
When they trialed this approach with 18 users over 30 days,
the researchers found that their gamification algorithm
increased participation probability to 73%, up from 53%
without gamification. Possible applications of this research arevery broad, including the real-time monitoring of weather
condition or highway congestion at various locations. Optimizing the gamification aspects of crowdsourcing apps could
greatly increase participation rates while not incurring
excessive payment costs.
Reference
Ueyama, Y., Tamai, M., Arakawa, Y., Yasumoto, K. 2014. Gamification-based incentive
mechanism for participatory sensing. Pervasive Computing and Communications
Workshops, 98–103.

The overview of the participatory sensing system (Ueyama et al., 2014, PERCOM).

56

Software Engineering

NAIST Research Highlights

An overview of the approach employed (Tantithamthavorn et al., 2016, ICSE).

The complexity of modern software has rapidly
outstripped the comprehension of any single coder. The
length of the source code that lies behind a major
computer program or website can easily run to tens of millionsof lines. It has become standard practice for organizations totest each module for bugs that could potentially lead to failure
or suboptimal performance under certain future conditions.
As part of this effort, the software quality assurance processusually employs specialized automated machine-learning
prediction models trained to identify software that may bevulnerable to defects. These tools are based on statistical ormachine-learning classification approaches that use predefined
search parameters, for example, the number of decision trees
to use in a random forest classifier. However, the optimal
settings for these parameters are not known ahead of time, andthere is reason to believe that defect prediction models may
suffer from reduced accuracy if they are trained under suboptimal parameter settings. Because the number of possible
parameter permutations is far too large to try exhaustively, amore systematic approach is needed.
To study this situation, a research team led by Kenichi
Matsumoto at NAIST used the automated parameter optimization tool called Caret using examples from 18 datasets,
including both proprietary and open-source software. They
found that changing parameter settings for the defect-detection
models can lead to significant improvement in performance.

Of the 26 machine-learning methods considered, Caret could
be used for 16 of them, including commonly used approaches
like random Forest, support vector machines, and artificial
neural networks.
Caret works by evaluating potential combinations of thecandidate parameter settings, up to a preset limit. The researchers used 100 repetitions to estimate the performance ofclassifiers that are trained using each of the candidate parameter settings. They found that using Caret improved the area
under the curve (the primary measure of model accuracy) of
defect prediction models by as much as 40 percentage points.
This approach also increased the chance that a top-performingclassifier would be identified by up to 83%. To ensure that theconclusions regarding the models were robust, out-of-sample
bootstrap validation was applied.
The results of this work can be applied throughout the fieldof machine learning. In particular, the accuracy gains due toparameter optimization can be beneficial for almost any kindof classification task. This project can help guide developers
determine how many resources to invest in this process basedon the prospective increase in performance.
Reference
Tantithamthavorn, C., McIntosh, S., Hassan, A.E., Matsumoto, K. 2016. Automatedparameter optimization of classification techniques for defect prediction models.
International Conference on Software Engineering, 321–332.

NAIST Research Highlights

57

Artificial Intelligence

30 YEARS OF HISTORY: 2011– 2021

Chemistry

30 YEARS OF HISTORY: 2011– 2021

Satoshi Nakamura

Tsuyoshi Kawai

Text and speech waveform
closed-loop deep learning

Shining a light on the structural basis
for highly sensitive photo-induced
cyclization in organic compounds

A machine learning system for speech that combines waveform-to-text and text-to-waveform algorithms
in a closed-loop architecture, may assist in future human/computer interfaces

A molecular design strategy highlights the intramolecular interactions that govern
photo-induced reactivity in a synthetic organic compound with higher light sensitivity
than the biological system responsible for night-visionL

Speech chain and related spoken language technologies (Tjandra et al., 2017, ASRU).

T

he neural processes used by the human brain to decode
incoming audible language signals and those that encode
outgoing speech are closely related. For example, it has
been observed that children with hearing difficulties often havetrouble speaking as well, due to, at least in part, the fact that they
do not receive the proper audible feedback to modulate their own
speech. However, the current machine learning algorithms forconverting a sound waveform into text (automatic speech
recognition) have developed almost entirely independently fromthose that do the reverse and produce a waveform from text (textto-speech synthesis). The ability to combine these two tasks intoa single workflow is desired for more sophisticated and seamlesscomputer interfaces, as with digital assistants like Siri or Alexa.
To address this need, a research team led by Satoshi
Nakamura at NAIST created a “closed loop” artificial intelligence system based on a sequence-to-sequence model. This
allowed the computer to operate using a combination of bothlabeled and unlabeled data. By using this deep neural network(DNN) approach, the computer was able to organically learn
the relationships between speech waveforms and snippets oftext, instead of relying on a complex model created by hand.
Based on this increased flexibility, the algorithm created morerobust and accurate models.
The researchers found that the key insight was the ability tojointly train the sequence-to-sequence automatic speech recognition model with the sequence-to-sequence text-to-speech

58

NAIST Research Highlights

synthesis model. Although the models can be trained
separately if the data have labels (supervised learning), theymust be trained together if the data are unlabeled (unsupervised learning). Although many labeled training sets areavailable, the vast majority of written and spoken language is
not prepared in this way. Thus, the option to operate on bothkinds of inputs is a distinct advantage.
When implementing this combined approach, the teamused a large corpus of text read by a single human speaker. The
speech waveforms were converted into spectrograms with a
2048-point fast Fourier Transform into 50-ms frame lengths.
This permitted the machine learning features, which could becorrelated by the algorithm with the corresponding text, to beefficiently extracted from the raw voice data.
The research in this study could enable the development ofan artificial intelligence program that more closely mimics theway the brain creates and processes sound. This is the first deeplearning model that attempts to integrate human speech
production with speech perception, and so paves the way forrevolutionary improvements in the ability of humans andcomputers to interface in a more natural fashion.
Reference
Tjandra, A., Sakti, S., Nakamura, S. 2017. Listening while speaking: Speech chain bydeep learning. IEEE Automatic Speech Recognition and Understanding Workshop,
301–308.

(hexane) and protic (methanol) solvents using UV-vis spectrosight-induced molecular isomerization (photochromism)
in nature contributes to animals’ night-vision
copy. Although the quantum yield for the ring-open to
ring-closed photoreaction was only 54% in methanol, it reachedcapabilities. Being able to control this photochromic
property in synthetic molecules, inspired by natural
98% in hexane. This was one of the largest photocyclization
systems, could broaden the scope of their potential applications
quantum yields reported at the time and confirmed that photonin photoswitches, eye-protection materials and otherabsorption quantitatively triggered photoisomerization in this
optical devices.
system. Meanwhile, the observed solvent dependence supported
the proposed mechanism of stabilization via noncovalent
To optimize the quantum yield and sensitivity of the photochromic reaction, it is crucial to understand the molecular
intramolecular interactions, such as H-bonding.
structural features that support the desired photoreactivity. In
The designed photochromic molecule exhibits significantly
2011, a research group led by Tsuyoshi Kawai at NAIST
greater sensitivity than the natural biological opsin-retinal
published a study detailing the synthesis, characterization andcomplex in animal night-vision systems because the researchers
photocyclization behavior of terarylene-based compounds.
were able to precisely control the ground-state conformation to
They revealed that a C 2 ground-state symmetry with
optimize its functional properties. The structure-function
insights described in this study will undoubtedly continue torationally designed intramolecular H-bonding and stericinteractions could promote the highly efficient photo-induced
inspire the design of synthetic systems that can be implemented
ring-closing reaction.
in next-generation optical devices.
Considering the weak, water-repellent, noncovalent interactions in the rhodopsin binding pocket, which hosts theReference
photo-active molecule in the natural system, it was reasonable to
Fukumoto, S., Nakashima, T., Kawai, T. 2011. Photon-quantitative reaction of aconclude that such an environment would be conducive to thedithiazolylarylene in solution. Angewandte Chemie International Edition, 50,
1565–1568.
photoisomerization process of a synthetic molecule under mild
conditions. Photochromic terarylene molecules contain
three ring moieties that can undergo reversible ringclosing/opening reactions upon UV/vis irradiation,
respectively, and their reported photocyclization
quantum yields (>60%) are comparable to the natural
system’s photoisomerization quantum yield (65%).
Thus, a dithiazolylarylene molecule was synthesized
and characterized in the solid state (by single-crystal
X-ray diffraction) and in solution. The designed
molecule adopted the desired geometrical conformation and exhibited weak intramolecular CH–N
hydrogen bonding, as well as S–N and CH3–π
interactions. These results were confirmed by temperature-dependent NMR studies and density functional
theory calculations, which also supported thecompound’s potential photoreactivity.
The solid crystal dithiazolylarylene turned blue
under UV light, so its photochromic performance
ORTEP drawing of the open-ring isomer (Fukumoto et al., 2011, Angew Chem Int Ed).
under 313-nm irradiation was also evaluated in aprotic

NAIST Research Highlights

59Artificial Intelligence

30 YEARS OF HISTORY: 2011– 2021

Chemistry

30 YEARS OF HISTORY: 2011– 2021

Satoshi Nakamura

Tsuyoshi Kawai

Text and speech waveform
closed-loop deep learning

Shining a light on the structural basis
for highly sensitive photo-induced
cyclization in organic compounds

A machine learning system for speech that combines waveform-to-text and text-to-waveform algorithms
in a closed-loop architecture, may assist in future human/computer interfaces

A molecular design strategy highlights the intramolecular interactions that govern
photo-induced reactivity in a synthetic organic compound with higher light sensitivity
than the biological system responsible for night-visionL

Speech chain and related spoken language technologies (Tjandra et al., 2017, ASRU).

T

he neural processes used by the human brain to decode
incoming audible language signals and those that encode
outgoing speech are closely related. For example, it has
been observed that children with hearing difficulties often havetrouble speaking as well, due to, at least in part, the fact that they
do not receive the proper audible feedback to modulate their own
speech. However, the current machine learning algorithms forconverting a sound waveform into text (automatic speech
recognition) have developed almost entirely independently fromthose that do the reverse and produce a waveform from text (textto-speech synthesis). The ability to combine these two tasks intoa single workflow is desired for more sophisticated and seamlesscomputer interfaces, as with digital assistants like Siri or Alexa.
To address this need, a research team led by Satoshi
Nakamura at NAIST created a “closed loop” artificial intelligence system based on a sequence-to-sequence model. This
allowed the computer to operate using a combination of bothlabeled and unlabeled data. By using this deep neural network(DNN) approach, the computer was able to organically learn
the relationships between speech waveforms and snippets oftext, instead of relying on a complex model created by hand.
Based on this increased flexibility, the algorithm created morerobust and accurate models.
The researchers found that the key insight was the ability tojointly train the sequence-to-sequence automatic speech recognition model with the sequence-to-sequence text-to-speech

58

NAIST Research Highlights

synthesis model. Although the models can be trained
separately if the data have labels (supervised learning), theymust be trained together if the data are unlabeled (unsupervised learning). Although many labeled training sets areavailable, the vast majority of written and spoken language is
not prepared in this way. Thus, the option to operate on bothkinds of inputs is a distinct advantage.
When implementing this combined approach, the teamused a large corpus of text read by a single human speaker. The
speech waveforms were converted into spectrograms with a
2048-point fast Fourier Transform into 50-ms frame lengths.
This permitted the machine learning features, which could becorrelated by the algorithm with the corresponding text, to beefficiently extracted from the raw voice data.
The research in this study could enable the development ofan artificial intelligence program that more closely mimics theway the brain creates and processes sound. This is the first deeplearning model that attempts to integrate human speech
production with speech perception, and so paves the way forrevolutionary improvements in the ability of humans andcomputers to interface in a more natural fashion.
Reference
Tjandra, A., Sakti, S., Nakamura, S. 2017. Listening while speaking: Speech chain bydeep learning. IEEE Automatic Speech Recognition and Understanding Workshop,
301–308.

(hexane) and protic (methanol) solvents using UV-vis spectrosight-induced molecular isomerization (photochromism)
in nature contributes to animals’ night-vision
copy. Although the quantum yield for the ring-open to
ring-closed photoreaction was only 54% in methanol, it reachedcapabilities. Being able to control this photochromic
property in synthetic molecules, inspired by natural
98% in hexane. This was one of the largest photocyclization
systems, could broaden the scope of their potential applications
quantum yields reported at the time and confirmed that photonin photoswitches, eye-protection materials and otherabsorption quantitatively triggered photoisomerization in this
optical devices.
system. Meanwhile, the observed solvent dependence supported
the proposed mechanism of stabilization via noncovalent
To optimize the quantum yield and sensitivity of the photochromic reaction, it is crucial to understand the molecular
intramolecular interactions, such as H-bonding.
structural features that support the desired photoreactivity. In
The designed photochromic molecule exhibits significantly
2011, a research group led by Tsuyoshi Kawai at NAIST
greater sensitivity than the natural biological opsin-retinal
published a study detailing the synthesis, characterization andcomplex in animal night-vision systems because the researchers
photocyclization behavior of terarylene-based compounds.
were able to precisely control the ground-state conformation to
They revealed that a C 2 ground-state symmetry with
optimize its functional properties. The structure-function
insights described in this study will undoubtedly continue torationally designed intramolecular H-bonding and stericinteractions could promote the highly efficient photo-induced
inspire the design of synthetic systems that can be implemented
ring-closing reaction.
in next-generation optical devices.
Considering the weak, water-repellent, noncovalent interactions in the rhodopsin binding pocket, which hosts theReference
photo-active molecule in the natural system, it was reasonable to
Fukumoto, S., Nakashima, T., Kawai, T. 2011. Photon-quantitative reaction of aconclude that such an environment would be conducive to thedithiazolylarylene in solution. Angewandte Chemie International Edition, 50,
1565–1568.
photoisomerization process of a synthetic molecule under mild
conditions. Photochromic terarylene molecules contain
three ring moieties that can undergo reversible ringclosing/opening reactions upon UV/vis irradiation,
respectively, and their reported photocyclization
quantum yields (>60%) are comparable to the natural
system’s photoisomerization quantum yield (65%).
Thus, a dithiazolylarylene molecule was synthesized
and characterized in the solid state (by single-crystal
X-ray diffraction) and in solution. The designed
molecule adopted the desired geometrical conformation and exhibited weak intramolecular CH–N
hydrogen bonding, as well as S–N and CH3–π
interactions. These results were confirmed by temperature-dependent NMR studies and density functional
theory calculations, which also supported thecompound’s potential photoreactivity.
The solid crystal dithiazolylarylene turned blue
under UV light, so its photochromic performance
ORTEP drawing of the open-ring isomer (Fukumoto et al., 2011, Angew Chem Int Ed).
under 313-nm irradiation was also evaluated in aprotic

NAIST Research Highlights

59

Biophysics

30 YEARS OF HISTORY: 2011– 2021

Hisao Yanagi

Femtosecond lasers:
A tool for understandingcell–cell adhesion pathologies

Chemically modified gain medium
developed for organic lasers

2000

C

ell–cell adhesion is foundational to neuroimmunity,
inflammatory diseases, and many other life processes.
However, some measurement methods for estimating
the strength of this adhesion disrupt native physiology; thus, it
can be difficult to apply corresponding measurements made in
the lab to general medical practice. A research team led by
Yoichiroh Hosokawa at NAIST used transient femtosecond
laser pulses to quantitate intercellular breaking forces in anoncontact manner, providing insight into otherwise intractable biological signal transduction pathways.
Multicellular organisms routinely modulate cell–celladhesion as a means to regulate physiological and pathologicaltissue functions. Quantitative estimates of adhesion strengths
are available from in vitro aggregation or pulling-force studies.
However, the in vivo applicability of such studies is limited
because they disrupt concurrent physiology. Hosokawa andhis team aimed to address the uncertainty in quantitative
estimates of in vivo cell–cell adhesion strengths through theirresearch using a combination of atomic force microscopy andfemtosecond laser pulses.
To solve this problem, the researchers first used an 800-nm,
120-femtosecond laser to apply a transient impulsive force onto
the water near the top of an atomic force microscopy cantilever.
By measuring the nanometer-scale oscillations of the cantilever, they were able to quantitate the corresponding impulsive
force. By using these data in experiments that measured theminimum impulsive force required to dissociate cell–celladhesion, the researchers were able to quantitate thisadhesion in an otherwise nondisruptive manner.
The researchers first checked their experimental approachagainst a conventional cell aggregation assay and a microspheremodel system. The cell aggregation assay was based on a typicalconnective tissue cell line NIH3T3 that expressed the adhesion
molecule CADM1. The laser didn't appear to damage the cells,
but did dissociate cell–cell interactions in a predictable manner.
The microsphere model system provided a useful measure of
the uncertainty of the impulsive force estimates (and thus thecalculated adhesion strengths). On the basis of several hundred
measurements of streptavidin-coated microspheres on a
biotin-coated substrate, the measurement accuracy was ~7%.
To demonstrate clear applicability to biologically pertinentcell cultures, the researchers next quantitated the adhesion in
two scenarios: either between leukocytes and endothelial cellmonolayers, or between kidney cell monolayers. In the former,
the impulse required to induce slipping along the monolayer
was on the order of 10−13 N·s. In the latter, the impulse required
to disrupt adhesion was on the order of 10−12 N·s. These
measurements are comparable to the best estimates derived
from in vitro work that nevertheless requires disruptive
mechanical contact.
These results provide a means of reliably measuring cell–celladhesion strengths without otherwise disrupting concurrent
physiology. Ongoing questions of how cell–cell adhesion
modulates the complex crosstalk that characterizes neuro-immune signaling can be addressed
quantitatively through thisresearch. Such knowledge could
assist medical practitioners totreat serious immune disorders
and characterize other cell-adhesion pathologies, such as thosewhich cause cancer.
© 2011 National Academy of Sciences

1991

30 YEARS OF HISTORY: 2011– 2021

Yoichiroh Hosokawa

Intercellular adhesion has been quantified in a nondisruptive manner, yielding
accurate estimates of physiological and pathological adhesion strengths

Femtosecond laser-induced intercellular dissociation of HL-60 leukocytes attached to HUVECs (A) andthe MDCK cell–cell interface (B) (Hosokawa et al., 2011, PNAS).

60

Nanotechnology

NAIST Research Highlights

Reference
Hosokawa, Y., Hagiyama, M., Iino, T.,
Murakami, Y., Ito, A. 2011. Noncontact
estimation of intercellular breaking force
using a femtosecond laser impulse
quantified by atomic force microscopy.
Proceedings of the National Academy of
Sciences of the United States of America, 108,
1777–1782.

X-Ray crystallography and photoluminescence spectroscopy are used to study chemicallymodified thiophene/phenylene co-oligomer crystals, which are found to be suitable as
the gain material for organic lasers

L

asers based on organic molecules have many advantages
over conventional devices.
For example, the intrinsic properties, including emission wavelengthand gain, are much easier to tune todesired values using chemical
modification. In addition, organic-based lasers can be fabricateddirectly on flexible substrates
without the need for extreme conditions, such as high temperatures or
toxic reagents. However, the integration of organic lasers into photonic
devices, including those used intelecommunication fiber optics that
support broadband internet service,
has been slow. New, optimized
molecules are needed before organic
lasers are able to be widely adopted.
One solution to this problem wasinvestigated by a research team led by
Fluorescence micrograph of single-crystal BP2T-OMe excited at 365 nm (Mizuno et al., 2012, Adv Mater).
Hisao Yanagi at NAIST using
thiophene/phenylene co-oligomer
occurred at 455 and 415 nm, while photoluminescent emission(TPCO) crystals. This class of polymers had already beenbands were observed at 525, 565 and 605 nm. These results
recognized for their ability to act as an efficient laser gain
suggested that the unusual luminescence bands were the result
medium. The gain medium is the most crucial part of a laserof the altered molecular orientation and crystal structure
because it is the location where energy is added so light amplificaused by the chemical substitutions.
cation can occur. For best performance, its light emission
It was also found that the regular orientation of the
wavelength bands should be narrow, and the energy losses
molecules in these organic crystals leads to significant anisotshould be low. It was theorized that small modifications to theropy – meaning the luminescence bands differed in strengthchemical formula could change the resulting crystal structure,depending on the direction of measurement. This was reflectedwhich in turn would impact its emission and absorption
in the optical properties in the form of polarized light emission.
properties. Single crystals of 5,5’‐bis(4’‐methoxybiOn the basis of this approach, crystals like BP2T-OMe seem
phenyl‐4‐yl)‐2,2’‐bithiophene were created, also called
closer to being incorporated as the gain medium of next-generBP2T-OMe, and its structure analyzed using X-ray diffraction.
ation organic lasers. This could lead to substantial reductions
They found that BP2T-OMe molecules crystallized into an
in cost for internet infrastructure, which could help close theorthorhombic form, as compared with the zigzag monoclinic“digital divide” by bringing high-speed telecommunication
structure of unsubstituted TPCO.
technology to remote areas.
The optical absorption and photoluminescence spectra
were then measured at room temperature. The bright green
Reference
fluorescent light emitted from the edges of the crystal indicatedMizuno, H., Haku, U., Marutani, Y., Ishizumi, A., Yanagi, H., Sasaki, F., Hotta, S. 2012.
that the photonic energy remained confined inside the crystal,
Single crystals of 5,5’‐bis(4’‐methoxybiphenyl‐4‐yl)‐2,2’‐bithiophene for organic
laser media. Advanced Materials, 24, 5744–5749.
as seen previously for other TPCO crystals. Absorption bands

NAIST Research Highlights

61

Biophysics

30 YEARS OF HISTORY: 2011– 2021

Hisao Yanagi

Femtosecond lasers:
A tool for understandingcell–cell adhesion pathologies

Chemically modified gain medium
developed for organic lasers

2000

C

ell–cell adhesion is foundational to neuroimmunity,
inflammatory diseases, and many other life processes.
However, some measurement methods for estimating
the strength of this adhesion disrupt native physiology; thus, it
can be difficult to apply corresponding measurements made in
the lab to general medical practice. A research team led by
Yoichiroh Hosokawa at NAIST used transient femtosecond
laser pulses to quantitate intercellular breaking forces in anoncontact manner, providing insight into otherwise intractable biological signal transduction pathways.
Multicellular organisms routinely modulate cell–celladhesion as a means to regulate physiological and pathologicaltissue functions. Quantitative estimates of adhesion strengths
are available from in vitro aggregation or pulling-force studies.
However, the in vivo applicability of such studies is limited
because they disrupt concurrent physiology. Hosokawa andhis team aimed to address the uncertainty in quantitative
estimates of in vivo cell–cell adhesion strengths through theirresearch using a combination of atomic force microscopy andfemtosecond laser pulses.
To solve this problem, the researchers first used an 800-nm,
120-femtosecond laser to apply a transient impulsive force onto
the water near the top of an atomic force microscopy cantilever.
By measuring the nanometer-scale oscillations of the cantilever, they were able to quantitate the corresponding impulsive
force. By using these data in experiments that measured theminimum impulsive force required to dissociate cell–celladhesion, the researchers were able to quantitate thisadhesion in an otherwise nondisruptive manner.
The researchers first checked their experimental approachagainst a conventional cell aggregation assay and a microspheremodel system. The cell aggregation assay was based on a typicalconnective tissue cell line NIH3T3 that expressed the adhesion
molecule CADM1. The laser didn't appear to damage the cells,
but did dissociate cell–cell interactions in a predictable manner.
The microsphere model system provided a useful measure of
the uncertainty of the impulsive force estimates (and thus thecalculated adhesion strengths). On the basis of several hundred
measurements of streptavidin-coated microspheres on a
biotin-coated substrate, the measurement accuracy was ~7%.
To demonstrate clear applicability to biologically pertinentcell cultures, the researchers next quantitated the adhesion in
two scenarios: either between leukocytes and endothelial cellmonolayers, or between kidney cell monolayers. In the former,
the impulse required to induce slipping along the monolayer
was on the order of 10−13 N·s. In the latter, the impulse required
to disrupt adhesion was on the order of 10−12 N·s. These
measurements are comparable to the best estimates derived
from in vitro work that nevertheless requires disruptive
mechanical contact.
These results provide a means of reliably measuring cell–celladhesion strengths without otherwise disrupting concurrent
physiology. Ongoing questions of how cell–cell adhesion
modulates the complex crosstalk that characterizes neuro-immune signaling can be addressed
quantitatively through thisresearch. Such knowledge could
assist medical practitioners totreat serious immune disorders
and characterize other cell-adhesion pathologies, such as thosewhich cause cancer.
© 2011 National Academy of Sciences

1991

30 YEARS OF HISTORY: 2011– 2021

Yoichiroh Hosokawa

Intercellular adhesion has been quantified in a nondisruptive manner, yielding
accurate estimates of physiological and pathological adhesion strengths

Femtosecond laser-induced intercellular dissociation of HL-60 leukocytes attached to HUVECs (A) andthe MDCK cell–cell interface (B) (Hosokawa et al., 2011, PNAS).

60

Nanotechnology

NAIST Research Highlights

Reference
Hosokawa, Y., Hagiyama, M., Iino, T.,
Murakami, Y., Ito, A. 2011. Noncontact
estimation of intercellular breaking force
using a femtosecond laser impulse
quantified by atomic force microscopy.
Proceedings of the National Academy of
Sciences of the United States of America, 108,
1777–1782.

X-Ray crystallography and photoluminescence spectroscopy are used to study chemicallymodified thiophene/phenylene co-oligomer crystals, which are found to be suitable as
the gain material for organic lasers

L

asers based on organic molecules have many advantages
over conventional devices.
For example, the intrinsic properties, including emission wavelengthand gain, are much easier to tune todesired values using chemical
modification. In addition, organic-based lasers can be fabricateddirectly on flexible substrates
without the need for extreme conditions, such as high temperatures or
toxic reagents. However, the integration of organic lasers into photonic
devices, including those used intelecommunication fiber optics that
support broadband internet service,
has been slow. New, optimized
molecules are needed before organic
lasers are able to be widely adopted.
One solution to this problem wasinvestigated by a research team led by
Fluorescence micrograph of single-crystal BP2T-OMe excited at 365 nm (Mizuno et al., 2012, Adv Mater).
Hisao Yanagi at NAIST using
thiophene/phenylene co-oligomer
occurred at 455 and 415 nm, while photoluminescent emission(TPCO) crystals. This class of polymers had already beenbands were observed at 525, 565 and 605 nm. These results
recognized for their ability to act as an efficient laser gain
suggested that the unusual luminescence bands were the result
medium. The gain medium is the most crucial part of a laserof the altered molecular orientation and crystal structure
because it is the location where energy is added so light amplificaused by the chemical substitutions.
cation can occur. For best performance, its light emission
It was also found that the regular orientation of the
wavelength bands should be narrow, and the energy losses
molecules in these organic crystals leads to significant anisotshould be low. It was theorized that small modifications to theropy – meaning the luminescence bands differed in strengthchemical formula could change the resulting crystal structure,depending on the direction of measurement. This was reflectedwhich in turn would impact its emission and absorption
in the optical properties in the form of polarized light emission.
properties. Single crystals of 5,5’‐bis(4’‐methoxybiOn the basis of this approach, crystals like BP2T-OMe seem
phenyl‐4‐yl)‐2,2’‐bithiophene were created, also called
closer to being incorporated as the gain medium of next-generBP2T-OMe, and its structure analyzed using X-ray diffraction.
ation organic lasers. This could lead to substantial reductions
They found that BP2T-OMe molecules crystallized into an
in cost for internet infrastructure, which could help close theorthorhombic form, as compared with the zigzag monoclinic“digital divide” by bringing high-speed telecommunication
structure of unsubstituted TPCO.
technology to remote areas.
The optical absorption and photoluminescence spectra
were then measured at room temperature. The bright green
Reference
fluorescent light emitted from the edges of the crystal indicatedMizuno, H., Haku, U., Marutani, Y., Ishizumi, A., Yanagi, H., Sasaki, F., Hotta, S. 2012.
that the photonic energy remained confined inside the crystal,
Single crystals of 5,5’‐bis(4’‐methoxybiphenyl‐4‐yl)‐2,2’‐bithiophene for organic
laser media. Advanced Materials, 24, 5744–5749.
as seen previously for other TPCO crystals. Absorption bands

NAIST Research Highlights

61

Energy Material

30 YEARS OF HISTORY: 2011– 2021

Applied Physics

30 YEARS OF HISTORY: 2011– 2021

Jun Ohta

Masakazu Nakamura

World’s smallest IR wireless energy
receiver developed for implantablebiodevices and smart sensors
A semiconductor photovoltaic platform that can absorb infrared light energy
is developed, which may help power implanted medical devices and smart objects
that will contribute to the “Internet of Things”

Control of charge and heat transports by molecular junctions for wearable thermoelectric generators.

Weaving carbon nanotubes into fabricfor harvesting human body heat
A fabrication process for carbon-nanotube composite threads that can form flexible
thermoelectric generator textiles is developed to recover waste body heatT

he process of recovering electricity from waste heat, suchas that produced based on the temperature difference
between a person’s body and the ambient environment,
is a promising avenue for energy harvesting. Thermoelectric
generators (TEGs) produce electricity directly from waste heatand offer the best approach for harnessing this process, and it
is hoped that they can be applied for use in self-powered,durable and low-cost wearable technologies.
However, development of TEGs for energy harvesting isburdened by several technical challenges. For instance, one
promising approach relies on solid-state TEG materials, whichconvert heat directly into voltage. Unfortunately, these materials require a rare combination of properties. In particular, a
TEG needs to have high electrical conductivity while simultaneously possessing low thermal conductivity. Moreover, thethickness required for optimal efficiency often prevents theresulting devices from being flexible enough to be woven into
clothing. Overcoming these limitations would allow small
wearable electronics to be powered by the user’s own bodyheat alone, which would reduce the need for batteries andstops for recharging.
To address these limitations, a research team led by
Masakazu Nakamura at NAIST used carbon-nanotube threads
to produce a flexible “thermoelectric fabric”. Using carbon
nanotube composite threads allowed the team to control the
thickness of the final material. The resulting thermal conductivity could be made to be less than 0.1 W/K·m, while the62

NAIST Research Highlights

thickness was large enough to produce a sufficiently large
temperature difference between the inner and outer surfaces.
The researchers found that this permitted the system to operate
even with the relatively small temperature difference between
the human body (about 37°C) and the surrounding air (usuallyaround 22°C).
To produce the fabric, carbon nanotubes were spun into a
thread along with binding polymers. The composite thread wasthen sewn into a felt fabric for structural stability. To create anelectrical device, alternating “stripes” of p-type and n-type
doped regions were made by exposing the fabric to chemicals
that removed or added electrons, respectively. The sample
remained intact even after 160 test cycles of bending andstretching. The touch of a finger was enough to generate avoltage in a prototype thermoelectric fabric. Within 4 seconds,
an electrical potential of 2.3 millivolts registered.
The proof of concept demonstrated by this research could
be extended to other flexible thermoelectric materials besides
carbon nanotubes, including conducting polymers, or organic/metal composites. This has great potential for a wide variety
of products – from biomedical devices to countertops – thatwould benefit from its own source of power.

W

ith the advent of small medical implants, there is anincreasing need for wireless power systems that canallow these devices to function without the need forbatteries or wires that cross the skin. Current solutions often
rely on electromagnetic induction with radio-frequency waves
to transmit energy from a source outside the body to the device.
However, these systems require large induction coils – often
bigger than the rest of the device – and suffer from low
efficiency. While this is sufficient for certain applications, suchas wireless charging of smartphones or powering RFID tags,
alternatives to electromagnetic induction for contactlessenergy transmission are needed. This is particularly true as weenter an era of the “Internet of Things (IoT)” in which many
more connected devices will need to be powered remotely.
To address this need, a research team led by Jun Ohta at
NAIST has created a complementary metal-oxide semiconductor (CMOS) photovoltaic receiver chip that works by receiving
infrared light. Like any other solar panel, the light energy canbe converted directly into an electrical voltage. For biomedical

applications, the wavelength of light must be carefully chosen
based on efficiency and safety considerations. Red or infrared
light sources placed on the body are preferred, because they
can pass more easily through tissue while also not causing cell
damage. For IoT devices, smart connected nodes could be
configured to run on indoor ambient light.
The researchers built a 1.25-mm × 1.25-mm prototype anddemonstrated the proof-of-concept by powering a blue LED
light. The integrated circuit eliminated the need for a voltage
booster by monitoring the voltage of the internal capacitor andswitching between the power-receiving photovoltaics and theload circuit as appropriate. An optogenetic neural stimulator
that had a volume of just one cubic millimeter was thenfabricated. This type of device is used in certain animal experiments, in which gene expression is turned on or off inside thebody by activating light-sensitive proteins. The team foundthat the stimulator could be powered solely by a commerciallyavailable IR flashlight.
This approach to making implantable medical devices and
IoT nodes more cost efficient andreliable holds great potential for
widespread adoption. For example, atemperature sensor placed on theexterior of food packaging to ensure
freshness could operate without
batteries based on the indoor
lighting. More sophisticated medical
devices for internal use, like apacemaker, could be continuallycharged by an IR LED the patient
wears over their heart.
By eliminating the need foron-device energy storage, nodes may
be substantially streamlined, andused in many more applications thanthey are today.
Reference

Reference
Ito, M., Koizumi, T., Kojima, H., Sato, T., Nakamura, M. 2017. From materials to device
design of a thermoelectric fabric for wearable energy harvesters. Journal of Materials
Chemistry A, 5, 12068.

Size and design of the 1 mm3 optogenetic stimulator.

Tokuda, T., Ishizu, T., Nattakarn, W., Haruta, M.,
Noda, T., Sasagawa, K., Sawan, M., Ohta, J. 2018.
1 mm3-sized optical neural stimulator based on
CMOS integrated photovoltaic power receiver.
AIP Advances, 108, 045018.

NAIST Research Highlights

63

Energy Material

30 YEARS OF HISTORY: 2011– 2021

Applied Physics

30 YEARS OF HISTORY: 2011– 2021

Jun Ohta

Masakazu Nakamura

World’s smallest IR wireless energy
receiver developed for implantablebiodevices and smart sensors
A semiconductor photovoltaic platform that can absorb infrared light energy
is developed, which may help power implanted medical devices and smart objects
that will contribute to the “Internet of Things”

Control of charge and heat transports by molecular junctions for wearable thermoelectric generators.

Weaving carbon nanotubes into fabricfor harvesting human body heat
A fabrication process for carbon-nanotube composite threads that can form flexible
thermoelectric generator textiles is developed to recover waste body heatT

he process of recovering electricity from waste heat, suchas that produced based on the temperature difference
between a person’s body and the ambient environment,
is a promising avenue for energy harvesting. Thermoelectric
generators (TEGs) produce electricity directly from waste heatand offer the best approach for harnessing this process, and it
is hoped that they can be applied for use in self-powered,durable and low-cost wearable technologies.
However, development of TEGs for energy harvesting isburdened by several technical challenges. For instance, one
promising approach relies on solid-state TEG materials, whichconvert heat directly into voltage. Unfortunately, these materials require a rare combination of properties. In particular, a
TEG needs to have high electrical conductivity while simultaneously possessing low thermal conductivity. Moreover, thethickness required for optimal efficiency often prevents theresulting devices from being flexible enough to be woven into
clothing. Overcoming these limitations would allow small
wearable electronics to be powered by the user’s own bodyheat alone, which would reduce the need for batteries andstops for recharging.
To address these limitations, a research team led by
Masakazu Nakamura at NAIST used carbon-nanotube threads
to produce a flexible “thermoelectric fabric”. Using carbon
nanotube composite threads allowed the team to control the
thickness of the final material. The resulting thermal conductivity could be made to be less than 0.1 W/K·m, while the62

NAIST Research Highlights

thickness was large enough to produce a sufficiently large
temperature difference between the inner and outer surfaces.
The researchers found that this permitted the system to operate
even with the relatively small temperature difference between
the human body (about 37°C) and the surrounding air (usuallyaround 22°C).
To produce the fabric, carbon nanotubes were spun into a
thread along with binding polymers. The composite thread wasthen sewn into a felt fabric for structural stability. To create anelectrical device, alternating “stripes” of p-type and n-type
doped regions were made by exposing the fabric to chemicals
that removed or added electrons, respectively. The sample
remained intact even after 160 test cycles of bending andstretching. The touch of a finger was enough to generate avoltage in a prototype thermoelectric fabric. Within 4 seconds,
an electrical potential of 2.3 millivolts registered.
The proof of concept demonstrated by this research could
be extended to other flexible thermoelectric materials besides
carbon nanotubes, including conducting polymers, or organic/metal composites. This has great potential for a wide variety
of products – from biomedical devices to countertops – thatwould benefit from its own source of power.

W

ith the advent of small medical implants, there is anincreasing need for wireless power systems that canallow these devices to function without the need forbatteries or wires that cross the skin. Current solutions often
rely on electromagnetic induction with radio-frequency waves
to transmit energy from a source outside the body to the device.
However, these systems require large induction coils – often
bigger than the rest of the device – and suffer from low
efficiency. While this is sufficient for certain applications, suchas wireless charging of smartphones or powering RFID tags,
alternatives to electromagnetic induction for contactlessenergy transmission are needed. This is particularly true as weenter an era of the “Internet of Things (IoT)” in which many
more connected devices will need to be powered remotely.
To address this need, a research team led by Jun Ohta at
NAIST has created a complementary metal-oxide semiconductor (CMOS) photovoltaic receiver chip that works by receiving
infrared light. Like any other solar panel, the light energy canbe converted directly into an electrical voltage. For biomedical

applications, the wavelength of light must be carefully chosen
based on efficiency and safety considerations. Red or infrared
light sources placed on the body are preferred, because they
can pass more easily through tissue while also not causing cell
damage. For IoT devices, smart connected nodes could be
configured to run on indoor ambient light.
The researchers built a 1.25-mm × 1.25-mm prototype anddemonstrated the proof-of-concept by powering a blue LED
light. The integrated circuit eliminated the need for a voltage
booster by monitoring the voltage of the internal capacitor andswitching between the power-receiving photovoltaics and theload circuit as appropriate. An optogenetic neural stimulator
that had a volume of just one cubic millimeter was thenfabricated. This type of device is used in certain animal experiments, in which gene expression is turned on or off inside thebody by activating light-sensitive proteins. The team foundthat the stimulator could be powered solely by a commerciallyavailable IR flashlight.
This approach to making implantable medical devices and
IoT nodes more cost efficient andreliable holds great potential for
widespread adoption. For example, atemperature sensor placed on theexterior of food packaging to ensure
freshness could operate without
batteries based on the indoor
lighting. More sophisticated medical
devices for internal use, like apacemaker, could be continuallycharged by an IR LED the patient
wears over their heart.
By eliminating the need foron-device energy storage, nodes may
be substantially streamlined, andused in many more applications thanthey are today.
Reference

Reference
Ito, M., Koizumi, T., Kojima, H., Sato, T., Nakamura, M. 2017. From materials to device
design of a thermoelectric fabric for wearable energy harvesters. Journal of Materials
Chemistry A, 5, 12068.

Size and design of the 1 mm3 optogenetic stimulator.

Tokuda, T., Ishizu, T., Nattakarn, W., Haruta, M.,
Noda, T., Sasagawa, K., Sawan, M., Ohta, J. 2018.
1 mm3-sized optical neural stimulator based on
CMOS integrated photovoltaic power receiver.
AIP Advances, 108, 045018.

NAIST Research Highlights

63

Outgrow your limits

Outgrow your limits

NAISTRESEARCHHIGHLIGHTS

http://w w w .naist.jp/en/

RE SE ARCH HIGHLIGHTS
30 YEARS OF HISTORY : 1991−2021

www.naist.jp/en

30
YEARS
OF
HISTORY:
1991−2021