別紙41 (課程博士(英文))Date ofsubmjssion (month day, year)

January l0小 2020

Dcpadment

Applied chen〕istry andS加den11D NU1刀berDB34Ⅱ

Life science
へPplicant's nameSUI〕e1刃isors

RyU牙O Tefo
Rika Numano

YasuhⅡ'O KakⅡnoto

Abstract (Doctor)
Title oflhesis

Development and application ofobse】'vation methods fbr functionaΠゆid domains andassembled stl'uctul'es in supp011ed lipid bilayel'S

Approx.80o words
In a111iving dlings, the outer and inner worlds of ce11S m"e sePω'ated by ce111nelnbranes
Slnce {1〕ey are dle outennost of the ce11S, they provide reaction aelds to recognize t11e alnbient

envh'01Ⅱnent, and to transport inforlnation and lnatelials into and out of ce11S.1hese play
ilnportant roles in the function of ce11S, such as ilnlnunity, infection, and lnetaboHsn〕. ce11

membranes have a blmolecular membl'ane compl、ising of amphゆ1)ilic Hpids, which sel've astl)e fundalnental skeleton, and c011tain lnelnbtane proteins and glycolipids i11 it. Localization
Of these ce11 1nembrane colnponents is key factors regulating d]e recognition andtransportation reactions. Artificial ce111nelnbrane systelns have been studied to elucidate tl〕efunctions of ceⅡ lnembranes. supported Hpid bilay飢'S (SLBS)釘'e a kind of m'tificia1 1ipid
Inelnblane situated at soHd‑Hquid int飢faces. Fluid and n、agile Hpid lnelnbranes m・e stabilizedh) tl〕e sLB systeln, and that highly sensitive analytical methods in tl〕e tield of int仇facial

Science n〕easurelnents m'e available. Especia11y sLBs have been used to h〕vestigate thefundalnental physicochelnical properties and lnolecular distribution of 11Pld bilay飢'n]̲elnbranes

In this ph.D. tl〕esis,1 ailned to investigate tl〕e functional dolnail〕s containing glycolipids

1＼VO・dilnensiona1 10calization, e.g. clustering and don〕ain forlnations, of σlycolipids andnlen〕̲bl'ane protelns eXⅨ't dleh' appropriate functions in the ce111nelnblane reactions of Hvh〕gOrganislns. Although extensive reseω'ches have been conducted on the diffusion andagglegation of Hplds and proteins ln ceⅡ lnelnbranes, glycochaln dynalnics and effects onInelnbrane properties m'e not weⅡ Understood. Elucidation of u)e behavior of ce111nelnbranes11) consid引'ation of glycochains on ce11 n〕en〕branes is expected to be useful for estabHshing
Inore acC山'ate ceH lnelnbrane n)odels and for developing new biodevices. For d〕is purpose,1developed obsa'vation lnethods for domains and assenlbled structures of glycoHpid, andSalnple prePω'ation lnethods for bact剖'ial nlode11nen〕branes, Additiona11y,1 found out a newappHcation of tl〕e glycoHpid localization to the dolnain conh'olin a h'eestanding bilay飢' 1ipid
Inenlbrane on a lni口'oweⅡ一fabricated si chipFirst,1 developed lnethod010gies for glycolipid・containil〕g sLB prepm'ation andObS磁'vation.1 investigated the lnorph010gy and properties of don〕alns induced by thehydrophilic・polylner・modi6ed lipid applying poly・et11ylene glyc01 (PEG)・modified lipid.1
fon11ed sLBs using a o‑10 ln01 % range of pEG・1nodified lipid concentration (CPEG).1
Studied its n〕orph010gy and auidity by auorescence lnicroscopy, tl〕e auorescence recoV飢'yaner photobleaching (FR'ヘ.P) 1nethod, and atolnic force lni仇'oscopy (AFNI). Fluorescencein〕ages and AFNltop0即'apl〕ies showed d〕at clust剖's of the pEG・1nodified Hpid and thePEG・1ipid・rich dolnains appem'ed depending on cpEG. Bod〕 10caHzed su'uctures 、V由'e inducedby the 11ydrophilic pEG chains, but gave different conu'ast in the AFNltopooraphies

Frequency・1nodulation AFN11'evealed a force・dependent appearance of the pEG・1ipid・richdolnain. This study demonsh"ated the hydrophilic・polyln圃'・induced Hpid localization, and itsPI〕ysical property that is crltical when the donlain is evaluated with AFM
Next,1 1nade bacterial nlodel sLBS, and evaluated their formation mechanisms for

Utilizing these lnodels to furth引' glycolipid and functiona1 1ipid observation. As a bacterial
model ceH lnelnbrane systeln, reconstituted 五Scherichia coli(五'. cohソ Ce111nelnbrane has beenStudied.1 forlned sLBs of 五'. coh exh'acted Hpids on mica and thenna11y oxidized si02/si
Substrates, and investigated their lnorph010gy and physical proP剖'ty with AFM andauorescence‑1nicroscope・based methods.1 found subsu'ate・dependent sLB fonnation, andtwo・ and three・d11nensiona11norph010gies dependlng on ca‑ concentration.1 also fon〕1ed
SLBs of phosphatidyletl〕anolamine (PE), phosphatidylglyoor01 (PG) and C雛'diolipin (CA),Which m'e dolninal〕t con〕ponents of五'. coh exu'acted phospholipids. The sLBs showed various
mmph010gies depending on the composition of pE, PG and cA. PG・ and cA・ dependent
holes, and pE dependent lnultiple lay引'S W由、e observed.1n this study,1 estabHshed fabrication
nlethod of 五'. coh extracted lipids sLB, and discoV引'ed two・ and three・dilnensional Hpidasselnblies depending on lipid species, substl"ate nlatel'ials, and ca2+ concentration
Then,1 h〕cluded diacylglyC引'01(DAG) and nlenlbrane protein inte即'ase (Nlplase) to d〕edeveloped sLBs of五, coh in order to establish lnelnbrane protein insertion lnode11nelnbrane
Melnbrane proteins m'e synthesized by riboson〕es and inserted il)to ce111nelnbranes, and thenP飢fonn their functions. Nlelnbrane protein insertion is thought to proceed by a uniV引'sal
Inolecular 111echaniS1Υl in aⅡ organislns. con'ect insertion of lnelnbrane proteins intomen〕branes is inlportant not only il〕 Hving organislns but also h〕 biotecl]n010gy. DAG presentin 五'. coli blocks t11e inadequate spontaneous insertion of lnelnbrane proteins. on the otherhand, Nlplase present in 五'. coliis known to prolnote tl〕e insertion oflnen〕brane proteins.1he仇'eation and conu'010fvarious artiticial ceⅡ lnelnblane systelns, induding Mplase and DAG,
Wi11 Contribute to tl)e developlnent of und引'standing and utilization of ce111nelnbranes.1
fonned and investigated sLB of五. coh' Hpids containing DAG or Mplase
Fina11y,1 discoV則'ed tl〕e glycolipid localization affects lnelnbrane dolnain forlnation on an〕icrowe11・fabricated substl'ate. FI'eestanding bilayer lipid 111elnbrane suspended by sLB is anadvanced sLB systeln, which is obtained by placing a Hpid bilay飢' on a substrate 、vith
Iniclometer ordel" holes. This systeln is designed to place indicators or sensors in the holes for
detecting the activities of the freestanding lnembl'ane.11〕 t11is freestanding region, specificIipid localizations m'e observed because of the colnpetitive il〕t引、action between tl)e supported
region and d〕e freestanding region. Recent study showed tl)at liquid disord飢'ed phasePref圃'entia11y exists in the freestandi11g region.1 attelnpted to change the properties of afreestanding 111elnbrane by using 仕le int引、action be{ween the hydrophilic polyn〕er chains oft11e glycolipid and tl〕e substrate. The bilay飢'1nelnbranes containing various concenu'ations ofganglioside GMl xvere preP磁'ed, and phase sepm'ation and don〕ain fonnation on d〕efreestanding lnen〕brane wa'e obsel'ved and evaluated using a nuorescence n〕i口、oscope. The
tilne course of the phase seP雛'ation state depended on GN11 Concentration, and the fonnation
Of functional domains on t11e n'eestanding lnelnbrane was successfU11y conu'011ed, This study
applied glycolipid locaHzation effectto engineering an a11ificial Hpid lnelnbrane systelnThis d〕esis conca'ns functional Hpid asselnbHes relatinσ to bacterial nlenlbranes,
exper11nenta11nethod010gies, and application for lnelnbrane engineering,1 expect that theseresearcl〕 works lead to break further veils of roles and lnechanism of functional Hpids inbiolnelnbrane reactions, and to contribute to progress il) tl〕e field of intmface chenlistry,
biochelnistry, and biodevices