TheBiophysical Society of Japan General IncorporatedAssociation Yuno Natsumei, Taro Toyotad'2 (iGrad. Art. Sbi., UVTiv. Tbk},o, !rsT PRESTO} In cells, biological macromo]ecules are contained at a volume fraction of20-30 vol %. Recently, this situation has much drawn attentlon as a crucia] faetor of the crowding effect of the cytosol. To construct ce]] models, giant vesicles (GVs) confining macromolecules, microspheres, or both have been adopted. However, in the conventional methods such as the

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... atural swelling method, the inner state (concentration, number density and so on) of macromolecules or microspheres is scarcely control]ed and non-uniform among the GVs. To forrn microsphere-containing GVs as desired, we employed the water-in-oil (WIO) emulsion centrifugation method reported by Pautot et al. (Langrnuir 2003, 19, 2870). because this method is based on WtO emulsion drop]et as a temp]ate of GV, By using po]ystyrene microspheres (diarneter = 1 pm) dispersions of 2.0-2S vol % to form WIO emulsions in liquid paTarnn, we obtained GVs that encapsulate microspheres with volume fractions in the range of O-50 vol % and their diameters were in range of 4-40 pm, A)though the distribution of the volume fraction of GVs was broad, it should be noted that the highest volume fraction of GV was hjgher than that of WtO emu]sien droplets. It is the advantage that we can achieve GVs confining microspheres at high number density rather than that of microspheres dispersion we prepared initially. This technique can be applied to the GV encapsu]ation of other microspheres or macromolecules that exhibit entropy-driven effects, such as the crowding effect, 3GI046 t・t'JV7U-GUVt[NA"nre"+MfiilXfhRTRACS Molecular computing system RTRACS encapsulated in oi]free giant unilameltar vesicte Koh-ichiroh Shohda, Tadashi Sugawura, Akira Suyama {Grad. St'h. Arts Sci., Univ. Tblyo) RTRACS (Reverse-transcription-and-TRanscription-based AutonomDus Computing System) is a molecu]ar computing system in which RNA molecules work as input and output.i'2 RTRACS is a reaction network system which consists of modularized molecu]ar ]ogic gates containing the Boo]ean gate such as AND, OR, etc. We intend to construct a giant unilameHar vesicle (GUV) encapsulating RTRACS as a minimal model of :iving cell. At present, the best preparation method ofGUV is the wio emutsion centrifuge method.3 However the GUV prepared with the centrifuge method potentia]]y contains the oil which surrounded the water droplet in the wlo emulsion. The oil changes the membrane properties, such as thickness, permeabi]ity, dynamics, etc. Henee we developed an oil-free GUV preparation method based on the lipid t"ilrn gentte hydration method in the presence of mono-and di-valent metal cations. The inner water pool of GUV js stained with TMRA-dextran (M.W, 10,OOO) as a vo]ume marker. The FAM-]abeled mo]ecular beacon probe which hybridizes with the output RNA molecule ofRTRACS was used as a reaetion marker. The dual-labeled GUV in which RTRACS runs wus ana]yzed by optical microscopy and flow cytometry. with a low pH buffer with a time-reso]ution of 1OO rns. We observed that at the initia] step the peak intensity ofthe L. phase gradually decreaseci but at the same The outermost layer of the huraan skin, stratum comeum (SC), consists of terminalty diffbrcntiated keratinocytes and highly ordered intercel]u]ar lipid ]ayers, The SC serves as a physicochemical barrier betvifeen the inner body and the outer external environment, Notwithstanding the extensive studies on structure of intercellular lipids which p]ays a crucial role for the skin barrier pTopenies, owing to ]ack of the structural evidencc under functioning ]ot of unresolved prob]em are ± eft open. Using X-ray and Electron di fTtactien methods we deepened understanding the healthy skin state and developing the percutaneous drugs. Both synchrotron X-ray difTtaction (XD) and electron difftaction (ED) methods are the very powerfu1 tool to analyze the intercellular lipid organization in SC. The XD has higher resotution than the ED, but requires jnvasive operatien because a 1arge amount ofskin sample is necessary to obtain reliable data. On the othcr hand, the ED makes it possjble to unalyze the struetures of the SC noninvasively, but rudiation damage by e]ectron bean" must be suppressed. By best use of these methods we wi]] report here a fundamenta] structural change of SC in the skin permeation of chemical agents, the efTect of temperature in the skin pcmneation, and the regional structurat differences on the body. We wi]] present these results and futuTe perspective for the SC structural study. Toshihiko(iFae. Sei., The modulation membranes bicontinuous s ¢ attering{TRthe kinetics d{o]eoy]phosphatidylserine 6K, and found that the Hii phase appeared after the pH change (the initial step), and then the H[i phase slowly converted into the QTiD phase (the second step) [2]. However, we could net fo]low the initial step due to the limited tirneresolution ef the method. In this report. we investigated the initial step ofthe low pH-induced L. to Q[iD phasetransitioninDOPSIMOintheabsenccofPEG-6KusingTR-SAXSwitha stopped flow apparatus, We succeeded in fo1]owing structural changes in the membranes after ]OO ms of the mixing of DOPSIMO-MLV in a neutral buffer ELI pH ffMEV6 DOPStMO gcota.e.Nbi 5S=-EvOva tNojrefiBopmorevee Initial Step of Low pH-Indueed Lameltar to Bicontinuous Cubic Phase Transition in Dio;eoylphosphatidylserinetMonoo]ein Okai, Tomeki Takaliashii, Taka-aki Tsuboii, Masahito Yamazaki2 Shizuoha Uhiv., 2Grad. Sch. Sci. 7lech., Shizuoka Univ.} of electrostatie interactions due to surface charges of lipid induces transitions between the L. phase and the inverse cubic (Qii) phase [1], Using time-resolved small ang]e X-ray SAXS) and a home-made rapid mixing method, we investigated of lew pH-induced L. to Q]iD phase transition in MLVs of (DOPS)/monoole{n(MO) in the presence of PEG-3H0900 Hiroaki Yajima satoi, Nobutaka Reseai'ch in,rt. GTPraattmatGDPtsattmacouadNe"TMMIEas(t6 ±Kza mmgltLarge Conformational Changes in Tublltin in the GTPand GD?-States Microtubules Obseryed by Cryo Electron Microscopy ' ], Toshihiko Ogura2, Ryo Nittai, Yasushi Okadat, Chikara Hirokawai (iGrad. SL'h. Med., Univ. 7bin,o,2Biomedical ,Ars7) Microtubu]es are dynamic polymeTs that stochastica]ly switch between growing and shrinking phases and their dynamics is regulated by GTP hydrolysis by Ptubulin,butitsmechanismremainsetusivebecausehigh-resolutionmicrotubule structures have on]y been revealed for the GDP-bound state. Here we sotved the cryo-EMstructureofmicrDtubulestabilizcdwithaGTPanalogueguanylyl5'-ct, P-methy]enediphosphonate (GMPCPP) at 8.8 A Tcsolution by deve]oping a novel cryo-EM image reconstruction algorithm. In contrast to the crystH[ structures of GTP-bound tubulin relatives such as T-tubulin and bacterial tubu]ins, gignificant changes were detected between GTPand GDP-bound states at the contacts between tubut[ns both along the proto filament and between neighboring protofilaments, contributing to the stabi]ity of the microtubule ]attiee. These find{ngs are consistent with the structura] p[asticity or lattice mode], and suggest the structural basis not only for the regulatory mechanism of microtubule dynamics, but also for the recognition of the nucleotide state of m{crotubu]e by severa[ microtubule-binding proteins, such as EBI or kinesin.