Studies of Membrane Fusion and Vesicle Fission of Biomembranes using Giant Unilamellar Vesicles and Cubic Phase and Their Applications

利用巨型单层囊泡和立方相进行生物膜膜融合和囊泡分裂的研究及其应用

• 批准号: 17310071
• 负责人: YAMAZAKI Masahito
• 金额: $ 7.51万
• 依托单位: Shizuoka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17310071/
• 关键词: Biophysics; Nanobioscience; Single Molecule Measurement; Giant Unilamellar Vesicle (GUV); Membrane fusion and Vesicle fission; Cubic phase of biomembranes; the single GUV method; Phase transition between Lα phase and cubic phase and cubic phases

(1) Using the single giant uniLαmelLαr vesicle (GUV) method, we found that low concentrations (less than critical micelle concentration (CMC), but above their threshold concentrations) of amphiphiles with a single long hydrocarbon chain (i.e., single long chain amphiphiles) such as lysophosphatidylcholine (lyso-PC), lyosophosphatidic acid, octylglucoside, induced the vesicle fission of GUVs of lipid membranes in the liquid-ordered (lo) phase such as dipalmitoylphosphatidylcholine(DPPC)/cholesterol(6/4) membranes. To elucidate the effect of the inclusion of DOPC (i.e., an Lα phase forming lipid) in the lo phase-membrane of GUVs on the lyso-PC-induced vesicle fission of the DPPC/chol (6/4)-GUV, we investigated effect of low concentrations of lyso-PC on GUVs of DPPC/DOPC/chol membranes. With an increase in DOPC concentration in the membrane, the threshold concentration of lyso-PC to induce the vesicle fission increased. At and above 30 mol% DOPC, no vesicle fission occurred. On the basis … More of these results, we have proposed a hypothesis of the mechanism of the single long chain amphiphile-induced vesicle fission of the GUV of the lo phase-membrane.(2) We investigated the effect of H+ (i.e. pH effect) on the phase and structure of membranes of a negatively charged lipid, dioleoylphosphatidylserine (DOPS) and an electrically neutral lipid, monoolein (MO) mixtures under a physiological ion concentration condition, using small-angle X-ray scattering (SAXS). At neutral pH, DOPS/MO membranes containing high concentrations of DOPS were in the Lα phase. First, the pH effect on the phase and structure of the multiLαmelLαr vesicles (MLVs) of the DOPS/MO membranes preformed at neutral pH was investigated by adding various low pH buffers into the MLV suspension. For 20%-DOPS/80%-MO-MLVs, at and below pH 2.9, a transition from the Lα to cubic (Q224) phase occurred within 1 h. This phase transition was reversible ; a subsequent increase in pH to a neutral one in the membrane suspension transformed the cubic phase into the original Lα phase. Second, we found that a decrease in pH transformed Lαrge uniLαmelLαr vesicles of DOPS/MO membranes into the cubic phase after the membrane fusion between the LUVs under simiLαr conditions. We have proposed the mechanism of the low-pH induced phase transition, and also made a quantitative analysis on the critical pH of the phase transition. This finding is the first demonstration that a change in pH can induce a reversible phase transition between the Lα and cubic phases of lipid membranes within 1 h. We reasonably consider that pH-induced interconversion between the cubic and the Lα phases would also occur in cells.(3)We have proposed a novel method, the single GUV method to probe functions and dynamics of biomembranes and interactions of substances with biomembranes. We have succeeded in revealing the pore formation induced by antimicrobial peptide, maginin2, and the interactions of antibacterial substance, (-)-epigallocatechin gallate(ECGg) with lipid membranes. Less

（1）利用单巨uniLαmelLαr囊泡（GUV）方法，我们发现低浓度（低于临界胶束浓度（CMC），但高于其阈值浓度）的具有单个长烃链的两亲物（即单长链两亲物），例如溶血磷脂酰胆碱（lyso-PC）、溶血磷脂酸、 辛基葡萄糖苷诱导液体有序 (lo) 相脂质膜 GUV 的囊泡分裂，例如二棕榈酰磷脂酰胆碱 (DPPC)/胆固醇 (6/4) 膜。为了阐明 GUV 的 lo 相膜中包含 DOPC（即 Lα 相形成脂质）对溶血 PC 诱导的 DPPC/chol (6/4)-GUV 囊泡裂变的影响，我们研究了低浓度的溶血 PC 对 DPPC/DOPC/chol 膜的 GUV 的影响。随着膜中DOPC浓度的增加，诱导囊泡分裂的lyso-PC的阈值浓度增加。在 30 mol% DOPC 及以上时，没有发生囊泡分裂。基于这些结果，我们提出了单长链两亲物诱导 lo 相膜 GUV 囊泡裂变机制的假设。(2) 我们研究了 H+（即 pH 效应）对带负电脂质、二油酰磷脂酰丝氨酸 (DOPS) 和电中性脂质膜的相和结构的影响， 使用小角 X 射线散射 (SAXS) 在生理离子浓度条件下分析单油酸甘油酯 (MO) 混合物。在中性 pH 条件下，含有高浓度 DOPS 的 DOPS/MO 膜处于 Lα 相。首先，通过在 MLV 悬浮液中添加各种低 pH 缓冲液，研究了 pH 对在中性 pH 下形成的 DOPS/MO 膜的多 LaαmelLαr 囊泡 (MLV) 的相和结构的影响。对于 20%-DOPS/80%-MO-MLV，在 pH 2.9 及以下时，1 小时内发生从 Lα 到立方 (Q224) 相的转变。这种相变是可逆的；随后，膜悬浮液中的 pH 值升高至中性，将立方相转变为原始的 Lα 相。其次，我们发现，在类似条件下，LUV 之间的膜融合后，pH 值的降低将 DOPS/MO 膜的大型 uniLαmelLαr 囊泡转变为立方相。我们提出了低pH诱导相变的机理，并对相变的临界pH值进行了定量分析。这一发现首次证明 pH 值的变化可以在 1 小时内诱导脂质膜的 Lα 相和立方相之间发生可逆相变。我们合理地认为pH诱导的立方相和Lα相之间的相互转化也会在细胞中发生。(3)我们提出了一种新方法，即单一GUV方法来探测生物膜的功能和动力学以及物质与生物膜的相互作用。我们成功揭示了抗菌肽maginin2诱导的孔形成以及抗菌物质(-)-表没食子儿茶素没食子酸酯(ECGg)与脂质膜的相互作用。较少的

项目成果

The "Le Chatelier's Principles"-Govemed Response of Actin Filaments to Osmotic Stress

“勒夏特列原理”-肌动蛋白丝对渗透压的调控反应

• 发表时间: 2006
• 期刊: J. Phys. Chem. B 110
• 作者: T. Ito;et. al.
• 通讯作者: et. al.

The "Le Chatelier's Principles"-Governed Response of Actin Filaments to Osmotic Stress.

“勒夏特列原理”——控制肌动蛋白丝对渗透压的反应。

• 发表时间: 2006
• 期刊: J. Phys. Chem. B 110
• 作者: Kosaku Kato;Yukiko Ohmori;Takeomi Mizutani;Hisashi Haga;Kazuyo Ohashi;Tadanao Ito;Kazushige Kawabata;Tadanao Ito et al.
• 通讯作者: Tadanao Ito et al.

Caonic DMPC/DMTAP Lipid Bilayers: Local Lateral Polarization of Phosphatidylcholine Headgroups

Caonic DMPC/DMTAP 脂质双层：磷脂酰胆碱头基的局部横向极化

• 发表时间: 2005
• 期刊: Langmuir 21
• 作者: V. Levadny;et. al.
• 通讯作者: et. al.

The Single GUV Method to Reveal Elementary Processes of Leakage of Internal Contents from Liposomes Induced by Antimicrobial Substances

单一 GUV 方法揭示抗菌物质引起的脂质体内部内容物泄漏的基本过程

• 发表时间: 2008
• 期刊: Advances in Planar Lipid Bilayers and Liposomes Elsevier
• 作者: Yoshihide Okamoto;Shah Md. Masum;Haruna Miyazawa;Masahito Yamazaki;Masahito Yamazaki;Masahito Yamazaki
• 通讯作者: Masahito Yamazaki

Formation of cubic phases from large unilamellar vesicles of dioleoylphosphatidylglycerol/monoolein membranes induced by low concentrations of Ca2+

• DOI: 10.1021/la051782i
• 发表时间: 2005-12-06
• 期刊: LANGMUIR
• 影响因子: 3.9
• 作者: Awad, TS;Okamoto, Y;Yamazaki, M
• 通讯作者: Yamazaki, M