Formation of a cell-model made of a liposome and cytoskeletal proteins

由脂质体和细胞骨架蛋白组成的细胞模型的形成

• 批准号: 08408029
• 负责人: HOTANI Hirokazu
• 金额: $ 20.67万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08408029/
• 关键词: cytoskeleton; liposome; microtubule; actin; cell model; dark-field microscopy; actin-binding protein; membrane protein

1. Morphological changes of liposomes.(1). Shape change caused by microtubule assemblyIn order to elucidate the role of the cytoskeleton in cellular morphogenesis, model systems using lipsomes encapsulating cytoskeletal proteins have been developed using tubulin or actin. Upon polymerization of the cytoskeleton, liposomes are transformed into characteristic shapes, which depend on the types of encapsulated cytoskeletal proteins. Two tubular projections developed from antipodal positions on spherical liposomes when microtubules polymerized. Both projections were straight, rigid, uniform in diameter and elongated in opposite directions. These observations indicate that the cytoskeleton can generate mechanical forces capable of transforming the phospholipid membrane.(2). Stabilization of bipolar liposomes by MAPs(3). Binding between microtubules and liposomes in the presence of MAPs2. Morphogenesis of liposomes encapsulating actin depends on the type of actin-crosslinkingWe characterized … More the transformation of liposomes encapsulating actin and its crosslinking proteins, fascin, α-actinin, or filamin, using real-time high-intensity dark-field microscopy. With increasing temperature, the encapsulated G-actin polymerized into actin filaments and formed bundles or gels, depending on the type of actin-crosslinking protein that was co-encapsulated, causing various morphological changes of liposomes. The differences in morphology among transformed liposomes indicate that actin-crosslinking proteins determine liposome shape by organizing their specific actin networks. Morphological analysis reveals that the crosslinking manner, i.e. distance and angular flexibility between adjacent crosslinked actin filaments, is essential for the morphogenesis rather than their binding affinity and stoichiometry to actin filaments.3. Opening-up of liposomes membranes by talinMorphological changes liposomes caused by interactions between liposomal membranes and talin, a cytoskeletal submembranous protein, were studied by direct, real-time observation using high-intensity dark-field microscopy. Surprisingly, when talin was added to a liposome solution, liposomes opened stable holes and were transformed into cup-shaped liposomes. The holes became larger with increasing talin concentration, and finally the cup-shaped liposomes were transformed into lipid bilayer sheets. These morphological changes were reversed by protein dilution, i.e., the sheets could be transformed back into closed spherical liposomes. We demonstrated that talin was localized mainly along the membrane verges, presumably avoiding exposure of its hydrophobic portion at the edge of the lipid bilayer. This is the first demonstration that a lipid bilayer can stably maintain a free verge in aqueous solution. This finding refutes the established dogma that all lipid bilayer membranes inevitably form closed vesicles, and suggests that talin is a useful tool for manipulating liposomes. Less

1.脂质体的形态变化。(1)．微管组装引起的形状变化为了阐明细胞骨架在细胞形态发生中的作用，已经使用微管蛋白或肌动蛋白开发了使用脂质体封装细胞骨架蛋白的模型系统。细胞骨架聚合后，脂质体转变为特征形状，这取决于封装的细胞骨架蛋白的类型。当微管聚合时，从球形脂质体上的对映位置形成两个管状突起。两个突出部分都是直的、刚性的、直径均匀且沿相反方向拉长。这些观察结果表明细胞骨架可以产生能够改变磷脂膜的机械力。(2)。通过 MAP 稳定双极脂质体 (3)。 MAPs2 存在下微管和脂质体之间的结合。包裹肌动蛋白的脂质体的形态发生取决于肌动蛋白交联的类型我们使用实时高强度暗场显微镜表征了包裹肌动蛋白及其交联蛋白、肌成束蛋白、α-肌动蛋白或细丝蛋白的脂质体的转化。随着温度升高，封装的G-肌动蛋白聚合成肌动蛋白丝并形成束或凝胶，这取决于共封装的肌动蛋白交联蛋白的类型，引起脂质体的各种形态变化。转化脂质体之间形态的差异表明肌动蛋白交联蛋白通过组织其特定的肌动蛋白网络来决定脂质体的形状。形态学分析表明，交联方式，即相邻交联肌动蛋白丝之间的距离和角度灵活性，对于形态发生至关重要，而不是它们与肌动蛋白丝的结合亲和力和化学计量。 3． talin 打开脂质体膜通过使用高强度暗场显微镜直接、实时观察，研究了由脂质体膜和talin（一种细胞骨架膜下蛋白）之间相互作用引起的脂质体形态变化。令人惊讶的是，当将talin添加到脂质体溶液中时，脂质体打开了稳定的孔并转变为杯状脂质体。随着talin浓度的增加，孔变得更大，最终杯状脂质体转变为脂质双层片。这些形态变化可以通过蛋白质稀释来逆转，即片层可以转回封闭的球形脂质体。我们证明talin主要位于膜边缘，大概避免了其疏水部分暴露在脂质双层边缘。这是首次证明脂质双层可以在水溶液中稳定地保持自由边缘。这一发现驳斥了所有脂质双层膜都不可避免地形成封闭囊泡的既定教条，并表明talin是操纵脂质体的有用工具。较少的

项目成果

Y. Suezaki et. al.: "A Statistical Mechanical Theory for the Adsorption of Protein to Liposomal Membranes."Biophys. Chem.. 80. 119-128 (1999)

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M.Honda et al.: "Morphogenesis of Liposomes Encapsulating Actin Depends on the Type of Actin-Crosslinking." J.Mol.Biol.287. 293-300 (1999)

M.Honda 等人：“包裹肌动蛋白的脂质体的形态发生取决于肌动蛋白交联的类型。”

Y. Suezaki: "A statistical mechanical theory for the adsorption of protein to liposomal membranes"Biophys. Chem.. 80. 119-128 (1999)

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M. Honda: "Morphogenesis of Liposomes Encapsulation Actin Depends on the Type of Actin-Crosslinking"J. Mol. Biol.. 287(2). 239-300 (1999)

M. Honda：“脂质体封装肌动蛋白的形态发生取决于肌动蛋白交联的类型”J。