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

1. 脂质体的形态变化。为了阐明细胞骨架在细胞形态发生中的作用，利用微管蛋白或肌动蛋白开发了利用脂质体包裹细胞骨架蛋白的模型系统。在细胞骨架聚合后，脂质体被转化为特征形状，这取决于被封装的细胞骨架蛋白的类型。当微管聚合时，两个管状突起从球形脂质体的对跖位置发展而来。这两种凸出物都是直的，坚硬的，直径均匀，在相反的方向上拉长。这些观察结果表明，细胞骨架可以产生能够转化磷脂膜的机械力。MAPs稳定双极性脂质体(3)。MAPs2存在时微管与脂质体之间的结合。脂质体包裹肌动蛋白的形态发生取决于肌动蛋白交联的类型。我们用实时高强度暗场显微镜观察了脂质体包裹肌动蛋白及其交联蛋白（束蛋白、α-肌动蛋白或丝蛋白）的转化。随着温度的升高，被包覆的g -肌动蛋白根据被包覆的肌动蛋白交联蛋白的类型，聚合成肌动蛋白丝并形成束状或凝胶状，引起脂质体的各种形态变化。转化脂质体的形态差异表明，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柔韧性，对形态发生至关重要，而不是它们与肌动蛋白丝的结合亲和力和化学计量。talin（一种细胞骨架亚膜蛋白）与脂质体膜相互作用引起脂质体的形态变化，采用高强度暗场显微镜直接实时观察。令人惊讶的是，当将talin加入到脂质体溶液中时，脂质体会打开稳定的孔并转化为杯状脂质体。随着talin浓度的增加，孔变大，最终杯状脂质体转化为脂质双层膜。这些形态变化被蛋白质稀释逆转，即，薄片可以转化回封闭的球形脂质体。我们证明talin主要沿膜边缘定位，可能避免暴露在脂质双分子层边缘的疏水部分。这是首次证明脂质双分子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。少

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