Functional analyses of dynamin GTPase on early steps of endocytosis

在本项目实施期间，取得了以下成果：1.动力蛋白突变体细胞的形态学和生化分析：我们分析了动力蛋白的三种显性负突变体K44 A、S45 N和T65 F。所有三种突变体都缺乏GTP和GDP的结合能力，并且在生物化学上无法区分。然而，电子显微镜分析显示每个突变体在转化细胞中显示出独特的形态中间体的积累。这些结果表明，这些突变的发动蛋白可能通过未知的发动蛋白的功能抑制内吞作用，而不是通过众所周知的GTP/GDP依赖的裂变步骤。2. Lysenin的超微结构研究：Lysenin是一种鞘磷脂特异性结合蛋白，具有溶血作用。我们已经确定了独特的六角寡聚体的鞘磷脂含有脂质体。在胞溶素处理的红细胞表面也发现了类似的结构。这些结果表明，胞溶素诱导溶血的孔形成寡聚体的细胞表面。3.突触囊泡形成的体外重建分析：我们成功地将脂质体与脑胞液、ATP和GTPγS孵育，重建了突触囊泡的形成。然后我们分析了脂质体的各种脂质组成对囊泡形成速率的影响。我们发现PIP 2积累形成囊泡。在PIP 2快速降解后，囊泡形成的积累持续存在。这些结果表明，PIP 2可以募集各种外壳蛋白的脂质体所需的囊泡出芽。

The following results were obtained in the term of current project. 1. Combined morphological and biochemical analyses of dynamin mutant cells : We have analyzed three dominant-negative mutants of dynamin, K44A, S45N, and T65F. All three mutants lacked binding capacity for both GTP and GDP, and biochemically indistinguishable. However, electron microscopic analyses revealed the each mutant showed the accumulation of unique morphological intermediates in transformed cells. These results indicated that these mutations in dynamin may inhibit the endocytosis through unknown dynamin's function not through well-known GTP/GDP-dependent fission steps. 2. Ultrastructural study of lysenin : Lysenin is a sphingomyelin-specific binding protein which induce hemolysis. We have identified the unique hexagonal oligomer on sphingomyelin-containing liposomes. The similar structures were found on the surface of lysenin-treated erythrocytes. These results indicated that lysenin induced hemolysis by pore-forming oligomer on the cell surface. 3. Analyses of in vitro reconstitution of synaptic vesicle formation : We have succeeded in reconstitution of the synaptic vesicle formation by incubating liposomes with brain cytosol, ATP and GTPγS. We then analyzed the effect of various lipid composion of liposomes on the rate of vesicle formation. We found that PIP2 accumulate the formation of vesicles. The accumulation of vesicle formation persisted after rapid degradation of PIP2. These results indicated that PIP2 may recruited various coat proteins to the liposomes required for vesicle budding.