Functional analyses of dynamin GTPase on early steps of endocytosis

在当前项目期间，获得了以下结果：1. dynamin突变细胞的形态学和生化分析：我们分析了三个dynamin的显性阴性突变体K44A、S45N和T65F。所有三个突变体都缺乏GTP和GDP的结合能力，并且在生物化学上无法区分。然而，电镜分析显示，每个突变体都在转化细胞中积累了独特的形态中间体。这些结果表明，这些动力蛋白突变可能通过未知的动力蛋白功能抑制内吞作用，而不是通过众所周知的GTP/ gdp依赖的裂变步骤。2. 溶菌素的超微结构研究：溶菌素是鞘磷脂特异性结合蛋白，可诱导溶血。我们在鞘磷脂脂质体上发现了独特的六方低聚物。在赖素处理的红细胞表面也发现了类似的结构。这些结果表明，溶菌素通过在细胞表面形成孔的低聚物诱导溶血。3. 突触囊泡形成的体外重建分析：我们通过脂质体与脑细胞质、ATP和GTPγS培养，成功地重建了突触囊泡的形成。然后，我们分析了脂质体的不同脂质组成对囊泡形成速率的影响。我们发现PIP2聚集形成囊泡。PIP2快速降解后，囊泡形成的积累持续存在。这些结果表明，PIP2可能募集了多种囊泡出芽所需的脂质体外壳蛋白。

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.