A Study on Regulatory Mechanisms of Endocytosis

关于胞吞作用的调控机制，有以下几个方面的研究。在GTP存在的情况下，用大的单层脂质体与脑细胞质或动力蛋白孵育可以重建动力蛋白依赖的内吞作用。在本实验系统中，使用敲除两性蛋白的脑细胞质，阐明两性蛋白1刺激动力蛋白GTPase活性，从而增强动力蛋白依赖性囊泡的形成。这种作用需要amphiphysin 1的BAR结构域和SH3结构域共同作用。大脂质体的低膜曲率也是amphiphysin 1.2刺激作用的必要条件。细胞周期蛋白依赖激酶5 （cyclin dependent kinase 5, cdk5）可磷酸化动力蛋白1和amphiphysin 1。在GTP存在的情况下，带磷酸化的动力蛋白和磷酸化的两栖素1的脂质体孵育导致很少的囊泡形成，而去磷酸化的蛋白则大量产生囊泡。因此，胞吞作用可能受到cdk5依赖性磷酸化的调控。dynamin2和dynamin3在支持细胞中有不同的定位，表明它们具有不同的功能。

Regarding to regulatory mechanisms of endocytosis, the followings were revealed.1.Regulation of endocytosis by Amphiphysin 1Dynamin-dependent endocytosis can be reconstituted in vitro by incubating large unilammelar liposomes with brain cytosol or dynamin in presence of GTP. Using amphipshyin knockout brain cytosol in this experimental system, it was clarified that amphiphysin 1 stimulates dynamin GTPase activity and thereby enhances dynamin-dependent vesicle formation. This effect required both BAR domain and SH3 domain of amphiphysin 1. Low membrane curvature of large liposomes was also requisite for the stimulatory effect of amphiphysin 1.2.Regulation of endocytosis cdk5-dependent phosphorylationBoth dynamin 1 and amphiphysin 1 are phosphorylated by cyclin dependent kinase 5 (cdk5). Incubation of liposomes with phosphorylated dynain and phosphorylated amphiphysin 1 in presence of GTP resulted in few vesicle formation, whereas dephophorylated proteins massively generated vesicles. Thus, endocytosis is likely to be regulated by cdk5-dependent phosphorylation.3.Localization of dynamin 2 and dynamin3Distinct localization of dynamin 2 and dynamin3 in Sertoli cells was revealed suggesting different functions of these isoforms.