A Study on Regulatory Mechanisms of Endocytosis

关于内吞作用的调控机制，揭示了以下几点：1.两栖动物蛋白对内吞作用的调节1在体外，通过将大的单细胞脂质体与脑胞浆或动力蛋白孵育在GTP的存在下，可以重建动力蛋白依赖性的内吞作用。在这个实验系统中，利用两栖动物基因敲除的脑胞浆，阐明了两栖动物蛋白1刺激动力蛋白GTP酶的活性，从而促进动力蛋白依赖的囊泡的形成。这种作用需要两栖动物蛋白1的BAR结构域和SH3结构域。大脂质体的低膜曲率也是两栖动物蛋白1.2刺激作用的必要条件。细胞内吞作用的调节依赖于CDK5。在GTP存在的情况下，脂质体与磷酸化的动力蛋白和磷酸化的两栖蛋白1孵育后形成的囊泡很少，而去磷酰化的蛋白质则大量地产生囊泡。因此，内吞作用很可能是通过依赖于CDK5的磷酸化来调节的。3.Dynamin 2和Dynamin3在Sertoli细胞中的定位不同，提示这些异构体具有不同的功能。

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.