Henley J R, Krueger E W, Oswald B J, McNiven M A
Department of Molecular Neuroscience, Mayo Clinic, Rochester, Minnesota 55905, USA.
J Cell Biol. 1998 Apr 6;141(1):85-99. doi: 10.1083/jcb.141.1.85.
The dynamins comprise an expanding family of ubiquitously expressed 100-kD GTPases that have been implicated in severing clathrin-coated pits during receptor-mediated endocytosis. Currently, it is unclear whether the different dynamin isoforms perform redundant functions or participate in distinct endocytic processes. To define the function of dynamin II in mammalian epithelial cells, we have generated and characterized peptide-specific antibodies to domains that either are unique to this isoform or conserved within the dynamin family. When microinjected into cultured hepatocytes these affinity-purified antibodies inhibited clathrin-mediated endocytosis and induced the formation of long plasmalemmal invaginations with attached clathrin-coated pits. In addition, clusters of distinct, nonclathrin-coated, flask-shaped invaginations resembling caveolae accumulated at the plasma membrane of antibody-injected cells. In support of this, caveola-mediated endocytosis of labeled cholera toxin B was inhibited in antibody-injected hepatocytes. Using immunoisolation techniques an anti-dynamin antibody isolated caveolar membranes directly from a hepatocyte postnuclear membrane fraction. Finally, double label immunofluorescence microscopy revealed a striking colocalization between dynamin and the caveolar coat protein caveolin. Thus, functional in vivo studies as well as ultrastructural and biochemical analyses indicate that dynamin mediates both clathrin-dependent endocytosis and the internalization of caveolae in mammalian cells.
发动蛋白构成了一个不断扩大的家族，这些普遍表达的100-kD GTP酶在受体介导的内吞作用过程中参与切断网格蛋白包被的小窝。目前尚不清楚不同的发动蛋白异构体是执行冗余功能还是参与不同的内吞过程。为了确定发动蛋白II在哺乳动物上皮细胞中的功能，我们制备并鉴定了针对该异构体特有的结构域或发动蛋白家族中保守结构域的肽特异性抗体。当将这些亲和纯化的抗体显微注射到培养的肝细胞中时，它们抑制了网格蛋白介导的内吞作用，并诱导形成了带有附着的网格蛋白包被小窝的长质膜内陷。此外，在注射抗体的细胞的质膜上积累了类似于小窝的独特的、非网格蛋白包被的烧瓶状内陷簇。与此相符的是，在注射抗体的肝细胞中，标记的霍乱毒素B的小窝介导的内吞作用受到抑制。使用免疫分离技术，一种抗发动蛋白抗体直接从肝细胞核后膜部分分离出小窝膜。最后，双标记免疫荧光显微镜显示发动蛋白与小窝包被蛋白小窝蛋白之间有明显的共定位。因此，体内功能研究以及超微结构和生化分析表明，发动蛋白在哺乳动物细胞中介导网格蛋白依赖性内吞作用和小窝的内化。
