Henley J R, Cao H, McNiven M A
Center for Basic Research in Digestive Diseases and Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA.
FASEB J. 1999 Dec;13 Suppl 2:S243-7. doi: 10.1096/fasebj.13.9002.s243.
Dynamin is a 100-kDa GTPase that has been implicated in endocytosis. To extend our understanding of its cellular functions, we have microinjected specific affinity-purified anti-dynamin antibodies into cultured mammalian epithelial cells. Using this approach, dynamin function can be inhibited specifically and rapidly in single cells. Effects of microinjected inhibitory antibodies on distinct endocytic processes and plasmalemmal morphology were then assayed by fluorescence microscopy (FM) and ultrastructural analysis. Micro-injected antibodies inhibit the clathrin-mediated endocytosis of fluorophore-labeled transferrin and cause a marked invagination of the plasma membrane. Many of these long plasmalemmal invaginations had clathrin-coated pits along their cytoplasmic surface. A number of distinct noncoated pits resembling plasmalemmal caveolae also accumulated in anti-dynamin antibody-injected cells. Further, the cellular uptake of cholera toxin B, which normally occurs by the internalization of caveolae, was inhibited in these cells. In support of these observations, immunoisolation techniques, double-label immuno-FM, and immunoelectron microscopy (immuno-EM) provided biochemical and morphological evidence that dynamin associates with plasmalemmal caveolae. Together, these observations indicate that dynamin mediates scission from the plasma membrane of both clathrin-coated pits and caveolae during distinct endocytic processes. These results demonstrate that dynamin isoforms are involved in an additional endocytic process that is distinct from clathrin-mediated endocytosis and provide significant insights into the molecular mechanisms governing the GTP-mediated internalization of caveolae. Evidence is provided demonstrating that dynamin isoforms have a differential distribution in mammalian cells. Targeting information for these isoforms is provided at least in part by regions of alternative splicing. Thus, the different dynamin isoforms may be localized to distinct cellular compartments but provide a similar scission function during the biogenesis of nascent cytoplasmic vesicles.
发动蛋白是一种100 kDa的GTP酶，与内吞作用有关。为了进一步了解其细胞功能，我们已将特异性亲和纯化的抗发动蛋白抗体显微注射到培养的哺乳动物上皮细胞中。使用这种方法，发动蛋白的功能可在单细胞中被特异性且快速地抑制。然后通过荧光显微镜（FM）和超微结构分析来检测显微注射的抑制性抗体对不同内吞过程和质膜形态的影响。显微注射的抗体抑制了荧光团标记的转铁蛋白的网格蛋白介导的内吞作用，并导致质膜明显内陷。许多这些长的质膜内陷在其细胞质表面有网格蛋白包被的小窝。一些类似质膜小窝的不同的无包被小窝也在注射了抗发动蛋白抗体的细胞中积累。此外，霍乱毒素B的细胞摄取（通常通过小窝的内化发生）在这些细胞中受到抑制。为支持这些观察结果，免疫分离技术、双标记免疫荧光显微镜和免疫电子显微镜（免疫EM）提供了生化和形态学证据，表明发动蛋白与质膜小窝相关联。总之，这些观察结果表明，发动蛋白在不同的内吞过程中介导网格蛋白包被小窝和小窝从质膜上的脱离。这些结果表明，发动蛋白异构体参与了一种不同于网格蛋白介导的内吞作用的额外内吞过程，并为控制小窝的GTP介导的内化的分子机制提供了重要见解。提供的证据表明发动蛋白异构体在哺乳动物细胞中有差异分布。这些异构体的靶向信息至少部分由可变剪接区域提供。因此，不同的发动蛋白异构体可能定位于不同的细胞区室，但在新生细胞质囊泡的生物发生过程中提供类似的切割功能。
