Participation of dynamin in the biogenesis of cytoplasmic vesicles

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.