Participation of dynamin in the biogenesis of cytoplasmic vesicles

Dynamin is a 100-kDa GTPase that has been implicated in endocytosis, To ext end our understanding of its cellular functions, we have microinjected spec ific affinity-purified anti-dynamin antibodies into cultured mammalian epit helial cells, Using this approach, dynamin function can be inhibited specif ically and rapidly in single cells. Effects of microinjected inhibitory ant ibodies on distinct endocytic processes and plasmalemmal morphology were th en assayed by fluorescence microscopy (FM) and ultrastructural analysis. Mi croinjected antibodies inhibit the clathrin-mediated endocytosis of fluorop hore-labeled transferrin and cause a marked imagination of the plasma membr ane. Many of these long plasmalemmal invaginations had clathrin-coated pits along their cytoplasmic surface. A number of distinct noncoated pits resem bling plasmalemmal caveolae also accumulated in anti-dynamin antibody-injec ted 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 im muno-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 distin ct 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 m echanisms governing the GTP-mediated internalization of caveolae, Evidence is provided demonstrating that dynamin isoforms have a differential distrib ution in mammalian cells. Targeting information for these isoforms is provi ded at least in part by regions of alternative splicing. Thus, the differen t dynamin isoforms may be localized to distinct cellular compartments but p rovide a similar scission function during the biogenesis of nascent cytopla smic vesicles.