Abstract. Dynarnin is the mammalian homologue to the Drosophila shibire gene product. Mutations in this 100-kD GTPase cause a pleiotropic defect in endocytosis. To further investigate its role, we generated stable HeLa cell lines expressing either wild-type dynamin or a mutant defective in GTP binding and hydrolysis driven by a tightly controlled, tetracyclineinducible promoter. Overexpression of wild-type dynamin had no effect. In contrast, coated pits failed to become constricted and coated vesicles failed to bud in cells overexpressing mutant dynamin so that endocytosis via both transferrin (Tfn) and EGF receptors was potently inhibited. Coated pit assembly, invagination, and the recruitment of receptors into coated pits were unaffected. Other vesicular transport pathways, including Tfn receptor recycling, Tfn receptor biosynthesis, and cathepsin D transport to lysosomes via Golgiderived coated vesicles, were unaffected. Bulk fluidphase uptake also continued at the same initial rates as wild type. EM immunolocalization showed that membrane-bound dynamin was specifically associated with clathrin-coated pits on the plasma membrane. Dynamin was also associated with isolated coated vesicles, suggesting that it plays a role in vesicle budding. Like the Drosophila shibire mutant, HeLa cells overexpressing mutant dynamin accumulated long tubules, many of which remained connected to the plasma membrane. We conclude that dynamin is specifically required for endocytic coated vesicle formarion, and that its GTP binding and hydrolysis activities are required to form constricted coated pits and, subsequently, for coated vesicle budding.
Epidermal growth factor receptor (EGFR) signaling was analyzed in mammalian cells conditionally defective for receptor-mediated endocytosis. EGF-dependent cell proliferation was enhanced in endocytosis-defective cells. However, early EGF-dependent signaling events were not uniformly up-regulated. A subset of signal transducers required the normal endocytic trafficking of EGFR for full activation. Thus, endocytic trafficking of activated EGFR plays a critical role not only in attenuating EGFR signaling but also in establishing and controlling specific signaling pathways.
Abstract. The role of human dynamin in receptormediated endocytosis was investigated by transient expression of GTP-binding domain mutants in mammalian cells. Using assays which detect intermediates in coated vesicle formation, the dynamin mutants were found to block endocytosis at a stage after the initiation of coat assembly and preceding the sequestration of ligands into deeply invaginated coated pits. Membrane transport from the ER to the Golgi complex was unaffected indicating that dynamin mutants specifically block early events in endocytosis. These results demonstrate that mutations in the GTP-binding domain of dynamin block Tfn-endocytosis in mammalian cells and suggest that a functional dynamin GTPase is required for receptor-mediated endocytosis via clathrincoated pits.ECEPTOR-mediated endocytosis occurs via the formation of clathrin-coated vesicles and involves coat assembly, receptor recruitment, coated pit invagination, and finally vesicle budding (Smythe and Warren, 1991;Robinson, 1992;Schmid, 1993). The major structural proteins of the coat, clathrin, and AP complexes have been well characterized (Pearse and Robinson, 1990;Keen, 1990;Brodsky, 1988). While proteins that regulate coated vesicle formation have not been identified (Schmid, 1992), a cellfree assay which reconstitutes reeeptor-mediated endocytosis has recently revealed a requirement for multiple GTPbinding proteins (Carter et al., 1993).Dynamin, a GTP-binding protein first isolated from bovine brain tissue (Shpetner and Vallee, 1989;Obar et al., 1990;Scaife and Margolis, 1990;Nakata et al., 1991) is a strong candidate for a GTPase that acts early in endocytosis. Dynamin is a member of a subfamily of structurally related GTP-binding proteins which share considerable sequence homology (43-66 %) in the amino-terminal GTPase domain, but more limited homology (<10-30%) in their COOHterminal regions. Other members of this family (for review see Collins, 1991) include the mammalian Mx-1 related, interferon-inducible proteins which confer viral resistance (Arnheiter and Meier, 1990), MGM1 which plays a role in yeast mitochondrial DNA maintenance (Jones and Fangman, 1992) and VPSlp, a protein involved in vacuolar sort-
The large GTPase dynamin is the first protein shown to catalyze membrane fission. Dynamin and its related proteins are essential to many cell functions, from endocytosis to organelle division and fusion, and it plays a critical role in many physiological functions such as synaptic transmission and muscle contraction. Research of the past three decades has focused on understanding how dynamin works. In this review, we present the basis for an emerging consensus on how dynamin functions. Three properties of dynamin are strongly supported by experimental data: first, dynamin oligomerizes into a helical polymer; second, dynamin oligomer constricts in the presence of GTP; and third, dynamin catalyzes membrane fission upon GTP hydrolysis. We present the two current models for fission, essentially diverging in how GTP energy is spent. We further discuss how future research might solve the remaining open questions presently under discussion.
Three cell-permeant compounds, cytochalasin D, latrunculin A and jasplakinolide, which perturb intracellular actin dynamics by distinct mechanisms, were used to probe the role of filamentous actin and actin assembly in clathrin-mediated endocytosis in mammalian cells. These compounds had variable effects on receptor-mediated endocytosis of transferrin that depended on both the cell line and the experimental protocol employed. Endocytosis in A431 cells assayed in suspension was inhibited by latrunculin A and jasplakinolide, but resistant to cytochalasin D, whereas neither compound inhibited endocytosis in adherent A431 cells. In contrast, endocytosis in adherent CHO cells was more sensitive to disruption of the actin cytoskeleton than endocytosis in CHO cells grown or assayed in suspension. Endocytosis in other cell types, including nonadherent K562 human erythroleukemic cells or adherent Cos-7 cells was unaffected by disruption of the actin cytoskeleton. While it remains possible that actin filaments can play an accessory role in receptor-mediated endocytosis, these discordant results indicate that actin assembly does not play an obligatory role in endocytic coated vesicle formation in cultured mammalian cells.
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