Immunocytochemical study of endocytotic structures accumulated in HeLa cells transformed with a temperature-sensitive mutant of dynamin.

Dynamin is a 100-kD GTPase, which is required for clathrin-mediated endocytosis. Recent studies have revealed that dynamin is closely involved in clathrin-coated vesicle formation. In this study we investigated the ultrastructure of endocytotic structures accumulated in HeLa cells that were transformed with a temperature-sensitive (ts) mutant of dynamin to clarify which step was blocked in dynts cells. Endocytosis of transferrin receptors was restricted at the level of surface-connected membrane structures. Tubular and vesicular membrane invaginations were accumulated in the cells' peripheral regions, suggesting that the endocytosis was blocked just before the pinching-off steps in coated vesicle formation. The "collared" tubes, which were reported to be localized in nerve terminals in shibirets1 flies and GTPgammaS-treated synaptosomes, were not observed in the dynts cells even at nonpermissive temperature. The distribution pattern of dynamin in deeply invaginated coated pits in dynts cells was similar to that in dynwt cells but not to that in dynK44A cells, which are other endocytosis-defective mutant cells. These morphological data suggest that dynts blocked the pinching-off steps in clathrin-coated vesicle formation, which may be caused by a different mechanism from that of dynK44A cells.