ARTICLE

Immunocytochemical Study of Endocytotic Structures Accumulated in HeLa Cells Transformed with a Temperature-sensitive Mutant of Dynamin

Correspondence to: Takeshi Baba, Dept. of Anatomy, Yamanashi Medical Univ., 1110 Shimokato, Tamaho, Yamanashi 409-3898, Japan.

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 dyn^ts 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 shibire^ts1 flies and GTPS-treated synaptosomes, were not observed in the dyn^ts cells even at nonpermissive temperature. The distribution pattern of dynamin in deeply invaginated coated pits in dyn^ts cells was similar to that in dyn^wt cells but not to that in dyn^K44A cells, which are other endocytosis-defective mutant cells. These morphological data suggest that dyn^ts blocked the pinching-off steps in clathrin-coated vesicle formation, which may be caused by a different mechanism from that of dyn^K44A cells. (J Histochem Cytochem 47:637–648, 1999)

Key Words: dynamin, endocytosis, immunocytochemistry, electron microscopy, coated pit, temperature-sensitive mutant

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