Actin assembly plays a variable, but not obligatory role in receptor-mediated endocytosis in mammalian cells.

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 jaspiakinolide, 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.

The actin cytoskeleton is required for receptor-mediated endocytosis in mammalian cells.

Actin filament organization is essential for endocytosis in yeast. In contrast, the actin-depolymerizing agent cytochalasin D has yielded ambiguous results as to a role for actin in receptor-mediated endocytosis in mammalian cells. We have therefore re-examined this issue using highly specific reagents known to sequester actin monomers. Two of these reagents, thymosin beta4 and DNase I, potently inhibited the sequestration of transferrin receptors into coated pits as measured in a cell-free system using perforated A431 cells. At low concentrations, thymosin beta4 but not DNase I was stimulatory. Importantly, the effects of both reagents were specifically neutralized by the addition of actin monomers. A role for the actin cytoskeleton was also detected in intact cells where latrunculin A, a drug that sequesters actin monomers, inhibited receptor-mediated endocytosis. Biochemical and morphological analyses suggest that these reagents inhibit later events in coated vesicle budding. These results provide new evidence that the actin cytoskeleton is required for receptor-mediated endocytosis in mammalian cells.