The Actin Cytoskeleton Is Required for Receptor-mediated Endocytosis in Mammalian Cells

Abstract

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 ␤4 and DNase I, potently inhibited the sequestration of transferrin receptors into coated pits as measured in a cellfree system using perforated A431 cells. At low concentrations, thymosin ␤4 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.The plasma membrane is directly linked to and functionally integrated with the underlying actin-based cytoskeleton which forms the cell "cortex." Thus, it might be anticipated that vesicular trafficking at the plasma membrane would require the active rearrangement of cortical actin filaments to remove a barrier to vesicular fusion or budding events. Alternatively, actin and actin-based motor proteins might be required to direct vesicle budding and fusion events through the cell cortex at the plasma membrane. Actin filaments can play both inhibitory and facilatory roles in exocytosis. For example, agoniststimulated secretion appears to require localized disassembly of F-actin at the cell periphery (1, 2). Furthermore, low concentrations of proteins which sequester actin monomers can trigger regulated secretion in permeabilized pancreatic acinar cells suggesting that actin filaments might act as a clamp preventing fusion of docked vesicles (3). In contrast, higher concentrations of these actin-sequestering proteins inhibit regulated secretion, suggesting that actin filament integrity might also play an as yet undefined, facilatory role in regulated exocytosis (3).Receptor-mediated endocytosis of the mating pheromone ␣-factor is potently inhibited in the yeast, S. cerevisiae, expressing mutations in either actin or the actin-binding protein, fimbrin (4). Yeast carrying mutations in three other genes, END3, END5/VRP1, and END7/RVS167, which disrupt actin organization, were also shown to be defective in endocytosis (5, 6). More recently a role for the type I myosin, myo5, in receptormediated endocytosis in yeast was revealed (7). Together these studies establish that actin filaments and actin-based motor proteins play an essential role in endocytosis in yeast.In contrast, the role of actin in endocytosis in mammalian cells remains poorly understood. Cytochalasin D, a drug that destabilizes actin filaments, inhibits receptor...

Birbeck Granules Are Subdomains of Endosomal Recycling Compartment in Human Epidermal Langerhans Cells, Which Form Where Langerin Accumulates

“…a large number of coated pits, which were labeled by goldconjugated anti-Langerin mAb, consistent with the recognized effect of these drugs on classical receptor-mediated endocytosis (Durrbach et al, 1996;Lamaze et al, 1997;Salamero et al, 2001). In addition, many of the BG-like structures were clathrin coated, or had labeled coated pits at their cytoplasmic terminal end.…”

“…Cytochalasin D and latrunculin A, known respectively to depolymerize and sequester F-actin filaments, have been shown to block the initial steps of endocytosis without affecting the recycling pathway in various cell types, including Fi LCs (Durrbach et al, 1996;Lamaze et al, 1997;Salamero et al, 2001). Having confirmed by immunofluorescence their expected effects on filamentous actin in this cell type (our unpublished data), we found that latrunculin A ( Figure 4B) and cytochalasin D (our unpublished data) were equally efficient in blocking the internalization of the mAb DCGM4 bound to Langerin at the surface of Fi LCs (compare Figure 4, B to A).…”

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“…Like phagocytosis, macropinocytosis clearly depends on actin polymerization at the plasma membrane. Direct dependence of macropinocytosis on actin is in marked contrast with clathrin-dependent micropinocytosis, which is not impaired by cytochalasins (30,49). Moreover, whereas macropinocytosis is suppressed by pharmacological inhibitors of PLC and PI3K, as well as by inhibition of the Na ϩ /H ϩ antiport, these pathways are usually not required for clathrin-dependent micropinocytosis (30,50,51).…”

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“…Actin plays a fundamental role in endocytosis in yeast, although actin's role in higher eukaryotic cells has yet to be fully appreciated. However, agents that destabilize actin filaments significantly reduce the rate of endocytosis in certain cell types (Lamaze et al, 1997;Fujimoto et al, 2000;Moskowitz et al, 2003).…”