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Vol. 17, Issue 1, 427-437, January 2006

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Actin Is Required for Endocytosis at the Apical Surface of Madin-Darby Canine Kidney Cells where ARF6 and Clathrin Regulate the Actin Cytoskeleton

Department of Pharmacology School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem-Ein Kerem Campus, Jerusalem 91120, Israel

Submitted May 11, 2005; Revised September 26, 2005; Accepted October 19, 2005
Monitoring Editor: Jean Gruenberg

In epithelial cell lines, apical but not basolateral clathrin-mediated endocytosis has been shown to be affected by actin-disrupting drugs. Using electron and fluorescence microscopy, as well as biochemical assays, we show that the amount of actin dedicated to endocytosis is limiting at the apical surface of epithelia. In part, this contributes to the low basal rate of clathrin-dependent endocytosis observed at this epithelial surface. ARF6 in its GTP-bound state triggers the recruitment of actin from the cell cortex to the clathrin-coated pit to enable dynamin-dependent endocytosis. In addition, we show that perturbation of the apical endocytic system by expression of a clathrin heavy-chain mutant results in the collapse of microvilli. This phenotype was completely reversed by the expression of an ARF6-GTP-locked mutant. These observations indicate that concomitant to actin recruitment, the apical clathrin endocytic system is deeply involved in the morphology of the apical plasma membrane.

Abbreviations used: pIgA, polymeric IgA; PM, plasma membrane; CME, clathrin-mediated endocytosis; CCP, clathrin-coated pit; GFP, green fluorescent protein; MDCK, Madin-Darby canine kidney; ARF, ADP ribosylation factor; Dx, doxycycline; WT, wild type.

Address correspondence to: Yoram Altschuler (yoram11{at}md.huji.ac.il).

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