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Am J Physiol Renal Physiol 289: F1134-F1143, 2005. First published June 14, 2005; doi:10.1152/ajprenal.00178.2005
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Association of CD2AP with dynamic actin on vesicles in podocytes

Thilo Welsch,1 Nicole Endlich,1 Gökmen Gökce,1 Elena Doroshenko,1 Jeremy C. Simpson,2 Wilhelm Kriz,1 Andrey S. Shaw,3 and Karlhans Endlich1

1Department of Anatomy and Cell Biology I, University of Heidelberg, and 2European Molecular Biology Laboratories, Heidelberg, Germany; and 3Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri

Submitted 25 April 2005 ; accepted in final form 6 June 2005

The docking protein CD2AP (CD2-associated protein) serves a nonredundant function in podocytes as CD2AP knockout mice die of renal failure at the age of 6–7 wk. Furthermore, haploinsufficiency due to mutation of the CD2AP gene is associated with focal segmental glomerulosclerosis in humans. Although CD2AP has been shown to interact with proteins regulating actin polymerization, with proteins of the slit diaphragm, and with the endocytic machinery, its critical function in podocytes remains unclear. In conditionally immortalized mouse podocytes, we demonstrate that CD2AP colocalizes with cortactin and F-actin in spots of ≤0.5-µm diameter. Confocal time-lapse microscopy in living podocytes expressing GFP-CD2AP or GFP-actin revealed that spots are motile, possess a limited lifetime, and are frequently associated with vesicles. A significant portion of spot-associated vesicles belongs to a later endosomal-sorting compartment, characterized by delayed uptake of fluorescent dextran (10 kDa) and by colocalization with Rab4, but not Rab5 and AP-2. Rapid accumulation of microinjected G-actin in spots and abrogation of spot motility by jasplakinolide demonstrate that spot movements depend on actin polymerization. Furthermore, a high turnover (half-time < 10 s) of CD2AP in spots was demonstrated by FRAP (fluorescence recovery after photobleaching). Our results demonstrate that CD2AP is associated with dynamic actin in a specific late endosomal compartment in podocytes, suggesting that CD2AP might be crucially involved in endosomal sorting and/or trafficking via regulation of actin assembly on vesicles.

glomerular epithelial cells; CMS; green fluorescent protein


Address for reprint requests and other correspondence: K. Endlich, Institut für Anatomie und Zellbiologie I, Universität Heidelberg, INF 307, D-69120 Heidelberg, Germany (e-mail: karlhans.endlich{at}urz.uni-heidelberg.de)




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