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Am J Physiol Renal Physiol (June 14, 2005). doi:10.1152/ajprenal.00178.2005
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Submitted on April 25, 2005
Accepted on June 6, 2005

Association of CD2AP with Dynamic Actin on Vesicles in Podocytes

Thilo Welsch1, Nicole Endlich1, Gokmen Gokce1, Elena Doroshenko1, Jeremy C. Simpson2, Wilhelm Kriz1, Andrey S. Shaw3, and Karlhans Endlich1*

1 Department of Anatomy and Cell Biology, University of Heidelberg, Heidelberg, Germany
2 EMBL, Heidelberg, Germany
3 Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA

* To whom correspondence should be addressed. E-mail: karlhans.endlich{at}urz.uni-heidelberg.de.

The docking protein CD2AP (CD2-associated protein) serves a non-redundant function in podocytes since CD2AP knockout mice die of renal failure at the age of 6-7 weeks. Furthermore, haploinsufficiency due to mutation of the CD2AP gene is associated with focal segmental glomerulosclerosis in humans. Though 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 on 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.




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