Indeed, podocyte junctions depend on P-cadherin and its conversation with catenins . expression in control podocytes (A) and in siRNA IQGAP1 transfected podocytes (B). Characteristics of FAK antibody: rabbit antibody, dilution 1/100, Upstate Biotechnology, Massachusetts, USA.(TIF) pone.0037695.s003.tif (2.2M) GUID:?0C828D8B-1618-4F68-937B-09BA59AEDF11 Physique S4: Nuclear staining in normal kidney tissue. In conditions previously described, IQGAP1 labelling (green) and nuclear staining with DAPI was performed. Labelling is usually representative of normal kidney. Scale bar, 50 m. Analysis was performed with a confocal microscope. Microscope sections, 0.5 m. Magnification, X40. Nephrin staining in normal kidney tissue: In conditions previously explained, nephrin labelling (reddish) was performed. In normal glomeruli, nephrin staining (reddish) was continuous round the glomerular basement membrane. Scale bar, 50 m. Analysis was performed with a confocal microscope. Microscope sections, 0.5 m. Magnification, X40.(TIF) pone.0037695.s004.tif (1.8M) GUID:?1515CF29-2D98-4918-AD2D-9DD1E5EA741C Physique S5: IQGAP1 glomerular expression and localization in normal kidney tissue. All sections from normal kidneys (6) are figured. In normal glomeruli, IQGAP1 labelling (green) was continuous, corresponding to podocytes. IQGAP1 was also expressed in parietal epithelial, endothelial and distal tubular cells. Nuclear staining with DAPI is also represented. Immunohistochemistry experiments were performed on paraffin sections from normal kidneys (6). Analysis was SR 146131 performed with confocal microscopy. Microscope sections, 0.5 m. Magnification, X40.(TIF) pone.0037695.s005.tif (2.0M) GUID:?264DEAD6-50A9-4714-90F2-29EF636C1EFB Abstract IQGAP1 is a scaffold protein that interacts with proteins of the cytoskeleton and the intercellular adhesion complex. In podocytes, Mouse monoclonal to CHUK IQGAP1 is usually associated with nephrin in the glomerular slit diaphragm (SD) complex, but its role remains ill-defined. In this work, we investigated the conversation of IQGAP1 with the cytoskeleton and SD proteins in podocytes in culture, and its role in podocyte migration and permeability. Expression, localization, and interactions between IQGAP1 and SD or cytoskeletal proteins were decided in cultured human podocytes by Western blot (WB), immunocytolocalization (IC), immunoprecipitation (IP), and In situ Proximity Ligation assay (IsPL). Involvement of IQGAP1 in migration and permeability was also assessed. IQGAP1 expression in normal kidney biopsies was analyzed by immunohistochemistry. IQGAP1 expression by podocytes increased during their differentiation. IC, IP, and IsPL experiments showed colocalizations and/or interactions between IQGAP1 and SD proteins (nephrin, MAGI-1, CD2AP, NCK 1/2, podocin), podocalyxin, and cytoskeletal proteins (-actinin-4). IQGAP1 silencing decreased podocyte migration and increased the permeability of a podocyte layer. Immunohistochemistry on normal human kidney confirmed IQGAP1 expression in podocytes and distal tubular epithelial cells and also showed an expression in glomerular parietal epithelial cells. In summary, our results suggest that IQGAP1, through its conversation with components of SD and cytoskeletal proteins, is usually involved in podocyte barrier properties. Introduction Podocytes are epithelial cells with long foot processes that interdigitate around glomerular capillaries. A specialized protein complex junction between foot processes forms the slit diaphragm which is crucial for glomerular integrity. Nephrin is the major structural component of the slit diaphragm . Other proteins such SR 146131 as podocin, phospholipase C1, CD2AP, -actinin-4, CLIC5 and the scaffold proteins NCK 1 and 2 are also important for maintaining the slit diaphragms integrity. Defects in the expression of one of these proteins induce proteinuria resulting from SR 146131 effacement of foot processes and loss of glomerular barrier integrity , , , , . IQGAP1, a scaffold protein involved in actin remodelling has been identified as being associated with nephrin in podocyte foot processes , . In humans, IQGAP1 is usually a 189 kDa protein , localized in the cytoplasm and also associated with the cell membrane , . IQGAP1 consists of several conversation SR 146131 domains: a calponin homology domain name which binds filamentous (F)-actin, a WW domain name interacting with ERK1-2 , an IQ domain name interacting with calmodulin  and MEK1-2 , and a GRD domain name (a Space related domain name, without GTPase activity) interacting with the Rho GTPases, Cdc42 and Rac1 , , . The C-terminus domain name interacts with CLIP170 , APC , -catenin  and E-cadherin , . By interacting with actin and the Rho GTPases Cdc42 and Rac1, IQGAP1 contributes to the actin network formation , , . Moreover, IQGAP1 connects the actin network to microtubules by binding to APC and CLIP170 , . IQGAP1 can also interact with E- and VE-cadherin  and -catenin at the cellular junctions of epithelial and endothelial cells. By interacting with these different proteins, IQGAP1 plays a major role in cellular migration, cytoskeleton business and linkage between actin cytoskeleton and microtubules. Apart from being associated with nephrin in the proximity of the glomerular slit diaphragm in human and rats , , IQGAP1 is also present in migrating junctional complexes during rat glomeruli development, with different cell localizations over.