Abstract
IQGAP1 is a scaffold protein that interacts with proteins of the cytoskeleton and the intercellular adhesion complex. In podocytes, IQGAP1 is associated with nephrin in the glomerular slit diaphragm (SD) complex, but its role remains ill-defined. In this work, we investigated the interaction 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 determined 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 studied by immunohistochemistry. IQGAP1 expression by podocytes increased during their in vitro 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 (α-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 interaction with components of SD and cytoskeletal proteins, is involved in podocyte barrier properties.
Highlights
Podocytes are epithelial cells with long foot processes that interdigitate around glomerular capillaries
Cells were initially grown at the permissive temperature of 33uC - day 0 (D0), and were incubated up to 16 days (D16) at the non permissive temperature of 37uC (Details on morphological differences are available in Figure S1) [25,26]
We found that in undifferentiated and differentiated podocytes IQGAP1 interacted with MAGI-1 and CD2AP, proteins already known as interacting with nephrin [27,28] (Figures 3B and 3C)
Summary
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 [1]. Other proteins such as podocin, phospholipase Ce1, CD2AP, a-actinin-4, CLIC5 and the scaffold proteins NCK 1 and 2 are important for maintaining the slit diaphragm’s integrity. IQGAP1, a scaffold protein involved in actin remodelling has been identified as being associated with nephrin in podocyte foot processes [7,8]. IQGAP1 is a 189 kDa protein [9], localized in the cytoplasm and associated with the cell membrane [7,8]. The C-terminus domain interacts with CLIP170 [16], APC [17], b-catenin [18] and E-cadherin [19,20]
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