Pdlim2 is a novel actin-regulating protein of podocyte foot processes
ABSTRACT The slit diaphragm and the apical and basal membrane domains of podocytes are connected to each other by an actin-based cytoskeleton critical to the maintenance of the glomerular filtration barrier. In an effort to discover novel regulatory proteins of the podocyte foot process, we identified and characterized pdlim2, a member of the actin-associated LIM protein subfamily of cytosolic proteins typified by an N-terminal PDZ domain and a C-terminal LIM domain. In the kidney, the pdlim2 protein is highly specific for the glomerulus and podocyte foot processes as shown by RT-PCR, western blotting, immunofluorescence, and immunoelectron microscopy. In cultured podocytes, pdlim2 was associated with stress fibers and cortical actin. Pdlim2 seems to regulate actin dynamics in podocytes since stress fibers were stabilized in its presence. Mechanistically, pdlim2 interacts with two actin-associated podocyte proteins, α-actinin-4 and angiomotin-like-1, as shown by immunoprecipitation and yeast two-hybrid analyses. By semi-quantitative immunoelectron microscopy, there was a reduced expression of pdlim2 in podocytes of patients with minimal change nephrotic syndrome and membranous nephropathy, whereas its expression was unchanged in patients with focal segmental glomerulosclerosis. Hence, pdlim2 is a novel actin-regulating protein of podocyte foot processes that may have a role in the pathogenesis of glomerular diseases.
SourceAvailable from: Xiongwu Wu[Show abstract] [Hide abstract]
ABSTRACT: Diseases of the kidney filtration barrier are a leading cause of ESRD. Most disorders affect the podocytes, polarized cells with a limited capacity for self-renewal that require tightly controlled signaling to maintain their integrity, viability, and function. Here, we provide an atlas of in vivo phosphorylated, glomerulus-expressed proteins, including podocyte-specific gene products, identified in an unbiased tandem mass spectrometry-based approach. We discovered 2449 phosphorylated proteins corresponding to 4079 identified high-confidence phosphorylated residues and performed a systematic bioinformatics analysis of this dataset. We discovered 146 phosphorylation sites on proteins abundantly expressed in podocytes. The prohibitin homology domain of the slit diaphragm protein podocin contained one such site, threonine 234 (T234), located within a phosphorylation motif that is mutated in human genetic forms of proteinuria. The T234 site resides at the interface of podocin dimers. Free energy calculation through molecular dynamic simulations revealed a role for T234 in regulating podocin dimerization. We show that phosphorylation critically regulates formation of high molecular weight complexes and that this may represent a general principle for the assembly of proteins containing prohibitin homology domains.Journal of the American Society of Nephrology 02/2014; 25(7). DOI:10.1681/ASN.2013070760 · 9.47 Impact Factor
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ABSTRACT: Background: Proteinuria is a cardinal sign of chronic kidney disease, which is a major healthcare problem that affects millions of people worldwide. Recent advances in molecular genetics and cell biology have revealed the podocyte as the primary functional regulator of the tri-layered glomerular filter. Since podocyte foot processes (FP) and their interposed slit diaphragms (SD) form the final barrier to protein loss, podocyte injury causes proteinuric kidney disease. Summary: A fundamental mechanism of proteinuric glomerular diseases is podocyte FP effacement and the loss of podocyte SD integrity, both of which involve the active rearrangement of the podocyte actin cytoskeleton. Initially, these early changes are reversible, but later can progress to cell detachment and death. Based on the importance of the actin cytoskeleton for podocyte development and the maintenance of the glomerular filter, podocyte research is heavily focused on studying actin’s molecular make-up and regulation. In this review we provide a comprehensive summary of the about 100 actin-associated proteins that have been described in podocytes to date, and we point out that so far only about one quarter of them have been shown to be functionally relevant for podocyte function in rodents or humans. Since actinmediated cell plasticity is a key feature of normal podocyte function, and alterations in actin dynamics appear to be a major driver in changing podocyte morphology and glomerular permeability, we discuss the current work on proteins and mechanisms that regulate actin polymerization and stress fiber contraction in podocyte FP in greater detail. Key Message: Without a doubt, the actin cytoskeleton is the key component of podocytes and proper glomerular filtration. Over the past 20 years many actin-associated proteins and actin-regulating mechanisms have been identified in podocytes. However, since most of these proteins are widely expressed and regulate actin in different cell types, it remains unclear if the podocyte actin cytoskeleton can be specifically targeted, and if and how actin-associated proteins can serve as novel drug targets in proteinuric kidney disease.Contributions to nephrology 07/2014; 2014(183):22-53. DOI:10.1159/000359923 · 1.53 Impact Factor
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ABSTRACT: Podocyte foot process effacement accompanied by actin cytoskeleton rearrangements is a cardinal feature of many progressive human proteinuric diseases. By microarray profiling of mouse glomerulus, SCHIP1 emerged as one of the most highly enriched transcripts. We detected Schip1 protein in the kidney glomerulus, specifically in podocytes foot processes. Functionally, Schip1 inactivation in zebrafish by morpholino knock-down results in foot process disorganization and podocyte loss leading to proteinuria. In cultured podocytes Schip1 localizes to cortical actin-rich regions of lamellipodia, where it forms a complex with Nherf2 and ezrin, proteins known to participate in actin remodeling stimulated by PDGFβ signaling. Mechanistically, overexpression of Schip1 in vitro causes accumulation of cortical F-actin with dissolution of transversal stress fibers and promotes cell migration in response to PDGF-BB stimulation. Upon actin disassembly by latrunculin A treatment, Schip1 remains associated with the residual F-actin-containing structures, suggesting a functional connection with actin cytoskeleton possibly via its interaction partners. A similar assay with cytochalasin D points to stabilization of cortical actin cytoskeleton in Schip1 overexpressing cells by attenuation of actin depolymerisation. Schip1 is a novel glomerular protein predominantly expressed in podocytes, necessary for the zebrafish pronephros development and function. Schip1 associates with the cortical actin cytoskeleton network and modulates its dynamics in response to PDGF signaling via interaction with the Nherf2/ezrin complex. Its implication in proteinuric diseases remains to be further investigated.PLoS ONE 05/2015; 10(3):e0122067. DOI:10.1371/journal.pone.0122067 · 3.53 Impact Factor