PALS1 is a key regulator of the lateral distribution of tight junction proteins in renal epithelial cells.

The evolutionarily conserved apical Crumbs (CRB) complex, consisting of the core components CRB3a (an isoform of CRB3), PALS1 and PATJ, plays a key role in epithelial cell-cell contact formation and cell polarization. Recently, we observed that deletion of one Pals1 allele in mice results in functional haploinsufficiency characterized by renal cysts. Here, to address the role of PALS1 at the cellular level, we generated CRISPR/Cas9-mediated PALS1-knockout MDCKII cell lines. The loss of PALS1 resulted in increased paracellular permeability, indicating an epithelial barrier defect. This defect was associated with a redistribution of several tight junction-associated proteins from bicellular to tricellular contacts. PALS1-dependent localization of tight junction proteins at bicellular junctions required its interaction with PATJ. Importantly, reestablishment of the tight junction belt upon transient F-actin depolymerization or upon Ca2+ removal was strongly delayed in PALS1-deficient cells. Additionally, the cytoskeleton regulator RhoA was redistributed from junctions into the cytosol under PALS1 knockout. Together, our data uncover a critical role of PALS1 in the coupling of tight junction proteins to the F-actin cytoskeleton, which ensures their correct distribution along bicellular junctions and the formation of tight epithelial barrier.

Loss of surface transport is a main cellular pathomechanism of CRB2 variants causing podocytopathies.

Crumbs2 (CRB2) is a central component of the renal filtration barrier and part of the slit diaphragm, a unique cell contact formed by glomerular podocytes. Some CRB2 variants cause recessive inherited forms of steroid-resistant nephrotic syndrome. However, the disease-causing potential of numerous CRB2 variants remains unknown. Here, we report the establishment of a live-cell imaging-based assay, allowing a quantitative evaluation of the pathogenic potential of so far non-categorized CRB2 variants. Based on in silico data analysis and protein prediction software, putative disease-associated CRB2 missense variants were selected, expressed as CRB2-GFP fusion proteins, and analyzed in reporter cell lines with BFP-labeled plasma membrane. We found that in comparison with PM-localized WT, disease-associated CRB2 variants remained predominantly at the ER. Accumulation at the ER was also present for several non-characterized CRB2 variants and variants in which putative disulfide bridge-forming cysteines were replaced. Strikingly, WT CRB2 retained inside the ER in cells lacking protein disulfide isomerase A3, indicating that posttranslational modification, especially the formation of disulfide bridges, is a crucial step for the CRB2 PM transport.

Crumbs2 Is an Essential Slit Diaphragm Protein of the Renal Filtration Barrier.

BACKGROUND: Crumbs2 is expressed at embryonic stages as well as in the retina, brain, and glomerular podocytes. Recent studies identified CRB2 mutations as a novel cause of steroid-resistant nephrotic syndrome (SRNS). METHODS: To study the function of Crb2 at the renal filtration barrier, mice lacking Crb2 exclusively in podocytes were generated. Gene expression and histologic studies as well as transmission and scanning electron microscopy were used to analyze these Crb2podKO knockout mice and their littermate controls. Furthermore, high-resolution expansion microscopy was used to investigate Crb2 distribution in murine glomeruli. For pull-down experiments, live cell imaging, and transcriptome analyses, cell lines were applied that inducibly express fluorescent protein-tagged CRB2 wild type and mutants. RESULTS: Crb2podKO mice developed proteinuria directly after birth that preceded a prominent development of disordered and effaced foot processes, upregulation of renal injury and inflammatory markers, and glomerulosclerosis. Pull-down assays revealed an interaction of CRB2 with Nephrin, mediated by their extracellular domains. Expansion microscopy showed that in mice glomeruli, Crb2 and Nephrin are organized in adjacent clusters. SRNS-associated CRB2 protein variants and a mutant that lacks a putative conserved O-glycosylation site were not transported to the cell surface. Instead, mutants accumulated in the ER, showed altered glycosylation pattern, and triggered an ER stress response. CONCLUSIONS: Crb2 is an essential component of the podocyte's slit diaphragm, interacting with Nephrin. Loss of slit diaphragm targeting and increasing ER stress are pivotal factors for onset and progression of CRB2-related SRNS.

LIM and SH3 protein 1 (LASP-1): A novel link between the slit membrane and actin cytoskeleton dynamics in podocytes.

The foot processes of podocytes exhibit a dynamic actin cytoskeleton, which maintains their complex cell structure and antagonizes the elastic forces of the glomerular capillary. Interdigitating secondary foot processes form a highly selective filter for proteins in the kidney, the slit membrane. Knockdown of slit membrane components such as Nephrin or Neph1 and cytoskeletal adaptor proteins such as CD2AP in mice leads to breakdown of the filtration barrier with foot process effacement, proteinuria, and early death of the mice. Less is known about the crosstalk between the slit membrane-associated proteins and cytoskeletal components inside the podocyte foot processes. Our study shows that LASP-1, an actin-binding protein, is highly expressed in podocytes. Electron microscopy studies demonstrate that LASP-1 is found at the slit membrane suggesting a role in anchoring slit membrane components to the actin cytoskeleton. Live cell imaging experiments with transfected podocytes reveal that LASP-1 is either part of a highly dynamic granular complex or a static, actin cytoskeleton-bound protein. We identify CD2AP as a novel LASP-1 binding partner that regulates its association with the actin cytoskeleton. Activation of the renin-angiotensin-aldosterone system, which is crucial for podocyte function, leads to phosphorylation and altered localization of LASP-1. In vivo studies using the Drosophila nephrocyte model indicate that Lasp is necessary for the slit membrane integrity and functional filtration.

Angiotensin II regulates phosphorylation of actin-associated proteins in human podocytes.

Within the kidney, angiotensin II (AngII) targets different cell types in the vasculature, tubuli, and glomeruli. An important part of the renal filtration barrier is composed of podocytes with their actin-rich foot processes. In this study, we used stable isotope labeling with amino acids in cell culture coupled to mass spectrometry to characterize relative changes in the phosphoproteome of human podocytes in response to short-term treatment with AngII. In 4 replicates, we identified a total of 17,956 peptides that were traceable to 2081 distinct proteins. Bioinformatic analyses revealed that among the increasingly phosphorylated peptides are predominantly peptides that are related to actin filaments, cytoskeleton, lamellipodia, mammalian target of rapamycin, and MAPK signaling. Among others, this screening approach highlighted the increased phosphorylation of actin-bundling protein, l-plastin (LCP1). AngII-dependent phosphorylation of LCP1 in cultured podocytes was mediated by the kinases ERK, p90 ribosomal S6 kinase, PKA, or PKC. LCP1 phosphorylation increased filopodia formation. In addition, treatment with AngII led to LCP1 redistribution to the cell margins, membrane ruffling, and formation of lamellipodia. Our data highlight the importance of AngII-triggered actin cytoskeleton-associated signal transduction in podocytes.-Schenk, L. K., Möller-Kerutt, A., Klosowski, R., Wolters, D., Schaffner-Reckinger, E., Weide, T., Pavenstädt, H., Vollenbröker, B. Angiotensin II regulates phosphorylation of actin-associated proteins in human podocytes.