SummaryPodocytes, highly specialized epithelial cells, build the outer part of the kidney filtration barrier and withstand high mechanical forces through a complex network of cellular protrusions. Here, we show that Arp2/3-dependent actin polymerization controls actomyosin contractility and focal adhesion maturation of podocyte protrusions and thereby regulates formation, maintenance, and capacity to adapt to mechanical requirements of the filtration barrier. We find that N-WASP-Arp2/3 define the development of complex arborized podocyte protrusions in vitro and in vivo. Loss of dendritic actin networks results in a pronounced activation of the actomyosin cytoskeleton and the generation of over-maturated but less efficient adhesion, leading to detachment of podocytes. Our data provide a model to explain podocyte protrusion morphology and their mechanical stability based on a tripartite relationship between actin polymerization, contractility, and adhesion.

The actin cytoskeleton is a main component of cells and it is crucially involved in many physiological processes, e.g. cell motility. Changes in the actin organization can be effected by diseases or vice versa. Due to the nonuniform pattern, it is difficult to quantify reasonable features of the actin cytoskeleton for a significantly high cell number. Here, we present an approach capable to fully segment and analyse the actin cytoskeleton of 2D fluorescence microscopic images with a special focus on stress fibres. The extracted feature data include length, width, orientation and intensity distributions of all traced stress fibres. Our approach combines morphological image processing techniques and a trace algorithm in an iterative manner, classifying the segmentation result with respect to the width of the stress fibres and in nonfibre-like actin. This approach enables us to capture experimentally induced processes like the condensation or the collapse of the actin cytoskeleton. We successfully applied the algorithm to F-actin images of cells that were treated with the actin polymerization inhibitor latrunculin A. Furthermore, we verified the robustness of our algorithm by a sensitivity analysis of the parameters, and we benchmarked our algorithm against established methods. In summary, we present a new approach to segment actin stress fibres over time to monitor condensation or collapse processes.

BackgroundPrevious research demonstrated that small Rho GTPases, modulators of the actin cytoskeleton, are drivers of podocyte foot-process effacement in glomerular diseases, such as FSGS. However, a comprehensive understanding of the regulatory networks of small Rho GTPases in podocytes is lacking.MethodsWe conducted an analysis of podocyte transcriptome and proteome datasets for Rho GTPases; mapped in vivo, podocyte-specific Rho GTPase affinity networks; and examined conditional knockout mice and murine disease models targeting Srgap1. To evaluate podocyte foot-process morphology, we used super-resolution microscopy and electron microscopy; in situ proximity ligation assays were used to determine the subcellular localization of the small GTPase-activating protein SRGAP1. We performed functional analysis of CRISPR/Cas9-generated SRGAP1 knockout podocytes in two-dimensional and three-dimensional cultures and quantitative interaction proteomics.ResultsWe demonstrated SRGAP1 localization to podocyte foot processes in vivo and to cellular protrusions in vitro. Srgap1fl/fl*Six2Cre but not Srgap1fl/fl*hNPHS2Cre knockout mice developed an FSGS-like phenotype at adulthood. Podocyte-specific deletion of Srgap1 by hNPHS2Cre resulted in increased susceptibility to doxorubicin-induced nephropathy. Detailed analysis demonstrated significant effacement of podocyte foot processes. Furthermore, SRGAP1-knockout podocytes showed excessive protrusion formation and disinhibition of the small Rho GTPase machinery in vitro. Evaluation of a SRGAP1-dependent interactome revealed the involvement of SRGAP1 with protrusive and contractile actin networks. Analysis of glomerular biopsy specimens translated these findings toward human disease by displaying a pronounced redistribution of SRGAP1 in FSGS.ConclusionsSRGAP1, a podocyte-specific RhoGAP, controls podocyte foot-process architecture by limiting the activity of protrusive, branched actin networks. Therefore, elucidating the complex regulatory small Rho GTPase affinity network points to novel targets for potentially precise intervention in glomerular diseases.

Podocyte loss and effacement of interdigitating podocyte foot processes are the major cause of a leaky filtration barrier and ESRD. Because the complex three-dimensional morphology of podocytes depends on the actin cytoskeleton, we studied the role in podocytes of the actin bundling protein palladin, which is highly expressed therein. We knocked down palladin in cultured podocytes by siRNA transfection or in zebrafish embryos by morpholino injection and studied the effects by immunofluorescence and live imaging. We also investigated kidneys of mice with podocyte-specific knockout of palladin (PodoPalld-/- mice) by immunofluorescence and ultrastructural analysis and kidney biopsy specimens from patients by immunostaining for palladin. Compared with control-treated podocytes, palladin-knockdown podocytes had reduced actin filament staining, smaller focal adhesions, and downregulation of the podocyte-specific proteins synaptopodin and -actinin-4. Furthermore, palladin-knockdown podocytes were more susceptible to disruption of the actin cytoskeleton with cytochalasin D, latrunculin A, or jasplakinolide and showed altered migration dynamics. In zebrafish embryos, palladin knockdown compromised the morphology and dynamics of epithelial cells at an early developmental stage. Compared with PodoPalld+/+ controls, PodoPalld-/- mice developed glomeruli with a disturbed morphology, an enlarged subpodocyte space, mild effacement, and significantly reduced expression of nephrin and vinculin. Furthermore, nephrotoxic serum injection led to significantly higher levels of proteinuria in PodoPalld-/- mice than in controls. Kidney biopsy specimens from patients with diabetic nephropathy and FSGS showed downregulation of palladin in podocytes as well. Palladin has an important role in podocyte function and.