Background Actin stress fibers are abundant in cultured cells, but little is known about them in vivo. In podocytes, much evidence suggests that mechanobiologic mechanisms underlie podocyte shape and adhesion in health and in injury, with structural changes to actin stress fibers potentially responsible for pathologic changes to cell morphology. However, this hypothesis is difficult to rigorously test in vivo due to challenges with visualization. A technology to image the actin cytoskeleton at high resolution is needed to better understand the role of structures such as actin stress fibers in podocytes. Methods We developed the first visualization technique capable of resolving the three-dimensional cytoskeletal network in mouse podocytes in detail while definitively identifying the proteins that comprise this network. This technique integrates membrane extraction, focused ion beam scanning electron microscopy, and machine learning image segmentation. Results Using isolated mouse glomeruli from healthy animals, we observed actin cables and intermediate filaments linking the interdigitated podocyte foot processes to newly described contractile actin structures located at the periphery of the podocyte cell body. Actin cables within foot processes formed a continuous, mesh-like, electron-dense sheet that incorporated the slit diaphragms. Conclusions Our new technique revealed, for the first time, the detailed three-dimensional organization of actin networks in healthy podocytes. In addition to being consistent with the gel compression hypothesis, which posits that foot processes connected by slit diaphragms act together to counterbalance the hydrodynamic forces across the glomerular filtration barrier, our data provide insight into how podocytes respond to mechanical cues from their surrounding environment.
Although actin stress fibers are abundant in cultured cells, little is known about these structures in vivo. In podocytes of the kidney glomerulus, much evidence suggests that mechanobiological mechanisms underlie injury, with changes to actin stress fiber structures potentially responsible for pathological changes to cell morphology. However, this hypothesis is difficult to rigorously test in vivo due to challenges with visualization. We therefore developed the first visualization technique capable of resolving the three-dimensional (3D) podocyte actin network with unprecedented detail in healthy and injured podocytes, and applied this technique to reveal the changes in the actin network that occur upon podocyte injury. Using isolated glomeruli from healthy mice as well as from three different mouse injury models (Cd2ap-/-, Lamb2-/- and the Col4a3-/- model of Alport syndrome), we applied our novel imaging technique that integrates membrane-extraction, focused ion bean scanning electron microscopy (FIB-SEM), and deep learning image segmentation. In healthy glomeruli, we observed actin cables that link the interdigitating podocyte foot processes to newly described actin structures located at the periphery of the cell body. The actin cables within the foot processes formed a continuous, mesh-like, electron dense sheet that incorporated the slit diaphragms required for kidney filtration. After injury, the actin network was markedly different, having lost its organization and presenting instead as a disorganized assemblage of actin condensates juxtaposed to the glomerular basement membrane. The new visualization method enabled us, for the first time, to observe the detailed 3D organization of actin networks in both healthy and injured podocytes. Shared features of actin condensations across all three injury models further suggested common mechanobiological pathways that govern changes to podocyte morphology after injury.
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