Three-Dimensional Visualization of the Podocyte Actin Network Using Integrated Membrane Extraction, Electron Microscopy, and Machine Learning.

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.

An ex vivo culture model of kidney podocyte injury reveals mechanosensitive, synaptopodin-templating, sarcomere-like structures.

Chronic kidney diseases are widespread and incurable. The biophysical mechanisms underlying them are unclear, in part because material systems for reconstituting the microenvironment of relevant kidney cells are limited. A critical question is how kidney podocytes (glomerular epithelial cells) regenerate foot processes of the filtration apparatus following injury. Recently identified sarcomere-like structures (SLSs) with periodically spaced myosin IIA and synaptopodin appear in injured podocytes in vivo. We hypothesized that SLSs template synaptopodin in the initial stages of recovery in response to microenvironmental stimuli and tested this hypothesis by developing an ex vivo culture system that allows control of the podocyte microenvironment. Results supported our hypothesis. SLSs in podocytes that migrated from isolated kidney glomeruli presented periodic synaptopodin-positive clusters that nucleated peripheral, foot process-like extensions. SLSs were mechanoresponsive to actomyosin inhibitors and substrate stiffness. Results suggest SLSs as mechanobiological mediators of podocyte recovery and as potential targets for therapeutic intervention.