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

Abstract

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.

Reciprocal regulation of SIRT1 and AMPK by Ginsenoside compound K impedes the conversion from plasma cells to mitigate for podocyte injury in MRL/lpr mice in a B cell-specific manner

“…However, increased dynamic motility could destabilize this state upon exposure to adriamycin-induced injury [ 45 ]. Moreover, the well-formed configuration of foot processes is contingent upon the soundness of the actin cytoskeleton, and the obliteration of podocytes emanates from the vulnerable cytoskeleton due to the loss of filamentous actin [ 46 ]. Previous studies have shown CK yields renoprotective effects in a dose-dependent manner, with the most significant improvements observed at a dosage of 40 mg/kg, alongside its anti-inflammatory actions [ 25 ].…”

A YAP/TAZ–ARHGAP29–RhoA Signaling Axis Regulates Podocyte Protrusions and Integrin Adhesions

“…Reorganization of the actin cytoskeleton and of cell-matrix adhesions towards a contractile and mechanically enforced RhoA-dependent actomyosin cytoskeleton is a common pattern in podocytopathy and considered a driving factor of glomerular disease [ 9 , 12 , 19 , 62 ]. Analysis in human kidney tissue showed increased ARHGAP29 expression in early glomerular disease stages ( Figure 6 ).…”

Cytoskeleton Rearrangement in Podocytopathies: An Update

“…In addition, mechanical forces can also influence the assembly and disassembly of AFs [ 10 ]. The actin cytoskeleton is strongly sensitive and responsive to the mechanical stress that podocytes face [ 20 ]. The AFs provide a structural and functional basis, thus building the cytoarchitecture of podocytes and enhancing adhesion to the extracellular matrix [ 7 , 16 ].…”