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

Chengqing Qu, Robyn Roth, Pongpratch Puapatanakul, Charles Loitman, Dina Hammad, Guy M. Genin, Jeffrey H. Miner, Hani Y. Suleiman

Research output: Contribution to journalArticlepeer-review

10 Scopus citations


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.

Original languageEnglish
Pages (from-to)155-173
Number of pages19
JournalJournal of the American Society of Nephrology
Issue number1
StatePublished - Jan 2022


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