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

Autor:	Qu C; Department of Mechanical Engineering, National Science Foundation Science and Technology Center for Engineering Mechanobiology, Washington University in St. Louis, St. Louis, Missouri., Roth R; Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri., Puapatanakul P; Division of Nephrology, Washington University School of Medicine, St. Louis, Missouri., Loitman C; Division of Nephrology, Washington University School of Medicine, St. Louis, Missouri., Hammad D; Division of Nephrology, Washington University School of Medicine, St. Louis, Missouri., Genin GM; Department of Mechanical Engineering, National Science Foundation Science and Technology Center for Engineering Mechanobiology, Washington University in St. Louis, St. Louis, Missouri., Miner JH; Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri.; Division of Nephrology, Washington University School of Medicine, St. Louis, Missouri., Suleiman HY; Division of Nephrology, Washington University School of Medicine, St. Louis, Missouri.
Jazyk:	angličtina
Zdroj:	Journal of the American Society of Nephrology : JASN [J Am Soc Nephrol] 2022 Jan; Vol. 33 (1), pp. 155-173. Date of Electronic Publication: 2021 Nov 10.
DOI:	10.1681/ASN.2021020182
Abstrakt:	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. (Copyright © 2022 by the American Society of Nephrology.)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu