Monitoring autophagy using super-resolution structured illumination and direct stochastic optical reconstruction microscopy.

Autor:	Lumkwana D; Microscopy and Imaging Translational Technology Platform, Cancer Research UK, University College London, London, United Kingdom., Engelbrecht L; Central Analytical Facility, Fluorescence Microscopy Unit, Stellenbosch University, South Africa., Loos B; Department of Physiological Sciences, Stellenbosch University, South Africa. Electronic address: bloos@sun.ac.za.
Jazyk:	angličtina
Zdroj:	Methods in cell biology [Methods Cell Biol] 2021; Vol. 165, pp. 139-152. Date of Electronic Publication: 2021 Jan 30.
DOI:	10.1016/bs.mcb.2020.12.005
Abstrakt:	Autophagy is a major protein degradation pathway responsible for the removal of primarily long-lived and misfolded proteins, contributing to cellular homeostasis. Autophagy dysfunction has been associated with the onset of various human pathologies. Visualizing key proteins that govern autophagy pathway activity, the molecular machinery and cargo is essential to elucidate roles and mechanisms of autophagy function. Although multiple fluorescence-based microscopy approaches exist to assess autophagy, the limit of resolution associated with light microscopy makes precise intracellular protein localization, interaction and molecular distribution challenging. Here we describe a detailed protocol for both super-resolution structured illumination microscopy (SR-SIM) as well as direct stochastic optical reconstruction microscopy (dSTORM) for the visualization of key proteins associated with the autophagy molecular machinery and cargo. The presented method enables to achieve increased resolving power to assess localization and molecular density profiles, typically not achievable with standard confocal or wide field fluorescence microcopy. (Copyright © 2021 Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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