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

Abstract

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.

Keywords: Autophagosome; Autophagy; Direct stochastic optical reconstruction; Fluorescence microscopy; Lysosome; Structured illumination.