Next-generation electron microscopy in autophagy research

Abstract

Autophagy is the process whereby cytosol, organelles, and inclusions are taken up in a double-membrane vesicle known as the autophagosome, and transported to the lysosome for destruction and recycling. Electron microscopy (EM) led to the discovery of autophagy in the 1950s and has been a central part of its characterization ever since. New capabilities in single particle EM studies of the autophagy machinery are beginning to provide exciting insights into the mechanisms of autophagosome initiation, growth, and substrate targeting. These include EM structures at various resolutions of part of the Atg1 protein kinase complex and all of the class III phosphatidylinositol 3-phosphate complex I that initiate autophagy; the mTORC1 complex that regulates autophagy initiation; the Ape1 particle, a major substrate for selective autophagy in yeast; and p62, a mammalian selective autophagy adaptors. Equally exciting are the prospects for increased resolution and insight into autophagosome formation in cells from advances in cryo-EM tomography and focused ion beam-scanning electron microscopy (FIB-SEM). This review considers recent accomplishments, prospects for progress, and remaining obstacles that still need to be overcome.

Mechanisms of Autophagy Initiation

“…These now-traditional approaches have yielded many important discoveries, yet they will not be able to break through to resolving the nanoscale mechanisms involved in phagophore initiation. Superresolution and cryo-electron microscopy methodologies are advancing quickly, and the advances are quickly being adopted in autophagy research (151). The imaging approaches will generate more insights that can be contemplated on the structural scale, and the structural biology studies are expanding in scope to approach the size scales reachable by superresolution imaging.…”

Insights into autophagosome biogenesis from structural and biochemical analyses of the ATG2AWIPI4 complex

“…In either case, there is currently insufficient information to produce a meaningful and complete structural model describing how ATG2, ATG9, and ATG18/WIPI are involved in membrane tethering at the ERphagophore junction. Higher resolution structural information of these protein complexes and their interactions with membranes, along with more precise localization of these proteins at the ER-phagophore junction will be required to elucidate such a model (48). Our negative stain EM analysis of the SUV-bound ATG2A-WIPI4 complex suggests that the primary regions in or adjacent to the N-terminus and ATG2_CAD are responsible for membrane binding and tethering (Fig.…”

Acidic pH environment induces autophagy in osteoblasts

“…The amount of LC3-II detected by western blot (WB) during the process of autophagy, reflects synthesis as well as consumption of the protein by the lysosomal compartment. In addition to protein levels of LC3, P62 is an important marker to study autophagic flux27282930. P62 is exclusively degraded after being bound directly to LC3 by the autophagy-lysosome pathway3132.…”