Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells.

Toshihiko Ogura,Tomoko Okada

doi:10.3390/ijms22041834

Abstract

Autophagy is an intracellular self-devouring system that plays a central role in cellular recycling. The formation of functional autophagosomes depends on several autophagy-related proteins, including the microtubule-associated proteins 1A/1B light chain 3 (LC3) and the conserved autophagy-related gene 12 (Atg12). We have recently developed a novel scanning electron-assisted dielectric microscope (SE-ADM) for nanoscale observations of intact cells. Here, we used the SE-ADM system to observe LC3- and Atg12-containing autophagosomes in cells labelled in the culture medium with antibodies conjugated to colloidal gold particles. We observed that, during autophagosome formation, Atg12 localized along the actin meshwork structure, whereas LC3 formed arcuate or circular alignments. Our system also showed a difference in the distribution of LC3 and Atg12; Atg12 was broadly distributed while LC3 was more localized. The difference in the spatial distribution demonstrated by our system explains the difference in the size of fluorescent spots due to the fluorescently labelled antibodies observed using optical microscopy. The direct SE-ADM observation of cells should thus be effective in analyses of autophagosome formation.

Highlights

When the light chain 3 (LC3) and actin fluorescence images were merged, LC3 was observed to colocalize with the actin meshwork structure as perinuclear actin (Figure 1C)
To analyze the localization of autophagy-related gene 12 (Atg12) and the meshwork actin involved in autophagosome formation, we investigated the distribution of Atg12 in the cells using the SEADM system (Figure 4)
We analyzed the localization of two autophagy-related proteins, LC3 and Atg12, in liquid medium using our newly developed scanning electron-assisted dielectric microscopy (SE-ADM) system

Summary

Introduction

Autophagy is a process of the self-devouring of unnecessary and dysfunctional cellular components such as protein aggregates and damaged organelles as well as invading bacteria, viruses, and protists [1,2,3,4,5]. Such materials are incorporated into and enclosed by a double-membraned autophagosome, which later fuses with the lysosome [2,3,6]. One category of these proteins includes ubiquitinlike proteins of the microtubule-associated proteins 1A/1B light chain 3 (LC3) family [9,10]

Methods

Results

Discussion

Conclusion