Autophagy-Related Proteins GABARAP and LC3B Label Structures of Similar Size but Different Shape in Super-Resolution Imaging.

Iman Abdollahzadeh,Thomas Gensch,Johnny Hendriks,Oliver H Weiergräber,Julia L Sanwald,Indra M Simons,Silke Hoffmann,Dieter Willbold

doi:10.3390/molecules24091833

Abstract

Subcellular structures containing autophagy-related proteins of the Atg8 protein family have been investigated with conventional wide-field fluorescence and single molecule localisation microscopy. Fusion proteins of GABARAP and LC3B, respectively, with EYFP were overexpressed in HEK293 cells. While size distributions of structures labelled by the two proteins were found to be similar, shape distributions appeared quite disparate, with EYFP-GABARAP favouring circular structures and elliptical structures being dominant for EYFP-LC3B. The latter also featured a nearly doubled fraction of U-shape structures. The experimental results point towards highly differential localisation of the two proteins, which appear to label structures representing distinct stages or even specific channels of vesicular trafficking pathways. Our data also demonstrate that the application of super-resolution techniques expands the possibilities of fluorescence-based methods in autophagy studies and in some cases can rectify conclusions obtained from conventional fluorescence microscopy with diffraction-limited resolution.

Highlights

Macroautophagy enables cells to replenish resources for energy metabolism and for anabolic reactions during periods of starvation, and to dispose of large structures that are not amenable to proteasomal degradation
We aimed to evaluate the impact of the improved lateral resolution in single-molecule localisation microscopy (SMLM), compared to conventional fluorescence microscopy, on the results of morphometric analysis
The shape and size distributions of enhanced yellow fluorescent protein (EYFP)-GABARAP and EYFP-LC3B containing structures, respectively, were investigated in fixed HEK293 cells, which were subjected to a standard protocol for enrichment of autophagic structures for 2 h right before fixation

Summary

Introduction

Macroautophagy (hereafter autophagy) enables cells to replenish resources for energy metabolism and for anabolic reactions during periods of starvation, and to dispose of large structures that are not amenable to proteasomal degradation. Autophagic cargo ranges from bulk cytosol to protein aggregates, damaged organelles, and even intracellular pathogens [1,2]. A hallmark of autophagy is the formation of double-membrane structures termed phagophores, which engulf cytoplasmic cargo and close to yield autophagosomes. The mature autophagosomes (several hundred nanometres in diameter) subsequently fuse with lysosomes, resulting in acidification and degradation of their contents by acid hydrolases. Genetic screening in yeast has led to the identification of more than 30 Atg genes, most of which are conserved in mammalian cells [3].

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