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

Molecules

JuliaL. Sanwald¹  IndraM. Simons¹  Iman Abdollahzadeh²  Thomas Gensch²  Johnny Hendriks²  Silke Hoffmann³  Dieter Willbold³  OliverH. Weiergräber³ 

[1] Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany;Institute of Complex Systems 4 (ICS-4, Cellular Biophysics), Forschungszentrum Jülich GmbH, Wilhelm-Johnen-Straße, 52428 Jülich, Germany;Institute of Complex Systems 6 (ICS-6, Structural Biochemistry), Forschungszentrum Jülich GmbH, Wilhelm-Johnen-Straße, 52428 Jülich, Germany;

关键词: Atg8;    autophagy;    EYFP blinking;    GABARAP;    LC3B;    shape distribution;    single molecule localisation microscopy;    SMLM;    super-resolution;   

DOI  :  10.3390/molecules24091833

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown