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

1
Institute of Complex Systems 4 (ICS-4, Cellular Biophysics), Forschungszentrum Jülich GmbH, Wilhelm-Johnen-Straße, 52428 Jülich, Germany
2
Institute of Complex Systems 6 (ICS-6, Structural Biochemistry), Forschungszentrum Jülich GmbH, Wilhelm-Johnen-Straße, 52428 Jülich, Germany
3
Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany
*
Authors to whom correspondence should be addressed.
Academic Editor: Jörg Fitter
Molecules 2019, 24(9), 1833; https://doi.org/10.3390/molecules24091833
Received: 20 March 2019 / Revised: 7 May 2019 / Accepted: 7 May 2019 / Published: 13 May 2019
(This article belongs to the Special Issue Single-Molecule Fluorescence Spectroscopy)
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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. View Full-Text
Keywords: Atg8; autophagy; EYFP blinking; GABARAP; LC3B; shape distribution; single molecule localisation microscopy; SMLM; super-resolution Atg8; autophagy; EYFP blinking; GABARAP; LC3B; shape distribution; single molecule localisation microscopy; SMLM; super-resolution
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
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Abdollahzadeh, I.; Hendriks, J.; Sanwald, J.L.; Simons, I.M.; Hoffmann, S.; Weiergräber, O.H.; Willbold, D.; Gensch, T. Autophagy-Related Proteins GABARAP and LC3B Label Structures of Similar Size but Different Shape in Super-Resolution Imaging. Molecules 2019, 24, 1833.

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