Fluorescence‐based ATG8 sensors monitor localization and function of LC3/GABARAP proteins |
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| Authors: | Alexandra Stolz Mateusz Putyrski Ivana Kutle Jessica Huber Chunxin Wang Viktória Major Sachdev S Sidhu Richard J Youle Vladimir V Rogov Volker Dötsch Andreas Ernst Ivan Dikic |
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| Affiliation: | 1. Institute of Biochemistry II, Goethe University, Frankfurt am Main, Germany;2. Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology TMP, Frankfurt am Main, Germany;3. Buchmann Institute for Molecular Life Sciences, Frankfurt am Main, Germany;4. Institute of Biophysical Chemistry, Goethe University, Frankfurt am Main, Germany;5. Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA;6. Banting and Best Department of Medical Research, The Donnelly Centre, University of Toronto, Toronto, ON, Canada;7. Department of Molecular Genetics, The Donnelly Centre, University of Toronto, Toronto, ON, Canada |
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| Abstract: | Autophagy is a cellular surveillance pathway that balances metabolic and energy resources and transports specific cargos, including damaged mitochondria, other broken organelles, or pathogens for degradation to the lysosome. Central components of autophagosomal biogenesis are six members of the LC3 and GABARAP family of ubiquitin‐like proteins (mATG8s). We used phage display to isolate peptides that possess bona fide LIR (LC3‐interacting region) properties and are selective for individual mATG8 isoforms. Sensitivity of the developed sensors was optimized by multiplication, charge distribution, and fusion with a membrane recruitment (FYVE) or an oligomerization (PB1) domain. We demonstrate the use of the engineered peptides as intracellular sensors that recognize specifically GABARAP, GABL1, GABL2, and LC3C, as well as a bispecific sensor for LC3A and LC3B. By using an LC3C‐specific sensor, we were able to monitor recruitment of endogenous LC3C to Salmonella during xenophagy, as well as to mitochondria during mitophagy. The sensors are general tools to monitor the fate of mATG8s and will be valuable in decoding the biological functions of the individual LC3/GABARAPs. |
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| Keywords: | ATG8 immunofluorescence LC3 phage display selective autophagy |
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