Probing aggrephagy using chemically-induced protein aggregates

Anne F. J. Janssen, Eugene A. Katrukha, Wendy Straaten, Pauline Verlhac, Fulvio Reggiori and Lukas C. Kapitein ()
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Anne F. J. Janssen: Utrecht University
Eugene A. Katrukha: Utrecht University
Wendy Straaten: Utrecht University
Pauline Verlhac: University Medical Center Groningen
Fulvio Reggiori: University Medical Center Groningen
Lukas C. Kapitein: Utrecht University

Nature Communications, 2018, vol. 9, issue 1, 1-10

Abstract: Abstract Selective types of autophagy mediate the clearance of specific cellular components and are essential to maintain cellular homeostasis. However, tools to directly induce and monitor such pathways are limited. Here we introduce the PIM (particles induced by multimerization) assay as a tool for the study of aggrephagy, the autophagic clearance of aggregates. The assay uses an inducible multimerization module to assemble protein clusters, which upon induction recruit ubiquitin, p62, and LC3 before being delivered to lysosomes. Moreover, use of a dual fluorescent tag allows for the direct observation of cluster delivery to the lysosome. Using flow cytometry and fluorescence microscopy, we show that delivery to the lysosome is partially dependent on p62 and ATG7. This assay will help in elucidating the spatiotemporal dynamics and control mechanisms underlying aggregate clearance by the autophagy–lysosomal system.

Date: 2018
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DOI: 10.1038/s41467-018-06674-4

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