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Nat Commun. 2018 Oct 12;9(1):4245. doi: 10.1038/s41467-018-06674-4.

Probing aggrephagy using chemically-induced protein aggregates.

Author information

1
Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
2
Department of Cell Biology, University of Groningen, University Medical Center Groningen, A. Deusinglaan 1, 9713 AV, Groningen, The Netherlands.
3
Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands. l.kapitein@uu.nl.

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.

PMID:
30315152
PMCID:
PMC6185936
DOI:
10.1038/s41467-018-06674-4
[Indexed for MEDLINE]
Free PMC Article

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