Probing aggrephagy using chemically-induced protein aggregates

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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.

Abstract

Autophagic clearance of aggregates, aggrephagy, is essential for cellular homeostasis but tools to induce and monitor it are limited. Here the authors present a fluorescence-based aggrephagy induction assay to study spatiotemporal dynamics and control mechanisms driving protein aggregate clearance.

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Aggresomes: A Cellular Response to Misfolded Proteins

Jennifer A. Johnston, Cristina Ward, Ron Kopito (1998)

Intracellular deposition of misfolded protein aggregates into ubiquitin-rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Why these aggregates form despite the existence of cellular machinery to recognize and degrade misfolded protein and how they are delivered to cytoplasmic inclusions are not known. We have investigated the intracellular fate of cystic fibrosis transmembrane conductance regulator (CFTR), an inefficiently folded integral membrane protein which is degraded by the cytoplasmic ubiquitin-proteasome pathway. Overexpression or inhibition of proteasome activity in transfected human embryonic kidney or Chinese hamster ovary cells led to the accumulation of stable, high molecular weight, detergent-insoluble, multiubiquitinated forms of CFTR. Using immunofluorescence and transmission electron microscopy with immunogold labeling, we demonstrate that undegraded CFTR molecules accumulate at a distinct pericentriolar structure which we have termed the aggresome. Aggresome formation is accompanied by redistribution of the intermediate filament protein vimentin to form a cage surrounding a pericentriolar core of aggregated, ubiquitinated protein. Disruption of microtubules blocks the formation of aggresomes. Similarly, inhibition of proteasome function also prevented the degradation of unassembled presenilin-1 molecules leading to their aggregation and deposition in aggresomes. These data lead us to propose that aggresome formation is a general response of cells which occurs when the capacity of the proteasome is exceeded by the production of aggregation-prone misfolded proteins.

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A role for NBR1 in autophagosomal degradation of ubiquitinated substrates.

Vladimir Kirkin, Trond Lamark, Yu-Shin Sou … (2009)

Autophagy is a catabolic process where cytosolic cellular components are delivered to the lysosome for degradation. Recent studies have indicated the existence of specific receptors, such as p62, which link ubiquitinated targets to autophagosomal degradation pathways. Here we show that NBR1 (neighbor of BRCA1 gene 1) is an autophagy receptor containing LC3- and ubiquitin (Ub)-binding domains. NBR1 is recruited to Ub-positive protein aggregates and degraded by autophagy depending on an LC3-interacting region (LIR) and LC3 family modifiers. Although NBR1 and p62 interact and form oligomers, they can function independently, as shown by autophagosomal clearance of NBR1 in p62-deficient cells. NBR1 was localized to Ub-positive inclusions in patients with liver dysfunction, and depletion of NBR1 abolished the formation of Ub-positive p62 bodies upon puromycin treatment of cells. We propose that NBR1 and p62 act as receptors for selective autophagosomal degradation of ubiquitinated targets.

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Discovery of Atg5/Atg7-independent alternative macroautophagy.

Yuya Nishida, Satoko Arakawa, Kenji Fujitani … (2009)

Macroautophagy is a process that leads to the bulk degradation of subcellular constituents by producing autophagosomes/autolysosomes. It is believed that Atg5 (ref. 4) and Atg7 (ref. 5) are essential genes for mammalian macroautophagy. Here we show, however, that mouse cells lacking Atg5 or Atg7 can still form autophagosomes/autolysosomes and perform autophagy-mediated protein degradation when subjected to certain stressors. Although lipidation of the microtubule-associated protein light chain 3 (LC3, also known as Map1lc3a) to form LC3-II is generally considered to be a good indicator of macroautophagy, it did not occur during the Atg5/Atg7-independent alternative process of macroautophagy. We also found that this alternative process of macroautophagy was regulated by several autophagic proteins, including Unc-51-like kinase 1 (Ulk1) and beclin 1. Unlike conventional macroautophagy, autophagosomes seemed to be generated in a Rab9-dependent manner by the fusion of isolation membranes with vesicles derived from the trans-Golgi and late endosomes. In vivo, Atg5-independent alternative macroautophagy was detected in several embryonic tissues. It also had a function in clearing mitochondria during erythroid maturation. These results indicate that mammalian macroautophagy can occur through at least two different pathways: an Atg5/Atg7-dependent conventional pathway and an Atg5/Atg7-independent alternative pathway.

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Author and article information

Contributors

Lukas C. Kapitein: l.kapitein@uu.nl

Journal

Journal ID (nlm-ta): Nat Commun

Journal ID (iso-abbrev): Nat Commun

Title: Nature Communications

Publisher: Nature Publishing Group UK (London )

ISSN (Electronic): 2041-1723

Publication date (Electronic): 12 October 2018

Publication date PMC-release: 12 October 2018

Publication date Collection: 2018

Volume: 9

Electronic Location Identifier: 4245

Affiliations

[1 ]ISNI 0000000120346234, GRID grid.5477.1, Division of Cell Biology, Department of Biology, Faculty of Science, , Utrecht University, ; Padualaan 8, 3584 CH Utrecht, The Netherlands

[2 ]ISNI 0000 0000 9558 4598, GRID grid.4494.d, Department of Cell Biology, University of Groningen, , University Medical Center Groningen, ; A. Deusinglaan 1, 9713 AV Groningen, The Netherlands

Author information

Eugene A. Katrukha http://orcid.org/0000-0002-9971-3603

Pauline Verlhac http://orcid.org/0000-0002-1059-4645

Fulvio Reggiori http://orcid.org/0000-0003-2652-2686

Article

Publisher ID: 6674

DOI: 10.1038/s41467-018-06674-4

PMC ID: 6185936

PubMed ID: 30315152

SO-VID: 3ee8087b-2f5c-493f-8651-e8cd17709343

License:

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

History

Date received : 22 November 2017

Date accepted : 13 September 2018

Funding

Funded by: SNF Sinergia (CRSII3_154421), Marie Skłodowska-Curie Cofund (713660), Marie Skłodowska-Curie ITN (765912) and ZonMW VICI (016.130.606)

Funded by: FundRef https://doi.org/10.13039/501100003246, Nederlandse Organisatie voor Wetenschappelijk Onderzoek (Netherlands Organisation for Scientific Research);

Award ID: (ALW-VIDI 864.12.008).

Award Recipient : Lukas C. Kapitein

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Probing aggrephagy using chemically-induced protein aggregates

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Most cited references 13

Aggresomes: A Cellular Response to Misfolded Proteins

A role for NBR1 in autophagosomal degradation of ubiquitinated substrates.

Discovery of Atg5/Atg7-independent alternative macroautophagy.

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Cited by 11

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