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An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure

Takahashi, Yoshinori, He, Haiyan, Tang, Zhenyuan, Hattori, Tatsuya, Liu, Ying, Young, Megan M., Serfass, Jacob M., Chen, Longgui, Gebru, Melat, Chen, Chong, Wills, Carson A., Atkinson, Jennifer M., Chen, Han, Abraham, Thomas, Wang, Hong-Gang·2018·Nature Communications
DOI 10.1038/s41467-018-05254-wPMC PMC6054611
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Abstract

The mechanism of phagophore closure remains unclear due to technical limitations in distinguishing unclosed and closed autophagosomal membranes. Here, we report the HaloTag-LC3 autophagosome completion assay that specifically detects phagophores, nascent autophagosomes, and mature autophagic structures. Using this assay, we identify the endosomal sorting complexes required for transport (ESCRT)-III component CHMP2A as a critical regulator of phagophore closure. During autophagy, CHMP2A translocates to the phagophore and regulates the separation of the inner and outer autophagosomal membranes to form double-membrane autophagosomes. Consistently, inhibition of the AAA-ATPase VPS4 activity impairs autophagosome completion. The ESCRT-mediated membrane abscission appears to be a critical step in forming functional autolysosomes by preventing mislocalization of lysosome-associated membrane glycoprotein 1 to the inner autophagosomal membrane. Collectively, our work reveals a function for the ESCRT machinery in the final step of autophagosome formation and provides a useful tool for quantitative analysis of autophagosome biogenesis and maturation.

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Crossref · 10.1038/s41467-018-05254-w

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  • GFP-LC3 has been demonstrated to be present in protein aggregates in an autophagy-unrelated manner and this association is dependent on its interaction with SQSTM1.

    “As a note of caution, GFP-LC3 has been demonstrated to be present in protein aggregates in an autophagy-unrelated manner and this association is dependent on its interaction with SQSTMI.”

    From Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition) 1 (2021) · cites this paper as [360]

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Research works printed but not linked: 36

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Metadata

Authors
Takahashi, Y., He, H., Tang, Z., Hattori, T., Liu, Y., Young, M. M., Serfass, J. M., Chen, L., Gebru, M., Chen, C., Wills, C. A., Atkinson, J. M., Chen, H., Abraham, T., & Wang, H.-G.
ORCID
Wang, Hong-Gang
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2018-07-20
Volume
9
Issue
1
ISSN
2041-1723
DOI
10.1038/s41467-018-05254-w
PMC
PMC6054611
Cited by
381
References
9
Language
en
Licence

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