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

Yoshinori Takahashi, Haiyan He, Zhenyuan Tang, Tatsuya Hattori, Ying Liu, Megan Marie Young, Jacob M. Serfass, Longgui Chen, Melat Gebru, Chong Chen, Carson A. Wills, Jennifer M. Atkinson, Han Chen, Thomas Abraham, Hong-Gang Wang

Research output: Contribution to journalArticle

23 Citations (Scopus)

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.

Original languageEnglish (US)
Article number2855
JournalNature communications
Volume9
Issue number1
DOIs
StatePublished - Dec 1 2018

Fingerprint

Endosomal Sorting Complexes Required for Transport
regulators
Autophagy
classifying
closures
Assays
membranes
Membranes
Lysosome-Associated Membrane Glycoproteins
biological evolution
lysosomes
machinery
Adenosine Triphosphatases
quantitative analysis
Autophagosomes
Chemical analysis

All Science Journal Classification (ASJC) codes

  • Chemistry(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Physics and Astronomy(all)

Cite this

Takahashi, Yoshinori ; He, Haiyan ; Tang, Zhenyuan ; Hattori, Tatsuya ; Liu, Ying ; Young, Megan Marie ; Serfass, Jacob M. ; Chen, Longgui ; Gebru, Melat ; Chen, Chong ; Wills, Carson A. ; Atkinson, Jennifer M. ; Chen, Han ; Abraham, Thomas ; Wang, Hong-Gang. / An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure. In: Nature communications. 2018 ; Vol. 9, No. 1.
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Takahashi, Y, He, H, Tang, Z, Hattori, T, Liu, Y, Young, MM, Serfass, JM, Chen, L, Gebru, M, Chen, C, Wills, CA, Atkinson, JM, Chen, H, Abraham, T & Wang, H-G 2018, 'An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure', Nature communications, vol. 9, no. 1, 2855. https://doi.org/10.1038/s41467-018-05254-w

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 Marie; Serfass, Jacob M.; Chen, Longgui; Gebru, Melat; Chen, Chong; Wills, Carson A.; Atkinson, Jennifer M.; Chen, Han; Abraham, Thomas; Wang, Hong-Gang.

In: Nature communications, Vol. 9, No. 1, 2855, 01.12.2018.

Research output: Contribution to journalArticle

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AU - Chen, Chong

AU - Wills, Carson A.

AU - Atkinson, Jennifer M.

AU - Chen, Han

AU - Abraham, Thomas

AU - Wang, Hong-Gang

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N2 - 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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