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

Author

Listed:
  • Yoshinori Takahashi

    (Penn State College of Medicine)

  • Haiyan He

    (Penn State College of Medicine)

  • Zhenyuan Tang

    (Penn State College of Medicine)

  • Tatsuya Hattori

    (Penn State College of Medicine)

  • Ying Liu

    (Penn State College of Medicine)

  • Megan M. Young

    (Penn State College of Medicine)

  • Jacob M. Serfass

    (Penn State College of Medicine)

  • Longgui Chen

    (Penn State College of Medicine)

  • Melat Gebru

    (Penn State College of Medicine)

  • Chong Chen

    (Penn State College of Medicine)

  • Carson A. Wills

    (Penn State College of Medicine)

  • Jennifer M. Atkinson

    (Penn State College of Medicine)

  • Han Chen

    (Penn State College of Medicine)

  • Thomas Abraham

    (Penn State College of Medicine
    Penn State College of Medicine)

  • Hong-Gang Wang

    (Penn State College of Medicine
    Penn State College of Medicine)

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.

Suggested Citation

  • Yoshinori Takahashi & Haiyan He & Zhenyuan Tang & Tatsuya Hattori & Ying Liu & Megan M. Young & Jacob M. Serfass & Longgui Chen & Melat Gebru & Chong Chen & Carson A. Wills & Jennifer M. Atkinson & Ha, 2018. "An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure," Nature Communications, Nature, vol. 9(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:9:y:2018:i:1:d:10.1038_s41467-018-05254-w
    DOI: 10.1038/s41467-018-05254-w
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    Cited by:

    1. Susanne G. Grein & Kyra A. Y. Defourny & Huib H. Rabouw & Soenita S. Goerdayal & Martijn J. C. Herwijnen & Richard W. Wubbolts & Maarten Altelaar & Frank J. M. Kuppeveld & Esther N. M. Nolte-‘t Hoen, 2022. "The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    2. Tomoyuki Hatano & Saravanan Palani & Dimitra Papatziamou & Ralf Salzer & Diorge P. Souza & Daniel Tamarit & Mehul Makwana & Antonia Potter & Alexandra Haig & Wenjue Xu & David Townsend & David Rochest, 2022. "Asgard archaea shed light on the evolutionary origins of the eukaryotic ubiquitin-ESCRT machinery," Nature Communications, Nature, vol. 13(1), pages 1-16, December.
    3. Nina Frey & Luigi Tortola & David Egli & Sharan Janjuha & Tanja Rothgangl & Kim Fabiano Marquart & Franziska Ampenberger & Manfred Kopf & Gerald Schwank, 2022. "Loss of Rnf31 and Vps4b sensitizes pancreatic cancer to T cell-mediated killing," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    4. Elodie Mailler & Carlos M. Guardia & Xiaofei Bai & Michal Jarnik & Chad D. Williamson & Yan Li & Nunziata Maio & Andy Golden & Juan S. Bonifacino, 2021. "The autophagy protein ATG9A enables lipid mobilization from lipid droplets," Nature Communications, Nature, vol. 12(1), pages 1-19, December.
    5. Takashi Nozawa & Hirotaka Toh & Junpei Iibushi & Kohei Kogai & Atsuko Minowa-Nozawa & Junko Satoh & Shinji Ito & Kazunori Murase & Ichiro Nakagawa, 2023. "Rab41-mediated ESCRT machinery repairs membrane rupture by a bacterial toxin in xenophagy," Nature Communications, Nature, vol. 14(1), pages 1-20, December.
    6. Agata N. Makar & Alina Boraman & Peter Mosen & Joanne E. Simpson & Jair Marques & Tim Michelberger & Stuart Aitken & Ann P. Wheeler & Dominic Winter & Alex Kriegsheim & Noor Gammoh, 2024. "The V-ATPase complex component RNAseK is required for lysosomal hydrolase delivery and autophagosome degradation," Nature Communications, Nature, vol. 15(1), pages 1-16, December.
    7. Yonglun Zeng & Baiying Li & Shuxian Huang & Hongbo Li & Wenhan Cao & Yixuan Chen & Guoyong Liu & Zhenping Li & Chao Yang & Lei Feng & Jiayang Gao & Sze Wan Lo & Jierui Zhao & Jinbo Shen & Yan Guo & Ca, 2023. "The plant unique ESCRT component FREE1 regulates autophagosome closure," Nature Communications, Nature, vol. 14(1), pages 1-17, December.
    8. Niccolò Mosesso & Niharika Savant Lerner & Tobias Bläske & Felix Groh & Shane Maguire & Marie Laura Niedermeier & Eliane Landwehr & Karin Vogel & Konstanze Meergans & Marie-Kristin Nagel & Malte Dresc, 2024. "Arabidopsis CaLB1 undergoes phase separation with the ESCRT protein ALIX and modulates autophagosome maturation," Nature Communications, Nature, vol. 15(1), pages 1-20, December.

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