TY - JOUR
T1 - An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure
AU - Takahashi, Yoshinori
AU - He, Haiyan
AU - Tang, Zhenyuan
AU - Hattori, Tatsuya
AU - Liu, Ying
AU - Young, Megan M.
AU - Serfass, Jacob M.
AU - Chen, Longgui
AU - Gebru, Melat
AU - Chen, Chong
AU - Wills, Carson A.
AU - Atkinson, Jennifer M.
AU - Chen, Han
AU - Abraham, Thomas
AU - Wang, Hong Gang
N1 - Funding Information:
We thank Rachael Mills for proofreading the manuscript. This work was supported by the Lois High Berstler Research Endowment Fund and the Four Diamonds Fund of the Pennsylvania State University College of Medicine. Confocal images were generated using the Leica SP8 microscope (NIH Shared Instrumentation grant S10OD010756-01A1) located in the Penn State Microscopy Imaging Core Facility.
Publisher Copyright:
© 2018, The Author(s).
PY - 2018/12/1
Y1 - 2018/12/1
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.
AB - 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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U2 - 10.1038/s41467-018-05254-w
DO - 10.1038/s41467-018-05254-w
M3 - Article
C2 - 30030437
AN - SCOPUS:85050602750
VL - 9
JO - Nature Communications
JF - Nature Communications
SN - 2041-1723
IS - 1
M1 - 2855
ER -