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

Nature Communications
Yoshinori TakahashiHong-Gang Wang

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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Methods Mentioned

BETA
lipidation
confocal microscopy
electron microscopy
fluorescence microscopy
transfection
PCR
light electron microscopy
confocal

Software Mentioned

Volocity
Imaris
Graph Pad Prism
SlideBook
Image Studio
Huygens

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