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

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 structu...

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Detalles Bibliográficos
Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054611/
https://www.ncbi.nlm.nih.gov/pubmed/30030437
http://dx.doi.org/10.1038/s41467-018-05254-w
Descripción
Sumario: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.