Assessment of EGFP-Q74 degradation for the measurement of autophagic flux

Yan Wang; Jiao Meng; Qi Wu; Guo Chen; Wei Xie; Zhen Zhang; Guido Kroemer; Oliver Kepp

doi:10.1016/bs.mcb.2020.10.018

Assessment of EGFP-Q74 degradation for the measurement of autophagic flux

Methods Cell Biol. 2021:165:31-38. doi: 10.1016/bs.mcb.2020.10.018. Epub 2021 Apr 27.

Authors

Yan Wang¹, Jiao Meng², Qi Wu³, Guo Chen³, Wei Xie³, Zhen Zhang⁴, Guido Kroemer⁵, Oliver Kepp⁶

Affiliations

¹ Department of Radiation Oncology, Fudan University Shanghai Cancer Center, Shanghai, China; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China; Cancer Institute, Fudan University Shanghai Cancer Center, Shanghai, China; Centre de Recherche des Cordeliers, Équipe 11 Labellisée par la Ligue Contre le Cancer, Université de Paris, Sorbonne Université, Inserm U1138, Institut Universitaire de France, Paris, France; Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France.
² Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China; Cancer Institute, Fudan University Shanghai Cancer Center, Shanghai, China.
³ Centre de Recherche des Cordeliers, Équipe 11 Labellisée par la Ligue Contre le Cancer, Université de Paris, Sorbonne Université, Inserm U1138, Institut Universitaire de France, Paris, France; Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France.
⁴ Department of Radiation Oncology, Fudan University Shanghai Cancer Center, Shanghai, China; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China. Electronic address: zhen_zhang@fudan.edu.cn.
⁵ Centre de Recherche des Cordeliers, Équipe 11 Labellisée par la Ligue Contre le Cancer, Université de Paris, Sorbonne Université, Inserm U1138, Institut Universitaire de France, Paris, France; Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France; Suzhou Institute for Systems Medicine, Chinese Academy of Medical Sciences, Suzhou, China; Pôle de Biologie, Hôpital Européen Georges-Pompidou, AP-HP, Paris, France; Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden. Electronic address: kroemer@orange.fr.
⁶ Centre de Recherche des Cordeliers, Équipe 11 Labellisée par la Ligue Contre le Cancer, Université de Paris, Sorbonne Université, Inserm U1138, Institut Universitaire de France, Paris, France; Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France. Electronic address: captain.olsen@gmail.com.

PMID: 34311869
DOI: 10.1016/bs.mcb.2020.10.018

Abstract

Autophagy plays a major role in physiological and pathological processes. The quantitation of the abundance of autophagy-specific substrates constitutes an efficient strategy for assessing autophagic activity. Here, we provide a detailed protocol for quantifying the decay of a fusion protein composed by enhanced green fluorescent protein (EGFP) and glutamine repeats (Q74) using regular or high-throughput fluorescence microscopy. This method provides a direct measurement of autophagic flux in a Huntington's disease model.

Keywords: Autophagy; EGFP-Q74; Fluorescence microscopy; Huntington's disease.

MeSH terms

Autophagy*
Green Fluorescent Proteins / genetics
Microscopy, Fluorescence

Substances

enhanced green fluorescent protein
Green Fluorescent Proteins