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In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice
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Assessment of mammalian endosomal microautophagy

Gregory J Krause1, Ana Maria Cuervo1

  • 1Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, New York, NY, United States; Institute for Aging Studies, Albert Einstein College of Medicine, New York, NY, United States.

Methods in Cell Biology
|July 6, 2021
PubMed

Insights

Endosomal microautophagy (eMI) selectively degrades proteins within late endosomes. New methods distinguish eMI from chaperone-mediated autophagy (CMA) by tracking hsc70-chaperoned protein uptake into endosomes versus lysosomes.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Endosomal microautophagy (eMI) selectively degrades cytosolic proteins within late endosomes/multi-vesicular bodies (LE/MVBs).
  • The initial step involves hsc70 chaperone recognizing a KFERQ-like motif on substrate proteins.
  • This recognition mechanism is shared with chaperone-mediated autophagy (CMA), necessitating pathway differentiation.

Purpose of the Study:

  • To provide detailed biochemical and imaging methods for tracking eMI activity.
  • To differentiate between eMI and CMA pathways in cellular and in vitro systems.
  • To enable researchers to determine if a protein is a substrate of eMI or CMA.

Main Methods:

  • In vitro biochemical assays using isolated LE/MVBs.
  • In cell culture studies utilizing fluorescent reporter systems.
  • Comparative analysis of protein uptake and degradation markers specific to eMI and CMA.

Main Results:

  • Established protocols for monitoring eMI activity in isolated LE/MVBs.
  • Developed fluorescent reporters to visualize and quantify protein trafficking in eMI.
  • Demonstrated approaches to distinguish eMI-specific protein substrates from CMA substrates.

Conclusions:

  • Biochemical and imaging techniques can effectively track and differentiate eMI from CMA.
  • Understanding the distinction between eMI and CMA is crucial for studying selective protein degradation.
  • These methods provide valuable tools for investigating the roles of eMI in cellular protein quality control.
Keywords:
AutophagyChaperonesLate endosomesMulti-vesicular bodiesOrganelle isolationProtein degradationProtein targetingProteostasis

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