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Related Concept Videos

Autophagy01:27

Autophagy

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Autophagy is a self-digesting process by which a cell protects itself from threats both within and outside the cell, ranging from abnormal proteins to invading bacteria. In this process, obsolete components of the cell and invading microbes are degraded by hydrolytic enzymes active in an acidic environment of the lysosomal lumen.
An autophagic pathway consists of a series of signaling events activated in response to diverse stress and physiological conditions such as food deprivation,...
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A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae
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Tracking Chaperone-Mediated Autophagy Flux with a pH-Resistant Fluorescent Reporter.

Ruotong Qi1, Xingyi Chen1, Zihan Li1

  • 1Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Fudan University, Shanghai 200438, China.

International Journal of Molecular Sciences
|January 11, 2025
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Summary

Researchers developed KFERQ-Gamillus, a novel probe to easily monitor chaperone-mediated autophagy (CMA) flux in living cells. This tool aids in understanding CMA

Keywords:
Gamilluschaperone-mediated autophagyfluxhalo

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Area of Science:

  • Cellular Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Chaperone-mediated autophagy (CMA) is a critical pathway for protein degradation.
  • Monitoring CMA flux is vital but technically challenging.
  • Existing methods lack efficiency and specificity.

Purpose of the Study:

  • To develop a novel, sensitive probe for monitoring CMA activity.
  • To enable real-time, quantitative assessment of CMA flux.
  • To facilitate high-throughput screening and research in CMA.

Main Methods:

  • Screening of green fluorescent proteins to develop a pH-resistant probe (KFERQ-Gamillus).
  • Utilizing KFERQ motif and LAMP2A for lysosomal targeting and degradation.
  • Creating a dual-reporter system (KFERQ-Gamillus-Halo) with Halo-tag.
  • Employing microscopy, image-based flow cytometry, and immunoblotting for detection.

Main Results:

  • KFERQ-Gamillus is activated under CMA-inducing conditions and specifically targets CMA.
  • The probe allows detection of CMA activity in living cells.
  • The KFERQ-Gamillus-Halo system distinguishes protein synthesis from degradation.
  • Quantitative and time-resolved monitoring of intracellular CMA flux is achieved.

Conclusions:

  • KFERQ-Gamillus and KFERQ-Gamillus-Halo are effective tools for monitoring CMA.
  • These probes offer quantitative, real-time assessment of CMA activity and flux.
  • The developed probes have significant potential for biomedical research and drug discovery.