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Related Experiment Video

Updated: Jun 18, 2025

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Published on: July 18, 2011

HaloTag as a substrate-based macroautophagy reporter.

Qiang Xiao^1,2, Gabrielle Cruz^1,2,3, Rachel Botham^1,2

¹Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037.

Proceedings of the National Academy of Sciences of the United States of America

|July 29, 2024

Summary

This summary is machine-generated.

Monitoring cellular autophagy, a key degradation process, can now be achieved by tracking TMR-HaloTag fluorescent conjugates. This method distinguishes autophagy activation from inhibition, aiding neurodegenerative disease research.

Keywords:

HaloTag autophagy lysosome macroautophagy reporter

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Published on: July 21, 2017

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Last Updated: Jun 18, 2025

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Published on: July 18, 2011

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

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Published on: July 27, 2013

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Published on: July 21, 2017

Area of Science:

Cell Biology
Molecular Biology
Neuroscience

Background:

Macroautophagy is a crucial cellular degradation pathway that enhances cell survival under stress and protects against neurodegenerative diseases.
Monitoring autophagy regulation in living cells is vital for understanding its physiological and pathological roles but remains technically challenging.

Purpose of the Study:

To develop a novel method for monitoring autophagy regulation in living cells.
To utilize TMR-HaloTag conjugates as fluorescent reporters for autophagy flux.

Main Methods:

Expression of HaloTag in cells of interest and reaction with tetramethylrhodamine (TMR) ligand.
Observation of fluorescent TMR-HaloTag conjugate accumulation in autophagosomes and lysosomes via fluorescence microscopy.
Assessment of TMR-HaloTag degradation products using SDS-PAGE and analysis of lysosomal puncta accumulation.

Main Results:

The rate of TMR-HaloTag conjugate accumulation in lysosomes correlates with the rate of autophagy.
Under basal conditions, TMR-HaloTag is primarily degraded by the proteasome (~95%), with slow and incomplete lysosomal degradation (~10%).
Autophagy activation is indicated by increased degraded TMR-HaloTag bands on SDS-PAGE and faster lysosomal puncta accumulation, distinguishing it from inhibition.
Proteasome inhibition causes TMR-HaloTag accumulation in lysosomes, suggesting crosstalk between autophagy and proteasomal degradation pathways.

Conclusions:

TMR-HaloTag fluorescence provides a viable method to monitor autophagy flux in living cells.
This technique allows for the differentiation between autophagy activation and inhibition.
The findings highlight the interplay between the proteasome and autophagy in cellular degradation.