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A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae
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Using CMAC Staining for Vacuole Characterization in Yeast.

José Ángel Clemente-Ramos1, Sara E Mole2

  • 1UCL Great Ormond Street Institute of Child Health, University College London, London, UK. s.mole@ucl.ac.uk.

Methods in Molecular Biology (Clifton, N.J.)
|October 13, 2025
PubMed
Summary

7-amino-4-chloromethylcoumarin (CMAC) staining effectively visualizes acidic yeast vacuoles. This method aids research into cellular processes, disease mechanisms, and potential drug targets.

Keywords:
7-amino-4-chloromethylcoumarinCMAC stainingFission yeastHomeostasisLysosomeSchizosaccharomyces pombeVacuole

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Area of Science:

  • Cell Biology
  • Microscopy
  • Yeast Genetics

Background:

  • Vacuoles are crucial acidic organelles in yeast cells, involved in homeostasis and various cellular processes.
  • Visualizing vacuole dynamics and morphology is essential for understanding cellular function and disease.
  • Existing methods may face challenges with cellular autofluorescence or throughput.

Purpose of the Study:

  • To highlight the utility of CMAC staining for yeast vacuole visualization.
  • To demonstrate CMAC's role in studying vacuole dynamics, morphology, and distribution.
  • To showcase CMAC's application in understanding cellular homeostasis and disease mechanisms.

Main Methods:

  • Utilizing 7-amino-4-chloromethylcoumarin (CMAC) as a fluorescent stain for acidic compartments.
  • Applying CMAC staining to living or fixed yeast cells for vacuole labeling.
  • Combining CMAC staining with automated imaging for high-throughput analysis.

Main Results:

  • CMAC selectively accumulates in acidic yeast vacuoles, providing clear contrast against autofluorescence.
  • The staining facilitates detailed visualization of vacuole dynamics, morphology, and distribution.
  • CMAC staining enhances imaging clarity and allows for efficient, automated analysis.

Conclusions:

  • CMAC staining is a valuable and effective tool for studying yeast vacuoles.
  • This method aids in understanding fundamental cellular processes like endocytic trafficking and autophagy.
  • CMAC staining has significant applications in drug screening and yeast models of lysosomal diseases, including neurodegenerative disorders.