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Updated: Oct 26, 2025
Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide
Published on: December 19, 2020
A novel tool for detecting lysosomal membrane permeabilization by high-throughput fluorescence microscopy
Karla Alvarez-Valadez1, Allan Sauvat2, Hélène Fohrer-Ting3
1Centre 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; Faculté de Médecine, Université Paris Saclay/Paris XI, Le Kremlin-Bicêtre, France.
Lysosomal membrane permeabilization (LMP) can be detected using Galectin 3, a marker that translocates to damaged lysosomes. This study presents a robust method to quantify LMP in individual lysosomes using fluorescence microscopy.
Area of Science:
- Cell Biology
- Molecular Biology
- Cellular Stress Response
Background:
- Lysosomes are central to cellular trafficking, degradation, nutrient sensing, and metabolic reprogramming.
- Lysosomes play critical roles in cellular adaptation to stress and are linked to cell death pathways.
- Lysosomal membrane permeabilization (LMP) can lead to cell death if cellular repair mechanisms fail.
Purpose of the Study:
- To develop a simple, fast, and robust protocol for detecting and quantifying LMP in individual lysosomes.
- To validate Galectin 3 as a specific marker for damaged lysosomes by assessing its colocalization with lysosomal markers.
- To enable high-throughput analysis of lysosomal damage and its effects on lysosome morphology and localization.
Main Methods:
- Utilized U2OS cells expressing mCherry-tagged Galectin 3 and mGFP-tagged LAMP1.
- Employed fluorescence microscopy for high-throughput detection and quantification of LMP.
- Verified Galectin 3 colocalization with lysosome-associated membrane protein 1 (LAMP1) to confirm lysosomal specificity.
Main Results:
- Successfully established a protocol for detecting LMP in individual lysosomes.
- Demonstrated that Galectin 3 translocation to the lysosomal lumen is a reliable indicator of LMP.
- Enabled simultaneous study of alterations in size, shape, and subcellular localization of intact and damaged lysosomes.
Conclusions:
- The developed method provides a reliable and efficient way to detect and quantify lysosomal membrane permeabilization.
- Galectin 3, when colocalized with LAMP1, serves as a valuable marker for identifying damaged lysosomes.
- This technique facilitates high-throughput analysis of lysosomal damage and its impact on cellular processes.
