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A novel tool for detecting lysosomal membrane permeabilization by high-throughput fluorescence microscopy

Abstract : Lysosomes are placed at the center of cellular trafficking and degradative pathways. They also function as a signaling platform for nutrient sensing and metabolic reprogramming. Lysosomes play crucial roles in cellular adaptation in response to stress and are tightly connected to a variety of cell death modalities. Several stimuli can initiate the permeabilization of the lysosome membrane, thus causing cell death when the cellular adaptive system fail to repair or replace damaged lysosomes. The induction of lysosomal membrane permeabilization (LMP) triggers the rapid translocation of Galectin 3/LGALS3 from the cytosol to the lysosomal lumen, making it a valuable marker of LMP. However, Galectin 3 can also be recruited to damaged endo/phagosomal membranes. To make sure that Galectin 3 labels damaged lysosomes, it is therefore important to verify its colocalization with lysosomal markers such as lysosome-associated membrane protein 1 (LAMP1). Here, we describe a simple, fast and robust protocol that allows the detection of LMP of individual lysosomes in U2OS cells expressing mCherry-tagged Galectin 3 and mGFP-tagged LAMP1. This method permits the high-throughput detection and quantification of damaged lysosomes by fluorescence microscopy. It also offers the advantage of studying, in the same experiment, the alterations in size, shape and subcellular localization of intact and damaged lysosomes.
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https://www.hal.inserm.fr/inserm-03653036
Contributor : Mojgan Djavaheri-Mergny Connect in order to contact the contributor
Submitted on : Wednesday, April 27, 2022 - 12:16:16 PM
Last modification on : Thursday, May 5, 2022 - 3:11:43 AM

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Karla Alvarez-Valadez, Allan Sauvat, Hélène Fohrer-Ting, Christophe Klein, Oliver Kepp, et al.. A novel tool for detecting lysosomal membrane permeabilization by high-throughput fluorescence microscopy. Monitoring Vesicular Trafficking in Cellular Responses to Stress - Part B, 165, Elsevier, pp.1-12, 2021, Methods in Cell Biology, ⟨10.1016/bs.mcb.2020.10.004⟩. ⟨inserm-03653036⟩

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