Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay

Abstract

Lysosomal membrane permeabilization (LMP) contributes to tissue involution, degenerative diseases, and cancer therapy. Its investigation has, however, been hindered by the lack of sensitive methods. Here, we characterize and validate the detection of galectin puncta at leaky lysosomes as a highly sensitive and easily manageable assay for LMP. LGALS1/galectin-1 and LGALS3/galectin-3 are best suited for this purpose due to their widespread expression, rapid translocation to leaky lysosomes and availability of high-affinity antibodies. Galectin staining marks individual leaky lysosomes early during lysosomal cell death and is useful when defining whether LMP is a primary or secondary cause of cell death. This sensitive method also reveals that cells can survive limited LMP and confirms a rapid formation of autophagic structures at the site of galectin puncta. Importantly, galectin staining detects individual leaky lysosomes also in paraffin-embedded tissues allowing us to demonstrate LMP in tumor xenografts in mice treated with cationic amphiphilic drugs and to identify a subpopulation of lysosomes that initiates LMP in involuting mouse mammary gland. The use of ectopic fluorescent galectins renders the galectin puncta assay suitable for automated screening and visualization of LMP in live cells and animals. Thus, the lysosomal galectin puncta assay opens up new possibilities to study LMP in cell death and its role in other cellular processes such as autophagy, senescence, aging, and inflammation.

Lysosome-Associated MiniSOG as a Photosensitizer for Mammalian Cells

“…Immediately upon blue-light illumination (GFP filter set, 3.9 W/ cm 2 , 5 min), the appearance of a number of mDsRed-Galectin1 red fluorescent puncta was observed (Supplementary Figure S3). Although these puncta were not as numerous as in the case of chemically-induced LMP (17), we concluded that at least some lysosomes underwent LMP in our experiments.…”

“…To test this hypothesis, we used an elegant method for LMP detection that was recently described (17). This assay is based on the translocation of fluorescently labeled cytoplasmic galectins (e.g., galectin 1 or 3) into leaky lysosomes that results in clearly visible punctate patterns in the cells.…”

A Unified Approach to Targeting the Lysosome's Degradative and Growth Signaling Roles

“…Furthermore, the difference in DNA damage induced by unmethylated DQs versus the corresponding methylated analogs increased with increasing linker length for each pair of compounds. The integrity of the lysosomal membrane was interrogated by detection of lectin galactoside binding soluble 3 (LGALS3)/galectin-3 localization, which was recently demonstrated to rapidly translocate to damaged lysosomes (21). When lysosomes are healthy, galectin-3 is uniformly dispersed throughout the cell.…”

Triggered recruitment of ESCRT machinery promotes endolysosomal repair

“…The lysosomotropic compound LLOME (L-leucyl-L-leucine O -methyl ester) is commonly used to rupture endolysosomes (3, 23, 24). LLOME rapidly permeates cellular membranes and accumulates in the lumen of acidified organelles, where it is condensed into a membranolytic polymer by the lysosomal enzyme cathepsin C (9, 23).…”