Fluorescence microscopic quantification of lysosomal cholesterol accumulation induced by cationic amphiphilic drugs.

Lysosomes are involved in the transport, degradation, and recycling of macromolecules through the autophagy and endocytosis pathways. Cholesterol is taken up by cells through the internalization of low-density lipoprotein (LDL) via LDL receptor-mediated endocytosis or micropinocytosis. Free cholesterol generated by the action of acid lipases contained in lysosomes can be transferred to other organelles. Dysfunctions in either cholesterol uptake or release from lysosomes can compromise the function and integrity of these organelles, thereby contributing to the pathogenesis of lysosomal storage disorders. We previously showed that some cationic amphiphilic drugs (CADs) mimic the phenotype of lysosomal storage disorders by inducing lysosomal cholesterol accumulation followed by lysosomal damage. Here, we describe two fluorescence microscopic methods for the visualization of cholesterol accumulation in lysosomes in response to the CAD leelamine. In the first method, the cell-permeable cholesterol analog labeled with the fluorophore BODIPY is used. In the second method, endogenous cholesterol-rich microdomains are labeled with filipin complex. Both methods imply the additional visualization of the lysosomal associated membrane protein 2 (LAMP2) by immunofluorescence. Finally, the role of lysosomal cholesterol accumulation in the induction of lysosomal membrane permeabilization (LMP) was assessed through a method based on the recruitment of Galectin-3 on damaged lysosomes.