Methods for Probing Lysosomal Membrane Permeabilization.

Abstract

Cell death triggered by lysosomal membrane permeabilization (LMP) is gaining increased interest as target for cancer therapy, but the death pathway also plays an important role in normal physiology (e.g., during involution of the mammary gland). LMP-induced cell death is triggered by release of hydrolases including cysteine cathepsin proteases from the lysosomal lumen into the cytosol. Limited release of proteases to the cytoplasm induces apoptosis or apoptosis-like cell death, whereas massive LMP results in rapid cellular necrosis. Here we introduce three complementary methods for quantifying and visualizing LMP: (i) monitoring LMP by immunocytochemistry, (ii) visualizing LMP by fluorescent dextran release, and (iii) quantification of LMP by activity measurements of lysosomal enzymes in digitonin-extracted cytosol.