title: Methods for the quantification of lysosomal membrane permeabilization: a hallmark of lysosomal cell death.
abstract: Lysosomal cell death is triggered by lysosomal membrane permeabilization (LMP) and subsequent release of lysosomal hydrolases from the lysosomal lumen into the cytosol. Once released into the cytosol, the lysosomal cathepsin proteases act as executioner proteases for the subsequent cell death-either autonomously without caspase activation or in concert with the classical apoptotic machinery. Lysosomal cell death usually remains functional in apoptosis-resistant cancer cells and thus holds great potential as a therapeutic strategy for circumventing apoptosis deficiency in cancers. Notably, lysosomal cell death also plays an important role in normal physiology, e.g., during the regression of the mammary gland. Here we present four complementary methods for the quantification and visualization of LMP during the onset of death: (1) enzymatic activity measurements of released lysosomal hydrolases in the cytosol after digitonin extraction, (2) direct visualization of LMP by monitoring the release of fluorescent dextran from lysosomes into the cytosol, (3) immunocytochemistry to detect cathepsins released into the cytosol, and (4) detection of the translocation of galectins to damaged lysosomes. The methods presented here can ideally be combined as needed to provide solid evidence for LMP after a given cytotoxic stimuli.
authors: Unit for Cell Death and Metabolism, Danish Cancer Society Research Center, Copenhagen, Denmark.
authors: Sonja
authors: S
authors: Aits
authors: Unit for Cell Death and Metabolism, Danish Cancer Society Research Center, Copenhagen, Denmark.
authors: Marja
authors: M
authors: Jäättelä
authors: Unit for Cell Death and Metabolism, Danish Cancer Society Research Center, Copenhagen, Denmark.
authors: Jesper
authors: J
authors: Nylandsted
mesh_terms: D017209:Apoptosis; D002403:Cathepsins / Q000378:metabolism; D002458:Cell Fractionation; D002463:Cell Membrane Permeability; D003600:Cytosol; D003902:Detergents / Q000494:pharmacology*; D003911:Dextrans / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology*; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000187:drug effects / Q000502:physiology*; D061986:MCF-7 Cells; D008856:Microscopy, Fluorescence
chemical_list: D003902:Detergents; D003911:Dextrans; D005452:Fluoresceins; D005456:Fluorescent Dyes; C052526:fluorescein-dextran; D002403:Cathepsins; D004072:Digitonin
keywords: Cathepsins; Death pathway; Galectins; LMP; Lysosomal cell death; Lysosomal membrane permeabilization; Method