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.

Phagolysosome acidification is required for silica and engineered nanoparticle-induced lysosome membrane permeabilization and resultant NLRP3 inflammasome activity

“…To assess LMP, we adapted recently reported methods that use digitonin extraction of the cytosol in order to measure leaked lysosomal cathepsin L and/or N -acetyl-β-D-glucosaminidase (NAG) activity (Supplementary Figure 1) (Aits et al , 2015). For these studies, we utilized BMdM to help determine LMP in vitro .…”

“…However, since Bafilomycin A1 can prevent cathepsin maturation possibly interfering with the assay, we measured leaked NAG as a second indicator of LMP. NAG activity is not necessarily dependent upon a lower lysosomal pH and has a longer half-life in the cytosol following LMP (Aits et al , 2015). NAG activity in the cytosolic fraction was significantly decreased with Bafilomycin A1 treatment for all particles (Figure 2B, 2C), confirming the necessity of phagolysosome acidification in particle-induced LMP.…”

“…Additionally, inhibitors that target lysosome acidification, such as Bafilomycin A1, can also affect the fluorescence of acidotropic dyes, influencing interpretation of mechanistic studies. In this study, we utilized methods adapted from Aits et al, which rely on the saponic qualities of digitionin for selective extraction of the cellular cytosolic fraction (Aits et al , 2015), allowing for direct quantification of LMP in primary macrophages following particle exposure. Furthermore, these methods allowed for a more direct investigation of the role of lysosomal acidification in particle-induced LMP.…”

“…LMP was assessed using methods modified from Aits et al (Aits et al , 2015). Isolated AM were plated in a 96 well plate at 1×10 5 cells per well and allowed to adhere for 2 hours.…”

Baixas doses de hidrocortisona possuem ação protetora no choque hemorrágico em ratos

“…Há ainda um possível efeito atribuído à hidrocortisona de estabilização das membranas lisossomais, que diminui a lesão celular e contribui para combater as lesões decorrentes do choque 27 . A microcirculação do intestino delgado está entre as que mais sofrem durante a hipoperfusão tecidual acarretando maiores alterações estruturais e comprometimento de suas funções protetoras, sendo considerado por muitos autores a via de disseminação de produtos citotóxicos e bactérias para a circulação e sistema linfático e assim órgãos distantes como os pulmões.…”