Methods for Probing Lysosomal Membrane Permeabilization

Significance Statement

The authors report three methods for quantifying and visualizing lysosomal membrane permeabilization:
(i) monitoring lysosomal membrane permeabilization by immunocytochemistry,
(ii) visualizing lysosomal membrane permeabilization by fluorescent dextran release,and
(iii) quantification of lysosomal membrane permeabilization by activity measurements of lysosomal enzymes in digitonin-extracted cytosol. Such methods can help researchers in advancing our knowledge in understanding cell death in normal physiology as well as in disease such as cancer.

First Report

A Method to Monitor Lysosomal Membrane Permeabilization by Immunocytochemistry

Cold Spring Harb Protoc. 2015;2015(10):pdb.prot086181.

Groth-Pedersen L¹, Jäättelä M², Nylandsted J².

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Department of Pediatrics and Adolescent Medicine, Rigshospitalet University Hospital, DK-2100 Copenhagen, Denmark; Unit for Cell Death and Metabolism, Center for Autophagy, Recycling and Disease, Danish Cancer Society Research Center, DK-2100 Copenhagen, Denmark.
Unit for Cell Death and Metabolism, Center for Autophagy, Recycling and Disease, Danish Cancer Society Research Center, DK-2100 Copenhagen, Denmark.

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Abstract

Programmed cell death involving lysosomal membrane permeabilization (LMP) is a common phenomenon-more the rule than the exception under various cytotoxic stimuli and stressful cellular conditions. The protocol presented here is based on immunocytochemical staining of cathepsin B or L to visualize translocation from the lysosomal lumen to the cytosol. In healthy cells, cathepsins appear in localized punctate structures representing intact lysosomes, whereas LMP results in a diffuse staining pattern throughout the cytoplasm. LMP can be triggered upstream, downstream, or independently of the classical apoptotic death pathway involving mitochondrial outer membrane permeabilization (MOMP). Co-staining with antibodies recognizing the active form of Bax allows investigation of the order of events between LMP and MOMP in death signaling.

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Second Report

Cold Spring Harb Protoc. 2015;2015(11):pdb.prot086165.

Quantification of Lysosomal Membrane Permeabilization by Cytosolic Cathepsin and β-N-Acetyl-Glucosaminidase Activity Measurements.

Jäättelä M¹, Nylandsted J¹.

Abstract
Programmed cell death involving lysosomal membrane permeabilization (LMP) is an alternative cell death pathway induced under various cellular conditions and by numerous cytotoxic stimuli. The method presented here to quantify LMP takes advantage of the detergent digitonin, which creates pores in cellular membranes by replacing cholesterol. The difference in cholesterol content between the plasma membrane (high) and lysosomal membrane (low) allows titration of digitonin to a concentration that permeabilizes the plasma membrane but leaves lysosomal membranes intact. The extent of LMP is determined by measuring the cytosolic activity of lysosomal hydrolases (e.g., cysteine cathepsins) and/or β-N-acetyl-glucosaminidase in the digitonin-extracted cytoplasm and comparing it to the total cellular enzyme activity. Digitonin extraction of the cytosol can be combined with precipitation of protein and/or western blot analysis for detection of lysosomal proteins (e.g., cathepsins).

Third Report

Cold Spring Harb Protoc. 2015;2015(10):pdb.prot086173.

Visualizing Lysosomal Membrane Permeabilization by Fluorescent Dextran Release.

Ellegaard AM¹, Jäättelä M¹, Nylandsted J¹.

Abstract
Lysosomal membrane permeabilization (LMP) is an effective programmed cell death pathway triggered in response to a variety of cytotoxic stimuli and cellular conditions. In the method presented here, LMP is monitored by first taking advantage of the steady endocytic capacity of cells to load fluorescent dextran into lysosomes, and then simply observing the translocation of lysosomally localized dextran into the cytosol after an LMP-inducing insult. Fluorescent dextran in healthy cells appears in punctate structures representing intact lysosomes, whereas after LMP, a diffuse staining pattern throughout the cytoplasm is observed. Using this method, LMP can be followed in real time using time-lapse imaging. The size of pores formed in the membrane during LMP by size exclusion can also be determined using dextrans of different sizes and colors.