Use of glycyl-l-phenylalanine 2-naphthylamide, a lysosome-disrupting cathepsin C substrate, to distinguish between lysosomes and prelysosomal endocytic vacuoles

Abstract

Lysosome-disrupting enzyme substrates have been used to distinguish between lysosomal and prelysosomal compartments along the endocytic pathway in isolated rat hepatocytes. The cells were incubated for various periods of time with 125I-labelled tyramine cellobiose (125I-TC) covalently coupled to asialoorosomucoid (AOM) (125I-TC-AOM); this molecule is internalized by receptor-mediated endocytosis and degraded in lysosomes, where the degradation products (acid-soluble, radio-labelled short peptides) accumulate, Glycyl-L-phenylalanine 2-naphthylamide (GPN) and methionine O-methyl ester (MOM), which are hydrolysed by lysosomal cathepsin C and a lysosomal esterase respectively, both diffused into hepatocytic lysosomes after electrodisruption of the cells. Intralysosomal accumulation of the hydrolysis products (amino acids) of these substrates caused osmotic lysis of more than 90% of the lysosomes, as measured by the release of acid-soluble radioactivity derived from 125I-TC-AOM degradation. The acid-soluble radioactivity coincided in sucrose-density gradients with a major peak of the lysosomal marker enzyme acid phosphatase at 1.18 g/ml; in addition a minor, presumably endosomal, acid phosphatase peak was observed around 1.14 g/ml. The major peak of acid phosphatase was almost completely released by GPN (and by MOM), while the minor peak seemed unaffected by GPN. Acid-insoluble radioactivity, presumably in endosomes, banded (after 1 h of 125I-TC-AOM uptake) as a major peak at 1.14 and a minor peak at 1.18 g/ml in sucrose gradients, and was not significantly released by GPN. GPN thus appears to be an excellent tool by which to distinguish between endosomes and lysosomes. MOM, on the other hand, released some radioactivity and acid phosphatase from endosomes as well as from lysosomes.

Molecular mechanisms of ATP secretion during immunogenic cell death

“…Quantitative data (means±S.E.M., n ¼ 3) are reported. ns, non-significant (Student's t-test) as compared with UNT cells treated with cumate only cathepsin C substrate, 47 drastically reduced the intensity of quinacrine fluorescence, while leaving the ER and mitochondria morphologically intact (Figures 5c and d). Further supporting the preference of quinacrine for lysosomes, untreated U2OS cells exhibited a preponderant co-localization between quinacrine and an LAMP1-red fluorescent protein (RFP) fusion protein, 48 which is specifically found in lysosomes, and such colocalization was reduced upon exposure to MTX and OXA but not to rapamycin (Figures 6a and b).…”

Lysosome-Sarcoplasmic Reticulum Junctions

“…2C, panels i and ii; n ϭ 6). To determine whether or not the labeled organelles were lysosome-related, cells were incubated with GPN, a substrate of the lysosomal exopeptidase cathepsin C that selectively disrupts lysosomes by osmotic lysis (26). To achieve this goal, cells were first incubated for 15 min with the selective inhibitor of myosin light-chain kinase 1-(5-chloronapthalene-1-sulfonyl)-1H-hexahydro-1,4,diazepine hydrochloride (ML-9 (27)) to prevent disruption of the spatial orientation of cells due to contraction by GPN.…”

Transport and release of colloidal 3-mercaptopropionic acid-coated CdSe–CdS/ZnS core-multishell quantum dots in human umbilical vein endothelial cells

“…GPN is known to permeabilize endosomes/lysosomes and thereafter induce Ca 2+ release and oscillations. 29,30 To quantify endosomal/lysosomal Ca 2+ content, we monitored cytosolic [Ca 2+ ] i in response to acute GPN addition to QD-treated HUVECs loaded with OGB-1. As expected, the addition of GPN resulted in a prompt increase in QD fluorescence followed by oscillations, most probably due to the release of Ca 2+ from endosomes/lysosomes ( Figure 9A).…”