Plasma‐membrane Repair is Regulated Extracellularly by Proteases Released from Lysosomes

Abstract

Eukaryotic cells repair wounds on their plasma-membrane (PM) within a few seconds in a process that involves at least three steps: Ca2+ influx, lysosomal exocytosis and lesion endocytosis. Previous work from our laboratory showed that lysosomal secretion of acid sphingomyelinase (ASM) promotes PM repair by triggering endocytosis of lesions. However, the role of other lysosomal enzymes such as proteases remained unknown. We present evidence for a role of secreted lysosomal proteases in PM repair. We show that HeLa and NRK cells permeabilized with the cytolysin streptolysin-O (SLO) in the presence of Ca2+ release cathepsins B, L and D into the extracellular medium, with a fast kinetics consistent with the time frame of PM repair. Dequenching of fluorescent DQ-BSA was detected extracellularly in the first seconds of repair, showing that proteolysis occurs during the process. PM repair, analyzed by flow cytometry and live microscopy after SLO injury, was enhanced by pre-treating cells with proteinase K and inhibited by the broad-specificity protease inhibitor alpha-2-macroglobulin. These results show, for the first time, that extracellular proteolytic events are involved in PM repair. Specific protease inhibitors and siRNA silencing for cathepsin B, L and D showed a role for these proteases during PM repair, including the resealing of mechanical wounds. Interestingly, ASM activity and subsequent PM repair were promoted by cysteine proteases and inhibited by aspartyl proteases, whereas serine proteases did not seem to be involved. Taken together, our data suggests that at least one of the mechanisms by whichlysosomal proteases regulate PM repair is through extracellular regulation of the lysosomal enzyme ASM.