PM repair is inhibited in cells deficient in the lysosomal cysteine proteases cathepsin B or cathepsin L.

<p>Time-lapse live imaging of FM1-43 influx was performed in HeLa cells exposed to SLO in the presence or absence of Ca²⁺, 48 h after transfection with control or cathepsin B siRNA <b>(A)</b> or 72 h after cathepsin L <b>(B)</b> siRNA. The insets show kinetic enzyme activity determinations using a specific fluorogenic substrate (left) or the protein levels of each enzyme detected by immunoblot with specific antibodies (right). Anti-tubulin antibodies were used in the same samples as loading controls. The data are representative of two independent experiments.</p

Protease inhibitors rapidly modulate the repair of mechanical wounds.

<p>NRK (left columns) or HeLa (right columns) cells were wounded by scraping and PM repair assays were performed in the absence <b>(A, E)</b> or in the presence of the following protease inhibitors: <b>(B, F)</b> E64 (100 μM); <b>(C, G)</b> Pepstatin-A (PEP-A,100 μM); <b>(D, H)</b> AEBSF (100 μM). After a resealing period of 2 min at 37°C the cell population was stained with PI and analyzed by FACS. The dotted black lines in (A, E) show the Ca²⁺-free permeabilization controls, which determined the gating (dashed line). The data are representative of at least three independent experiments.</p

Active lysosomal proteases are released with differential kinetics during PM wounding and repair.

<p>NRK cells were pre-incubated on ice with SLO (100 ng/ml) followed by addition of Ca²⁺-containing media pre-warmed at 37°C to trigger pore-formation and lysosomal exocytosis. The supernatant was collected at the indicated periods of time and analyzed for activity of the cysteine proteases cathepsins B and L and the aspartyl protease cathepsin D. Results are expressed as percentage of the total activity present in the cells (determined by assaying enzymatic activity in whole cell lysates), and correspond to the mean +/- SD of three independent experiments.</p

Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

<div><p>Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca^2+-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D.</p></div

ASM secreted during cell injury associates with the PM and is proteolytically modulated.

<p><b>(A)</b> Effect of protease inhibitors on ASM activity released from wounded cells. NRK cells were permeabilized with SLO (100 ng/ml) in Ca²⁺ media containing (green) or not (blue) a protease inhibitor cocktail and incubated at 37°C for the indicated time. As controls, cells not treated with SLO (NT) were incubated with (purple) or without (red) inhibitors. Supernatant samples were placed on ice, protease inhibitors were added and ASM activity was assayed. Similar results were obtained with HeLa cells (not shown). <b>(B)</b> Effect of inhibitors on ASM activity released from wounded cells. NRK cells were permeabilized with SLO (100 ng/ml) in Ca²⁺-containing media with 100 μM E64, 100 μM pepstatin-A (PEP-A) or no inhibitors, and incubated at 37°C for 30 s. The data represent the mean +/- SD of triplicate assays. * P = 0.039, Student’s t test. <b>(C)</b> Detection of cell-associated ASM during the first seconds after SLO wounding. NRK cells were permeabilized with SLO (100 ng/ml), incubated at 37°C in Ca²⁺-containing media for the indicated time, washed and immunofluorescence was performed with rabbit anti-ASM antibodies, followed by imaging under identical settings. <b>(D)</b> Detection of the active 65 kDa form of ASM in supernatant and membrane-associated fractions of wounded cells. NRK cells were permeabilized or not with SLO (100 ng/ml) with or without 100 μM E64 or 100 μM pepstatin-A for the indicated periods of time, and samples of the supernatant or of material removed from the cell surface by an acid wash were analyzed by Western blot with rabbit anti-ASM antibodies. The data are representative of at least three independent experiments. <b>(E)</b> Detection of the 65 kDa ASM form in the supernatant and membrane-associated fractions of wounded cells depleted or not in cathepsin D (CATD). NRK cells were permeabilized or not with SLO and analyzed as described in (D). The data are representative of at least three independent experiments.</p

Deficiency in the lysosomal aspartyl protease cathepsin D facilitates PM repair.

<p><b>(A)</b> Cathepsin D activity determined kinetically using a specific fluorogenic substrate (left) and cathepsin D protein (precursor and mature forms) levels detected by immunoblot with specific antibodies (right) in HeLa cell lysates 36 h after transfection with control (blue) or cathepsin D (red) siRNA. Anti-tubulin antibodies were used in the same samples as loading controls. <b>(B,C)</b> FACS quantification of PI staining in HeLa cells treated with control (blue) or cathepsin D (red) siRNA after wounding by scraping in the absence <b>(B)</b> or presence <b>(C)</b> of Ca²⁺. After a resealing period of 2 min at 37°C the cell population was stained with PI and analyzed by FACS. The dashed line indicates the gating based on the Ca²⁺-free permeabilization control. The data are representative of two independent experiments.</p

Protease inhibitors rapidly modulate the repair of SLO wounds.

<p>NRK (left column) or HeLa (right column) cells were wounded with SLO (NRK 150 ng/ml, HeLa 250 ng/ml) and PM repair assays were performed in the absence <b>(A, E)</b> or in the presence of the following protease inhibitors: <b>(B, F)</b> E64 (100 μM); <b>(C, G)</b> Pepstatin-A (PEP-A,100 μM); <b>(D, H)</b> AEBSF (100 μM). After a resealing period of 2 min at 37°C the cell population was stained with PI and analyzed by FACS. In (A, E) the green histograms show the FACS profile of not wounded cells, and the dotted black histograms show the Ca²⁺-free permeabilization controls, which determined the gating (dashed line). The data are representative of at least three independent experiments.</p

Rapid extracellular proteolysis triggered by SLO wounding is required for PM repair.

<p><b>(A)</b> Cleavage of extracellularly added DQ-BSA during PM wounding and repair. HeLa cells treated or not with 200 ng/ml SLO were incubated with DQ-BSA for 2 min at the indicated temperature, followed by measuring the de-quenching generated fluorescence. The data represent the mean +/- SD of triplicate assays. ** P = 0.00297, Student’s t test. <b>(B)</b> De-quenched DQ-BSA associated with the surface of wounded cells. NRK cells treated or not with 50 ng/ml SLO in the presence or absence of Ca²⁺ were incubated with DQ-BSA followed by fluorescence deconvolution imaging (top panel). Bars = 10 μm. The mean cell-associated fluorescence intensity was determined and expressed as fold increase relative to the starting level (bottom panel). The data represent the mean +/- SD of fluorescence intensity values associated with 24–26 individual cells, and are representative of two independent experiments. <b>(C)</b> FACS quantification of PI staining in NRK cells permeabilized with SLO (200 ng/ml) in the presence (red) or absence (blue) of the protease inhibitor alpha-2-macroglobulin (20 μg/ml). The dotted histogram shows the Ca²⁺-free permeabilization control, which determined the gating (dashed line). The inset shows the percentage of cells that excluded PI after 2 min at 37°C. The data represent the mean +/- SD of three independent experiments. <b>(D)</b> Time-lapse live imaging of FM1-43 influx into NRK cells exposed to SLO (350 ng/ml) in the presence or absence of Ca²⁺ and the protease inhibitor alpha-2-macroglobulin (20 μg/ml). The data represent the mean +/- SEM of 27–52 cells per condition.</p

Proposed mechanism for rapid modulation of PM repair by secreted lysosomal proteases.

<p>Ca²⁺ influx through wounds in the PM rapidly triggers exocytosis of lysosomes, releasing the proteases cathepsins B, L and D along with the lipase ASM. Cathepsins B and L are active extracellularly 5–10 s after PM wounding, while cathepsin D activity appears after a delay of 30–60 s. At early time points, cathepsins B and L (and possibly additional cysteine proteases) may cleave cell surface proteins and contribute to membrane access and/or activation of ASM. ASM hydrolizes sphingomyelin on the outer leaflet of the PM generating ceramide, which promotes lesion endocytosis and PM repair [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152583#pone.0152583.ref013" target="_blank">13</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152583#pone.0152583.ref030" target="_blank">30</a>]. ASM-generated ceramide may enhance cathepsin D activity, which plays a role in down-regulating ASM. Around 1 min after wounding the PM integrity is restored, and lysosomal hydrolases are no longer active extracellularly.</p