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Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes.

Castro-Gomes T, Corrotte M, Tam C, Andrews NW - PLoS ONE (2016)

Bottom Line: However, whether lysosomal proteases released during cell injury participate in resealing is unknown.Conversely, surface protein degradation facilitates plasma membrane resealing.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, 20742, United States of America.

ABSTRACT
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.

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Related in: MedlinePlus

Protease inhibitors rapidly modulate the repair of mechanical wounds.NRK (left columns) or HeLa (right columns) cells were wounded by scraping and PM repair assays were performed in the absence (A, E) or in the presence of the following protease inhibitors: (B, F) E64 (100 μM); (C, G) Pepstatin-A (PEP-A,100 μM); (D, H) 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.
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pone.0152583.g004: Protease inhibitors rapidly modulate the repair of mechanical wounds.NRK (left columns) or HeLa (right columns) cells were wounded by scraping and PM repair assays were performed in the absence (A, E) or in the presence of the following protease inhibitors: (B, F) E64 (100 μM); (C, G) Pepstatin-A (PEP-A,100 μM); (D, H) 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.

Mentions: Next, we investigated the effect of cysteine, aspartyl and serine protease inhibitors on the ability of cells to reseal their PM. In the presence of the cysteine protease inhibitor E64, the ability of cells to repair wounds triggered by SLO was reduced compared to controls in both NRK (Fig 3A and 3B) and HeLa cells (Fig 3E and 3F). Interestingly, the aspartyl protease inhibitor pepstatin-A had the opposite effect, facilitating PM repair in both cell types (Fig 3A–3C and 3A–3G). Under the same conditions, the serine protease inhibitor AEBSF had no significant effect (Fig 3A–3D and 3A–3H). Very similar results were obtained when the cells were wounded by scraping from the dish, indicating that the observed effect of protease inhibitors is not restricted to wounding with the pore-forming protein SLO (Fig 4). A similar effect of the inhibitors was also observed using a live microscopy kinetic assay of FM1-43 influx (S1 Fig).


Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes.

Castro-Gomes T, Corrotte M, Tam C, Andrews NW - PLoS ONE (2016)

Protease inhibitors rapidly modulate the repair of mechanical wounds.NRK (left columns) or HeLa (right columns) cells were wounded by scraping and PM repair assays were performed in the absence (A, E) or in the presence of the following protease inhibitors: (B, F) E64 (100 μM); (C, G) Pepstatin-A (PEP-A,100 μM); (D, H) 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814109&req=5

pone.0152583.g004: Protease inhibitors rapidly modulate the repair of mechanical wounds.NRK (left columns) or HeLa (right columns) cells were wounded by scraping and PM repair assays were performed in the absence (A, E) or in the presence of the following protease inhibitors: (B, F) E64 (100 μM); (C, G) Pepstatin-A (PEP-A,100 μM); (D, H) 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.
Mentions: Next, we investigated the effect of cysteine, aspartyl and serine protease inhibitors on the ability of cells to reseal their PM. In the presence of the cysteine protease inhibitor E64, the ability of cells to repair wounds triggered by SLO was reduced compared to controls in both NRK (Fig 3A and 3B) and HeLa cells (Fig 3E and 3F). Interestingly, the aspartyl protease inhibitor pepstatin-A had the opposite effect, facilitating PM repair in both cell types (Fig 3A–3C and 3A–3G). Under the same conditions, the serine protease inhibitor AEBSF had no significant effect (Fig 3A–3D and 3A–3H). Very similar results were obtained when the cells were wounded by scraping from the dish, indicating that the observed effect of protease inhibitors is not restricted to wounding with the pore-forming protein SLO (Fig 4). A similar effect of the inhibitors was also observed using a live microscopy kinetic assay of FM1-43 influx (S1 Fig).

Bottom Line: However, whether lysosomal proteases released during cell injury participate in resealing is unknown.Conversely, surface protein degradation facilitates plasma membrane resealing.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, 20742, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus