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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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Deficiency in the lysosomal aspartyl protease cathepsin D facilitates PM repair.(A) 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,C) 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) or presence (C) 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.
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pone.0152583.g007: Deficiency in the lysosomal aspartyl protease cathepsin D facilitates PM repair.(A) 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,C) 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) or presence (C) 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.

Mentions: To directly address the role of major lysosomal proteases in the regulation of PM repair, we used RNAi to deplete HeLa cells in cathepsins B, L and D and performed wounding and resealing assays. To make sure that our siRNA procedure reduced the amount of active enzyme in the cells, in addition to western blots we also performed specific enzyme activity assays in the lysates. Closely mirroring what we previously observed with E64 and pepstatin-A (Figs 3 and 4), siRNA-mediated depletion of cathepsins B and L activity reduced PM repair (Fig 6A and 6B), whereas cathepsin D activity depletion improved the ability of cells to reseal (Fig 7A–7C). Thus, although we cannot rule out that cytosolic proteases released from injured cells also contribute to the observed extracellular proteolytic activity and influence PM repair [21, 42], our results implicate the lysosomal cysteine proteases cathepsin B and L in facilitating PM resealing, and cathepsin D as a negative regulator of the process.


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

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

Deficiency in the lysosomal aspartyl protease cathepsin D facilitates PM repair.(A) 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,C) 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) or presence (C) 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0152583.g007: Deficiency in the lysosomal aspartyl protease cathepsin D facilitates PM repair.(A) 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,C) 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) or presence (C) 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.
Mentions: To directly address the role of major lysosomal proteases in the regulation of PM repair, we used RNAi to deplete HeLa cells in cathepsins B, L and D and performed wounding and resealing assays. To make sure that our siRNA procedure reduced the amount of active enzyme in the cells, in addition to western blots we also performed specific enzyme activity assays in the lysates. Closely mirroring what we previously observed with E64 and pepstatin-A (Figs 3 and 4), siRNA-mediated depletion of cathepsins B and L activity reduced PM repair (Fig 6A and 6B), whereas cathepsin D activity depletion improved the ability of cells to reseal (Fig 7A–7C). Thus, although we cannot rule out that cytosolic proteases released from injured cells also contribute to the observed extracellular proteolytic activity and influence PM repair [21, 42], our results implicate the lysosomal cysteine proteases cathepsin B and L in facilitating PM resealing, and cathepsin D as a negative regulator of the process.

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