Limits...
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.

Show MeSH

Related in: MedlinePlus

Rapid extracellular proteolysis triggered by SLO wounding is required for PM repair.(A) 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) 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. (C) 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. (D) 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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4814109&req=5

pone.0152583.g001: Rapid extracellular proteolysis triggered by SLO wounding is required for PM repair.(A) 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) 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. (C) 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. (D) 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.

Mentions: Our initial studies detected extracellular proteolysis at early stages of the PM repair process, in cells wounded by the pore-forming toxin streptolysin O (SLO). In the presence of Ca2+, SLO pore formation at 37°C resulted in extracellular de-quenching of the proteolytically activated fluorogenic substrate DQ-BSA. Proteolysis-dependent fluorescence was detected on HeLa cells in suspension and also on attached NRK cells, taking advantage of the ability of DQ-BSA to bind extracellularly to the cell surface (Fig 1A and 1B). When analyzed by live fluorescence microscopy, a marked increase in DQ-BSA fluorescence was increasingly observed on the surface of NRK cells wounded with SLO in the presence of Ca2+, a condition that allows lysosomal exocytosis and PM repair [9] (Fig 1B). In both cell types, proteolytic de-quenching of DQ-BSA was significantly lower under conditions that do not trigger lysosomal exocytosis, such as in the absence of pore formation (no SLO, or SLO 4°C) or in the absence of Ca2+. The PM-associated DQ-BSA proteolysis occurred rapidly, being detectable just a few seconds after cell injury (Fig 1B). These results indicate that proteases are released from cells and are active extracellularly during the process of PM wounding and repair.


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

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

Rapid extracellular proteolysis triggered by SLO wounding is required for PM repair.(A) 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) 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. (C) 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. (D) 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0152583.g001: Rapid extracellular proteolysis triggered by SLO wounding is required for PM repair.(A) 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) 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. (C) 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. (D) 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.
Mentions: Our initial studies detected extracellular proteolysis at early stages of the PM repair process, in cells wounded by the pore-forming toxin streptolysin O (SLO). In the presence of Ca2+, SLO pore formation at 37°C resulted in extracellular de-quenching of the proteolytically activated fluorogenic substrate DQ-BSA. Proteolysis-dependent fluorescence was detected on HeLa cells in suspension and also on attached NRK cells, taking advantage of the ability of DQ-BSA to bind extracellularly to the cell surface (Fig 1A and 1B). When analyzed by live fluorescence microscopy, a marked increase in DQ-BSA fluorescence was increasingly observed on the surface of NRK cells wounded with SLO in the presence of Ca2+, a condition that allows lysosomal exocytosis and PM repair [9] (Fig 1B). In both cell types, proteolytic de-quenching of DQ-BSA was significantly lower under conditions that do not trigger lysosomal exocytosis, such as in the absence of pore formation (no SLO, or SLO 4°C) or in the absence of Ca2+. The PM-associated DQ-BSA proteolysis occurred rapidly, being detectable just a few seconds after cell injury (Fig 1B). These results indicate that proteases are released from cells and are active extracellularly during the process of PM wounding and repair.

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