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

Proposed mechanism for rapid modulation of PM repair by secreted lysosomal proteases.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 [13, 30]. 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0152583.g009: Proposed mechanism for rapid modulation of PM repair by secreted lysosomal proteases.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 [13, 30]. 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.

Mentions: Earlier investigations of the mechanism of PM repair have mostly focused on intracellular events, occurring at the cytoplasmic side of the membrane wound. In this study, we found that extracellular activity of proteases released from lysosomes play a role in regulating PM repair. First, we detected an increase in extracellular proteolysis during the first seconds after cell wounding and found that this process promotes PM repair. Second, active cysteine lysosomal proteases were released into the supernatant shortly after cell wounding, coinciding with an E64-sensitive solubilization of cell-associated proteins observed during the same period. Third, inhibition of cysteine proteases or RNAi-mediated silencing of the cathepsins B and L interfered with the ability of cells to reseal after injury, suggesting that these lysosomal cysteine proteases may facilitate PM repair by hydrolyzing proteins associated with the cell surface. Fourth, aspartyl protease inhibitors or RNAi-mediated silencing of cathepsin D enhanced PM repair, an effect that was associated with cell surface accumulation of active ASM, a lysosomal enzyme that promotes PM repair [20, 33]. Collectively, our results reveal that proteases released from wounded cells through lysosomal exocytosis can regulate PM repair, apparently by initially facilitating the process and subsequently down-regulating it (a proposed mechanism is shown in Fig 9).


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

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

Proposed mechanism for rapid modulation of PM repair by secreted lysosomal proteases.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 [13, 30]. 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0152583.g009: Proposed mechanism for rapid modulation of PM repair by secreted lysosomal proteases.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 [13, 30]. 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.
Mentions: Earlier investigations of the mechanism of PM repair have mostly focused on intracellular events, occurring at the cytoplasmic side of the membrane wound. In this study, we found that extracellular activity of proteases released from lysosomes play a role in regulating PM repair. First, we detected an increase in extracellular proteolysis during the first seconds after cell wounding and found that this process promotes PM repair. Second, active cysteine lysosomal proteases were released into the supernatant shortly after cell wounding, coinciding with an E64-sensitive solubilization of cell-associated proteins observed during the same period. Third, inhibition of cysteine proteases or RNAi-mediated silencing of the cathepsins B and L interfered with the ability of cells to reseal after injury, suggesting that these lysosomal cysteine proteases may facilitate PM repair by hydrolyzing proteins associated with the cell surface. Fourth, aspartyl protease inhibitors or RNAi-mediated silencing of cathepsin D enhanced PM repair, an effect that was associated with cell surface accumulation of active ASM, a lysosomal enzyme that promotes PM repair [20, 33]. Collectively, our results reveal that proteases released from wounded cells through lysosomal exocytosis can regulate PM repair, apparently by initially facilitating the process and subsequently down-regulating it (a proposed mechanism is shown in Fig 9).

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