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Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation.

Mills JC, Stone NL, Erhardt J, Pittman RN - J. Cell Biol. (1998)

Bottom Line: A screen of kinase inhibitors performed on synchronized blebbing cells indicated that only myosin light chain kinase (MLCK) inhibitors decreased blebbing.Based on these results, a working model is proposed for how actin/myosin II interactions cause cell contraction and membrane blebbing.Our results provide the first evidence that MLC phosphorylation is critical for apoptotic membrane blebbing and also implicate Rho signaling in these active morphological changes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
The evolutionarily conserved execution phase of apoptosis is defined by characteristic changes occurring during the final stages of death; specifically cell shrinkage, dynamic membrane blebbing, condensation of chromatin, and DNA fragmentation. Mechanisms underlying these hallmark features of apoptosis have previously been elusive, largely because the execution phase is a rapid event whose onset is asynchronous across a population of cells. In the present study, a model system is described for using the caspase inhibitor, z-VAD-FMK, to block apoptosis and generate a synchronous population of cells actively extruding and retracting membrane blebs. This model system allowed us to determine signaling mechanisms underlying this characteristic feature of apoptosis. A screen of kinase inhibitors performed on synchronized blebbing cells indicated that only myosin light chain kinase (MLCK) inhibitors decreased blebbing. Immunoprecipitation of myosin II demonstrated that myosin regulatory light chain (MLC) phosphorylation was increased in blebbing cells and that MLC phosphorylation was prevented by inhibitors of MLCK. MLC phosphorylation is also mediated by the small G protein, Rho. C3 transferase inhibited apoptotic membrane blebbing, supporting a role for a Rho family member in this process. Finally, blebbing was also inhibited by disruption of the actin cytoskeleton. Based on these results, a working model is proposed for how actin/myosin II interactions cause cell contraction and membrane blebbing. Our results provide the first evidence that MLC phosphorylation is critical for apoptotic membrane blebbing and also implicate Rho signaling in these active morphological changes. The model system described here should facilitate future studies of MLCK, Rho, and other signal transduction pathways activated during the execution phase of apoptosis.

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Inhibitors of  MLCK stop apoptotic membrane blebbing. (a) Addition  of either kinase inhibitor (1  μM STS, 4 μM KT-5926, 5  μM KN-93, 5 μM KT-5823,  or 5 μM bisindolylmaleimide 1) or equivalent volume  of DMSO 24 h after serum  deprivation and z-VAD-FMK  treatment. Cells were counted  immediately before addition  of kinase inhibitor, and the  same fields counted 1 h after  addition of inhibitor. Percent  inhibition of blebbing was  calculated by comparing the  portion of cells blebbing before and after treatment.  Data represent the mean ±  SEM for three experiments.  (b) The MLCK inhibitor, ML-9, inhibits blebbing of cells deprived of serum and treated with 100 μM z-VAD-FMK or 100  μM BaF (a general caspase inhibitor) in a dose-dependent manner. Cells were counted as above, in the presence of 20 or 80 μM  ML-9. Data represent the mean ± SEM for three experiments.
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Figure 5: Inhibitors of MLCK stop apoptotic membrane blebbing. (a) Addition of either kinase inhibitor (1 μM STS, 4 μM KT-5926, 5 μM KN-93, 5 μM KT-5823, or 5 μM bisindolylmaleimide 1) or equivalent volume of DMSO 24 h after serum deprivation and z-VAD-FMK treatment. Cells were counted immediately before addition of kinase inhibitor, and the same fields counted 1 h after addition of inhibitor. Percent inhibition of blebbing was calculated by comparing the portion of cells blebbing before and after treatment. Data represent the mean ± SEM for three experiments. (b) The MLCK inhibitor, ML-9, inhibits blebbing of cells deprived of serum and treated with 100 μM z-VAD-FMK or 100 μM BaF (a general caspase inhibitor) in a dose-dependent manner. Cells were counted as above, in the presence of 20 or 80 μM ML-9. Data represent the mean ± SEM for three experiments.

Mentions: To determine which kinase(s) STS inhibits to block blebbing, a panel of more specific inhibitors was screened. PC12 cells, deprived of serum and treated with z-VAD-FMK for 24 h, were treated for 1 h with either STS, KT5926 (an inhibitor of MLCK), KN-93 (an inhibitor of CAMKII), KT5823 (an inhibitor of protein kinase G), bisindolylmaleimide 1 (an inhibitor of protein kinase C) or H-89 (an inhibitor of protein kinase A). Only kinase inhibitors that inhibit MLCK (i.e., STS and KT5926) have a statistically significant effect on blebbing—STS decreases blebbing by 89%, and KT5926 by 39% (Fig. 5 a). The PKA inhibitor, H89, induces rapid death of cells at a concentration similar to that used in other studies (30 μM; Chijiwa et al., 1990) and has no effect on blebbing at a slightly lower concentration (7.5 μM).


Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation.

Mills JC, Stone NL, Erhardt J, Pittman RN - J. Cell Biol. (1998)

Inhibitors of  MLCK stop apoptotic membrane blebbing. (a) Addition  of either kinase inhibitor (1  μM STS, 4 μM KT-5926, 5  μM KN-93, 5 μM KT-5823,  or 5 μM bisindolylmaleimide 1) or equivalent volume  of DMSO 24 h after serum  deprivation and z-VAD-FMK  treatment. Cells were counted  immediately before addition  of kinase inhibitor, and the  same fields counted 1 h after  addition of inhibitor. Percent  inhibition of blebbing was  calculated by comparing the  portion of cells blebbing before and after treatment.  Data represent the mean ±  SEM for three experiments.  (b) The MLCK inhibitor, ML-9, inhibits blebbing of cells deprived of serum and treated with 100 μM z-VAD-FMK or 100  μM BaF (a general caspase inhibitor) in a dose-dependent manner. Cells were counted as above, in the presence of 20 or 80 μM  ML-9. Data represent the mean ± SEM for three experiments.
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Figure 5: Inhibitors of MLCK stop apoptotic membrane blebbing. (a) Addition of either kinase inhibitor (1 μM STS, 4 μM KT-5926, 5 μM KN-93, 5 μM KT-5823, or 5 μM bisindolylmaleimide 1) or equivalent volume of DMSO 24 h after serum deprivation and z-VAD-FMK treatment. Cells were counted immediately before addition of kinase inhibitor, and the same fields counted 1 h after addition of inhibitor. Percent inhibition of blebbing was calculated by comparing the portion of cells blebbing before and after treatment. Data represent the mean ± SEM for three experiments. (b) The MLCK inhibitor, ML-9, inhibits blebbing of cells deprived of serum and treated with 100 μM z-VAD-FMK or 100 μM BaF (a general caspase inhibitor) in a dose-dependent manner. Cells were counted as above, in the presence of 20 or 80 μM ML-9. Data represent the mean ± SEM for three experiments.
Mentions: To determine which kinase(s) STS inhibits to block blebbing, a panel of more specific inhibitors was screened. PC12 cells, deprived of serum and treated with z-VAD-FMK for 24 h, were treated for 1 h with either STS, KT5926 (an inhibitor of MLCK), KN-93 (an inhibitor of CAMKII), KT5823 (an inhibitor of protein kinase G), bisindolylmaleimide 1 (an inhibitor of protein kinase C) or H-89 (an inhibitor of protein kinase A). Only kinase inhibitors that inhibit MLCK (i.e., STS and KT5926) have a statistically significant effect on blebbing—STS decreases blebbing by 89%, and KT5926 by 39% (Fig. 5 a). The PKA inhibitor, H89, induces rapid death of cells at a concentration similar to that used in other studies (30 μM; Chijiwa et al., 1990) and has no effect on blebbing at a slightly lower concentration (7.5 μM).

Bottom Line: A screen of kinase inhibitors performed on synchronized blebbing cells indicated that only myosin light chain kinase (MLCK) inhibitors decreased blebbing.Based on these results, a working model is proposed for how actin/myosin II interactions cause cell contraction and membrane blebbing.Our results provide the first evidence that MLC phosphorylation is critical for apoptotic membrane blebbing and also implicate Rho signaling in these active morphological changes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
The evolutionarily conserved execution phase of apoptosis is defined by characteristic changes occurring during the final stages of death; specifically cell shrinkage, dynamic membrane blebbing, condensation of chromatin, and DNA fragmentation. Mechanisms underlying these hallmark features of apoptosis have previously been elusive, largely because the execution phase is a rapid event whose onset is asynchronous across a population of cells. In the present study, a model system is described for using the caspase inhibitor, z-VAD-FMK, to block apoptosis and generate a synchronous population of cells actively extruding and retracting membrane blebs. This model system allowed us to determine signaling mechanisms underlying this characteristic feature of apoptosis. A screen of kinase inhibitors performed on synchronized blebbing cells indicated that only myosin light chain kinase (MLCK) inhibitors decreased blebbing. Immunoprecipitation of myosin II demonstrated that myosin regulatory light chain (MLC) phosphorylation was increased in blebbing cells and that MLC phosphorylation was prevented by inhibitors of MLCK. MLC phosphorylation is also mediated by the small G protein, Rho. C3 transferase inhibited apoptotic membrane blebbing, supporting a role for a Rho family member in this process. Finally, blebbing was also inhibited by disruption of the actin cytoskeleton. Based on these results, a working model is proposed for how actin/myosin II interactions cause cell contraction and membrane blebbing. Our results provide the first evidence that MLC phosphorylation is critical for apoptotic membrane blebbing and also implicate Rho signaling in these active morphological changes. The model system described here should facilitate future studies of MLCK, Rho, and other signal transduction pathways activated during the execution phase of apoptosis.

Show MeSH
Related in: MedlinePlus