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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: 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.

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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Staurosporine inhibits apoptotic blebbing in  PC12 and COS-7 cells. Serum removal and z-VAD-FMK treatment were performed as described in the  legend to Fig. 1. Cells were  recorded for at least 10 min  before addition of 1 μM STS.  (a) Time-lapse images of a  representative field of PC12  cells 24 h after removal of serum and treatment with 100  μM z-VAD-FMK. 5-min intervals are shown before and  after addition of 1 μM STS  (added at time zero). Note  that 10 min after adding STS,  blebbing ceased. (b) Time-lapse images of a representative field of COS-7 cells 48 h  after removal of serum and  treatment with 100 μM  z-VAD-FMK. 13-min intervals are shown before and after addition of 1 μM STS (added at  time zero). Approximately 26 min after STS addition, blebbing  ceased. Bars: (a) 25 μM; (b) 50 μM.
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Figure 4: Staurosporine inhibits apoptotic blebbing in PC12 and COS-7 cells. Serum removal and z-VAD-FMK treatment were performed as described in the legend to Fig. 1. Cells were recorded for at least 10 min before addition of 1 μM STS. (a) Time-lapse images of a representative field of PC12 cells 24 h after removal of serum and treatment with 100 μM z-VAD-FMK. 5-min intervals are shown before and after addition of 1 μM STS (added at time zero). Note that 10 min after adding STS, blebbing ceased. (b) Time-lapse images of a representative field of COS-7 cells 48 h after removal of serum and treatment with 100 μM z-VAD-FMK. 13-min intervals are shown before and after addition of 1 μM STS (added at time zero). Approximately 26 min after STS addition, blebbing ceased. Bars: (a) 25 μM; (b) 50 μM.

Mentions: As all other agents used to induce apoptosis in our laboratory (e.g., growth factor withdrawal, UV irradiation, H2O2, ceramide, and menadione) cause membrane blebbing, the absence of this feature of apoptosis in STS-treated cells was intriguing. Two hypotheses could explain this observation: (1) STS initiates apoptosis downstream of blebbing; or (2) STS initiates apoptosis upstream of blebbing but prevents blebbing by concomitantly inhibiting a necessary downstream kinase. To differentiate between these alternatives, cultures of PC12 and COS-7 cells were deprived of serum, and treated with z-VAD-FMK until a substantial portion of cells were actively blebbing (24 h of serum deprivation for PC12 cells and 48 h for COS-7 cells). Then STS was added and cells were followed using time-lapse videomicroscopy. Both PC12 and COS-7 cells stop blebbing within minutes after addition of STS (Fig. 4) but remain viable for hours. In other experiments, blebbing of UV-irradiated PC12 cells was also inhibited by STS treatment (data not shown), suggesting that the effect of STS is not limited to trophic factor withdrawal induced apoptotic blebbing.


Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation.

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

Staurosporine inhibits apoptotic blebbing in  PC12 and COS-7 cells. Serum removal and z-VAD-FMK treatment were performed as described in the  legend to Fig. 1. Cells were  recorded for at least 10 min  before addition of 1 μM STS.  (a) Time-lapse images of a  representative field of PC12  cells 24 h after removal of serum and treatment with 100  μM z-VAD-FMK. 5-min intervals are shown before and  after addition of 1 μM STS  (added at time zero). Note  that 10 min after adding STS,  blebbing ceased. (b) Time-lapse images of a representative field of COS-7 cells 48 h  after removal of serum and  treatment with 100 μM  z-VAD-FMK. 13-min intervals are shown before and after addition of 1 μM STS (added at  time zero). Approximately 26 min after STS addition, blebbing  ceased. Bars: (a) 25 μM; (b) 50 μM.
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Figure 4: Staurosporine inhibits apoptotic blebbing in PC12 and COS-7 cells. Serum removal and z-VAD-FMK treatment were performed as described in the legend to Fig. 1. Cells were recorded for at least 10 min before addition of 1 μM STS. (a) Time-lapse images of a representative field of PC12 cells 24 h after removal of serum and treatment with 100 μM z-VAD-FMK. 5-min intervals are shown before and after addition of 1 μM STS (added at time zero). Note that 10 min after adding STS, blebbing ceased. (b) Time-lapse images of a representative field of COS-7 cells 48 h after removal of serum and treatment with 100 μM z-VAD-FMK. 13-min intervals are shown before and after addition of 1 μM STS (added at time zero). Approximately 26 min after STS addition, blebbing ceased. Bars: (a) 25 μM; (b) 50 μM.
Mentions: As all other agents used to induce apoptosis in our laboratory (e.g., growth factor withdrawal, UV irradiation, H2O2, ceramide, and menadione) cause membrane blebbing, the absence of this feature of apoptosis in STS-treated cells was intriguing. Two hypotheses could explain this observation: (1) STS initiates apoptosis downstream of blebbing; or (2) STS initiates apoptosis upstream of blebbing but prevents blebbing by concomitantly inhibiting a necessary downstream kinase. To differentiate between these alternatives, cultures of PC12 and COS-7 cells were deprived of serum, and treated with z-VAD-FMK until a substantial portion of cells were actively blebbing (24 h of serum deprivation for PC12 cells and 48 h for COS-7 cells). Then STS was added and cells were followed using time-lapse videomicroscopy. Both PC12 and COS-7 cells stop blebbing within minutes after addition of STS (Fig. 4) but remain viable for hours. In other experiments, blebbing of UV-irradiated PC12 cells was also inhibited by STS treatment (data not shown), suggesting that the effect of STS is not limited to trophic factor withdrawal induced apoptotic blebbing.

Bottom Line: 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.

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