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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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The number of  blebbing cells increase over  time after serum removal in  the presence of z-VAD-FMK.  (a) Blebbing cells were  scored at 0 and 24 h after  serum removal and initiation  of z-VAD-FMK treatment.  The percentage of blebbing  cells was calculated by dividing the number of blebbing  cells by the total number of  living cells per field. At 24 h,  ∼50% of serum-deprived,  z-VAD-FMK–treated cells  were blebbing (SF + z-VAD,  dark striped bars). SF, serum  free; C, control N2 medium  plus DMSO; and C + z-VAD  N2 medium plus 100 μM  z-VAD-FMK. Data represent  the mean ± SEM for three  experiments. (b) The percentage of blebbing cells was calculated every 1.5 h from a time-lapse video recording of a field of cells treated with 100 μM  z-VAD-FMK after removal of serum. The results depicted are  representative of three separate experiments.
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Figure 3: The number of blebbing cells increase over time after serum removal in the presence of z-VAD-FMK. (a) Blebbing cells were scored at 0 and 24 h after serum removal and initiation of z-VAD-FMK treatment. The percentage of blebbing cells was calculated by dividing the number of blebbing cells by the total number of living cells per field. At 24 h, ∼50% of serum-deprived, z-VAD-FMK–treated cells were blebbing (SF + z-VAD, dark striped bars). SF, serum free; C, control N2 medium plus DMSO; and C + z-VAD N2 medium plus 100 μM z-VAD-FMK. Data represent the mean ± SEM for three experiments. (b) The percentage of blebbing cells was calculated every 1.5 h from a time-lapse video recording of a field of cells treated with 100 μM z-VAD-FMK after removal of serum. The results depicted are representative of three separate experiments.

Mentions: An additional striking observation in these initial experiments is the greatly increased percentage of cells dynamically extruding and retracting membrane blebs in serum-deprived cultures after treatment with z-VAD-FMK (Fig. 2 a), compared to serum-deprived cells in the absence of z-VAD-FMK (data not shown). To determine the increased number of blebbing cells in serum-deprived, z-VAD-FMK–treated cultures, cells were counted and scored for blebbing at time zero and at 24 h (Fig. 3 a). At time zero, low percentages of cells with blebs are present in all groups, which probably reflects the normal background death in these cultures. After 24 h in serum-free medium without z-VAD-FMK, only 10% of cells are blebbing, presumably because these cells undergo the asynchronous death characteristic of apoptosis. Consequently, only a small percentage of these cells are blebbing at any one time (Fig. 3 a). Control cells maintained for 24 h in serum-free medium supplemented with N2 components have a low background death and very few cells are blebbing (Fig. 3 a). Inhibition of caspases in the absence of an apoptotic stimulus does not cause blebbing by itself, as only a small percentage of z-VAD-FMK–treated cells are blebbing in medium containing N2 components at 24 h (Fig. 3 a). The small increase in blebbing in the N2 z-VAD-FMK condition compared to N2 alone at 24 h is consistent with inhibition of normal background death by z-VAD-FMK. After serum removal and treatment with z-VAD-FMK, there is a marked, nearly fivefold increase in the fraction of cells blebbing at 24 h (Fig. 3 a), supporting a previous report that z-VAD-FMK blocks death after onset of blebbing (McCarthy et al., 1997). This raised the possibility of using the increased number of cells stopped early in the execution phase of apoptosis to investigate the mechanisms underlying dynamic membrane blebbing.


Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation.

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

The number of  blebbing cells increase over  time after serum removal in  the presence of z-VAD-FMK.  (a) Blebbing cells were  scored at 0 and 24 h after  serum removal and initiation  of z-VAD-FMK treatment.  The percentage of blebbing  cells was calculated by dividing the number of blebbing  cells by the total number of  living cells per field. At 24 h,  ∼50% of serum-deprived,  z-VAD-FMK–treated cells  were blebbing (SF + z-VAD,  dark striped bars). SF, serum  free; C, control N2 medium  plus DMSO; and C + z-VAD  N2 medium plus 100 μM  z-VAD-FMK. Data represent  the mean ± SEM for three  experiments. (b) The percentage of blebbing cells was calculated every 1.5 h from a time-lapse video recording of a field of cells treated with 100 μM  z-VAD-FMK after removal of serum. The results depicted are  representative of three separate experiments.
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Related In: Results  -  Collection

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Figure 3: The number of blebbing cells increase over time after serum removal in the presence of z-VAD-FMK. (a) Blebbing cells were scored at 0 and 24 h after serum removal and initiation of z-VAD-FMK treatment. The percentage of blebbing cells was calculated by dividing the number of blebbing cells by the total number of living cells per field. At 24 h, ∼50% of serum-deprived, z-VAD-FMK–treated cells were blebbing (SF + z-VAD, dark striped bars). SF, serum free; C, control N2 medium plus DMSO; and C + z-VAD N2 medium plus 100 μM z-VAD-FMK. Data represent the mean ± SEM for three experiments. (b) The percentage of blebbing cells was calculated every 1.5 h from a time-lapse video recording of a field of cells treated with 100 μM z-VAD-FMK after removal of serum. The results depicted are representative of three separate experiments.
Mentions: An additional striking observation in these initial experiments is the greatly increased percentage of cells dynamically extruding and retracting membrane blebs in serum-deprived cultures after treatment with z-VAD-FMK (Fig. 2 a), compared to serum-deprived cells in the absence of z-VAD-FMK (data not shown). To determine the increased number of blebbing cells in serum-deprived, z-VAD-FMK–treated cultures, cells were counted and scored for blebbing at time zero and at 24 h (Fig. 3 a). At time zero, low percentages of cells with blebs are present in all groups, which probably reflects the normal background death in these cultures. After 24 h in serum-free medium without z-VAD-FMK, only 10% of cells are blebbing, presumably because these cells undergo the asynchronous death characteristic of apoptosis. Consequently, only a small percentage of these cells are blebbing at any one time (Fig. 3 a). Control cells maintained for 24 h in serum-free medium supplemented with N2 components have a low background death and very few cells are blebbing (Fig. 3 a). Inhibition of caspases in the absence of an apoptotic stimulus does not cause blebbing by itself, as only a small percentage of z-VAD-FMK–treated cells are blebbing in medium containing N2 components at 24 h (Fig. 3 a). The small increase in blebbing in the N2 z-VAD-FMK condition compared to N2 alone at 24 h is consistent with inhibition of normal background death by z-VAD-FMK. After serum removal and treatment with z-VAD-FMK, there is a marked, nearly fivefold increase in the fraction of cells blebbing at 24 h (Fig. 3 a), supporting a previous report that z-VAD-FMK blocks death after onset of blebbing (McCarthy et al., 1997). This raised the possibility of using the increased number of cells stopped early in the execution phase of apoptosis to investigate the mechanisms underlying dynamic membrane 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