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Altered cytochrome c display precedes apoptotic cell death in Drosophila.

Varkey J, Chen P, Jemmerson R, Abrams JM - J. Cell Biol. (1999)

Bottom Line: We found that an overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis.In cell-free studies, cytosolic caspase activation was triggered by mitochondria from apoptotic cells but identical preparations from healthy cells were inactive.Our observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neuroscience, University of Texas, Southwestern Medical Center, Dallas, Texas 75235-9039, USA.

ABSTRACT
Drosophila affords a genetically well-defined system to study apoptosis in vivo. It offers a powerful extension to in vitro models that have implicated a requirement for cytochrome c in caspase activation and apoptosis. We found that an overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis. The altered configuration is manifested by display of an otherwise hidden epitope and occurs without release of the protein into the cytosol. Conditional expression of the Drosophila death activators, reaper or grim, provoked apoptogenic cytochrome c display and, surprisingly, caspase activity was necessary and sufficient to induce this alteration. In cell-free studies, cytosolic caspase activation was triggered by mitochondria from apoptotic cells but identical preparations from healthy cells were inactive. Our observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases.

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Altered cytochrome c  display is provoked by expression of the apoptosis activators,  rpr or grim. L2 cells were stably  transfected with plasmids expressing rpr (A and B) and grim  (C and D) driven by the metallothionein promoter. Upon activation of this conditional promoter by copper (B and D,  respectively) cells exhibited pronounced labeling with mAb 2G8  and underwent apoptosis. Uninduced rpr (A) and grim (C) cells,  or cells transfected with pMTAL  vector alone uninduced (E) or  induced (F) did not exhibit apoptotic morphology nor did they  exhibit staining with the antibody. Specificity of this staining was further substantiated when rpr (G) and grim (H) expressing cells were stained with preadsorbed  2G8 mAb in parallel and did not exhibit cytochrome c immunoreactivity. The punctate and relatively large dimensions associated with  immunoreactivity might reflect aggregations of mitochondria during apoptosis (see Li et al., 1998).
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Figure 3: Altered cytochrome c display is provoked by expression of the apoptosis activators, rpr or grim. L2 cells were stably transfected with plasmids expressing rpr (A and B) and grim (C and D) driven by the metallothionein promoter. Upon activation of this conditional promoter by copper (B and D, respectively) cells exhibited pronounced labeling with mAb 2G8 and underwent apoptosis. Uninduced rpr (A) and grim (C) cells, or cells transfected with pMTAL vector alone uninduced (E) or induced (F) did not exhibit apoptotic morphology nor did they exhibit staining with the antibody. Specificity of this staining was further substantiated when rpr (G) and grim (H) expressing cells were stained with preadsorbed 2G8 mAb in parallel and did not exhibit cytochrome c immunoreactivity. The punctate and relatively large dimensions associated with immunoreactivity might reflect aggregations of mitochondria during apoptosis (see Li et al., 1998).

Mentions: Previous studies on Drosophila SL2 cells have shown that conditional expression of rpr or grim triggers apoptosis in cultured cells and in transgenic animals (White et al., 1994, 1996; Chen et al., 1996b; Nordstrom et al., 1996). Transiently transfected SL2 cells were induced for rpr or grim and, at various time intervals after induction, the preparations were examined for cytochrome c immunoreactivity with mAb 2G8. As seen in Fig. 3, apoptotic cultures (Fig. 3, B and D) exhibited profound staining with the antibody. In contrast, uninduced healthy cells (Fig. 3, A and C) and cells transfected with the vector (pMt) alone in the absence or presence of the inducing agent copper (Fig. 3, E and F) showed no signs of cytochrome c display. As before, mAb 2G8 preadsorbed with cytochrome c–Sepharose 4B (Fig. 3, F and H) validated specificity of immunoreactivity for cytochrome c. It should also be emphasized that routine labeling of apoptotic SL2 cells (or egg chambers) with a variety of antibodies directed against different antigens does not behave like mAb 2G8, and we can readily exclude the possibility that the pre-apoptotic condition somehow promotes enhanced immunodetection.


Altered cytochrome c display precedes apoptotic cell death in Drosophila.

Varkey J, Chen P, Jemmerson R, Abrams JM - J. Cell Biol. (1999)

Altered cytochrome c  display is provoked by expression of the apoptosis activators,  rpr or grim. L2 cells were stably  transfected with plasmids expressing rpr (A and B) and grim  (C and D) driven by the metallothionein promoter. Upon activation of this conditional promoter by copper (B and D,  respectively) cells exhibited pronounced labeling with mAb 2G8  and underwent apoptosis. Uninduced rpr (A) and grim (C) cells,  or cells transfected with pMTAL  vector alone uninduced (E) or  induced (F) did not exhibit apoptotic morphology nor did they  exhibit staining with the antibody. Specificity of this staining was further substantiated when rpr (G) and grim (H) expressing cells were stained with preadsorbed  2G8 mAb in parallel and did not exhibit cytochrome c immunoreactivity. The punctate and relatively large dimensions associated with  immunoreactivity might reflect aggregations of mitochondria during apoptosis (see Li et al., 1998).
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Related In: Results  -  Collection

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Figure 3: Altered cytochrome c display is provoked by expression of the apoptosis activators, rpr or grim. L2 cells were stably transfected with plasmids expressing rpr (A and B) and grim (C and D) driven by the metallothionein promoter. Upon activation of this conditional promoter by copper (B and D, respectively) cells exhibited pronounced labeling with mAb 2G8 and underwent apoptosis. Uninduced rpr (A) and grim (C) cells, or cells transfected with pMTAL vector alone uninduced (E) or induced (F) did not exhibit apoptotic morphology nor did they exhibit staining with the antibody. Specificity of this staining was further substantiated when rpr (G) and grim (H) expressing cells were stained with preadsorbed 2G8 mAb in parallel and did not exhibit cytochrome c immunoreactivity. The punctate and relatively large dimensions associated with immunoreactivity might reflect aggregations of mitochondria during apoptosis (see Li et al., 1998).
Mentions: Previous studies on Drosophila SL2 cells have shown that conditional expression of rpr or grim triggers apoptosis in cultured cells and in transgenic animals (White et al., 1994, 1996; Chen et al., 1996b; Nordstrom et al., 1996). Transiently transfected SL2 cells were induced for rpr or grim and, at various time intervals after induction, the preparations were examined for cytochrome c immunoreactivity with mAb 2G8. As seen in Fig. 3, apoptotic cultures (Fig. 3, B and D) exhibited profound staining with the antibody. In contrast, uninduced healthy cells (Fig. 3, A and C) and cells transfected with the vector (pMt) alone in the absence or presence of the inducing agent copper (Fig. 3, E and F) showed no signs of cytochrome c display. As before, mAb 2G8 preadsorbed with cytochrome c–Sepharose 4B (Fig. 3, F and H) validated specificity of immunoreactivity for cytochrome c. It should also be emphasized that routine labeling of apoptotic SL2 cells (or egg chambers) with a variety of antibodies directed against different antigens does not behave like mAb 2G8, and we can readily exclude the possibility that the pre-apoptotic condition somehow promotes enhanced immunodetection.

Bottom Line: We found that an overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis.In cell-free studies, cytosolic caspase activation was triggered by mitochondria from apoptotic cells but identical preparations from healthy cells were inactive.Our observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neuroscience, University of Texas, Southwestern Medical Center, Dallas, Texas 75235-9039, USA.

ABSTRACT
Drosophila affords a genetically well-defined system to study apoptosis in vivo. It offers a powerful extension to in vitro models that have implicated a requirement for cytochrome c in caspase activation and apoptosis. We found that an overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis. The altered configuration is manifested by display of an otherwise hidden epitope and occurs without release of the protein into the cytosol. Conditional expression of the Drosophila death activators, reaper or grim, provoked apoptogenic cytochrome c display and, surprisingly, caspase activity was necessary and sufficient to induce this alteration. In cell-free studies, cytosolic caspase activation was triggered by mitochondria from apoptotic cells but identical preparations from healthy cells were inactive. Our observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases.

Show MeSH