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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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p35 and peptide-based caspase inhibitors block altered  cytochrome c display. Cells stably transfected and induced for  Mt-rpr exhibited binding of mAb 2G8 as seen in A while cells  cotransfected with Mt-rpr and Mt-p35 did not label with mAb  2G8 (B). Similarly, Mt-rpr cells induced with copper and treated  with caspase inhibitors, DEVD-fmk (C) and zVAD-fmk (D), did  not stain with mAb 2G8. Expression of rpr and p35 proteins in  these cells was verified in separate studies.
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Figure 6: p35 and peptide-based caspase inhibitors block altered cytochrome c display. Cells stably transfected and induced for Mt-rpr exhibited binding of mAb 2G8 as seen in A while cells cotransfected with Mt-rpr and Mt-p35 did not label with mAb 2G8 (B). Similarly, Mt-rpr cells induced with copper and treated with caspase inhibitors, DEVD-fmk (C) and zVAD-fmk (D), did not stain with mAb 2G8. Expression of rpr and p35 proteins in these cells was verified in separate studies.

Mentions: The Drosophila death activators, rpr and grim, activate one or more caspases to elicit apoptosis (Chen et al., 1996b; Nordstrom et al., 1996; White et al., 1996; Fraser et al., 1997). To study the temporal relation of cytochrome c display with respect to caspase activity, we cotransfected SL2 cells with pMt-rpr and pMt-p35. 6 h after induction, cells induced for rpr alone showed pronounced labeling with mAb 2G8 (Fig. 6 A) whereas cells expressing rpr together with p35 were prevented from apoptosis and did not bind the mAb (Fig. 6 B). These observations suggested that apoptogenic cytochrome c display required caspase activity, a presumption that was further substantiated when rpr-expressing cells were treated with the peptide caspase inhibitors zDEVD-fmk and zVAD-fmk. As seen for p35-blocked cells, these inhibitors similarly prevented mAb 2G8 labeling and subsequent apoptosis (Fig. 6, C and D). Parallel results were observed in grim-expressing cells (not shown).


Altered cytochrome c display precedes apoptotic cell death in Drosophila.

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

p35 and peptide-based caspase inhibitors block altered  cytochrome c display. Cells stably transfected and induced for  Mt-rpr exhibited binding of mAb 2G8 as seen in A while cells  cotransfected with Mt-rpr and Mt-p35 did not label with mAb  2G8 (B). Similarly, Mt-rpr cells induced with copper and treated  with caspase inhibitors, DEVD-fmk (C) and zVAD-fmk (D), did  not stain with mAb 2G8. Expression of rpr and p35 proteins in  these cells was verified in separate studies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132929&req=5

Figure 6: p35 and peptide-based caspase inhibitors block altered cytochrome c display. Cells stably transfected and induced for Mt-rpr exhibited binding of mAb 2G8 as seen in A while cells cotransfected with Mt-rpr and Mt-p35 did not label with mAb 2G8 (B). Similarly, Mt-rpr cells induced with copper and treated with caspase inhibitors, DEVD-fmk (C) and zVAD-fmk (D), did not stain with mAb 2G8. Expression of rpr and p35 proteins in these cells was verified in separate studies.
Mentions: The Drosophila death activators, rpr and grim, activate one or more caspases to elicit apoptosis (Chen et al., 1996b; Nordstrom et al., 1996; White et al., 1996; Fraser et al., 1997). To study the temporal relation of cytochrome c display with respect to caspase activity, we cotransfected SL2 cells with pMt-rpr and pMt-p35. 6 h after induction, cells induced for rpr alone showed pronounced labeling with mAb 2G8 (Fig. 6 A) whereas cells expressing rpr together with p35 were prevented from apoptosis and did not bind the mAb (Fig. 6 B). These observations suggested that apoptogenic cytochrome c display required caspase activity, a presumption that was further substantiated when rpr-expressing cells were treated with the peptide caspase inhibitors zDEVD-fmk and zVAD-fmk. As seen for p35-blocked cells, these inhibitors similarly prevented mAb 2G8 labeling and subsequent apoptosis (Fig. 6, C and D). Parallel results were observed in grim-expressing cells (not shown).

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