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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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Immunoprecipita-tion of Drosophila cytochrome c. Western blot analyses were used to visualize  immunoprecipitated proteins that were probed with  anti–cytochrome c mAb 7H8.  Lanes 1 and 2 show whole  cell suspension of healthy  SL2 cells (S) and grim-expressing cells 4 h after induction  (G). Lanes 3 and 4 were precleared lysates used for immunoprecipitation (see Materials and Methods). Lanes 5–8 were immunoprecipitates from SL2 or grim-expressing cell lysates using two  anti–cytochrome c mAbs, 6H2 and 2G8. The same two cytochrome c bands were also observed in lanes 1–4 on shorter exposures.
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Figure 1: Immunoprecipita-tion of Drosophila cytochrome c. Western blot analyses were used to visualize immunoprecipitated proteins that were probed with anti–cytochrome c mAb 7H8. Lanes 1 and 2 show whole cell suspension of healthy SL2 cells (S) and grim-expressing cells 4 h after induction (G). Lanes 3 and 4 were precleared lysates used for immunoprecipitation (see Materials and Methods). Lanes 5–8 were immunoprecipitates from SL2 or grim-expressing cell lysates using two anti–cytochrome c mAbs, 6H2 and 2G8. The same two cytochrome c bands were also observed in lanes 1–4 on shorter exposures.

Mentions: Although two cytochrome c genes, DC4 and DC3, have been described in Drosophila melanogaster (Limbach and Wu, 1985) studies at the level of protein (Inoue et al., 1986) and at the level of RNA (Limbach and Wu, 1985) suggest that DC4, which shows >86% identity with its rat counterpart, is either the predominant or only form of actively expressed product. We screened an existing panel of mAbs, directed against mammalian versions of cytochrome c, as possible probes for in situ analyses of the fly counterpart. The potential utility of these mAbs was assessed by immunoprecipitations of SL2 cell lysates, probed with a third anti–cytochrome c mAb, 7H8, to detect denatured cytochrome c in Western blots. The results in Fig. 1 show that two mAbs, 6H2 (Goshorn et al., 1991) and 2G8 (Mueller and Jemmerson, 1996), recognized Drosophila cytochrome c. Both antibodies detected a doublet that comigrated with mammalian cytochrome c at ∼13 kD. While mAb 2G8 preferentially precipitated the upper band, mAb 6H2 had about equal affinity for both forms of cytochrome c. No obvious correlation between the relative abundance of the two cytochrome c bands and apoptosis was observed (see below). The same banding pattern was also observed when immunoprecipitation was performed using purified Drosophila cytochrome c (Liu, J., and R. Jemmerson, unpublished observation).


Altered cytochrome c display precedes apoptotic cell death in Drosophila.

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

Immunoprecipita-tion of Drosophila cytochrome c. Western blot analyses were used to visualize  immunoprecipitated proteins that were probed with  anti–cytochrome c mAb 7H8.  Lanes 1 and 2 show whole  cell suspension of healthy  SL2 cells (S) and grim-expressing cells 4 h after induction  (G). Lanes 3 and 4 were precleared lysates used for immunoprecipitation (see Materials and Methods). Lanes 5–8 were immunoprecipitates from SL2 or grim-expressing cell lysates using two  anti–cytochrome c mAbs, 6H2 and 2G8. The same two cytochrome c bands were also observed in lanes 1–4 on shorter exposures.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132929&req=5

Figure 1: Immunoprecipita-tion of Drosophila cytochrome c. Western blot analyses were used to visualize immunoprecipitated proteins that were probed with anti–cytochrome c mAb 7H8. Lanes 1 and 2 show whole cell suspension of healthy SL2 cells (S) and grim-expressing cells 4 h after induction (G). Lanes 3 and 4 were precleared lysates used for immunoprecipitation (see Materials and Methods). Lanes 5–8 were immunoprecipitates from SL2 or grim-expressing cell lysates using two anti–cytochrome c mAbs, 6H2 and 2G8. The same two cytochrome c bands were also observed in lanes 1–4 on shorter exposures.
Mentions: Although two cytochrome c genes, DC4 and DC3, have been described in Drosophila melanogaster (Limbach and Wu, 1985) studies at the level of protein (Inoue et al., 1986) and at the level of RNA (Limbach and Wu, 1985) suggest that DC4, which shows >86% identity with its rat counterpart, is either the predominant or only form of actively expressed product. We screened an existing panel of mAbs, directed against mammalian versions of cytochrome c, as possible probes for in situ analyses of the fly counterpart. The potential utility of these mAbs was assessed by immunoprecipitations of SL2 cell lysates, probed with a third anti–cytochrome c mAb, 7H8, to detect denatured cytochrome c in Western blots. The results in Fig. 1 show that two mAbs, 6H2 (Goshorn et al., 1991) and 2G8 (Mueller and Jemmerson, 1996), recognized Drosophila cytochrome c. Both antibodies detected a doublet that comigrated with mammalian cytochrome c at ∼13 kD. While mAb 2G8 preferentially precipitated the upper band, mAb 6H2 had about equal affinity for both forms of cytochrome c. No obvious correlation between the relative abundance of the two cytochrome c bands and apoptosis was observed (see below). The same banding pattern was also observed when immunoprecipitation was performed using purified Drosophila cytochrome c (Liu, J., and R. Jemmerson, unpublished observation).

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
Related in: MedlinePlus