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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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Labeling for cytochrome c precedes programmed cell  death in ovaries. Drosophila egg chambers were examined with  mAb 2G8. (A) The mAb labels cytochrome c specifically in nurse  cells at stage 10B. Characteristically punctate staining is found  throughout the nurse cell cytoplasm only at this stage. (B) Single  egg chamber magnified (brackets denote stage 10B egg chambers). Note that earlier stages are not stained. (C) With mAb  2G8 preadsorbed on cytochrome c–Sepharose 4B, this stage did  not exhibit labeling. Cytoplasmic and nuclear integrity of nurse  cells at this stage was substantiated by the cytoplasmic marker,  rhodamine phalloidin, and nuclear staining technique, TUNEL  (D and E), respectively (see text). Apoptotic nuclei, labeled by  TUNEL, are evident at a later stage in oogenesis (G), when no  cytochrome c labeling could be seen (F).
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Figure 2: Labeling for cytochrome c precedes programmed cell death in ovaries. Drosophila egg chambers were examined with mAb 2G8. (A) The mAb labels cytochrome c specifically in nurse cells at stage 10B. Characteristically punctate staining is found throughout the nurse cell cytoplasm only at this stage. (B) Single egg chamber magnified (brackets denote stage 10B egg chambers). Note that earlier stages are not stained. (C) With mAb 2G8 preadsorbed on cytochrome c–Sepharose 4B, this stage did not exhibit labeling. Cytoplasmic and nuclear integrity of nurse cells at this stage was substantiated by the cytoplasmic marker, rhodamine phalloidin, and nuclear staining technique, TUNEL (D and E), respectively (see text). Apoptotic nuclei, labeled by TUNEL, are evident at a later stage in oogenesis (G), when no cytochrome c labeling could be seen (F).

Mentions: To directly demonstrate the involvement of cytochrome c during apoptosis, Drosophila ovaries were stained with the anti–cytochrome c mAbs described above and egg chambers at all stages of development were analyzed. In preliminary experiments, mAb 2G8 detected cytochrome c in apoptotic cells prompting extensive studies with this mAb. As shown in Fig. 2, A and B, only nurse cells at stage 10B exhibited pronounced cytochrome c immunoreactivity distributed as characteristically punctate labeling of the cytoplasm in a pattern consistent with localization to mitochondria (see Fig. 4 B). Such staining was not seen in nurse cells before this stage, nor was this overt immunoreactivity seen in any other cell type of the Drosophila egg chamber. Moreover, this staining was specific for cytochrome c immunoreactivity because no labeling of nurse cells (Fig. 2 C) was observed if the antibody was first preadsorbed with cytochrome c covalently bound to Sepharose 4B (Urbanski and Margoliash, 1977).


Altered cytochrome c display precedes apoptotic cell death in Drosophila.

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

Labeling for cytochrome c precedes programmed cell  death in ovaries. Drosophila egg chambers were examined with  mAb 2G8. (A) The mAb labels cytochrome c specifically in nurse  cells at stage 10B. Characteristically punctate staining is found  throughout the nurse cell cytoplasm only at this stage. (B) Single  egg chamber magnified (brackets denote stage 10B egg chambers). Note that earlier stages are not stained. (C) With mAb  2G8 preadsorbed on cytochrome c–Sepharose 4B, this stage did  not exhibit labeling. Cytoplasmic and nuclear integrity of nurse  cells at this stage was substantiated by the cytoplasmic marker,  rhodamine phalloidin, and nuclear staining technique, TUNEL  (D and E), respectively (see text). Apoptotic nuclei, labeled by  TUNEL, are evident at a later stage in oogenesis (G), when no  cytochrome c labeling could be seen (F).
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Related In: Results  -  Collection

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Figure 2: Labeling for cytochrome c precedes programmed cell death in ovaries. Drosophila egg chambers were examined with mAb 2G8. (A) The mAb labels cytochrome c specifically in nurse cells at stage 10B. Characteristically punctate staining is found throughout the nurse cell cytoplasm only at this stage. (B) Single egg chamber magnified (brackets denote stage 10B egg chambers). Note that earlier stages are not stained. (C) With mAb 2G8 preadsorbed on cytochrome c–Sepharose 4B, this stage did not exhibit labeling. Cytoplasmic and nuclear integrity of nurse cells at this stage was substantiated by the cytoplasmic marker, rhodamine phalloidin, and nuclear staining technique, TUNEL (D and E), respectively (see text). Apoptotic nuclei, labeled by TUNEL, are evident at a later stage in oogenesis (G), when no cytochrome c labeling could be seen (F).
Mentions: To directly demonstrate the involvement of cytochrome c during apoptosis, Drosophila ovaries were stained with the anti–cytochrome c mAbs described above and egg chambers at all stages of development were analyzed. In preliminary experiments, mAb 2G8 detected cytochrome c in apoptotic cells prompting extensive studies with this mAb. As shown in Fig. 2, A and B, only nurse cells at stage 10B exhibited pronounced cytochrome c immunoreactivity distributed as characteristically punctate labeling of the cytoplasm in a pattern consistent with localization to mitochondria (see Fig. 4 B). Such staining was not seen in nurse cells before this stage, nor was this overt immunoreactivity seen in any other cell type of the Drosophila egg chamber. Moreover, this staining was specific for cytochrome c immunoreactivity because no labeling of nurse cells (Fig. 2 C) was observed if the antibody was first preadsorbed with cytochrome c covalently bound to Sepharose 4B (Urbanski and Margoliash, 1977).

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