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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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Distribution of Drosophila cytochrome c during  apoptosis. (A) Healthy SL2  cells and apoptotic rpr- or  grim-expressing cells were  fractionated 4 h after induction (see Materials and Methods). Cytochrome c in the total cell lysates (lanes 1–3),  heavy membrane fractions  (lanes 4–6), and S-100 cytosol  (lanes 7–9) was visualized by  Western blot analyses using  mAb 7H8. Note that cytochrome c efflux into the cytosol of apoptotic cells was not  observed, and that cytochrome c was substantially  enriched in heavy membrane  fractions which contain mitochondria. (B) Two separate  panels of confocal immunofluorescence microscopy images showing the colocalization of mitochondria and 2G8  immunoreactivity. Cells were  incubated in 50 nM rhodamine 123 before fixing (see  Materials and Methods) and  viewed for rhodamine and  fluorescein. Coincidence is  indicated by absence of green only labeling. (C) High magnification view of a single cell showing punctate 2G8 immunoreactivity, visualized by conventional microscopy.
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Figure 4: Distribution of Drosophila cytochrome c during apoptosis. (A) Healthy SL2 cells and apoptotic rpr- or grim-expressing cells were fractionated 4 h after induction (see Materials and Methods). Cytochrome c in the total cell lysates (lanes 1–3), heavy membrane fractions (lanes 4–6), and S-100 cytosol (lanes 7–9) was visualized by Western blot analyses using mAb 7H8. Note that cytochrome c efflux into the cytosol of apoptotic cells was not observed, and that cytochrome c was substantially enriched in heavy membrane fractions which contain mitochondria. (B) Two separate panels of confocal immunofluorescence microscopy images showing the colocalization of mitochondria and 2G8 immunoreactivity. Cells were incubated in 50 nM rhodamine 123 before fixing (see Materials and Methods) and viewed for rhodamine and fluorescein. Coincidence is indicated by absence of green only labeling. (C) High magnification view of a single cell showing punctate 2G8 immunoreactivity, visualized by conventional microscopy.

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)

Distribution of Drosophila cytochrome c during  apoptosis. (A) Healthy SL2  cells and apoptotic rpr- or  grim-expressing cells were  fractionated 4 h after induction (see Materials and Methods). Cytochrome c in the total cell lysates (lanes 1–3),  heavy membrane fractions  (lanes 4–6), and S-100 cytosol  (lanes 7–9) was visualized by  Western blot analyses using  mAb 7H8. Note that cytochrome c efflux into the cytosol of apoptotic cells was not  observed, and that cytochrome c was substantially  enriched in heavy membrane  fractions which contain mitochondria. (B) Two separate  panels of confocal immunofluorescence microscopy images showing the colocalization of mitochondria and 2G8  immunoreactivity. Cells were  incubated in 50 nM rhodamine 123 before fixing (see  Materials and Methods) and  viewed for rhodamine and  fluorescein. Coincidence is  indicated by absence of green only labeling. (C) High magnification view of a single cell showing punctate 2G8 immunoreactivity, visualized by conventional microscopy.
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Figure 4: Distribution of Drosophila cytochrome c during apoptosis. (A) Healthy SL2 cells and apoptotic rpr- or grim-expressing cells were fractionated 4 h after induction (see Materials and Methods). Cytochrome c in the total cell lysates (lanes 1–3), heavy membrane fractions (lanes 4–6), and S-100 cytosol (lanes 7–9) was visualized by Western blot analyses using mAb 7H8. Note that cytochrome c efflux into the cytosol of apoptotic cells was not observed, and that cytochrome c was substantially enriched in heavy membrane fractions which contain mitochondria. (B) Two separate panels of confocal immunofluorescence microscopy images showing the colocalization of mitochondria and 2G8 immunoreactivity. Cells were incubated in 50 nM rhodamine 123 before fixing (see Materials and Methods) and viewed for rhodamine and fluorescein. Coincidence is indicated by absence of green only labeling. (C) High magnification view of a single cell showing punctate 2G8 immunoreactivity, visualized by conventional microscopy.
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
Related in: MedlinePlus