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The two cytochrome c species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells.

Dorstyn L, Mills K, Lazebnik Y, Kumar S - J. Cell Biol. (2004)

Bottom Line: Here, we report that silencing expression of either or both DC3 and DC4 had no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells.In cell-free studies, recombinant DC3 or DC4 failed to activate caspases in Drosophila cell lysates, but remarkably induced caspase activation in extracts from human cells.Overall, our results argue that DARK-mediated DRONC activation occurs independently of cytochrome c.

View Article: PubMed Central - PubMed

Affiliation: Hanson Institute, Adelaide, Australia 5000.

ABSTRACT
In Drosophila, activation of the apical caspase DRONC requires the apoptotic protease-activating factor homologue, DARK. However, unlike caspase activation in mammals, DRONC activation is not accompanied by the release of cytochrome c from mitochondria. Drosophila encodes two cytochrome c proteins, Cytc-p (DC4) the predominantly expressed species, and Cytc-d (DC3), which is implicated in caspase activation during spermatogenesis. Here, we report that silencing expression of either or both DC3 and DC4 had no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells. We find that loss of function mutations in dc3 and dc4, do not affect caspase activation during Drosophila development and that ectopic expression of DC3 or DC4 in Drosophila cells does not induce caspase activation. In cell-free studies, recombinant DC3 or DC4 failed to activate caspases in Drosophila cell lysates, but remarkably induced caspase activation in extracts from human cells. Overall, our results argue that DARK-mediated DRONC activation occurs independently of cytochrome c.

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Localization of DC3 and DC4 in healthy and apoptotic cells. (A) Vector, dc3-HA-, or dc4-HA-transfected BG2 cells were treated with CHX for 4 h where indicated and costained with MitoTracker green and anti-HA antibody. (B) Untreated or (C) CHX treated BG2 cells transfected with vector alone (top), dc3-HA (middle), or dc4-HA (bottom) were costained with anti-DRICE and anti-HA antibodies. The last column displays merged images showing costaining of DC3 and DC4 with mitochondria (A) and partial colocalization with DRICE (C). Bar, 5 μm.
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fig3: Localization of DC3 and DC4 in healthy and apoptotic cells. (A) Vector, dc3-HA-, or dc4-HA-transfected BG2 cells were treated with CHX for 4 h where indicated and costained with MitoTracker green and anti-HA antibody. (B) Untreated or (C) CHX treated BG2 cells transfected with vector alone (top), dc3-HA (middle), or dc4-HA (bottom) were costained with anti-DRICE and anti-HA antibodies. The last column displays merged images showing costaining of DC3 and DC4 with mitochondria (A) and partial colocalization with DRICE (C). Bar, 5 μm.

Mentions: To verify the results obtained by cell fractionation, we assessed the localization of DC3 and DC4 in these BG2 cell lines. We found that the majority of DC3 and DC4 protein colocalize with MitoTracker, in both live and apoptotic cells (Fig. 3 A), indicating that these proteins remain in the mitochondria during apoptosis. A portion of DC3 was detectable outside of mitochondria in live and apoptotic cells (Fig. 3 A) consistent with its detection in light membrane and cytosol fractions (Fig. 2 D). We used an antibody that detects processed DRICE protein and confirmed that ectopic expression of DC3 or DC4 did not cause DRICE activation. Consistent with our previous observations (Dorstyn et al., 2002) we also found that in cells treated with cycloheximide, active DRICE partially colocalized with mitochondria (Fig. 3 C). However, considering that neither of the cytochromes are required for DRICE activation, the observed association may be unrelated to caspase activation.


The two cytochrome c species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells.

Dorstyn L, Mills K, Lazebnik Y, Kumar S - J. Cell Biol. (2004)

Localization of DC3 and DC4 in healthy and apoptotic cells. (A) Vector, dc3-HA-, or dc4-HA-transfected BG2 cells were treated with CHX for 4 h where indicated and costained with MitoTracker green and anti-HA antibody. (B) Untreated or (C) CHX treated BG2 cells transfected with vector alone (top), dc3-HA (middle), or dc4-HA (bottom) were costained with anti-DRICE and anti-HA antibodies. The last column displays merged images showing costaining of DC3 and DC4 with mitochondria (A) and partial colocalization with DRICE (C). Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172470&req=5

fig3: Localization of DC3 and DC4 in healthy and apoptotic cells. (A) Vector, dc3-HA-, or dc4-HA-transfected BG2 cells were treated with CHX for 4 h where indicated and costained with MitoTracker green and anti-HA antibody. (B) Untreated or (C) CHX treated BG2 cells transfected with vector alone (top), dc3-HA (middle), or dc4-HA (bottom) were costained with anti-DRICE and anti-HA antibodies. The last column displays merged images showing costaining of DC3 and DC4 with mitochondria (A) and partial colocalization with DRICE (C). Bar, 5 μm.
Mentions: To verify the results obtained by cell fractionation, we assessed the localization of DC3 and DC4 in these BG2 cell lines. We found that the majority of DC3 and DC4 protein colocalize with MitoTracker, in both live and apoptotic cells (Fig. 3 A), indicating that these proteins remain in the mitochondria during apoptosis. A portion of DC3 was detectable outside of mitochondria in live and apoptotic cells (Fig. 3 A) consistent with its detection in light membrane and cytosol fractions (Fig. 2 D). We used an antibody that detects processed DRICE protein and confirmed that ectopic expression of DC3 or DC4 did not cause DRICE activation. Consistent with our previous observations (Dorstyn et al., 2002) we also found that in cells treated with cycloheximide, active DRICE partially colocalized with mitochondria (Fig. 3 C). However, considering that neither of the cytochromes are required for DRICE activation, the observed association may be unrelated to caspase activation.

Bottom Line: Here, we report that silencing expression of either or both DC3 and DC4 had no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells.In cell-free studies, recombinant DC3 or DC4 failed to activate caspases in Drosophila cell lysates, but remarkably induced caspase activation in extracts from human cells.Overall, our results argue that DARK-mediated DRONC activation occurs independently of cytochrome c.

View Article: PubMed Central - PubMed

Affiliation: Hanson Institute, Adelaide, Australia 5000.

ABSTRACT
In Drosophila, activation of the apical caspase DRONC requires the apoptotic protease-activating factor homologue, DARK. However, unlike caspase activation in mammals, DRONC activation is not accompanied by the release of cytochrome c from mitochondria. Drosophila encodes two cytochrome c proteins, Cytc-p (DC4) the predominantly expressed species, and Cytc-d (DC3), which is implicated in caspase activation during spermatogenesis. Here, we report that silencing expression of either or both DC3 and DC4 had no effect on apoptosis or activation of DRONC and DRICE in Drosophila cells. We find that loss of function mutations in dc3 and dc4, do not affect caspase activation during Drosophila development and that ectopic expression of DC3 or DC4 in Drosophila cells does not induce caspase activation. In cell-free studies, recombinant DC3 or DC4 failed to activate caspases in Drosophila cell lysates, but remarkably induced caspase activation in extracts from human cells. Overall, our results argue that DARK-mediated DRONC activation occurs independently of cytochrome c.

Show MeSH
Related in: MedlinePlus