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Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals, enhancing cytochrome c release to the cytosol.

Breckenridge DG, Stojanovic M, Marcellus RC, Shore GC - J. Cell Biol. (2003)

Bottom Line: Cell.Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway.Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6.

ABSTRACT
Stimulation of cell surface death receptors activates caspase-8, which targets a limited number of substrates including BAP31, an integral membrane protein of the endoplasmic reticulum (ER). Recently, we reported that a caspase-resistant BAP31 mutant inhibited several features of Fas-induced apoptosis, including the release of cytochrome c (cyt.c) from mitochondria (Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Mol. Cell. Biol. 20:6731-6740), implicating ER-mitochondria crosstalk in this pathway. Here, we report that the p20 caspase cleavage fragment of BAP31 can direct pro-apoptotic signals between the ER and mitochondria. Adenoviral expression of p20 caused an early release of Ca2+ from the ER, concomitant uptake of Ca2+ into mitochondria, and mitochondrial recruitment of Drp1, a dynamin-related protein that mediates scission of the outer mitochondrial membrane, resulting in dramatic fragmentation and fission of the mitochondrial network. Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway. Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.

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Drp1 mediates p20-induced mitochondrial fission. (A) Recruitment of endogenous Drp1 to mitochondria. HeLa cells were mock infected (top) or infected with Adp20 (bottom) in the presence of zVAD-fmk, and 17 h post-infection, cells were fixed, double stained with anti-Drp1 (green) and anti-TOM20 (red) antibodies, and imaged by confocal immunofluorescence microscopy. Enlargement of the merged overlay revealed that clusters of Drp1 relocate along mitochondrial filaments before the onset of fission. (B) H1299 cells, H1299 cells treated with TG, or H1299 b5-BCL-2 cells were infected with Adp20+zVAD for 18 h and the mitochondrial fraction was isolated and analyzed for the presence of Drp1 by SDS-PAGE and immunoblotting. The blot was reprobed with anti-TOM20 antibody to demonstrate equal protein loading.
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fig6: Drp1 mediates p20-induced mitochondrial fission. (A) Recruitment of endogenous Drp1 to mitochondria. HeLa cells were mock infected (top) or infected with Adp20 (bottom) in the presence of zVAD-fmk, and 17 h post-infection, cells were fixed, double stained with anti-Drp1 (green) and anti-TOM20 (red) antibodies, and imaged by confocal immunofluorescence microscopy. Enlargement of the merged overlay revealed that clusters of Drp1 relocate along mitochondrial filaments before the onset of fission. (B) H1299 cells, H1299 cells treated with TG, or H1299 b5-BCL-2 cells were infected with Adp20+zVAD for 18 h and the mitochondrial fraction was isolated and analyzed for the presence of Drp1 by SDS-PAGE and immunoblotting. The blot was reprobed with anti-TOM20 antibody to demonstrate equal protein loading.

Mentions: Recently, fragmentation of mitochondria during staurosporin-induced apoptosis was demonstrated to be dependent on mitochondrial fission mediated by Drp1 (Frank et al., 2001). Therefore, we investigated whether Drp1-dependent fission played a role in the p20 pathway. We began by examining the subcellular localization of Drp1 because GFP-tagged Drp1 was shown to redistribute from a predominately cytosolic location to predicted sites of division along mitochondrial tubules after treatment with staurosporin (Frank et al., 2001). Fig. 6 A documents that endogenous Drp1 was recruited to mitochondria before the onset of mitochondrial fragmentation in HeLa cells treated with Adp20. HeLa cells were used for this experiment because their mitochondria form long, clearly defined tubules ideal for colocalization studies; however, the results were also confirmed in Rat1 and H1299 cells. In mock-infected HeLa cells, Drp1 was distributed throughout the cytosol and showed only minor colocalization with mitochondria stained with TOM20 (Fig. 6 A), likely because Drp1 normally cycles on and off mitochondria continuously (Frank et al., 2001; Smirnova et al., 2001). In contrast, Drp1 showed a strong colocalization with mitochondria in HeLa cells infected with Adp20 + zVAD-fmk for 17 h (Fig. 6 A, bottom panels). Enlargement of the merged image revealed that Drp1 formed clusters along the surface of mitochondrial tubules before the onset of fragmentation. Interestingly, in Caenorhabditis elegans, similar clusters of GFP-Drp1 on mitochondrial tubules were shown to coincide with future sites of membrane scission (Labrousse et al., 1999). Pretreatment of H1299 cells with TG or expression of b5-BCL-2 reduced the amount of endogenous Drp1 recovered in the mitochondrial fraction after p20 expression (Fig. 6 B, compare lane 2 with lanes 3 and 4) and inhibited mitochondrial fission (Fig. 4 C).


Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals, enhancing cytochrome c release to the cytosol.

Breckenridge DG, Stojanovic M, Marcellus RC, Shore GC - J. Cell Biol. (2003)

Drp1 mediates p20-induced mitochondrial fission. (A) Recruitment of endogenous Drp1 to mitochondria. HeLa cells were mock infected (top) or infected with Adp20 (bottom) in the presence of zVAD-fmk, and 17 h post-infection, cells were fixed, double stained with anti-Drp1 (green) and anti-TOM20 (red) antibodies, and imaged by confocal immunofluorescence microscopy. Enlargement of the merged overlay revealed that clusters of Drp1 relocate along mitochondrial filaments before the onset of fission. (B) H1299 cells, H1299 cells treated with TG, or H1299 b5-BCL-2 cells were infected with Adp20+zVAD for 18 h and the mitochondrial fraction was isolated and analyzed for the presence of Drp1 by SDS-PAGE and immunoblotting. The blot was reprobed with anti-TOM20 antibody to demonstrate equal protein loading.
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fig6: Drp1 mediates p20-induced mitochondrial fission. (A) Recruitment of endogenous Drp1 to mitochondria. HeLa cells were mock infected (top) or infected with Adp20 (bottom) in the presence of zVAD-fmk, and 17 h post-infection, cells were fixed, double stained with anti-Drp1 (green) and anti-TOM20 (red) antibodies, and imaged by confocal immunofluorescence microscopy. Enlargement of the merged overlay revealed that clusters of Drp1 relocate along mitochondrial filaments before the onset of fission. (B) H1299 cells, H1299 cells treated with TG, or H1299 b5-BCL-2 cells were infected with Adp20+zVAD for 18 h and the mitochondrial fraction was isolated and analyzed for the presence of Drp1 by SDS-PAGE and immunoblotting. The blot was reprobed with anti-TOM20 antibody to demonstrate equal protein loading.
Mentions: Recently, fragmentation of mitochondria during staurosporin-induced apoptosis was demonstrated to be dependent on mitochondrial fission mediated by Drp1 (Frank et al., 2001). Therefore, we investigated whether Drp1-dependent fission played a role in the p20 pathway. We began by examining the subcellular localization of Drp1 because GFP-tagged Drp1 was shown to redistribute from a predominately cytosolic location to predicted sites of division along mitochondrial tubules after treatment with staurosporin (Frank et al., 2001). Fig. 6 A documents that endogenous Drp1 was recruited to mitochondria before the onset of mitochondrial fragmentation in HeLa cells treated with Adp20. HeLa cells were used for this experiment because their mitochondria form long, clearly defined tubules ideal for colocalization studies; however, the results were also confirmed in Rat1 and H1299 cells. In mock-infected HeLa cells, Drp1 was distributed throughout the cytosol and showed only minor colocalization with mitochondria stained with TOM20 (Fig. 6 A), likely because Drp1 normally cycles on and off mitochondria continuously (Frank et al., 2001; Smirnova et al., 2001). In contrast, Drp1 showed a strong colocalization with mitochondria in HeLa cells infected with Adp20 + zVAD-fmk for 17 h (Fig. 6 A, bottom panels). Enlargement of the merged image revealed that Drp1 formed clusters along the surface of mitochondrial tubules before the onset of fragmentation. Interestingly, in Caenorhabditis elegans, similar clusters of GFP-Drp1 on mitochondrial tubules were shown to coincide with future sites of membrane scission (Labrousse et al., 1999). Pretreatment of H1299 cells with TG or expression of b5-BCL-2 reduced the amount of endogenous Drp1 recovered in the mitochondrial fraction after p20 expression (Fig. 6 B, compare lane 2 with lanes 3 and 4) and inhibited mitochondrial fission (Fig. 4 C).

Bottom Line: Cell.Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway.Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6.

ABSTRACT
Stimulation of cell surface death receptors activates caspase-8, which targets a limited number of substrates including BAP31, an integral membrane protein of the endoplasmic reticulum (ER). Recently, we reported that a caspase-resistant BAP31 mutant inhibited several features of Fas-induced apoptosis, including the release of cytochrome c (cyt.c) from mitochondria (Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Mol. Cell. Biol. 20:6731-6740), implicating ER-mitochondria crosstalk in this pathway. Here, we report that the p20 caspase cleavage fragment of BAP31 can direct pro-apoptotic signals between the ER and mitochondria. Adenoviral expression of p20 caused an early release of Ca2+ from the ER, concomitant uptake of Ca2+ into mitochondria, and mitochondrial recruitment of Drp1, a dynamin-related protein that mediates scission of the outer mitochondrial membrane, resulting in dramatic fragmentation and fission of the mitochondrial network. Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway. Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.

Show MeSH
Related in: MedlinePlus