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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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Expression of a Drp1K38E dominant-negative mutant inhibits p20 induced disruption of the mitochondrial network. (A) Rat1 fibroblasts were transiently transfected with CFP-Drp1 or CFP- Drp1K38E, then either mock infected or infected with Adp20 in the presence of zVAD-fmk. 24 h post-infection cells were fixed, stained with anti-TOM20, and analyzed by fluorescence microscopy. Cells expressing CFP-Drp1 or CFP- Drp1K38E were identified under the cyan filter and are indicated with an arrow. (B) CFP- Drp1K38E inhibits cyt.c release. H1299 cells were treated as in B for 36 h, and immunofluorescence microscopy was used to assess the distribution of cyt.c in cells positive for CFP fluorescence. Shown is the mean ± SD of four independent experiments. (C) H1299 cells were transiently cotransfected with the indicated constructs and 36 h post-transfection cell lysates were collected and processed for DEVDase activity, shown is the mean ± SD of three independent experiments.
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fig7: Expression of a Drp1K38E dominant-negative mutant inhibits p20 induced disruption of the mitochondrial network. (A) Rat1 fibroblasts were transiently transfected with CFP-Drp1 or CFP- Drp1K38E, then either mock infected or infected with Adp20 in the presence of zVAD-fmk. 24 h post-infection cells were fixed, stained with anti-TOM20, and analyzed by fluorescence microscopy. Cells expressing CFP-Drp1 or CFP- Drp1K38E were identified under the cyan filter and are indicated with an arrow. (B) CFP- Drp1K38E inhibits cyt.c release. H1299 cells were treated as in B for 36 h, and immunofluorescence microscopy was used to assess the distribution of cyt.c in cells positive for CFP fluorescence. Shown is the mean ± SD of four independent experiments. (C) H1299 cells were transiently cotransfected with the indicated constructs and 36 h post-transfection cell lysates were collected and processed for DEVDase activity, shown is the mean ± SD of three independent experiments.

Mentions: To confirm that p20 mediates its sensitizing effect on mitochondria through Drp1, we examined the effect of a dominant-negative Drp1 mutant on p20-induced mitochondrial changes. Mutation of a conserved lysine (K38) in the GTP binding domain of Drp1 is predicted to reduce GTPase activity (Smirnova et al., 1998; Bleazard et al., 1999), and expression of such a mutant inhibits OMM scission (Labrousse et al., 1999). Ectopic expression of CFP-Drp1K38E in Rat1 cells offset the normal balance between mitochondrial fission and fusion and increased the connectivity of mitochondria compared with untransfected cells or cells transfected with wild-type CFP-Drp1 (Fig. 7 A, top panels; transfected [CFP-positive] cells are indicated by arrows). Overexpression of wild-type Drp1 does not induce fission in mammalian cells (Smirnova et al., 1998; Frank et al., 2001), and accordingly, Rat1 cells transiently transfected with CFP-Drp1 exhibited a normal mitochondrial phenotype and underwent fragmentation in response to Adp20 (Fig. 7 A). Cells transfected with CFP-Drp1K38E, on the other hand, resisted Adp20-induced mitochondrial fission and the highly interconnected network remained intact. As shown in Fig. 7 (B and C), CFP-Drp1K38E also inhibited p20-induced cyt.c release and caspase activation. Based on morphological criteria, as well as the recruitment of endogenous Drp-1 to mitochondria and dominant interference by the Drp1K38E mutant, we conclude that p20 activates Drp1-dependent mitochondrial fission, sensitizing this organelle for cyt.c release.


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)

Expression of a Drp1K38E dominant-negative mutant inhibits p20 induced disruption of the mitochondrial network. (A) Rat1 fibroblasts were transiently transfected with CFP-Drp1 or CFP- Drp1K38E, then either mock infected or infected with Adp20 in the presence of zVAD-fmk. 24 h post-infection cells were fixed, stained with anti-TOM20, and analyzed by fluorescence microscopy. Cells expressing CFP-Drp1 or CFP- Drp1K38E were identified under the cyan filter and are indicated with an arrow. (B) CFP- Drp1K38E inhibits cyt.c release. H1299 cells were treated as in B for 36 h, and immunofluorescence microscopy was used to assess the distribution of cyt.c in cells positive for CFP fluorescence. Shown is the mean ± SD of four independent experiments. (C) H1299 cells were transiently cotransfected with the indicated constructs and 36 h post-transfection cell lysates were collected and processed for DEVDase activity, shown is the mean ± SD of three independent experiments.
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Related In: Results  -  Collection

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fig7: Expression of a Drp1K38E dominant-negative mutant inhibits p20 induced disruption of the mitochondrial network. (A) Rat1 fibroblasts were transiently transfected with CFP-Drp1 or CFP- Drp1K38E, then either mock infected or infected with Adp20 in the presence of zVAD-fmk. 24 h post-infection cells were fixed, stained with anti-TOM20, and analyzed by fluorescence microscopy. Cells expressing CFP-Drp1 or CFP- Drp1K38E were identified under the cyan filter and are indicated with an arrow. (B) CFP- Drp1K38E inhibits cyt.c release. H1299 cells were treated as in B for 36 h, and immunofluorescence microscopy was used to assess the distribution of cyt.c in cells positive for CFP fluorescence. Shown is the mean ± SD of four independent experiments. (C) H1299 cells were transiently cotransfected with the indicated constructs and 36 h post-transfection cell lysates were collected and processed for DEVDase activity, shown is the mean ± SD of three independent experiments.
Mentions: To confirm that p20 mediates its sensitizing effect on mitochondria through Drp1, we examined the effect of a dominant-negative Drp1 mutant on p20-induced mitochondrial changes. Mutation of a conserved lysine (K38) in the GTP binding domain of Drp1 is predicted to reduce GTPase activity (Smirnova et al., 1998; Bleazard et al., 1999), and expression of such a mutant inhibits OMM scission (Labrousse et al., 1999). Ectopic expression of CFP-Drp1K38E in Rat1 cells offset the normal balance between mitochondrial fission and fusion and increased the connectivity of mitochondria compared with untransfected cells or cells transfected with wild-type CFP-Drp1 (Fig. 7 A, top panels; transfected [CFP-positive] cells are indicated by arrows). Overexpression of wild-type Drp1 does not induce fission in mammalian cells (Smirnova et al., 1998; Frank et al., 2001), and accordingly, Rat1 cells transiently transfected with CFP-Drp1 exhibited a normal mitochondrial phenotype and underwent fragmentation in response to Adp20 (Fig. 7 A). Cells transfected with CFP-Drp1K38E, on the other hand, resisted Adp20-induced mitochondrial fission and the highly interconnected network remained intact. As shown in Fig. 7 (B and C), CFP-Drp1K38E also inhibited p20-induced cyt.c release and caspase activation. Based on morphological criteria, as well as the recruitment of endogenous Drp-1 to mitochondria and dominant interference by the Drp1K38E mutant, we conclude that p20 activates Drp1-dependent mitochondrial fission, sensitizing this organelle for cyt.c release.

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