Limits...
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.

Show MeSH

Related in: MedlinePlus

p20 induces fragmentation of the mitochondrial network as an early event. (A) Mitochondrial restructuring and fragmentation occur in the absence of cell shrinkage. Rat1 fibroblasts were infected with Adp20 in the presence of 50 μM zVAD-fmk (to prevent caspase activation and cell detachment) for 20 h, fixed, and double stained with anti-tubulin and anti-TOM20 antibodies. (B) Mitochondrial fragmentation occurs before activation of BAX and cyt.c release. As in A, except cells were infected for 25 h and double stained with anti-cyt.c antibody (arrows, lower left) and the active conformation-specific anti-BAX-NT antibody (aa 1–21, Upstate Biotechnology; arrows, lower right).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172754&req=5

fig5: p20 induces fragmentation of the mitochondrial network as an early event. (A) Mitochondrial restructuring and fragmentation occur in the absence of cell shrinkage. Rat1 fibroblasts were infected with Adp20 in the presence of 50 μM zVAD-fmk (to prevent caspase activation and cell detachment) for 20 h, fixed, and double stained with anti-tubulin and anti-TOM20 antibodies. (B) Mitochondrial fragmentation occurs before activation of BAX and cyt.c release. As in A, except cells were infected for 25 h and double stained with anti-cyt.c antibody (arrows, lower left) and the active conformation-specific anti-BAX-NT antibody (aa 1–21, Upstate Biotechnology; arrows, lower right).

Mentions: The observation that p20 caused mitochondrial fragmentation was extended in Fig. 5. p20 induced an early fragmentation of the mitochondrial network into small punctiform organelles in all cell types tested, including H1299, Rat1, and HeLa cells (Figs. 4 and 5; unpublished data). The gross morphological changes in the mitochondrial network could be observed 15–16 h after Adp20 infection (i.e., 2–3 h after the onset of Ca2+ release), a time when p20 sensitized mitochondria to caspase-8–induced cyt.c release (Fig. 3). Induction of mitochondrial fragmentation by p20 occurred in the absence of zVAD-fmk–sensitive caspase activation and cell shrinkage or disruption of microtubules. For example, Fig. 5 A shows that Rat1 fibroblasts expressing p20 + zVAD-fmk for 20 h and costained with anti-tubulin and anti-TOM20 antibodies displayed a normal microtubule distribution despite having fragmented mitochondria. The punctiform mitochondria could be observed in living cells stained with MitoTracker® Red (unpublished data), indicating that fragmented mitochondria maintain membrane potential and were not an artifact of fixation. Co-staining of Rat1 fibroblasts expressing p20 + zVAD-fmk with antibody to cyt.c and antibody selective for the active conformation of BAX (Desagher et al., 1999) revealed that the transition of mitochondria into punctiform organelles preceded cyt.c release and activation of BAX. As exemplified in Fig. 5 B, most cells expressing p20 + zVAD-fmk for 25 h displayed fragmented mitochondria but showed no signs of cyt.c release or BAX conformation-specific immunoreactivity. BAX immunoreactivity could only be observed in apoptotic cells that had released cyt.c from mitochondria, and all cells that had undergone cyt.c release stained positive for BAX. These results suggest that p20 induces early fragmentation of mitochondria, which precedes BAX activation and cyt.c release. Given that disintegration of the mitochondrial network has been demonstrated to contribute to apoptotic progression (Desagher and Martinou, 2000; Frank et al., 2001) but that BAX/BAK activation and cyt.c release are normally stimulated by BH3-only molecules (Korsmeyer et al., 2000), it is likely that p20 mediates its sensitizing effect by inducing early fragmentation of mitochondria.


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)

p20 induces fragmentation of the mitochondrial network as an early event. (A) Mitochondrial restructuring and fragmentation occur in the absence of cell shrinkage. Rat1 fibroblasts were infected with Adp20 in the presence of 50 μM zVAD-fmk (to prevent caspase activation and cell detachment) for 20 h, fixed, and double stained with anti-tubulin and anti-TOM20 antibodies. (B) Mitochondrial fragmentation occurs before activation of BAX and cyt.c release. As in A, except cells were infected for 25 h and double stained with anti-cyt.c antibody (arrows, lower left) and the active conformation-specific anti-BAX-NT antibody (aa 1–21, Upstate Biotechnology; arrows, lower right).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172754&req=5

fig5: p20 induces fragmentation of the mitochondrial network as an early event. (A) Mitochondrial restructuring and fragmentation occur in the absence of cell shrinkage. Rat1 fibroblasts were infected with Adp20 in the presence of 50 μM zVAD-fmk (to prevent caspase activation and cell detachment) for 20 h, fixed, and double stained with anti-tubulin and anti-TOM20 antibodies. (B) Mitochondrial fragmentation occurs before activation of BAX and cyt.c release. As in A, except cells were infected for 25 h and double stained with anti-cyt.c antibody (arrows, lower left) and the active conformation-specific anti-BAX-NT antibody (aa 1–21, Upstate Biotechnology; arrows, lower right).
Mentions: The observation that p20 caused mitochondrial fragmentation was extended in Fig. 5. p20 induced an early fragmentation of the mitochondrial network into small punctiform organelles in all cell types tested, including H1299, Rat1, and HeLa cells (Figs. 4 and 5; unpublished data). The gross morphological changes in the mitochondrial network could be observed 15–16 h after Adp20 infection (i.e., 2–3 h after the onset of Ca2+ release), a time when p20 sensitized mitochondria to caspase-8–induced cyt.c release (Fig. 3). Induction of mitochondrial fragmentation by p20 occurred in the absence of zVAD-fmk–sensitive caspase activation and cell shrinkage or disruption of microtubules. For example, Fig. 5 A shows that Rat1 fibroblasts expressing p20 + zVAD-fmk for 20 h and costained with anti-tubulin and anti-TOM20 antibodies displayed a normal microtubule distribution despite having fragmented mitochondria. The punctiform mitochondria could be observed in living cells stained with MitoTracker® Red (unpublished data), indicating that fragmented mitochondria maintain membrane potential and were not an artifact of fixation. Co-staining of Rat1 fibroblasts expressing p20 + zVAD-fmk with antibody to cyt.c and antibody selective for the active conformation of BAX (Desagher et al., 1999) revealed that the transition of mitochondria into punctiform organelles preceded cyt.c release and activation of BAX. As exemplified in Fig. 5 B, most cells expressing p20 + zVAD-fmk for 25 h displayed fragmented mitochondria but showed no signs of cyt.c release or BAX conformation-specific immunoreactivity. BAX immunoreactivity could only be observed in apoptotic cells that had released cyt.c from mitochondria, and all cells that had undergone cyt.c release stained positive for BAX. These results suggest that p20 induces early fragmentation of mitochondria, which precedes BAX activation and cyt.c release. Given that disintegration of the mitochondrial network has been demonstrated to contribute to apoptotic progression (Desagher and Martinou, 2000; Frank et al., 2001) but that BAX/BAK activation and cyt.c release are normally stimulated by BH3-only molecules (Korsmeyer et al., 2000), it is likely that p20 mediates its sensitizing effect by inducing early fragmentation of mitochondria.

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