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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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p20-induced mitochondrial fragmentation is mediated by an ER-mitochondria Ca2+ signal. (A) Top, p20 induces a time dependent release of Ca2+ from the ER. H1299 cells were infected with Adp20 in the presence of 50 μM zVAD-fmk and at the indicated times post-infection, cells were loaded with Fura2-AM in Ca2+-free buffer and ER calcium stores were measured as the sudden difference in Fura2 fluorescence recorded after the addition of TG (see Materials and methods). Shown is the mean and SD of five independent experiments. Bottom, elevated [Ca2+]m after Adp20 infection. HeLa cells were treated as in A, except cells were loaded with Rhod2-AM and the [Ca2+]m was estimated as described in the Materials and methods. (B) p20 induces dramatic fragmentation of mitochondria, which is inhibited by predepletion of ER Ca2+ stores with TG. H1299 cells were infected with 50 μM Adp20 + zVAD-fmk in the absence or presence of 50 nm TG for 24 h, and mitochondria were visualized by anti-cyt.c staining. Representative images are shown. (C) Reducing ER Ca2+ stores, chelating cytosolic Ca2+, or preventing mitochondrial Ca2+ uptake inhibits p20-induced fragmentation of mitochondria. As in B, but H1229 cells, or H1299 cells pretreated with 50 nm TG, 2 μM BAPTA-AM, or 20 μM Ru360, or H1299 b5-BCL-2 cells were infected with Adp20 + zVAD-fmk for 24 h and the number of cells showing signs of mitochondrial fragmentation was quantified. Shown is the mean ± SD of five independent experiments.
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fig4: p20-induced mitochondrial fragmentation is mediated by an ER-mitochondria Ca2+ signal. (A) Top, p20 induces a time dependent release of Ca2+ from the ER. H1299 cells were infected with Adp20 in the presence of 50 μM zVAD-fmk and at the indicated times post-infection, cells were loaded with Fura2-AM in Ca2+-free buffer and ER calcium stores were measured as the sudden difference in Fura2 fluorescence recorded after the addition of TG (see Materials and methods). Shown is the mean and SD of five independent experiments. Bottom, elevated [Ca2+]m after Adp20 infection. HeLa cells were treated as in A, except cells were loaded with Rhod2-AM and the [Ca2+]m was estimated as described in the Materials and methods. (B) p20 induces dramatic fragmentation of mitochondria, which is inhibited by predepletion of ER Ca2+ stores with TG. H1299 cells were infected with 50 μM Adp20 + zVAD-fmk in the absence or presence of 50 nm TG for 24 h, and mitochondria were visualized by anti-cyt.c staining. Representative images are shown. (C) Reducing ER Ca2+ stores, chelating cytosolic Ca2+, or preventing mitochondrial Ca2+ uptake inhibits p20-induced fragmentation of mitochondria. As in B, but H1229 cells, or H1299 cells pretreated with 50 nm TG, 2 μM BAPTA-AM, or 20 μM Ru360, or H1299 b5-BCL-2 cells were infected with Adp20 + zVAD-fmk for 24 h and the number of cells showing signs of mitochondrial fragmentation was quantified. Shown is the mean ± SD of five independent experiments.

Mentions: Next, we sought to identify the early ER signaling events after p20 expression. Release of Ca2+ from the ER occurs as an early event during many forms of apoptosis, including the Fas pathway, and Ca2+ has been implicated as a second messenger between ER and mitochondria during apoptosis (Hajnoczky et al., 2000; Breckenridge and Shore, 2002). We tested whether p20 expression altered ER Ca2+ homeostasis by loading Adp20-infected cells with the Ca2+-sensitive fluorescent indicator Fura2-AM and measuring the increase in cytosolic Ca2+ that results from thapsigargin (TG)-induced depletion of ER stores. TG invokes a rapid emptying of ER Ca2+ stores to the cytosol by irreversibly inhibiting SERCA pumps that normally maintain the concentration of ER Ca2+ ([Ca2+]ER) several orders of magnitude above that of the cytosol ([Ca2+]c). Fig. 4 A reveals that expression of p20 in H1299 cells in the presence of zVAD-fmk caused an early, time-dependent decrease in ER Ca2+ stores. The kinetics of ER Ca2+ release was concomitant with an increase in the concentration of mitochondrial Ca2+ ([Ca2+]m), measured by Rhod2 fluorescence. These changes in ER and mitochondrial Ca2+ levels could be measured as early as 12–14 h post-infection (i.e., 2–4 h after p20 protein appears; Fig. 2 A), making them the earliest events we observed in the p20 pathway.


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-induced mitochondrial fragmentation is mediated by an ER-mitochondria Ca2+ signal. (A) Top, p20 induces a time dependent release of Ca2+ from the ER. H1299 cells were infected with Adp20 in the presence of 50 μM zVAD-fmk and at the indicated times post-infection, cells were loaded with Fura2-AM in Ca2+-free buffer and ER calcium stores were measured as the sudden difference in Fura2 fluorescence recorded after the addition of TG (see Materials and methods). Shown is the mean and SD of five independent experiments. Bottom, elevated [Ca2+]m after Adp20 infection. HeLa cells were treated as in A, except cells were loaded with Rhod2-AM and the [Ca2+]m was estimated as described in the Materials and methods. (B) p20 induces dramatic fragmentation of mitochondria, which is inhibited by predepletion of ER Ca2+ stores with TG. H1299 cells were infected with 50 μM Adp20 + zVAD-fmk in the absence or presence of 50 nm TG for 24 h, and mitochondria were visualized by anti-cyt.c staining. Representative images are shown. (C) Reducing ER Ca2+ stores, chelating cytosolic Ca2+, or preventing mitochondrial Ca2+ uptake inhibits p20-induced fragmentation of mitochondria. As in B, but H1229 cells, or H1299 cells pretreated with 50 nm TG, 2 μM BAPTA-AM, or 20 μM Ru360, or H1299 b5-BCL-2 cells were infected with Adp20 + zVAD-fmk for 24 h and the number of cells showing signs of mitochondrial fragmentation was quantified. Shown is the mean ± SD of five independent experiments.
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Related In: Results  -  Collection

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fig4: p20-induced mitochondrial fragmentation is mediated by an ER-mitochondria Ca2+ signal. (A) Top, p20 induces a time dependent release of Ca2+ from the ER. H1299 cells were infected with Adp20 in the presence of 50 μM zVAD-fmk and at the indicated times post-infection, cells were loaded with Fura2-AM in Ca2+-free buffer and ER calcium stores were measured as the sudden difference in Fura2 fluorescence recorded after the addition of TG (see Materials and methods). Shown is the mean and SD of five independent experiments. Bottom, elevated [Ca2+]m after Adp20 infection. HeLa cells were treated as in A, except cells were loaded with Rhod2-AM and the [Ca2+]m was estimated as described in the Materials and methods. (B) p20 induces dramatic fragmentation of mitochondria, which is inhibited by predepletion of ER Ca2+ stores with TG. H1299 cells were infected with 50 μM Adp20 + zVAD-fmk in the absence or presence of 50 nm TG for 24 h, and mitochondria were visualized by anti-cyt.c staining. Representative images are shown. (C) Reducing ER Ca2+ stores, chelating cytosolic Ca2+, or preventing mitochondrial Ca2+ uptake inhibits p20-induced fragmentation of mitochondria. As in B, but H1229 cells, or H1299 cells pretreated with 50 nm TG, 2 μM BAPTA-AM, or 20 μM Ru360, or H1299 b5-BCL-2 cells were infected with Adp20 + zVAD-fmk for 24 h and the number of cells showing signs of mitochondrial fragmentation was quantified. Shown is the mean ± SD of five independent experiments.
Mentions: Next, we sought to identify the early ER signaling events after p20 expression. Release of Ca2+ from the ER occurs as an early event during many forms of apoptosis, including the Fas pathway, and Ca2+ has been implicated as a second messenger between ER and mitochondria during apoptosis (Hajnoczky et al., 2000; Breckenridge and Shore, 2002). We tested whether p20 expression altered ER Ca2+ homeostasis by loading Adp20-infected cells with the Ca2+-sensitive fluorescent indicator Fura2-AM and measuring the increase in cytosolic Ca2+ that results from thapsigargin (TG)-induced depletion of ER stores. TG invokes a rapid emptying of ER Ca2+ stores to the cytosol by irreversibly inhibiting SERCA pumps that normally maintain the concentration of ER Ca2+ ([Ca2+]ER) several orders of magnitude above that of the cytosol ([Ca2+]c). Fig. 4 A reveals that expression of p20 in H1299 cells in the presence of zVAD-fmk caused an early, time-dependent decrease in ER Ca2+ stores. The kinetics of ER Ca2+ release was concomitant with an increase in the concentration of mitochondrial Ca2+ ([Ca2+]m), measured by Rhod2 fluorescence. These changes in ER and mitochondrial Ca2+ levels could be measured as early as 12–14 h post-infection (i.e., 2–4 h after p20 protein appears; Fig. 2 A), making them the earliest events we observed in the p20 pathway.

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