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Bcl-X(L) specifically activates Bak to induce swelling and restructuring of the endoplasmic reticulum.

Klee M, Pimentel-Muiños FX - J. Cell Biol. (2005)

Bottom Line: Experiments with Bak and Bax-deficient mouse embryonic fibroblasts show that endogenous Bak mediates the effect, whereas Bax is mainly irrelevant.These results reveal a previously unidentified role of Bak in regulating reticular conformation.Because this activity is absent in Bax, it constitutes one of the first examples of functional divergence between the two multidomain homologues.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación del Cáncer, Universidad de Salamanca-CSIC, Salamanca, 37007 Spain.

ABSTRACT
Bcl-2 family members Bak and Bax constitute a mitochondrial gateway for multiple death pathways. Both proteins are also present in the endoplasmic reticulum where they control apoptosis through the regulation of calcium levels. We show here that reticular Bak has the additional capacity of modulating the structure of this organelle. Coexpression of Bak and Bcl-X(L) provokes extensive swelling and vacuolization of reticular cisternae. A Bak version lacking the BH3 domain suffices to induce this phenotype, and reticular targeting of this mutant retains the activity. Expression of upstream BH3-only activators in similar conditions recapitulates ER swelling and vacuolization if ryanodine receptor calcium channel activity is inhibited. Experiments with Bak and Bax-deficient mouse embryonic fibroblasts show that endogenous Bak mediates the effect, whereas Bax is mainly irrelevant. These results reveal a previously unidentified role of Bak in regulating reticular conformation. Because this activity is absent in Bax, it constitutes one of the first examples of functional divergence between the two multidomain homologues.

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Involvement of RyR calcium channel activity in the regulation of cytoplasmic vacuolization induced by Bak. (A) Treatment with dantrolene potentiates vacuolization. 293T cells were transfected with a mix of the indicated plasmids and a plasmid expressing GFP. Where indicated, dantrolene (50 μM) was added 1 h after transfection. 36 h after transfection, cells were fixed, mounted, and analyzed by confocal microscopy. Shown are representative fields. The same magnification was used for both control and treated cells. (B) Caffeine treatment reduces vacuolization. 293T cells were transfected as in A. 36 h later, caffeine (20 mM) was added as indicated for 3.5 h, and cells were subsequently fixed and mounted. Quantitation of vacuolated cells was done as in Fig. 1 B. (C) Swollen ER cisternae and normal ER show similar calcium concentrations. 293T cells were transfected with the indicated plasmids in the presence of plasmids expressing cytosolic RFP and a reticular ratiometric calcium indicator (erYC3.3). 36 h after transfection, cells were analyzed in vivo by confocal microscopy to obtain FRET intensities of erYC3.3. In the top panel, RFP was used to locate a vacuolated cell in proximity to an unvacuolated neighbor with a similar level of transfection, and emission FRET ratios (535 nm/480 nm) were obtained for both cells. Shown are emissions in red (RFP), green (erYC3.3, 535 nm), and FRET ratios. The middle panel shows a profile of FRET intensities across the indicated yellow line, including both vacuolated (Vacuole) and unvacuolated (Normal) ER. The bottom panel shows a profile of ER FRET intensities of a cell transfected with an irrelevant vector and presenting similar erYC3.3 expression levels as cells in the top panel (measured by emission at 535 nm). (D) Treatment with thapsigargin and EGTA reduces erYC3.3 FRET intensities both in swollen and unaltered ER. 293T cells were cotransfected with Bak, Bcl-XL, and erYC3.3. 36 h after transfection, both unvacuolated (Normal) and vacuolated (Vacuolae) cells were analyzed in vivo to obtain confocal FRET levels in the ER. After capturing FRET images for each untreated cell, 2 mM EGTA and 200 μM thapsigargin were simultaneously added to the culture, and reticular FRET images acquired after 15 min. This time point showed the maximum effect. To represent the data, mean values of FRET intensities across a chosen section of the ER were obtained for each cell in control and treated conditions. Shown are data averaged for at least five cells analyzed in each experimental condition, along with the corresponding SDs.
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fig6: Involvement of RyR calcium channel activity in the regulation of cytoplasmic vacuolization induced by Bak. (A) Treatment with dantrolene potentiates vacuolization. 293T cells were transfected with a mix of the indicated plasmids and a plasmid expressing GFP. Where indicated, dantrolene (50 μM) was added 1 h after transfection. 36 h after transfection, cells were fixed, mounted, and analyzed by confocal microscopy. Shown are representative fields. The same magnification was used for both control and treated cells. (B) Caffeine treatment reduces vacuolization. 293T cells were transfected as in A. 36 h later, caffeine (20 mM) was added as indicated for 3.5 h, and cells were subsequently fixed and mounted. Quantitation of vacuolated cells was done as in Fig. 1 B. (C) Swollen ER cisternae and normal ER show similar calcium concentrations. 293T cells were transfected with the indicated plasmids in the presence of plasmids expressing cytosolic RFP and a reticular ratiometric calcium indicator (erYC3.3). 36 h after transfection, cells were analyzed in vivo by confocal microscopy to obtain FRET intensities of erYC3.3. In the top panel, RFP was used to locate a vacuolated cell in proximity to an unvacuolated neighbor with a similar level of transfection, and emission FRET ratios (535 nm/480 nm) were obtained for both cells. Shown are emissions in red (RFP), green (erYC3.3, 535 nm), and FRET ratios. The middle panel shows a profile of FRET intensities across the indicated yellow line, including both vacuolated (Vacuole) and unvacuolated (Normal) ER. The bottom panel shows a profile of ER FRET intensities of a cell transfected with an irrelevant vector and presenting similar erYC3.3 expression levels as cells in the top panel (measured by emission at 535 nm). (D) Treatment with thapsigargin and EGTA reduces erYC3.3 FRET intensities both in swollen and unaltered ER. 293T cells were cotransfected with Bak, Bcl-XL, and erYC3.3. 36 h after transfection, both unvacuolated (Normal) and vacuolated (Vacuolae) cells were analyzed in vivo to obtain confocal FRET levels in the ER. After capturing FRET images for each untreated cell, 2 mM EGTA and 200 μM thapsigargin were simultaneously added to the culture, and reticular FRET images acquired after 15 min. This time point showed the maximum effect. To represent the data, mean values of FRET intensities across a chosen section of the ER were obtained for each cell in control and treated conditions. Shown are data averaged for at least five cells analyzed in each experimental condition, along with the corresponding SDs.

Mentions: High calcium levels in the cytoplasm have been shown to induce ER compartmentalization and vacuolization (Subramanian and Meyer, 1997), pointing out to a role of calcium in the regulation of ER structure. Because Bcl-2 family members have the ability to alter ER calcium homeostasis (Nutt et al., 2002; Scorrano et al., 2003; Zong et al., 2003), the observed reticular swelling induced by Bak might be related to this activity. To test this hypothesis we used chemicals that influence calcium homeostasis in different ways. Chelation of extracellular calcium by EGTA, or treatment with the SERCA pump inhibitor thapsigargin, which allows a passive calcium leakage from ER stores (Breckenridge et al., 2003), had no effect in the degree of vacuolization (unpublished data). Dantrolene is a commercial drug that inhibits ER calcium release through the RyR calcium channels (Zhao et al., 2001; Fill and Copello, 2002). This drug has been shown to reduce the cytosolic concentration of calcium in some systems (Jacobs et al., 1991). The presence of dantrolene during expression of transfected Bak and Bcl-XL, or Bak-ΔBH3 alone, had the unexpected effect of exacerbating the vacuolated phenotype (Fig. 6 A). Vacuolae tended to occupy the vast majority of cytoplasmic space in the presence of dantrolene, whereas in its absence they were typically smaller (Fig. 6 A). In contrast, no change was observed in the cytoplasm of cells cotransfected with Bax and Bcl-XL or either Bak or Bax in the presence of apoptotic blockers like the caspase inhibitor p35 or a dominant-negative version of caspase-9 (unpublished data). This suggests that dantrolene only shows an effect in the presence of an underlying vacuolating stimulus.


Bcl-X(L) specifically activates Bak to induce swelling and restructuring of the endoplasmic reticulum.

Klee M, Pimentel-Muiños FX - J. Cell Biol. (2005)

Involvement of RyR calcium channel activity in the regulation of cytoplasmic vacuolization induced by Bak. (A) Treatment with dantrolene potentiates vacuolization. 293T cells were transfected with a mix of the indicated plasmids and a plasmid expressing GFP. Where indicated, dantrolene (50 μM) was added 1 h after transfection. 36 h after transfection, cells were fixed, mounted, and analyzed by confocal microscopy. Shown are representative fields. The same magnification was used for both control and treated cells. (B) Caffeine treatment reduces vacuolization. 293T cells were transfected as in A. 36 h later, caffeine (20 mM) was added as indicated for 3.5 h, and cells were subsequently fixed and mounted. Quantitation of vacuolated cells was done as in Fig. 1 B. (C) Swollen ER cisternae and normal ER show similar calcium concentrations. 293T cells were transfected with the indicated plasmids in the presence of plasmids expressing cytosolic RFP and a reticular ratiometric calcium indicator (erYC3.3). 36 h after transfection, cells were analyzed in vivo by confocal microscopy to obtain FRET intensities of erYC3.3. In the top panel, RFP was used to locate a vacuolated cell in proximity to an unvacuolated neighbor with a similar level of transfection, and emission FRET ratios (535 nm/480 nm) were obtained for both cells. Shown are emissions in red (RFP), green (erYC3.3, 535 nm), and FRET ratios. The middle panel shows a profile of FRET intensities across the indicated yellow line, including both vacuolated (Vacuole) and unvacuolated (Normal) ER. The bottom panel shows a profile of ER FRET intensities of a cell transfected with an irrelevant vector and presenting similar erYC3.3 expression levels as cells in the top panel (measured by emission at 535 nm). (D) Treatment with thapsigargin and EGTA reduces erYC3.3 FRET intensities both in swollen and unaltered ER. 293T cells were cotransfected with Bak, Bcl-XL, and erYC3.3. 36 h after transfection, both unvacuolated (Normal) and vacuolated (Vacuolae) cells were analyzed in vivo to obtain confocal FRET levels in the ER. After capturing FRET images for each untreated cell, 2 mM EGTA and 200 μM thapsigargin were simultaneously added to the culture, and reticular FRET images acquired after 15 min. This time point showed the maximum effect. To represent the data, mean values of FRET intensities across a chosen section of the ER were obtained for each cell in control and treated conditions. Shown are data averaged for at least five cells analyzed in each experimental condition, along with the corresponding SDs.
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fig6: Involvement of RyR calcium channel activity in the regulation of cytoplasmic vacuolization induced by Bak. (A) Treatment with dantrolene potentiates vacuolization. 293T cells were transfected with a mix of the indicated plasmids and a plasmid expressing GFP. Where indicated, dantrolene (50 μM) was added 1 h after transfection. 36 h after transfection, cells were fixed, mounted, and analyzed by confocal microscopy. Shown are representative fields. The same magnification was used for both control and treated cells. (B) Caffeine treatment reduces vacuolization. 293T cells were transfected as in A. 36 h later, caffeine (20 mM) was added as indicated for 3.5 h, and cells were subsequently fixed and mounted. Quantitation of vacuolated cells was done as in Fig. 1 B. (C) Swollen ER cisternae and normal ER show similar calcium concentrations. 293T cells were transfected with the indicated plasmids in the presence of plasmids expressing cytosolic RFP and a reticular ratiometric calcium indicator (erYC3.3). 36 h after transfection, cells were analyzed in vivo by confocal microscopy to obtain FRET intensities of erYC3.3. In the top panel, RFP was used to locate a vacuolated cell in proximity to an unvacuolated neighbor with a similar level of transfection, and emission FRET ratios (535 nm/480 nm) were obtained for both cells. Shown are emissions in red (RFP), green (erYC3.3, 535 nm), and FRET ratios. The middle panel shows a profile of FRET intensities across the indicated yellow line, including both vacuolated (Vacuole) and unvacuolated (Normal) ER. The bottom panel shows a profile of ER FRET intensities of a cell transfected with an irrelevant vector and presenting similar erYC3.3 expression levels as cells in the top panel (measured by emission at 535 nm). (D) Treatment with thapsigargin and EGTA reduces erYC3.3 FRET intensities both in swollen and unaltered ER. 293T cells were cotransfected with Bak, Bcl-XL, and erYC3.3. 36 h after transfection, both unvacuolated (Normal) and vacuolated (Vacuolae) cells were analyzed in vivo to obtain confocal FRET levels in the ER. After capturing FRET images for each untreated cell, 2 mM EGTA and 200 μM thapsigargin were simultaneously added to the culture, and reticular FRET images acquired after 15 min. This time point showed the maximum effect. To represent the data, mean values of FRET intensities across a chosen section of the ER were obtained for each cell in control and treated conditions. Shown are data averaged for at least five cells analyzed in each experimental condition, along with the corresponding SDs.
Mentions: High calcium levels in the cytoplasm have been shown to induce ER compartmentalization and vacuolization (Subramanian and Meyer, 1997), pointing out to a role of calcium in the regulation of ER structure. Because Bcl-2 family members have the ability to alter ER calcium homeostasis (Nutt et al., 2002; Scorrano et al., 2003; Zong et al., 2003), the observed reticular swelling induced by Bak might be related to this activity. To test this hypothesis we used chemicals that influence calcium homeostasis in different ways. Chelation of extracellular calcium by EGTA, or treatment with the SERCA pump inhibitor thapsigargin, which allows a passive calcium leakage from ER stores (Breckenridge et al., 2003), had no effect in the degree of vacuolization (unpublished data). Dantrolene is a commercial drug that inhibits ER calcium release through the RyR calcium channels (Zhao et al., 2001; Fill and Copello, 2002). This drug has been shown to reduce the cytosolic concentration of calcium in some systems (Jacobs et al., 1991). The presence of dantrolene during expression of transfected Bak and Bcl-XL, or Bak-ΔBH3 alone, had the unexpected effect of exacerbating the vacuolated phenotype (Fig. 6 A). Vacuolae tended to occupy the vast majority of cytoplasmic space in the presence of dantrolene, whereas in its absence they were typically smaller (Fig. 6 A). In contrast, no change was observed in the cytoplasm of cells cotransfected with Bax and Bcl-XL or either Bak or Bax in the presence of apoptotic blockers like the caspase inhibitor p35 or a dominant-negative version of caspase-9 (unpublished data). This suggests that dantrolene only shows an effect in the presence of an underlying vacuolating stimulus.

Bottom Line: Experiments with Bak and Bax-deficient mouse embryonic fibroblasts show that endogenous Bak mediates the effect, whereas Bax is mainly irrelevant.These results reveal a previously unidentified role of Bak in regulating reticular conformation.Because this activity is absent in Bax, it constitutes one of the first examples of functional divergence between the two multidomain homologues.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación del Cáncer, Universidad de Salamanca-CSIC, Salamanca, 37007 Spain.

ABSTRACT
Bcl-2 family members Bak and Bax constitute a mitochondrial gateway for multiple death pathways. Both proteins are also present in the endoplasmic reticulum where they control apoptosis through the regulation of calcium levels. We show here that reticular Bak has the additional capacity of modulating the structure of this organelle. Coexpression of Bak and Bcl-X(L) provokes extensive swelling and vacuolization of reticular cisternae. A Bak version lacking the BH3 domain suffices to induce this phenotype, and reticular targeting of this mutant retains the activity. Expression of upstream BH3-only activators in similar conditions recapitulates ER swelling and vacuolization if ryanodine receptor calcium channel activity is inhibited. Experiments with Bak and Bax-deficient mouse embryonic fibroblasts show that endogenous Bak mediates the effect, whereas Bax is mainly irrelevant. These results reveal a previously unidentified role of Bak in regulating reticular conformation. Because this activity is absent in Bax, it constitutes one of the first examples of functional divergence between the two multidomain homologues.

Show MeSH
Related in: MedlinePlus