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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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Coexpression of BimEL or tBid with Bcl-XL induces ER swelling in the presence of dantrolene. (A) Quantitation of dantrolene-treated cells showing cytoplasmic vacuolization after transfection with BimEL or tBid, as a function of cotransfection with Bcl-XL. 293T cells were transfected with a mix of the indicated constructs in the presence of plasmids expressing GFP and the apoptotic inhibitor p35. Dantrolene (50 μM) was added 1 h after transfection to all experimental points. 36 h later the proportion of GFP-expressing cells showing cytoplasmic vacuolization was determined as in Fig. 1 B. (B) ER swelling is the cause of cytoplasmic vacuolization induced by cotransfection of BimEL or tBid with Bcl-XL, in the presence of dantrolene. 293T cells were transfected with plasmids expressing either Bim or tBid (as indicated), in combination with Bcl-XL, GFP, and erRFP expression plasmids. Dantrolene (50 μM) was added 1 h after transfection. 36 h after transfection, cells were analyzed by in vivo confocal microscopy. (C) 293T cells express endogenous Bak and Bax. Cells were either transfected with the indicated DNAs or left untransfected, and lysed 24 h after transfection. Shown are Western blots probed with antibodies against Bak or Bax (top), and subsequently reprobed with an anti-AU mAb (bottom) as indicated. Lanes corresponding to transfected cells provide a size reference to identify the endogenous protein. Re-probing with an anti-AU mAb excludes sample contaminations across wells.
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fig7: Coexpression of BimEL or tBid with Bcl-XL induces ER swelling in the presence of dantrolene. (A) Quantitation of dantrolene-treated cells showing cytoplasmic vacuolization after transfection with BimEL or tBid, as a function of cotransfection with Bcl-XL. 293T cells were transfected with a mix of the indicated constructs in the presence of plasmids expressing GFP and the apoptotic inhibitor p35. Dantrolene (50 μM) was added 1 h after transfection to all experimental points. 36 h later the proportion of GFP-expressing cells showing cytoplasmic vacuolization was determined as in Fig. 1 B. (B) ER swelling is the cause of cytoplasmic vacuolization induced by cotransfection of BimEL or tBid with Bcl-XL, in the presence of dantrolene. 293T cells were transfected with plasmids expressing either Bim or tBid (as indicated), in combination with Bcl-XL, GFP, and erRFP expression plasmids. Dantrolene (50 μM) was added 1 h after transfection. 36 h after transfection, cells were analyzed by in vivo confocal microscopy. (C) 293T cells express endogenous Bak and Bax. Cells were either transfected with the indicated DNAs or left untransfected, and lysed 24 h after transfection. Shown are Western blots probed with antibodies against Bak or Bax (top), and subsequently reprobed with an anti-AU mAb (bottom) as indicated. Lanes corresponding to transfected cells provide a size reference to identify the endogenous protein. Re-probing with an anti-AU mAb excludes sample contaminations across wells.

Mentions: Bak and Bax are physiologically activated by BH3-only homologues like BimEL or Bid (Bouillet and Strasser, 2002). To explore if these upstream effectors are capable of generating a vacuolated phenotype, we transfected cells with plasmids expressing either full-length BimEL or a truncated version of Bid (tBid) known to be constitutively active (Li et al., 1998). Coexpression of BimEL or tBid with Bcl-XL induced a low but detectable number of cells (∼1%) to show cytoplasmic vacuolae (unpublished data), suggesting an incipient ER swelling. Given the potentiating effect of dantrolene (Fig. 6 A), we wondered if the drug could turn this weak effect into a vacuolated phenotype easier to identify. The presence of dantrolene from early on after transfection with BimEL or tBid in combination with Bcl-XL resulted in a substantial number of vacuolated cells (Fig. 7 A), whereas in the absence of Bcl-XL both molecules induced a more modest phenotype (Fig. 7 A). Cotransfection with erRFP confirmed the reticular origin of the induced vacuolae (Fig. 7 B). Therefore, upstream apoptotic activators can cause ER changes that remain undetectable with the techniques used unless amplified by inhibition of RyR channels. In light of our previous results this effect could be mediated by endogenous Bak, which, consistent with this model, is present in 293T cells (Fig. 7 C).


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

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

Coexpression of BimEL or tBid with Bcl-XL induces ER swelling in the presence of dantrolene. (A) Quantitation of dantrolene-treated cells showing cytoplasmic vacuolization after transfection with BimEL or tBid, as a function of cotransfection with Bcl-XL. 293T cells were transfected with a mix of the indicated constructs in the presence of plasmids expressing GFP and the apoptotic inhibitor p35. Dantrolene (50 μM) was added 1 h after transfection to all experimental points. 36 h later the proportion of GFP-expressing cells showing cytoplasmic vacuolization was determined as in Fig. 1 B. (B) ER swelling is the cause of cytoplasmic vacuolization induced by cotransfection of BimEL or tBid with Bcl-XL, in the presence of dantrolene. 293T cells were transfected with plasmids expressing either Bim or tBid (as indicated), in combination with Bcl-XL, GFP, and erRFP expression plasmids. Dantrolene (50 μM) was added 1 h after transfection. 36 h after transfection, cells were analyzed by in vivo confocal microscopy. (C) 293T cells express endogenous Bak and Bax. Cells were either transfected with the indicated DNAs or left untransfected, and lysed 24 h after transfection. Shown are Western blots probed with antibodies against Bak or Bax (top), and subsequently reprobed with an anti-AU mAb (bottom) as indicated. Lanes corresponding to transfected cells provide a size reference to identify the endogenous protein. Re-probing with an anti-AU mAb excludes sample contaminations across wells.
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Related In: Results  -  Collection

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fig7: Coexpression of BimEL or tBid with Bcl-XL induces ER swelling in the presence of dantrolene. (A) Quantitation of dantrolene-treated cells showing cytoplasmic vacuolization after transfection with BimEL or tBid, as a function of cotransfection with Bcl-XL. 293T cells were transfected with a mix of the indicated constructs in the presence of plasmids expressing GFP and the apoptotic inhibitor p35. Dantrolene (50 μM) was added 1 h after transfection to all experimental points. 36 h later the proportion of GFP-expressing cells showing cytoplasmic vacuolization was determined as in Fig. 1 B. (B) ER swelling is the cause of cytoplasmic vacuolization induced by cotransfection of BimEL or tBid with Bcl-XL, in the presence of dantrolene. 293T cells were transfected with plasmids expressing either Bim or tBid (as indicated), in combination with Bcl-XL, GFP, and erRFP expression plasmids. Dantrolene (50 μM) was added 1 h after transfection. 36 h after transfection, cells were analyzed by in vivo confocal microscopy. (C) 293T cells express endogenous Bak and Bax. Cells were either transfected with the indicated DNAs or left untransfected, and lysed 24 h after transfection. Shown are Western blots probed with antibodies against Bak or Bax (top), and subsequently reprobed with an anti-AU mAb (bottom) as indicated. Lanes corresponding to transfected cells provide a size reference to identify the endogenous protein. Re-probing with an anti-AU mAb excludes sample contaminations across wells.
Mentions: Bak and Bax are physiologically activated by BH3-only homologues like BimEL or Bid (Bouillet and Strasser, 2002). To explore if these upstream effectors are capable of generating a vacuolated phenotype, we transfected cells with plasmids expressing either full-length BimEL or a truncated version of Bid (tBid) known to be constitutively active (Li et al., 1998). Coexpression of BimEL or tBid with Bcl-XL induced a low but detectable number of cells (∼1%) to show cytoplasmic vacuolae (unpublished data), suggesting an incipient ER swelling. Given the potentiating effect of dantrolene (Fig. 6 A), we wondered if the drug could turn this weak effect into a vacuolated phenotype easier to identify. The presence of dantrolene from early on after transfection with BimEL or tBid in combination with Bcl-XL resulted in a substantial number of vacuolated cells (Fig. 7 A), whereas in the absence of Bcl-XL both molecules induced a more modest phenotype (Fig. 7 A). Cotransfection with erRFP confirmed the reticular origin of the induced vacuolae (Fig. 7 B). Therefore, upstream apoptotic activators can cause ER changes that remain undetectable with the techniques used unless amplified by inhibition of RyR channels. In light of our previous results this effect could be mediated by endogenous Bak, which, consistent with this model, is present in 293T cells (Fig. 7 C).

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