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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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Cytoplasmic vacuolae are swollen ER cisternae. (A) Colocalization of erRFP with endogenous calreticulin. 293T cells were transfected with a plasmid encoding ER-targeted RFP (erRFP). 48 h later cells were fixed and stained with an anti-calreticulin antibody followed by an Alexa 488 (green)–coupled antibody, and analyzed by confocal microscopy. Shown is one representative example. (B) Vacuolae induced by transfection of Bak and Bcl-XL, or Bak-ΔBH3, are filled with coexpressed erRFP. 293T, Cos, and HeLa cells were cotransfected with the indicated constructs and plasmids expressing GFP and erRFP. 24 h (293T cells, as indicated), or 48 h after transfection, cells were analyzed in vivo by confocal microscopy. Live cells were used since fixation resulted in the disappearance of erRFP fluorescence from vacuolae in mounted preparations. White arrows indicate reticular cisternae that remain unaffected at early time points in 293T cells.
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fig4: Cytoplasmic vacuolae are swollen ER cisternae. (A) Colocalization of erRFP with endogenous calreticulin. 293T cells were transfected with a plasmid encoding ER-targeted RFP (erRFP). 48 h later cells were fixed and stained with an anti-calreticulin antibody followed by an Alexa 488 (green)–coupled antibody, and analyzed by confocal microscopy. Shown is one representative example. (B) Vacuolae induced by transfection of Bak and Bcl-XL, or Bak-ΔBH3, are filled with coexpressed erRFP. 293T, Cos, and HeLa cells were cotransfected with the indicated constructs and plasmids expressing GFP and erRFP. 24 h (293T cells, as indicated), or 48 h after transfection, cells were analyzed in vivo by confocal microscopy. Live cells were used since fixation resulted in the disappearance of erRFP fluorescence from vacuolae in mounted preparations. White arrows indicate reticular cisternae that remain unaffected at early time points in 293T cells.

Mentions: ER swelling is a morphological change associated with multiple cell death modalities (Van Cruchten and Van Den Broeck, 2002). To determine if vacuolae were topologically related to reticular cisternae, we created a version of RFP targeted to the ER lumen (erRFP). Control experiments established that erRFP completely colocalized with the ER marker calreticulin in normal cells (Fig. 4 A). Transfection of Bak in the presence of Bcl-XL, or Bak-ΔBH3 alone, in combination with this construct showed that the induced vacuolae were completely filled with erRFP, clearly establishing their reticular origin (Fig. 4 B). A substantial fraction of the ER remained unchanged at early stages of the swelling process (Fig. 4 B, 293T cells, 24 h), but reticular cisternae seemed to be progressively incorporated into the altered structures as transfection evolved (Fig. 4 B). These results reveal the potential of Bak to regulate ER structure. Because vacuolae can often occupy a substantial area of the cell, their generation likely involves a swelling process, although some contribution of ER tubulae fusion cannot be ruled out.


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

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

Cytoplasmic vacuolae are swollen ER cisternae. (A) Colocalization of erRFP with endogenous calreticulin. 293T cells were transfected with a plasmid encoding ER-targeted RFP (erRFP). 48 h later cells were fixed and stained with an anti-calreticulin antibody followed by an Alexa 488 (green)–coupled antibody, and analyzed by confocal microscopy. Shown is one representative example. (B) Vacuolae induced by transfection of Bak and Bcl-XL, or Bak-ΔBH3, are filled with coexpressed erRFP. 293T, Cos, and HeLa cells were cotransfected with the indicated constructs and plasmids expressing GFP and erRFP. 24 h (293T cells, as indicated), or 48 h after transfection, cells were analyzed in vivo by confocal microscopy. Live cells were used since fixation resulted in the disappearance of erRFP fluorescence from vacuolae in mounted preparations. White arrows indicate reticular cisternae that remain unaffected at early time points in 293T cells.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171806&req=5

fig4: Cytoplasmic vacuolae are swollen ER cisternae. (A) Colocalization of erRFP with endogenous calreticulin. 293T cells were transfected with a plasmid encoding ER-targeted RFP (erRFP). 48 h later cells were fixed and stained with an anti-calreticulin antibody followed by an Alexa 488 (green)–coupled antibody, and analyzed by confocal microscopy. Shown is one representative example. (B) Vacuolae induced by transfection of Bak and Bcl-XL, or Bak-ΔBH3, are filled with coexpressed erRFP. 293T, Cos, and HeLa cells were cotransfected with the indicated constructs and plasmids expressing GFP and erRFP. 24 h (293T cells, as indicated), or 48 h after transfection, cells were analyzed in vivo by confocal microscopy. Live cells were used since fixation resulted in the disappearance of erRFP fluorescence from vacuolae in mounted preparations. White arrows indicate reticular cisternae that remain unaffected at early time points in 293T cells.
Mentions: ER swelling is a morphological change associated with multiple cell death modalities (Van Cruchten and Van Den Broeck, 2002). To determine if vacuolae were topologically related to reticular cisternae, we created a version of RFP targeted to the ER lumen (erRFP). Control experiments established that erRFP completely colocalized with the ER marker calreticulin in normal cells (Fig. 4 A). Transfection of Bak in the presence of Bcl-XL, or Bak-ΔBH3 alone, in combination with this construct showed that the induced vacuolae were completely filled with erRFP, clearly establishing their reticular origin (Fig. 4 B). A substantial fraction of the ER remained unchanged at early stages of the swelling process (Fig. 4 B, 293T cells, 24 h), but reticular cisternae seemed to be progressively incorporated into the altered structures as transfection evolved (Fig. 4 B). These results reveal the potential of Bak to regulate ER structure. Because vacuolae can often occupy a substantial area of the cell, their generation likely involves a swelling process, although some contribution of ER tubulae fusion cannot be ruled out.

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