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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 ER-localized Bak in the generation of reticular swelling. (A) Perivacuolar localization of AU-Bak, AU-Bcl-XL, and AU-Bak-ΔBH3. 293T cells were transfected with the indicated constructs along with GFP. 36 h after transfection cells were fixed, stained with an anti-AU mAb followed by an anti–mouse-Cy3 (red) antibody, and mounted for confocal microscopy. N and V denote nuclei and vacuolae, respectively. (B) A version of Bak-ΔBH3 specifically targeted to the ER (Bak-ΔBH3-cb5) retains the capacity to induce ER swelling, whereas mitochondrial targeting (Bak-ΔBH3-ActA) results in a decreased activity. 293T or HeLa cells (as shown) were transfected with the indicated expression plasmids along with GFP. Z-VAD.fmk (100 μM) was added 1 h after transfection to reduce death. 36 h after transfection, cytoplasmic vacuolization was scored as in Fig. 1 B.
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fig5: Involvement of ER-localized Bak in the generation of reticular swelling. (A) Perivacuolar localization of AU-Bak, AU-Bcl-XL, and AU-Bak-ΔBH3. 293T cells were transfected with the indicated constructs along with GFP. 36 h after transfection cells were fixed, stained with an anti-AU mAb followed by an anti–mouse-Cy3 (red) antibody, and mounted for confocal microscopy. N and V denote nuclei and vacuolae, respectively. (B) A version of Bak-ΔBH3 specifically targeted to the ER (Bak-ΔBH3-cb5) retains the capacity to induce ER swelling, whereas mitochondrial targeting (Bak-ΔBH3-ActA) results in a decreased activity. 293T or HeLa cells (as shown) were transfected with the indicated expression plasmids along with GFP. Z-VAD.fmk (100 μM) was added 1 h after transfection to reduce death. 36 h after transfection, cytoplasmic vacuolization was scored as in Fig. 1 B.

Mentions: To determine the subcellular localization of transfected Bak with respect to dilated ER cisternae we used AU-tagged versions of the different molecules. Anti-AU immunofluorescence stainings of cells transfected with AU-Bak in combination with untagged Bcl-XL, or AU-Bak-ΔBH3 alone, revealed that vacuolae were often coated with a punctated pattern of AU reactivity (Fig. 5 A). This was particularly obvious in areas of direct apposition with the nucleus (Fig. 5 A). A variation of the same experiment showed a similar distribution of Bcl-XL (Fig. 5 A). These results indicate the existence of a topological link between effector molecules and swollen cisternae.


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 ER-localized Bak in the generation of reticular swelling. (A) Perivacuolar localization of AU-Bak, AU-Bcl-XL, and AU-Bak-ΔBH3. 293T cells were transfected with the indicated constructs along with GFP. 36 h after transfection cells were fixed, stained with an anti-AU mAb followed by an anti–mouse-Cy3 (red) antibody, and mounted for confocal microscopy. N and V denote nuclei and vacuolae, respectively. (B) A version of Bak-ΔBH3 specifically targeted to the ER (Bak-ΔBH3-cb5) retains the capacity to induce ER swelling, whereas mitochondrial targeting (Bak-ΔBH3-ActA) results in a decreased activity. 293T or HeLa cells (as shown) were transfected with the indicated expression plasmids along with GFP. Z-VAD.fmk (100 μM) was added 1 h after transfection to reduce death. 36 h after transfection, cytoplasmic vacuolization was scored as in Fig. 1 B.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Involvement of ER-localized Bak in the generation of reticular swelling. (A) Perivacuolar localization of AU-Bak, AU-Bcl-XL, and AU-Bak-ΔBH3. 293T cells were transfected with the indicated constructs along with GFP. 36 h after transfection cells were fixed, stained with an anti-AU mAb followed by an anti–mouse-Cy3 (red) antibody, and mounted for confocal microscopy. N and V denote nuclei and vacuolae, respectively. (B) A version of Bak-ΔBH3 specifically targeted to the ER (Bak-ΔBH3-cb5) retains the capacity to induce ER swelling, whereas mitochondrial targeting (Bak-ΔBH3-ActA) results in a decreased activity. 293T or HeLa cells (as shown) were transfected with the indicated expression plasmids along with GFP. Z-VAD.fmk (100 μM) was added 1 h after transfection to reduce death. 36 h after transfection, cytoplasmic vacuolization was scored as in Fig. 1 B.
Mentions: To determine the subcellular localization of transfected Bak with respect to dilated ER cisternae we used AU-tagged versions of the different molecules. Anti-AU immunofluorescence stainings of cells transfected with AU-Bak in combination with untagged Bcl-XL, or AU-Bak-ΔBH3 alone, revealed that vacuolae were often coated with a punctated pattern of AU reactivity (Fig. 5 A). This was particularly obvious in areas of direct apposition with the nucleus (Fig. 5 A). A variation of the same experiment showed a similar distribution of Bcl-XL (Fig. 5 A). These results indicate the existence of a topological link between effector molecules and swollen cisternae.

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