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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 Bak and Bcl-XL induces cytoplasmic vacuolization. (A) Confocal analysis of cytoplasmic vacuolization induced by coexpression of Bak and Bcl-XL. 293T cells were transfected with a mix of the indicated expression plasmids in combination with a plasmid expressing cytosolic GFP. 36 h after transfection, cells were fixed, mounted, and analyzed by confocal microscopy. (B) Time course of vacuolization induced by coexpression of Bak and Bcl-XL. 293T cells were cotransfected with Bak, Bcl-XL, and GFP as in A. At the indicated time points the proportion of GFP-expressing cells showing a detectable level of cytoplasmic vacuolization was determined in vivo by blindly counting green cells under an inverted fluorescence microscope. At least 400 cells were scored for each experimental point. Columns represent the percentage of GFP-expressing cells showing a vacuolated cytoplasm. Error bars show SDs of percentages obtained by counting at least eight different fields. (C) Quantitation of cells showing cytoplasmic vacuolization after transfection with Bak or Bax in combination with Bcl-XL. 293T cells were transfected as in A, and analyzed as in B, 36 h after transfection. (D) Confocal analysis of cytoplasmic vacuolization induced by Bak and Bcl-XL in different cell types. The indicated cells were transfected with the same mix of plasmids as in A. 24 h (293T, as indicated) or 36 h (293T, HeLa, and Cos) later, cells were fixed, stained with DAPI, and mounted for confocal analysis. (E) Bcl-XL inhibits Bak and Bax-induced apoptosis to the same extent. 293T cells were transfected with the indicated expression plasmids. 24 h after transfection, cells were lysed and equal amounts of proteins were subjected to Western blotting using an anti-PARP antibody. (F) AU-Bak and AU-Bax are expressed to comparable levels. 293T cells were transfected as in E with the exception that a vector expressing the viral caspase inhibitor p35 was introduced in all experimental points to block death. 24 h after transfection, cells were lysed and equal amounts of proteins were resolved by PAGE and blotted with an anti-AU mAb.
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fig1: Coexpression of Bak and Bcl-XL induces cytoplasmic vacuolization. (A) Confocal analysis of cytoplasmic vacuolization induced by coexpression of Bak and Bcl-XL. 293T cells were transfected with a mix of the indicated expression plasmids in combination with a plasmid expressing cytosolic GFP. 36 h after transfection, cells were fixed, mounted, and analyzed by confocal microscopy. (B) Time course of vacuolization induced by coexpression of Bak and Bcl-XL. 293T cells were cotransfected with Bak, Bcl-XL, and GFP as in A. At the indicated time points the proportion of GFP-expressing cells showing a detectable level of cytoplasmic vacuolization was determined in vivo by blindly counting green cells under an inverted fluorescence microscope. At least 400 cells were scored for each experimental point. Columns represent the percentage of GFP-expressing cells showing a vacuolated cytoplasm. Error bars show SDs of percentages obtained by counting at least eight different fields. (C) Quantitation of cells showing cytoplasmic vacuolization after transfection with Bak or Bax in combination with Bcl-XL. 293T cells were transfected as in A, and analyzed as in B, 36 h after transfection. (D) Confocal analysis of cytoplasmic vacuolization induced by Bak and Bcl-XL in different cell types. The indicated cells were transfected with the same mix of plasmids as in A. 24 h (293T, as indicated) or 36 h (293T, HeLa, and Cos) later, cells were fixed, stained with DAPI, and mounted for confocal analysis. (E) Bcl-XL inhibits Bak and Bax-induced apoptosis to the same extent. 293T cells were transfected with the indicated expression plasmids. 24 h after transfection, cells were lysed and equal amounts of proteins were subjected to Western blotting using an anti-PARP antibody. (F) AU-Bak and AU-Bax are expressed to comparable levels. 293T cells were transfected as in E with the exception that a vector expressing the viral caspase inhibitor p35 was introduced in all experimental points to block death. 24 h after transfection, cells were lysed and equal amounts of proteins were resolved by PAGE and blotted with an anti-AU mAb.

Mentions: Previous reports indicate that the enforced expression of Bak and Bax triggers spontaneous cell death (Chittenden et al., 1995b; Xiang et al., 1996), an activity opposed by anti-apoptotic homologues like Bcl-XL (Chittenden et al., 1995a; Simonen et al., 1997). To look for functional differences between Bak and Bax we took advantage of this overexpression approach, and transfected both molecules under various conditions into 293T cells. A striking difference arose when the induced apoptotic process was inhibited by Bcl-XL. Simultaneous expression of Bak and Bcl-XL provoked a prominent cytoplasmic vacuolization revealed by the exclusion of cytosolic GFP (Fig. 1 A), whereas this phenotype was absent in cells coexpressing Bax and Bcl-XL (Fig. 1 A). Time course experiments showed that the effect was first detectable between 12 and 16 h after transfection (Fig. 1 B), peaking at around 36 h after transfection (Fig. 1 B). At this time point there was no detectable vacuolization in cells transfected with Bax in the presence of Bcl-XL, Bcl-XL alone, or empty vector (Fig. 1 C). At later times cells lost viability as evidenced by an increased permeability to propidium iodide (unpublished data), a death process that progressed without detectable caspase-dependent proteolytic processing of poly(ADP-ribose) polymerase (PARP; unpublished data). This vacuolating capacity of Bak and Bcl-XL was independent of the cell type, because different cell lines were similarly susceptible (Fig. 1 D). DNA staining with DAPI showed that at least one prominent cytoplasmic vacuole was usually localized in close contact with the periphery of the nucleus (Fig. 1 D). The different potential of transfected Bak and Bax in this assay was not due to a dissimilar inhibitory capability of Bcl-XL, because the proteolytic processing of PARP was equally blocked by Bcl-XL in both cases (Fig. 1 E). In addition, no significant differences in Bak and Bax expression levels could account for the observed phenomena (Fig. 1 F).


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 Bak and Bcl-XL induces cytoplasmic vacuolization. (A) Confocal analysis of cytoplasmic vacuolization induced by coexpression of Bak and Bcl-XL. 293T cells were transfected with a mix of the indicated expression plasmids in combination with a plasmid expressing cytosolic GFP. 36 h after transfection, cells were fixed, mounted, and analyzed by confocal microscopy. (B) Time course of vacuolization induced by coexpression of Bak and Bcl-XL. 293T cells were cotransfected with Bak, Bcl-XL, and GFP as in A. At the indicated time points the proportion of GFP-expressing cells showing a detectable level of cytoplasmic vacuolization was determined in vivo by blindly counting green cells under an inverted fluorescence microscope. At least 400 cells were scored for each experimental point. Columns represent the percentage of GFP-expressing cells showing a vacuolated cytoplasm. Error bars show SDs of percentages obtained by counting at least eight different fields. (C) Quantitation of cells showing cytoplasmic vacuolization after transfection with Bak or Bax in combination with Bcl-XL. 293T cells were transfected as in A, and analyzed as in B, 36 h after transfection. (D) Confocal analysis of cytoplasmic vacuolization induced by Bak and Bcl-XL in different cell types. The indicated cells were transfected with the same mix of plasmids as in A. 24 h (293T, as indicated) or 36 h (293T, HeLa, and Cos) later, cells were fixed, stained with DAPI, and mounted for confocal analysis. (E) Bcl-XL inhibits Bak and Bax-induced apoptosis to the same extent. 293T cells were transfected with the indicated expression plasmids. 24 h after transfection, cells were lysed and equal amounts of proteins were subjected to Western blotting using an anti-PARP antibody. (F) AU-Bak and AU-Bax are expressed to comparable levels. 293T cells were transfected as in E with the exception that a vector expressing the viral caspase inhibitor p35 was introduced in all experimental points to block death. 24 h after transfection, cells were lysed and equal amounts of proteins were resolved by PAGE and blotted with an anti-AU mAb.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171806&req=5

fig1: Coexpression of Bak and Bcl-XL induces cytoplasmic vacuolization. (A) Confocal analysis of cytoplasmic vacuolization induced by coexpression of Bak and Bcl-XL. 293T cells were transfected with a mix of the indicated expression plasmids in combination with a plasmid expressing cytosolic GFP. 36 h after transfection, cells were fixed, mounted, and analyzed by confocal microscopy. (B) Time course of vacuolization induced by coexpression of Bak and Bcl-XL. 293T cells were cotransfected with Bak, Bcl-XL, and GFP as in A. At the indicated time points the proportion of GFP-expressing cells showing a detectable level of cytoplasmic vacuolization was determined in vivo by blindly counting green cells under an inverted fluorescence microscope. At least 400 cells were scored for each experimental point. Columns represent the percentage of GFP-expressing cells showing a vacuolated cytoplasm. Error bars show SDs of percentages obtained by counting at least eight different fields. (C) Quantitation of cells showing cytoplasmic vacuolization after transfection with Bak or Bax in combination with Bcl-XL. 293T cells were transfected as in A, and analyzed as in B, 36 h after transfection. (D) Confocal analysis of cytoplasmic vacuolization induced by Bak and Bcl-XL in different cell types. The indicated cells were transfected with the same mix of plasmids as in A. 24 h (293T, as indicated) or 36 h (293T, HeLa, and Cos) later, cells were fixed, stained with DAPI, and mounted for confocal analysis. (E) Bcl-XL inhibits Bak and Bax-induced apoptosis to the same extent. 293T cells were transfected with the indicated expression plasmids. 24 h after transfection, cells were lysed and equal amounts of proteins were subjected to Western blotting using an anti-PARP antibody. (F) AU-Bak and AU-Bax are expressed to comparable levels. 293T cells were transfected as in E with the exception that a vector expressing the viral caspase inhibitor p35 was introduced in all experimental points to block death. 24 h after transfection, cells were lysed and equal amounts of proteins were resolved by PAGE and blotted with an anti-AU mAb.
Mentions: Previous reports indicate that the enforced expression of Bak and Bax triggers spontaneous cell death (Chittenden et al., 1995b; Xiang et al., 1996), an activity opposed by anti-apoptotic homologues like Bcl-XL (Chittenden et al., 1995a; Simonen et al., 1997). To look for functional differences between Bak and Bax we took advantage of this overexpression approach, and transfected both molecules under various conditions into 293T cells. A striking difference arose when the induced apoptotic process was inhibited by Bcl-XL. Simultaneous expression of Bak and Bcl-XL provoked a prominent cytoplasmic vacuolization revealed by the exclusion of cytosolic GFP (Fig. 1 A), whereas this phenotype was absent in cells coexpressing Bax and Bcl-XL (Fig. 1 A). Time course experiments showed that the effect was first detectable between 12 and 16 h after transfection (Fig. 1 B), peaking at around 36 h after transfection (Fig. 1 B). At this time point there was no detectable vacuolization in cells transfected with Bax in the presence of Bcl-XL, Bcl-XL alone, or empty vector (Fig. 1 C). At later times cells lost viability as evidenced by an increased permeability to propidium iodide (unpublished data), a death process that progressed without detectable caspase-dependent proteolytic processing of poly(ADP-ribose) polymerase (PARP; unpublished data). This vacuolating capacity of Bak and Bcl-XL was independent of the cell type, because different cell lines were similarly susceptible (Fig. 1 D). DNA staining with DAPI showed that at least one prominent cytoplasmic vacuole was usually localized in close contact with the periphery of the nucleus (Fig. 1 D). The different potential of transfected Bak and Bax in this assay was not due to a dissimilar inhibitory capability of Bcl-XL, because the proteolytic processing of PARP was equally blocked by Bcl-XL in both cases (Fig. 1 E). In addition, no significant differences in Bak and Bax expression levels could account for the observed phenomena (Fig. 1 F).

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