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Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis.

Nechushtan A, Smith CL, Lamensdorf I, Yoon SH, Youle RJ - J. Cell Biol. (2001)

Bottom Line: Bax leaves the mitochondrial membranes and coalesces into large clusters containing thousands of Bax molecules that remain adjacent to mitochondria.We found the formation of Bax and Bak apoptotic clusters to be caspase independent and inhibited completely and specifically by Bcl-X(L), correlating cluster formation with cytotoxic activity.Our results reveal the importance of a novel structure formed by certain Bcl-2 family members during the process of cell death.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Section, Surgical Neurology Branch, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Bax is a member of the Bcl-2 family of proteins known to regulate mitochondria-dependent programmed cell death. Early in apoptosis, Bax translocates from the cytosol to the mitochondrial membrane. We have identified by confocal and electron microscopy a novel step in the Bax proapoptotic mechanism immediately subsequent to mitochondrial translocation. Bax leaves the mitochondrial membranes and coalesces into large clusters containing thousands of Bax molecules that remain adjacent to mitochondria. Bak, a close homologue of Bax, colocalizes in these apoptotic clusters in contrast to other family members, Bid and Bad, which circumscribe the outer mitochondrial membrane throughout cell death progression. We found the formation of Bax and Bak apoptotic clusters to be caspase independent and inhibited completely and specifically by Bcl-X(L), correlating cluster formation with cytotoxic activity. Our results reveal the importance of a novel structure formed by certain Bcl-2 family members during the process of cell death.

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Bcl-XL does not prevent mitochondrial targeting of Bak or Bax-S184V but inhibits their proapoptotic activity. Cos-7 cells were transiently cotransfected with the pGL3 luciferase reporter construct, Bcl-XL, and CFP-Bax-S184V (A) or CFP-Bak (B) in 1:16:4 ratios. The ECFP-C1 expression vector was used as a control. After 24 h of incubation, cells were harvested and processed for luciferase activity. Results were quantitated with a scintillation counter and displayed as 106 counts of light intensity. The error bars show a mean standard error determined from six independent measurements. (C and D) Healthy Cos-7 cells transiently expressing CFP-S184V and CFP-Bak with and without YFP-Bcl-XL were incubated with Mitotracker red CMXRos (Mito) to reveal mitochondria and visualized by laser scanning confocal microscopy. If Bcl-XL was to inhibit binding of Bax-S184V or Bak to mitochondria as a protection mechanism, both Bak and Bax-S184V proteins (green) would show a higher degree of cytosolic distribution. Both Bax-S184V (C, green) and Bak (D, green) localized to mitochondria (blue) similarly in the presence or absence of Bcl-XL. Bars, 25 μm.
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Figure 7: Bcl-XL does not prevent mitochondrial targeting of Bak or Bax-S184V but inhibits their proapoptotic activity. Cos-7 cells were transiently cotransfected with the pGL3 luciferase reporter construct, Bcl-XL, and CFP-Bax-S184V (A) or CFP-Bak (B) in 1:16:4 ratios. The ECFP-C1 expression vector was used as a control. After 24 h of incubation, cells were harvested and processed for luciferase activity. Results were quantitated with a scintillation counter and displayed as 106 counts of light intensity. The error bars show a mean standard error determined from six independent measurements. (C and D) Healthy Cos-7 cells transiently expressing CFP-S184V and CFP-Bak with and without YFP-Bcl-XL were incubated with Mitotracker red CMXRos (Mito) to reveal mitochondria and visualized by laser scanning confocal microscopy. If Bcl-XL was to inhibit binding of Bax-S184V or Bak to mitochondria as a protection mechanism, both Bak and Bax-S184V proteins (green) would show a higher degree of cytosolic distribution. Both Bax-S184V (C, green) and Bak (D, green) localized to mitochondria (blue) similarly in the presence or absence of Bcl-XL. Bars, 25 μm.

Mentions: Bcl-XL has been shown to inhibit apoptosis induced by different stimuli in various cell types. Bcl-XL prevents the cell death induced by overexpression of Bax or Bak and reduces the rate of cell loss during STS treatment (Chittenden et al. 1995; Vander Heiden et al. 1997; Wolter et al. 1997; Griffiths et al. 1999). Furthermore, Bcl-XL does not require a direct interaction with Bax to inhibit its promotion of cell death (Cheng et al. 1996). To analyze the biological significance of cluster formation, we asked what effect Bcl-XL might have on this process. Bcl-XL is known to inhibit Bax translocation to mitochondria during apoptosis (Wolter et al. 1997). Thus, to specifically test the role of Bcl-XL on cluster formation we focused on Bax-S184V because this protein constitutively associates with mitochondria. The S184V mutant form of Bax is constitutively targeted to the mitochondria in healthy cells and has a proapoptotic activity similar to wild-type Bax (Nechushtan et al. 1999). We have found that, like Bax, Bax-S184V also forms mitochondrial-associated clusters in apoptotic cells (Fig. 6 A). When we coexpressed Bax-S184V and Bcl-XL in Cos-7 cells, we found that Bcl-XL completely prevented the cluster formation of the Bax mutant with or without treatment of STS (Fig. 6 B). Cells cotransfected with Bcl-XL and Bax-S184V showed a marked reduction in apoptosis compared with cells singly transfected with Bax-S184V (Fig. 7 A). CFP-Bax-S184V targeted the mitochondria regardless of the absence or presence of overexpressed YFP-Bcl-XL, showing that Bcl-XL does not inhibit the mitochondria-binding step (Fig. 7 C). However, the presence of Bcl-XL completely inhibited coalescence into clusters as is observed in cells singly transfected with Bax (Fig. 1) or Bax-S184V (Fig. 6 A). Analysis of CFP-Bcl-XL showed that during apoptosis, Bcl-XL always circumscribes the mitochondria (data not shown). Bak, like Bax-S184V, is bound to mitochondria in healthy cells. Cotransfection of Bcl-XL and Bak caused no change in Bak localization (Fig. 7 D). However, when apoptosis was induced by incubation with STS, no Bak cluster formation occurred (data not shown) as occurs in the absence of Bcl-XL (Fig. 3). Cotransfection with Bcl-XL also markedly inhibited Bak-stimulated apoptosis (Fig. 7 B). Furthermore, analysis of CFP-Bcl-XL showed that Bcl-XL always circumscribes the mitochondria in the presence of either Bax-S184V or Bak (Fig. 7C and Fig. D) and remains on the mitochondria during apoptosis (data not shown). The observation that Bcl-XL specifically blocks cluster formation and the bioactivity of Bax-S184V and Bak indicates that the clusters are the biologically active proapoptotic structures.


Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis.

Nechushtan A, Smith CL, Lamensdorf I, Yoon SH, Youle RJ - J. Cell Biol. (2001)

Bcl-XL does not prevent mitochondrial targeting of Bak or Bax-S184V but inhibits their proapoptotic activity. Cos-7 cells were transiently cotransfected with the pGL3 luciferase reporter construct, Bcl-XL, and CFP-Bax-S184V (A) or CFP-Bak (B) in 1:16:4 ratios. The ECFP-C1 expression vector was used as a control. After 24 h of incubation, cells were harvested and processed for luciferase activity. Results were quantitated with a scintillation counter and displayed as 106 counts of light intensity. The error bars show a mean standard error determined from six independent measurements. (C and D) Healthy Cos-7 cells transiently expressing CFP-S184V and CFP-Bak with and without YFP-Bcl-XL were incubated with Mitotracker red CMXRos (Mito) to reveal mitochondria and visualized by laser scanning confocal microscopy. If Bcl-XL was to inhibit binding of Bax-S184V or Bak to mitochondria as a protection mechanism, both Bak and Bax-S184V proteins (green) would show a higher degree of cytosolic distribution. Both Bax-S184V (C, green) and Bak (D, green) localized to mitochondria (blue) similarly in the presence or absence of Bcl-XL. Bars, 25 μm.
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Figure 7: Bcl-XL does not prevent mitochondrial targeting of Bak or Bax-S184V but inhibits their proapoptotic activity. Cos-7 cells were transiently cotransfected with the pGL3 luciferase reporter construct, Bcl-XL, and CFP-Bax-S184V (A) or CFP-Bak (B) in 1:16:4 ratios. The ECFP-C1 expression vector was used as a control. After 24 h of incubation, cells were harvested and processed for luciferase activity. Results were quantitated with a scintillation counter and displayed as 106 counts of light intensity. The error bars show a mean standard error determined from six independent measurements. (C and D) Healthy Cos-7 cells transiently expressing CFP-S184V and CFP-Bak with and without YFP-Bcl-XL were incubated with Mitotracker red CMXRos (Mito) to reveal mitochondria and visualized by laser scanning confocal microscopy. If Bcl-XL was to inhibit binding of Bax-S184V or Bak to mitochondria as a protection mechanism, both Bak and Bax-S184V proteins (green) would show a higher degree of cytosolic distribution. Both Bax-S184V (C, green) and Bak (D, green) localized to mitochondria (blue) similarly in the presence or absence of Bcl-XL. Bars, 25 μm.
Mentions: Bcl-XL has been shown to inhibit apoptosis induced by different stimuli in various cell types. Bcl-XL prevents the cell death induced by overexpression of Bax or Bak and reduces the rate of cell loss during STS treatment (Chittenden et al. 1995; Vander Heiden et al. 1997; Wolter et al. 1997; Griffiths et al. 1999). Furthermore, Bcl-XL does not require a direct interaction with Bax to inhibit its promotion of cell death (Cheng et al. 1996). To analyze the biological significance of cluster formation, we asked what effect Bcl-XL might have on this process. Bcl-XL is known to inhibit Bax translocation to mitochondria during apoptosis (Wolter et al. 1997). Thus, to specifically test the role of Bcl-XL on cluster formation we focused on Bax-S184V because this protein constitutively associates with mitochondria. The S184V mutant form of Bax is constitutively targeted to the mitochondria in healthy cells and has a proapoptotic activity similar to wild-type Bax (Nechushtan et al. 1999). We have found that, like Bax, Bax-S184V also forms mitochondrial-associated clusters in apoptotic cells (Fig. 6 A). When we coexpressed Bax-S184V and Bcl-XL in Cos-7 cells, we found that Bcl-XL completely prevented the cluster formation of the Bax mutant with or without treatment of STS (Fig. 6 B). Cells cotransfected with Bcl-XL and Bax-S184V showed a marked reduction in apoptosis compared with cells singly transfected with Bax-S184V (Fig. 7 A). CFP-Bax-S184V targeted the mitochondria regardless of the absence or presence of overexpressed YFP-Bcl-XL, showing that Bcl-XL does not inhibit the mitochondria-binding step (Fig. 7 C). However, the presence of Bcl-XL completely inhibited coalescence into clusters as is observed in cells singly transfected with Bax (Fig. 1) or Bax-S184V (Fig. 6 A). Analysis of CFP-Bcl-XL showed that during apoptosis, Bcl-XL always circumscribes the mitochondria (data not shown). Bak, like Bax-S184V, is bound to mitochondria in healthy cells. Cotransfection of Bcl-XL and Bak caused no change in Bak localization (Fig. 7 D). However, when apoptosis was induced by incubation with STS, no Bak cluster formation occurred (data not shown) as occurs in the absence of Bcl-XL (Fig. 3). Cotransfection with Bcl-XL also markedly inhibited Bak-stimulated apoptosis (Fig. 7 B). Furthermore, analysis of CFP-Bcl-XL showed that Bcl-XL always circumscribes the mitochondria in the presence of either Bax-S184V or Bak (Fig. 7C and Fig. D) and remains on the mitochondria during apoptosis (data not shown). The observation that Bcl-XL specifically blocks cluster formation and the bioactivity of Bax-S184V and Bak indicates that the clusters are the biologically active proapoptotic structures.

Bottom Line: Bax leaves the mitochondrial membranes and coalesces into large clusters containing thousands of Bax molecules that remain adjacent to mitochondria.We found the formation of Bax and Bak apoptotic clusters to be caspase independent and inhibited completely and specifically by Bcl-X(L), correlating cluster formation with cytotoxic activity.Our results reveal the importance of a novel structure formed by certain Bcl-2 family members during the process of cell death.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Section, Surgical Neurology Branch, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Bax is a member of the Bcl-2 family of proteins known to regulate mitochondria-dependent programmed cell death. Early in apoptosis, Bax translocates from the cytosol to the mitochondrial membrane. We have identified by confocal and electron microscopy a novel step in the Bax proapoptotic mechanism immediately subsequent to mitochondrial translocation. Bax leaves the mitochondrial membranes and coalesces into large clusters containing thousands of Bax molecules that remain adjacent to mitochondria. Bak, a close homologue of Bax, colocalizes in these apoptotic clusters in contrast to other family members, Bid and Bad, which circumscribe the outer mitochondrial membrane throughout cell death progression. We found the formation of Bax and Bak apoptotic clusters to be caspase independent and inhibited completely and specifically by Bcl-X(L), correlating cluster formation with cytotoxic activity. Our results reveal the importance of a novel structure formed by certain Bcl-2 family members during the process of cell death.

Show MeSH
Related in: MedlinePlus