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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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Bak clusters and colocalizes with Bax in dying cells. Healthy and apoptotic Cos-7 cells transiently coexpressing CFP-Bak with either YFP-20 (A) or YFP-Bax (B) were examined by laser scanning confocal microscopy. Bak (red) was associated with mitochondria in healthy cells (control) and formed clusters adjacent to mitochondria labeled with YFP-20 (green). Bak had a distinctive distribution from Bax in healthy cells (B, control) but colocalized with Bax in dying cells (B, STS). Mitochondria were labeled with Mitotracker red CMXRos in B (blue). (C) Silver-enhanced immunogold labeling of CFP-Bak in Cos-7 cells showing specific labeling of gold particles around the mitochondria outer membrane in healthy cells (control) and in large clusters associated with the mitochondria in apoptotic cells (STS). Apoptosis was induced by addition of 1 μM STS for 5 h. Bars: (A and B) 25 μm; (C) 0.5 μm.
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Figure 3: Bak clusters and colocalizes with Bax in dying cells. Healthy and apoptotic Cos-7 cells transiently coexpressing CFP-Bak with either YFP-20 (A) or YFP-Bax (B) were examined by laser scanning confocal microscopy. Bak (red) was associated with mitochondria in healthy cells (control) and formed clusters adjacent to mitochondria labeled with YFP-20 (green). Bak had a distinctive distribution from Bax in healthy cells (B, control) but colocalized with Bax in dying cells (B, STS). Mitochondria were labeled with Mitotracker red CMXRos in B (blue). (C) Silver-enhanced immunogold labeling of CFP-Bak in Cos-7 cells showing specific labeling of gold particles around the mitochondria outer membrane in healthy cells (control) and in large clusters associated with the mitochondria in apoptotic cells (STS). Apoptosis was induced by addition of 1 μM STS for 5 h. Bars: (A and B) 25 μm; (C) 0.5 μm.

Mentions: Among the proapoptotic members of the Bcl-2 family, Bak is closest to Bax in terms of sequence homology, size (Adams and Cory 1998), and biological activity. Studying mice lacking both Bax and Bak genes (bax−/− bak−/−) showed overlapping roles of these proteins in the regulation of apoptosis (Lindsten et al. 2000). Like Bax, Bak has been shown to induce the loss of mitochondrial membrane potential and lead to cytochrome c release, whereas Bcl-2 and Bcl-XL prevent these events (Eskes et al. 1998; Shimizu et al. 1999; Chatterjee et al. 2000; Wei et al. 2000). However, Bax exists in the cytosol of healthy cells and translocates to the mitochondria during apoptosis, whereas Bak constitutively resides on mitochondrial membranes in healthy cells. To determine whether Bak forms similar clusters during apoptosis, cells were cotransfected with CFP-Bak and either YFP-20 as a marker for mitochondria or YFP-Bax to visualize Bax apoptotic clusters. Bak is located on mitochondria in healthy cells and fully colocalizes with YFP-20 (Fig. 3 A, top row) in contrast to Bax, which resides in the cytosol (Fig. 3 B, top row, second panel). However, upon induction of apoptosis by STS, Bak segregates from YFP-20 and the mitochondria and coalesces into mitochondria-associated clusters (Fig. 3 A, bottom row). These clusters have the same characteristics as those formed by Bax in terms of size, location, and number. Bak and Bax colocalize in the same clusters adjacent to mitochondria during apoptosis (Fig. 3 B, bottom row, overlay). The localization of Bak and Bax is significantly different from that of the mitochondria (Fig. 3 B, bottom row, overlay, blue staining). To further investigate these apoptotic clusters of Bak revealed by the fluorescence images of living cells, we examined Cos-7 cells expressing CFP-Bak using EM (Fig. 3). Bak circumscribes the outer mitochondrial membrane in healthy cells (Fig. 3 C, control) and coalesces in large multimeric clusters in apoptotic ones (Fig. 3 C, STS). The number and distribution of the Bak clusters are similar to those found for Bax (Fig. 2).


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)

Bak clusters and colocalizes with Bax in dying cells. Healthy and apoptotic Cos-7 cells transiently coexpressing CFP-Bak with either YFP-20 (A) or YFP-Bax (B) were examined by laser scanning confocal microscopy. Bak (red) was associated with mitochondria in healthy cells (control) and formed clusters adjacent to mitochondria labeled with YFP-20 (green). Bak had a distinctive distribution from Bax in healthy cells (B, control) but colocalized with Bax in dying cells (B, STS). Mitochondria were labeled with Mitotracker red CMXRos in B (blue). (C) Silver-enhanced immunogold labeling of CFP-Bak in Cos-7 cells showing specific labeling of gold particles around the mitochondria outer membrane in healthy cells (control) and in large clusters associated with the mitochondria in apoptotic cells (STS). Apoptosis was induced by addition of 1 μM STS for 5 h. Bars: (A and B) 25 μm; (C) 0.5 μm.
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Figure 3: Bak clusters and colocalizes with Bax in dying cells. Healthy and apoptotic Cos-7 cells transiently coexpressing CFP-Bak with either YFP-20 (A) or YFP-Bax (B) were examined by laser scanning confocal microscopy. Bak (red) was associated with mitochondria in healthy cells (control) and formed clusters adjacent to mitochondria labeled with YFP-20 (green). Bak had a distinctive distribution from Bax in healthy cells (B, control) but colocalized with Bax in dying cells (B, STS). Mitochondria were labeled with Mitotracker red CMXRos in B (blue). (C) Silver-enhanced immunogold labeling of CFP-Bak in Cos-7 cells showing specific labeling of gold particles around the mitochondria outer membrane in healthy cells (control) and in large clusters associated with the mitochondria in apoptotic cells (STS). Apoptosis was induced by addition of 1 μM STS for 5 h. Bars: (A and B) 25 μm; (C) 0.5 μm.
Mentions: Among the proapoptotic members of the Bcl-2 family, Bak is closest to Bax in terms of sequence homology, size (Adams and Cory 1998), and biological activity. Studying mice lacking both Bax and Bak genes (bax−/− bak−/−) showed overlapping roles of these proteins in the regulation of apoptosis (Lindsten et al. 2000). Like Bax, Bak has been shown to induce the loss of mitochondrial membrane potential and lead to cytochrome c release, whereas Bcl-2 and Bcl-XL prevent these events (Eskes et al. 1998; Shimizu et al. 1999; Chatterjee et al. 2000; Wei et al. 2000). However, Bax exists in the cytosol of healthy cells and translocates to the mitochondria during apoptosis, whereas Bak constitutively resides on mitochondrial membranes in healthy cells. To determine whether Bak forms similar clusters during apoptosis, cells were cotransfected with CFP-Bak and either YFP-20 as a marker for mitochondria or YFP-Bax to visualize Bax apoptotic clusters. Bak is located on mitochondria in healthy cells and fully colocalizes with YFP-20 (Fig. 3 A, top row) in contrast to Bax, which resides in the cytosol (Fig. 3 B, top row, second panel). However, upon induction of apoptosis by STS, Bak segregates from YFP-20 and the mitochondria and coalesces into mitochondria-associated clusters (Fig. 3 A, bottom row). These clusters have the same characteristics as those formed by Bax in terms of size, location, and number. Bak and Bax colocalize in the same clusters adjacent to mitochondria during apoptosis (Fig. 3 B, bottom row, overlay). The localization of Bak and Bax is significantly different from that of the mitochondria (Fig. 3 B, bottom row, overlay, blue staining). To further investigate these apoptotic clusters of Bak revealed by the fluorescence images of living cells, we examined Cos-7 cells expressing CFP-Bak using EM (Fig. 3). Bak circumscribes the outer mitochondrial membrane in healthy cells (Fig. 3 C, control) and coalesces in large multimeric clusters in apoptotic ones (Fig. 3 C, STS). The number and distribution of the Bak clusters are similar to those found for Bax (Fig. 2).

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