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Bcl-xL regulates mitochondrial energetics by stabilizing the inner membrane potential.

Chen YB, Aon MA, Hsu YT, Soane L, Teng X, McCaffery JM, Cheng WC, Qi B, Li H, Alavian KN, Dayhoff-Brannigan M, Zou S, Pineda FJ, O'Rourke B, Ko YH, Pedersen PL, Kaczmarek LK, Jonas EA, Hardwick JM - J. Cell Biol. (2011)

Bottom Line: Computational, biochemical, and genetic evidence indicated that Bcl-x(L) reduces futile ion flux across the inner mitochondrial membrane to prevent a wasteful drain on cellular resources, thereby preventing an energetic crisis during stress.Given that F(1)F(O)-ATP synthase directly affects mitochondrial membrane potential and having identified the mitochondrial ATP synthase β subunit in a screen for Bcl-x(L)-binding partners, we tested and found that Bcl-x(L) failed to protect β subunit-deficient yeast.Thus, by bolstering mitochondrial energetic capacity, Bcl-x(L) may contribute importantly to cell survival independently of other Bcl-2 family proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.

ABSTRACT
Mammalian Bcl-x(L) protein localizes to the outer mitochondrial membrane, where it inhibits apoptosis by binding Bax and inhibiting Bax-induced outer membrane permeabilization. Contrary to expectation, we found by electron microscopy and biochemical approaches that endogenous Bcl-x(L) also localized to inner mitochondrial cristae. Two-photon microscopy of cultured neurons revealed large fluctuations in inner mitochondrial membrane potential when Bcl-x(L) was genetically deleted or pharmacologically inhibited, indicating increased total ion flux into and out of mitochondria. Computational, biochemical, and genetic evidence indicated that Bcl-x(L) reduces futile ion flux across the inner mitochondrial membrane to prevent a wasteful drain on cellular resources, thereby preventing an energetic crisis during stress. Given that F(1)F(O)-ATP synthase directly affects mitochondrial membrane potential and having identified the mitochondrial ATP synthase β subunit in a screen for Bcl-x(L)-binding partners, we tested and found that Bcl-x(L) failed to protect β subunit-deficient yeast. Thus, by bolstering mitochondrial energetic capacity, Bcl-x(L) may contribute importantly to cell survival independently of other Bcl-2 family proteins.

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Bcl-xL–deficient mitochondria have fluctuatingmembrane potentials. (A) Continuous recordings (3.5-sintervals) of TMRM intensities per ROI of individual cortical neuronsfor at least 250 s (DIV4–5; Fig.1). Traces are representative of multiple neurons in threeindependent experiments. (B) Fluorescence intensity (arbitrary units[a.u.]) of TMRM stain per pixel was determined for the markedmitochondria-rich region of a single neuron inbcl-x–deficient (cKO) and control (Cont)cultures shown in Fig. 1 C. (C)SDs were calculated for mean TMRM fluorescence intensities per pixel for200 individual mitochondria derived from 20 different cells per genotypemonitored every 2.5 s for at least 90 s. Each symbol represents onemitochondrion. An F-test for variance comparing control andbcl-x–deficient neuronal mitochondria wasperformed; P < 0.0001. (D) Continuous recordings of intracellularcalcium levels at 4-s intervals in DIV3–4 cortical neurons.Initial intracellular calcium levels from four independent experimentsare graphed. *, P = 0.039. (E) Mitochondrial membranepotential fluctuation increases with ABT-737. Fluorescence intensitieswere measured in small puncta (estimated to be one mitochondrion) nearthe soma in cultured rat hippocampal neurons (DIV14–16) stainedwith 5 nM TMRE. Relative fluorescence intensities (collected at 1/s) forthe same puncta/mitochondria treated with 0.1% DMSO before and afteraddition of 1 µM ABT-737 (in 0.1% DMSO) for 10 min are shown. (F)SDs of TMRE intensity measurements as in E; data are for 30 measurementsfor each of 12 puncta in six cells in two independent experiments andare similar to three additional experiments with protocol variations.Paired t test was used; *, P = 0.02. (G)SD of TMRE as in F, except 4 d after transfection with shRNA vector withscrambled (n = 10) orbcl-x–specific shRNAs (n= 17). Fluorescent images were taken every 3 s for 8 min. Pairedt test was used; **, P =0.00027. (F and G) Data are presented as the mean ± SD.
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fig4: Bcl-xL–deficient mitochondria have fluctuatingmembrane potentials. (A) Continuous recordings (3.5-sintervals) of TMRM intensities per ROI of individual cortical neuronsfor at least 250 s (DIV4–5; Fig.1). Traces are representative of multiple neurons in threeindependent experiments. (B) Fluorescence intensity (arbitrary units[a.u.]) of TMRM stain per pixel was determined for the markedmitochondria-rich region of a single neuron inbcl-x–deficient (cKO) and control (Cont)cultures shown in Fig. 1 C. (C)SDs were calculated for mean TMRM fluorescence intensities per pixel for200 individual mitochondria derived from 20 different cells per genotypemonitored every 2.5 s for at least 90 s. Each symbol represents onemitochondrion. An F-test for variance comparing control andbcl-x–deficient neuronal mitochondria wasperformed; P < 0.0001. (D) Continuous recordings of intracellularcalcium levels at 4-s intervals in DIV3–4 cortical neurons.Initial intracellular calcium levels from four independent experimentsare graphed. *, P = 0.039. (E) Mitochondrial membranepotential fluctuation increases with ABT-737. Fluorescence intensitieswere measured in small puncta (estimated to be one mitochondrion) nearthe soma in cultured rat hippocampal neurons (DIV14–16) stainedwith 5 nM TMRE. Relative fluorescence intensities (collected at 1/s) forthe same puncta/mitochondria treated with 0.1% DMSO before and afteraddition of 1 µM ABT-737 (in 0.1% DMSO) for 10 min are shown. (F)SDs of TMRE intensity measurements as in E; data are for 30 measurementsfor each of 12 puncta in six cells in two independent experiments andare similar to three additional experiments with protocol variations.Paired t test was used; *, P = 0.02. (G)SD of TMRE as in F, except 4 d after transfection with shRNA vector withscrambled (n = 10) orbcl-x–specific shRNAs (n= 17). Fluorescent images were taken every 3 s for 8 min. Pairedt test was used; **, P =0.00027. (F and G) Data are presented as the mean ± SD.

Mentions: Because the mitochondrial F1FO ATP synthase is an importantcontrol point for proton flux across the inner mitochondrial membrane,mitochondrial membrane potential was further evaluated by time-lapse imaging(3.5-s intervals). TMRM intensity in mitochondria-enriched regions fluctuatesmodestly in control neurons, which is consistent with an earlier study (Vergun et al., 2003). However,bcl-x–deficient neurons exhibited a strikingfluctuation in TMRM fluorescence intensity over irregular intervals in time(Fig. 4 A), across a singlebcl-x knockout cell (Fig. 4B), and in individual mitochondria (Fig. 4 C). Thus, the increase in mean mitochondrial potential inbcl-x–deficient neurons (Fig. 1 B) represents the mean of a time-varying potentialthat fluctuates predominantly to higher (more negative) potentials thancontrols. Therefore, the presence of Bcl-xL stabilizes the innermitochondrial membrane potential.


Bcl-xL regulates mitochondrial energetics by stabilizing the inner membrane potential.

Chen YB, Aon MA, Hsu YT, Soane L, Teng X, McCaffery JM, Cheng WC, Qi B, Li H, Alavian KN, Dayhoff-Brannigan M, Zou S, Pineda FJ, O'Rourke B, Ko YH, Pedersen PL, Kaczmarek LK, Jonas EA, Hardwick JM - J. Cell Biol. (2011)

Bcl-xL–deficient mitochondria have fluctuatingmembrane potentials. (A) Continuous recordings (3.5-sintervals) of TMRM intensities per ROI of individual cortical neuronsfor at least 250 s (DIV4–5; Fig.1). Traces are representative of multiple neurons in threeindependent experiments. (B) Fluorescence intensity (arbitrary units[a.u.]) of TMRM stain per pixel was determined for the markedmitochondria-rich region of a single neuron inbcl-x–deficient (cKO) and control (Cont)cultures shown in Fig. 1 C. (C)SDs were calculated for mean TMRM fluorescence intensities per pixel for200 individual mitochondria derived from 20 different cells per genotypemonitored every 2.5 s for at least 90 s. Each symbol represents onemitochondrion. An F-test for variance comparing control andbcl-x–deficient neuronal mitochondria wasperformed; P < 0.0001. (D) Continuous recordings of intracellularcalcium levels at 4-s intervals in DIV3–4 cortical neurons.Initial intracellular calcium levels from four independent experimentsare graphed. *, P = 0.039. (E) Mitochondrial membranepotential fluctuation increases with ABT-737. Fluorescence intensitieswere measured in small puncta (estimated to be one mitochondrion) nearthe soma in cultured rat hippocampal neurons (DIV14–16) stainedwith 5 nM TMRE. Relative fluorescence intensities (collected at 1/s) forthe same puncta/mitochondria treated with 0.1% DMSO before and afteraddition of 1 µM ABT-737 (in 0.1% DMSO) for 10 min are shown. (F)SDs of TMRE intensity measurements as in E; data are for 30 measurementsfor each of 12 puncta in six cells in two independent experiments andare similar to three additional experiments with protocol variations.Paired t test was used; *, P = 0.02. (G)SD of TMRE as in F, except 4 d after transfection with shRNA vector withscrambled (n = 10) orbcl-x–specific shRNAs (n= 17). Fluorescent images were taken every 3 s for 8 min. Pairedt test was used; **, P =0.00027. (F and G) Data are presented as the mean ± SD.
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Related In: Results  -  Collection

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fig4: Bcl-xL–deficient mitochondria have fluctuatingmembrane potentials. (A) Continuous recordings (3.5-sintervals) of TMRM intensities per ROI of individual cortical neuronsfor at least 250 s (DIV4–5; Fig.1). Traces are representative of multiple neurons in threeindependent experiments. (B) Fluorescence intensity (arbitrary units[a.u.]) of TMRM stain per pixel was determined for the markedmitochondria-rich region of a single neuron inbcl-x–deficient (cKO) and control (Cont)cultures shown in Fig. 1 C. (C)SDs were calculated for mean TMRM fluorescence intensities per pixel for200 individual mitochondria derived from 20 different cells per genotypemonitored every 2.5 s for at least 90 s. Each symbol represents onemitochondrion. An F-test for variance comparing control andbcl-x–deficient neuronal mitochondria wasperformed; P < 0.0001. (D) Continuous recordings of intracellularcalcium levels at 4-s intervals in DIV3–4 cortical neurons.Initial intracellular calcium levels from four independent experimentsare graphed. *, P = 0.039. (E) Mitochondrial membranepotential fluctuation increases with ABT-737. Fluorescence intensitieswere measured in small puncta (estimated to be one mitochondrion) nearthe soma in cultured rat hippocampal neurons (DIV14–16) stainedwith 5 nM TMRE. Relative fluorescence intensities (collected at 1/s) forthe same puncta/mitochondria treated with 0.1% DMSO before and afteraddition of 1 µM ABT-737 (in 0.1% DMSO) for 10 min are shown. (F)SDs of TMRE intensity measurements as in E; data are for 30 measurementsfor each of 12 puncta in six cells in two independent experiments andare similar to three additional experiments with protocol variations.Paired t test was used; *, P = 0.02. (G)SD of TMRE as in F, except 4 d after transfection with shRNA vector withscrambled (n = 10) orbcl-x–specific shRNAs (n= 17). Fluorescent images were taken every 3 s for 8 min. Pairedt test was used; **, P =0.00027. (F and G) Data are presented as the mean ± SD.
Mentions: Because the mitochondrial F1FO ATP synthase is an importantcontrol point for proton flux across the inner mitochondrial membrane,mitochondrial membrane potential was further evaluated by time-lapse imaging(3.5-s intervals). TMRM intensity in mitochondria-enriched regions fluctuatesmodestly in control neurons, which is consistent with an earlier study (Vergun et al., 2003). However,bcl-x–deficient neurons exhibited a strikingfluctuation in TMRM fluorescence intensity over irregular intervals in time(Fig. 4 A), across a singlebcl-x knockout cell (Fig. 4B), and in individual mitochondria (Fig. 4 C). Thus, the increase in mean mitochondrial potential inbcl-x–deficient neurons (Fig. 1 B) represents the mean of a time-varying potentialthat fluctuates predominantly to higher (more negative) potentials thancontrols. Therefore, the presence of Bcl-xL stabilizes the innermitochondrial membrane potential.

Bottom Line: Computational, biochemical, and genetic evidence indicated that Bcl-x(L) reduces futile ion flux across the inner mitochondrial membrane to prevent a wasteful drain on cellular resources, thereby preventing an energetic crisis during stress.Given that F(1)F(O)-ATP synthase directly affects mitochondrial membrane potential and having identified the mitochondrial ATP synthase β subunit in a screen for Bcl-x(L)-binding partners, we tested and found that Bcl-x(L) failed to protect β subunit-deficient yeast.Thus, by bolstering mitochondrial energetic capacity, Bcl-x(L) may contribute importantly to cell survival independently of other Bcl-2 family proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.

ABSTRACT
Mammalian Bcl-x(L) protein localizes to the outer mitochondrial membrane, where it inhibits apoptosis by binding Bax and inhibiting Bax-induced outer membrane permeabilization. Contrary to expectation, we found by electron microscopy and biochemical approaches that endogenous Bcl-x(L) also localized to inner mitochondrial cristae. Two-photon microscopy of cultured neurons revealed large fluctuations in inner mitochondrial membrane potential when Bcl-x(L) was genetically deleted or pharmacologically inhibited, indicating increased total ion flux into and out of mitochondria. Computational, biochemical, and genetic evidence indicated that Bcl-x(L) reduces futile ion flux across the inner mitochondrial membrane to prevent a wasteful drain on cellular resources, thereby preventing an energetic crisis during stress. Given that F(1)F(O)-ATP synthase directly affects mitochondrial membrane potential and having identified the mitochondrial ATP synthase β subunit in a screen for Bcl-x(L)-binding partners, we tested and found that Bcl-x(L) failed to protect β subunit-deficient yeast. Thus, by bolstering mitochondrial energetic capacity, Bcl-x(L) may contribute importantly to cell survival independently of other Bcl-2 family proteins.

Show MeSH
Related in: MedlinePlus