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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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Altered mitochondrial parameters inbcl-x–deficient neurons. (A)Immunofluorescence microscopy of control(bcl-x+/flox;NEX+/cre)and cKO(bcl-xflox/flox;NEX+/cre)cortical cultures (DIV6) stained with anti–cytochromec (green; 7H8.2C12 [1:80; BD] and goatanti–mouse Alexa Fluor 488 [Invitrogen]) to detect mitochondriaand costained for Cre recombinase (red; anti-Cre [1:2,000; EMD] and Cy3goat anti–rabbit [1:1,000; Jackson ImmunoResearch Laboratories,Inc.). Images were captured with a real-time camera (DiagnosticInstruments, Inc.) and a microscope (Eclipse E800; Nikon). Bars, 4µm. (B) Summary of mean fluorescence intensities ± SEMfrom two-photon microscopy images of live cortical cultures recordedsimultaneously in three channels to assess ΔΨm(100 nM TMRM), ROS accumulation (2 µM CM-DCF), and NAD(P)H(intrinsic fluorescence) in one mitochondria-enriched ROI per cell formultiple cells (control, n = 50; cKO,n = 56) from multiple cultures (control,n = 9; cKO, n = 11),with each culture prepared from a different embryo. a.u., arbitraryunit. Student’s t test was used; *, P< 10−6; **, P <10−10; ***, P <10−12. (C) Examples of two-photon microscopyimages marking example ROI. Fluorescence intensity is scaled withpseudocolors (filled arrows). N, nucleus. Bars, 10 µm. (D) Adiagram of three major determinants of mitochondrial membrane potential(dashed boxes). Electron flow from NADH to O2 (red arrow) viathe ETC complexes (I–IV), proton (H+) pathsacross the membrane (blue arrows), ATP/ADP + Pi exchange via ANTand phosphate carrier (PC; gray arrow), and inner mitochondrial membrane(IM) and outer mitochondrial membrane (OM). OSCP, oligomycinsensitivity–conferring protein.
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fig1: Altered mitochondrial parameters inbcl-x–deficient neurons. (A)Immunofluorescence microscopy of control(bcl-x+/flox;NEX+/cre)and cKO(bcl-xflox/flox;NEX+/cre)cortical cultures (DIV6) stained with anti–cytochromec (green; 7H8.2C12 [1:80; BD] and goatanti–mouse Alexa Fluor 488 [Invitrogen]) to detect mitochondriaand costained for Cre recombinase (red; anti-Cre [1:2,000; EMD] and Cy3goat anti–rabbit [1:1,000; Jackson ImmunoResearch Laboratories,Inc.). Images were captured with a real-time camera (DiagnosticInstruments, Inc.) and a microscope (Eclipse E800; Nikon). Bars, 4µm. (B) Summary of mean fluorescence intensities ± SEMfrom two-photon microscopy images of live cortical cultures recordedsimultaneously in three channels to assess ΔΨm(100 nM TMRM), ROS accumulation (2 µM CM-DCF), and NAD(P)H(intrinsic fluorescence) in one mitochondria-enriched ROI per cell formultiple cells (control, n = 50; cKO,n = 56) from multiple cultures (control,n = 9; cKO, n = 11),with each culture prepared from a different embryo. a.u., arbitraryunit. Student’s t test was used; *, P< 10−6; **, P <10−10; ***, P <10−12. (C) Examples of two-photon microscopyimages marking example ROI. Fluorescence intensity is scaled withpseudocolors (filled arrows). N, nucleus. Bars, 10 µm. (D) Adiagram of three major determinants of mitochondrial membrane potential(dashed boxes). Electron flow from NADH to O2 (red arrow) viathe ETC complexes (I–IV), proton (H+) pathsacross the membrane (blue arrows), ATP/ADP + Pi exchange via ANTand phosphate carrier (PC; gray arrow), and inner mitochondrial membrane(IM) and outer mitochondrial membrane (OM). OSCP, oligomycinsensitivity–conferring protein.

Mentions: To explore the function of Bcl-xL in healthy neurons, severalmitochondrial parameters were analyzed by two-photon laser-scanning fluorescencemicroscopy, comparing control and bcl-x conditional knockout(cKO) cortical neuron cultures (Berman et al.,2009). Both unfloxed and bcl-x–floxedlittermates express neuron-specific knockin NEX-Cre recombinase starting aroundembryonic day 12 (E12) to delete bcl-x. Staining for Crerecombinase serves as a positive marker for the survival ofbcl-x–deficient (and control unfloxed) corticalneurons (Fig. 1 A; Berman et al., 2009). Mitochondrial membrane potential(ΔΨm) was assessed in immature cortical cultureswith the potentiometric dye tetramethylrhodamine methyl ester (TMRM; nonquenchmode), revealing higher fluorescence intensity in the mitochondria-enrichedregions of bcl-x knockout cortical neurons (Fig. 1, B and C [left]). This is not aresult of increased mitochondrial biomass becausebcl-x–deficient neurons have lower, not higher,mitochondrial biomass in these and other cell types based on several criteria(Kowaltowski et al., 2002; Berman et al., 2009). Thus, it appearsthat bcl-x deficiency may result in a higher mitochondrialmembrane 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)

Altered mitochondrial parameters inbcl-x–deficient neurons. (A)Immunofluorescence microscopy of control(bcl-x+/flox;NEX+/cre)and cKO(bcl-xflox/flox;NEX+/cre)cortical cultures (DIV6) stained with anti–cytochromec (green; 7H8.2C12 [1:80; BD] and goatanti–mouse Alexa Fluor 488 [Invitrogen]) to detect mitochondriaand costained for Cre recombinase (red; anti-Cre [1:2,000; EMD] and Cy3goat anti–rabbit [1:1,000; Jackson ImmunoResearch Laboratories,Inc.). Images were captured with a real-time camera (DiagnosticInstruments, Inc.) and a microscope (Eclipse E800; Nikon). Bars, 4µm. (B) Summary of mean fluorescence intensities ± SEMfrom two-photon microscopy images of live cortical cultures recordedsimultaneously in three channels to assess ΔΨm(100 nM TMRM), ROS accumulation (2 µM CM-DCF), and NAD(P)H(intrinsic fluorescence) in one mitochondria-enriched ROI per cell formultiple cells (control, n = 50; cKO,n = 56) from multiple cultures (control,n = 9; cKO, n = 11),with each culture prepared from a different embryo. a.u., arbitraryunit. Student’s t test was used; *, P< 10−6; **, P <10−10; ***, P <10−12. (C) Examples of two-photon microscopyimages marking example ROI. Fluorescence intensity is scaled withpseudocolors (filled arrows). N, nucleus. Bars, 10 µm. (D) Adiagram of three major determinants of mitochondrial membrane potential(dashed boxes). Electron flow from NADH to O2 (red arrow) viathe ETC complexes (I–IV), proton (H+) pathsacross the membrane (blue arrows), ATP/ADP + Pi exchange via ANTand phosphate carrier (PC; gray arrow), and inner mitochondrial membrane(IM) and outer mitochondrial membrane (OM). OSCP, oligomycinsensitivity–conferring protein.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3198165&req=5

fig1: Altered mitochondrial parameters inbcl-x–deficient neurons. (A)Immunofluorescence microscopy of control(bcl-x+/flox;NEX+/cre)and cKO(bcl-xflox/flox;NEX+/cre)cortical cultures (DIV6) stained with anti–cytochromec (green; 7H8.2C12 [1:80; BD] and goatanti–mouse Alexa Fluor 488 [Invitrogen]) to detect mitochondriaand costained for Cre recombinase (red; anti-Cre [1:2,000; EMD] and Cy3goat anti–rabbit [1:1,000; Jackson ImmunoResearch Laboratories,Inc.). Images were captured with a real-time camera (DiagnosticInstruments, Inc.) and a microscope (Eclipse E800; Nikon). Bars, 4µm. (B) Summary of mean fluorescence intensities ± SEMfrom two-photon microscopy images of live cortical cultures recordedsimultaneously in three channels to assess ΔΨm(100 nM TMRM), ROS accumulation (2 µM CM-DCF), and NAD(P)H(intrinsic fluorescence) in one mitochondria-enriched ROI per cell formultiple cells (control, n = 50; cKO,n = 56) from multiple cultures (control,n = 9; cKO, n = 11),with each culture prepared from a different embryo. a.u., arbitraryunit. Student’s t test was used; *, P< 10−6; **, P <10−10; ***, P <10−12. (C) Examples of two-photon microscopyimages marking example ROI. Fluorescence intensity is scaled withpseudocolors (filled arrows). N, nucleus. Bars, 10 µm. (D) Adiagram of three major determinants of mitochondrial membrane potential(dashed boxes). Electron flow from NADH to O2 (red arrow) viathe ETC complexes (I–IV), proton (H+) pathsacross the membrane (blue arrows), ATP/ADP + Pi exchange via ANTand phosphate carrier (PC; gray arrow), and inner mitochondrial membrane(IM) and outer mitochondrial membrane (OM). OSCP, oligomycinsensitivity–conferring protein.
Mentions: To explore the function of Bcl-xL in healthy neurons, severalmitochondrial parameters were analyzed by two-photon laser-scanning fluorescencemicroscopy, comparing control and bcl-x conditional knockout(cKO) cortical neuron cultures (Berman et al.,2009). Both unfloxed and bcl-x–floxedlittermates express neuron-specific knockin NEX-Cre recombinase starting aroundembryonic day 12 (E12) to delete bcl-x. Staining for Crerecombinase serves as a positive marker for the survival ofbcl-x–deficient (and control unfloxed) corticalneurons (Fig. 1 A; Berman et al., 2009). Mitochondrial membrane potential(ΔΨm) was assessed in immature cortical cultureswith the potentiometric dye tetramethylrhodamine methyl ester (TMRM; nonquenchmode), revealing higher fluorescence intensity in the mitochondria-enrichedregions of bcl-x knockout cortical neurons (Fig. 1, B and C [left]). This is not aresult of increased mitochondrial biomass becausebcl-x–deficient neurons have lower, not higher,mitochondrial biomass in these and other cell types based on several criteria(Kowaltowski et al., 2002; Berman et al., 2009). Thus, it appearsthat bcl-x deficiency may result in a higher mitochondrialmembrane 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