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Mitochondrial ceramide-rich macrodomains functionalize Bax upon irradiation.

Lee H, Rotolo JA, Mesicek J, Penate-Medina T, Rimner A, Liao WC, Yin X, Ragupathi G, Ehleiter D, Gulbins E, Zhai D, Reed JC, Haimovitz-Friedman A, Fuks Z, Kolesnick R - PLoS ONE (2011)

Bottom Line: Our recent studies in the C. elegans germline indicate that mitochondrial ceramide generation is obligate for radiation-induced apoptosis, although a mechanism for ceramide action was not delineated.Here we demonstrate that ceramide, generated in the mitochondrial outer membrane of mammalian cells upon irradiation, forms a platform into which Bax inserts, oligomerizes and functionalizes as a pore.We posit conceptualization of ceramide as a membrane-based stress calibrator, driving membrane macrodomain organization, which in mitochondria regulates intensity of Bax-induced MOMP, and is pharmacologically tractable in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.

ABSTRACT

Background: Evidence indicates that Bax functions as a "lipidic" pore to regulate mitochondrial outer membrane permeabilization (MOMP), the apoptosis commitment step, through unknown membrane elements. Here we show mitochondrial ceramide elevation facilitates MOMP-mediated cytochrome c release in HeLa cells by generating a previously-unrecognized mitochondrial ceramide-rich macrodomain (MCRM), which we visualize and isolate, into which Bax integrates.

Methodology/principal findings: MCRMs, virtually non-existent in resting cells, form upon irradiation coupled to ceramide synthase-mediated ceramide elevation, optimizing Bax insertion/oligomerization and MOMP. MCRMs are detected by confocal microscopy in intact HeLa cells and isolated biophysically as a light membrane fraction from HeLa cell lysates. Inhibiting ceramide generation using a well-defined natural ceramide synthase inhibitor, Fumonisin B1, prevented radiation-induced Bax insertion, oligomerization and MOMP. MCRM deconstruction using purified mouse hepatic mitochondria revealed ceramide alone is non-apoptogenic. Rather Bax integrates into MCRMs, oligomerizing therein, conferring 1-2 log enhanced cytochrome c release. Consistent with this mechanism, MCRM Bax isolates as high molecular weight "pore-forming" oligomers, while non-MCRM membrane contains exclusively MOMP-incompatible monomeric Bax.

Conclusions/significance: Our recent studies in the C. elegans germline indicate that mitochondrial ceramide generation is obligate for radiation-induced apoptosis, although a mechanism for ceramide action was not delineated. Here we demonstrate that ceramide, generated in the mitochondrial outer membrane of mammalian cells upon irradiation, forms a platform into which Bax inserts, oligomerizes and functionalizes as a pore. We posit conceptualization of ceramide as a membrane-based stress calibrator, driving membrane macrodomain organization, which in mitochondria regulates intensity of Bax-induced MOMP, and is pharmacologically tractable in vitro and in vivo.

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Ceramide induces formation of a mitochondrial ceramide-rich macrodomain (MCRM).(A) Ionizing radiation (10 Gy) induces co-localization of endogenous Bax with MCRMs in HeLa cells. Mitochondria were isolated from HeLa cells 34 h after irradiation and immunostained as described in Supporting Information Text S1. Data represent typical stainings from 1 of 4 similar studies in which 2000 mitochondria were analyzed each. (B) Addition of exogenous C16-ceramide induces co-localization of endogenous full-length Bax with MCRMs in HeLa cells. Mitochondria were isolated from HeLa cells using percoll gradient and treated with ceramide as Figure 3A. After 30 min incubation, mitochondria were fixed and stained with MitoTracker (blue), while ceramide and Bax were localized using anti-ceramide IgM (red) or anti-Bax IgG (green), respectively. Control IgM and IgG did not yield detectable signals (not shown). These data represent 1 of 3 similar studies. (C) Bax translocates into a radiation-generated HeLa MCRM. Upper panel: 34 h post-irradiation, HeLa mitochondria were isolated as in Materials and Methods and incubated with 0.15% Triton X-100 in MBS buffer for 30 min on ice. 40 µl mitochondrial homogenate (3.3 µg/µl) were subjected to 5–30% mini-discontinuous sucrose density gradient centrifugation as described in Materials and Methods. 20 µl aliquots of 80 µl fractions were analyzed by immunoblotting using the indicated antibodies. The protein level of each fraction was assessed using the Bio-Rad Dc protein assay kit (PE, Pellet). Data are from 1 of 4 studies, consisting of 2 independent gradients per study. The gradient shown displays our clearest example of Bax translocation into light membranes. Lower panel: Bax in each fraction, revealed by immunoblotting and quantified using NIH Image software, was normalized to protein content for all 8 gradients. (D) MCRM Bax exists as high molecular weight oligomers. Mitochondria from 10 Gy-irradiated HeLa cells, disrupted by either (a) 1% CHAPS and sonication or (b) dounce homogenization in 0.15% Triton X-100, were subjected to 5–30% discontinuous sucrose gradient for MCRM isolation as in Experimental Procedures. Light (MCRM; fractions 6,7) and heavy fractions (solubilized proteins; fractions 11,12) were analyzed by gel filtration on Sephacryl S-200 column as in Figure 2C. 500 µl of each eluted fraction were concentrated by 20% TCA precipitation for immunoblotting. Data are from 3 independent studies.
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pone-0019783-g005: Ceramide induces formation of a mitochondrial ceramide-rich macrodomain (MCRM).(A) Ionizing radiation (10 Gy) induces co-localization of endogenous Bax with MCRMs in HeLa cells. Mitochondria were isolated from HeLa cells 34 h after irradiation and immunostained as described in Supporting Information Text S1. Data represent typical stainings from 1 of 4 similar studies in which 2000 mitochondria were analyzed each. (B) Addition of exogenous C16-ceramide induces co-localization of endogenous full-length Bax with MCRMs in HeLa cells. Mitochondria were isolated from HeLa cells using percoll gradient and treated with ceramide as Figure 3A. After 30 min incubation, mitochondria were fixed and stained with MitoTracker (blue), while ceramide and Bax were localized using anti-ceramide IgM (red) or anti-Bax IgG (green), respectively. Control IgM and IgG did not yield detectable signals (not shown). These data represent 1 of 3 similar studies. (C) Bax translocates into a radiation-generated HeLa MCRM. Upper panel: 34 h post-irradiation, HeLa mitochondria were isolated as in Materials and Methods and incubated with 0.15% Triton X-100 in MBS buffer for 30 min on ice. 40 µl mitochondrial homogenate (3.3 µg/µl) were subjected to 5–30% mini-discontinuous sucrose density gradient centrifugation as described in Materials and Methods. 20 µl aliquots of 80 µl fractions were analyzed by immunoblotting using the indicated antibodies. The protein level of each fraction was assessed using the Bio-Rad Dc protein assay kit (PE, Pellet). Data are from 1 of 4 studies, consisting of 2 independent gradients per study. The gradient shown displays our clearest example of Bax translocation into light membranes. Lower panel: Bax in each fraction, revealed by immunoblotting and quantified using NIH Image software, was normalized to protein content for all 8 gradients. (D) MCRM Bax exists as high molecular weight oligomers. Mitochondria from 10 Gy-irradiated HeLa cells, disrupted by either (a) 1% CHAPS and sonication or (b) dounce homogenization in 0.15% Triton X-100, were subjected to 5–30% discontinuous sucrose gradient for MCRM isolation as in Experimental Procedures. Light (MCRM; fractions 6,7) and heavy fractions (solubilized proteins; fractions 11,12) were analyzed by gel filtration on Sephacryl S-200 column as in Figure 2C. 500 µl of each eluted fraction were concentrated by 20% TCA precipitation for immunoblotting. Data are from 3 independent studies.

Mentions: After addition of C16-ceramide, and/or recombinant full-length (FL) Bax (Figure 4C) or BaxΔC (Figure 4D), mitochondria were stained with MitoTracker (blue), and mitochondrial ceramide and Bax were localized using anti-ceramide IgM (red) or anti-Bax IgG (green), respectively. Control IgM and IgG did not yield detectable signals (see Figure 5A). Consistent with the Western analysis (Figure S6), Figures 4C and 4D show that endogenous Bax was not immunodetected in naïve mitochondria (top lanes), while barely detectable ceramide-containing domains were observed at low incidence (top lanes). Very little added recombinant full length Bax or BaxΔC co-localized with these small ceramide-rich domains (second lanes in Figures 4C and 4D, respectively). Addition of exogenous C16-ceramide alone to naïve mitochondria resulted in formation of large MCRMs in 65–75% of mitochondria (third lanes and bottom lanes in Figures 4C and 4D), with recombinant full length Bax or BaxΔC co-localizing in ∼70% of these newly-formed platforms (identified as whitish-yellow merged signals in the bottom lanes). Further, more than half of the C16-ceramide uptake, 60±5% of the total, went to manufacturing these newly-formed MCRMs. MCRM formation was not an artifact of fixation preceding staining as reported for the Forssman antigen on the apical surface of MDCKII cells [58], as reversing this order did not impact MCRM detection (Figure S10).


Mitochondrial ceramide-rich macrodomains functionalize Bax upon irradiation.

Lee H, Rotolo JA, Mesicek J, Penate-Medina T, Rimner A, Liao WC, Yin X, Ragupathi G, Ehleiter D, Gulbins E, Zhai D, Reed JC, Haimovitz-Friedman A, Fuks Z, Kolesnick R - PLoS ONE (2011)

Ceramide induces formation of a mitochondrial ceramide-rich macrodomain (MCRM).(A) Ionizing radiation (10 Gy) induces co-localization of endogenous Bax with MCRMs in HeLa cells. Mitochondria were isolated from HeLa cells 34 h after irradiation and immunostained as described in Supporting Information Text S1. Data represent typical stainings from 1 of 4 similar studies in which 2000 mitochondria were analyzed each. (B) Addition of exogenous C16-ceramide induces co-localization of endogenous full-length Bax with MCRMs in HeLa cells. Mitochondria were isolated from HeLa cells using percoll gradient and treated with ceramide as Figure 3A. After 30 min incubation, mitochondria were fixed and stained with MitoTracker (blue), while ceramide and Bax were localized using anti-ceramide IgM (red) or anti-Bax IgG (green), respectively. Control IgM and IgG did not yield detectable signals (not shown). These data represent 1 of 3 similar studies. (C) Bax translocates into a radiation-generated HeLa MCRM. Upper panel: 34 h post-irradiation, HeLa mitochondria were isolated as in Materials and Methods and incubated with 0.15% Triton X-100 in MBS buffer for 30 min on ice. 40 µl mitochondrial homogenate (3.3 µg/µl) were subjected to 5–30% mini-discontinuous sucrose density gradient centrifugation as described in Materials and Methods. 20 µl aliquots of 80 µl fractions were analyzed by immunoblotting using the indicated antibodies. The protein level of each fraction was assessed using the Bio-Rad Dc protein assay kit (PE, Pellet). Data are from 1 of 4 studies, consisting of 2 independent gradients per study. The gradient shown displays our clearest example of Bax translocation into light membranes. Lower panel: Bax in each fraction, revealed by immunoblotting and quantified using NIH Image software, was normalized to protein content for all 8 gradients. (D) MCRM Bax exists as high molecular weight oligomers. Mitochondria from 10 Gy-irradiated HeLa cells, disrupted by either (a) 1% CHAPS and sonication or (b) dounce homogenization in 0.15% Triton X-100, were subjected to 5–30% discontinuous sucrose gradient for MCRM isolation as in Experimental Procedures. Light (MCRM; fractions 6,7) and heavy fractions (solubilized proteins; fractions 11,12) were analyzed by gel filtration on Sephacryl S-200 column as in Figure 2C. 500 µl of each eluted fraction were concentrated by 20% TCA precipitation for immunoblotting. Data are from 3 independent studies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3113798&req=5

pone-0019783-g005: Ceramide induces formation of a mitochondrial ceramide-rich macrodomain (MCRM).(A) Ionizing radiation (10 Gy) induces co-localization of endogenous Bax with MCRMs in HeLa cells. Mitochondria were isolated from HeLa cells 34 h after irradiation and immunostained as described in Supporting Information Text S1. Data represent typical stainings from 1 of 4 similar studies in which 2000 mitochondria were analyzed each. (B) Addition of exogenous C16-ceramide induces co-localization of endogenous full-length Bax with MCRMs in HeLa cells. Mitochondria were isolated from HeLa cells using percoll gradient and treated with ceramide as Figure 3A. After 30 min incubation, mitochondria were fixed and stained with MitoTracker (blue), while ceramide and Bax were localized using anti-ceramide IgM (red) or anti-Bax IgG (green), respectively. Control IgM and IgG did not yield detectable signals (not shown). These data represent 1 of 3 similar studies. (C) Bax translocates into a radiation-generated HeLa MCRM. Upper panel: 34 h post-irradiation, HeLa mitochondria were isolated as in Materials and Methods and incubated with 0.15% Triton X-100 in MBS buffer for 30 min on ice. 40 µl mitochondrial homogenate (3.3 µg/µl) were subjected to 5–30% mini-discontinuous sucrose density gradient centrifugation as described in Materials and Methods. 20 µl aliquots of 80 µl fractions were analyzed by immunoblotting using the indicated antibodies. The protein level of each fraction was assessed using the Bio-Rad Dc protein assay kit (PE, Pellet). Data are from 1 of 4 studies, consisting of 2 independent gradients per study. The gradient shown displays our clearest example of Bax translocation into light membranes. Lower panel: Bax in each fraction, revealed by immunoblotting and quantified using NIH Image software, was normalized to protein content for all 8 gradients. (D) MCRM Bax exists as high molecular weight oligomers. Mitochondria from 10 Gy-irradiated HeLa cells, disrupted by either (a) 1% CHAPS and sonication or (b) dounce homogenization in 0.15% Triton X-100, were subjected to 5–30% discontinuous sucrose gradient for MCRM isolation as in Experimental Procedures. Light (MCRM; fractions 6,7) and heavy fractions (solubilized proteins; fractions 11,12) were analyzed by gel filtration on Sephacryl S-200 column as in Figure 2C. 500 µl of each eluted fraction were concentrated by 20% TCA precipitation for immunoblotting. Data are from 3 independent studies.
Mentions: After addition of C16-ceramide, and/or recombinant full-length (FL) Bax (Figure 4C) or BaxΔC (Figure 4D), mitochondria were stained with MitoTracker (blue), and mitochondrial ceramide and Bax were localized using anti-ceramide IgM (red) or anti-Bax IgG (green), respectively. Control IgM and IgG did not yield detectable signals (see Figure 5A). Consistent with the Western analysis (Figure S6), Figures 4C and 4D show that endogenous Bax was not immunodetected in naïve mitochondria (top lanes), while barely detectable ceramide-containing domains were observed at low incidence (top lanes). Very little added recombinant full length Bax or BaxΔC co-localized with these small ceramide-rich domains (second lanes in Figures 4C and 4D, respectively). Addition of exogenous C16-ceramide alone to naïve mitochondria resulted in formation of large MCRMs in 65–75% of mitochondria (third lanes and bottom lanes in Figures 4C and 4D), with recombinant full length Bax or BaxΔC co-localizing in ∼70% of these newly-formed platforms (identified as whitish-yellow merged signals in the bottom lanes). Further, more than half of the C16-ceramide uptake, 60±5% of the total, went to manufacturing these newly-formed MCRMs. MCRM formation was not an artifact of fixation preceding staining as reported for the Forssman antigen on the apical surface of MDCKII cells [58], as reversing this order did not impact MCRM detection (Figure S10).

Bottom Line: Our recent studies in the C. elegans germline indicate that mitochondrial ceramide generation is obligate for radiation-induced apoptosis, although a mechanism for ceramide action was not delineated.Here we demonstrate that ceramide, generated in the mitochondrial outer membrane of mammalian cells upon irradiation, forms a platform into which Bax inserts, oligomerizes and functionalizes as a pore.We posit conceptualization of ceramide as a membrane-based stress calibrator, driving membrane macrodomain organization, which in mitochondria regulates intensity of Bax-induced MOMP, and is pharmacologically tractable in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.

ABSTRACT

Background: Evidence indicates that Bax functions as a "lipidic" pore to regulate mitochondrial outer membrane permeabilization (MOMP), the apoptosis commitment step, through unknown membrane elements. Here we show mitochondrial ceramide elevation facilitates MOMP-mediated cytochrome c release in HeLa cells by generating a previously-unrecognized mitochondrial ceramide-rich macrodomain (MCRM), which we visualize and isolate, into which Bax integrates.

Methodology/principal findings: MCRMs, virtually non-existent in resting cells, form upon irradiation coupled to ceramide synthase-mediated ceramide elevation, optimizing Bax insertion/oligomerization and MOMP. MCRMs are detected by confocal microscopy in intact HeLa cells and isolated biophysically as a light membrane fraction from HeLa cell lysates. Inhibiting ceramide generation using a well-defined natural ceramide synthase inhibitor, Fumonisin B1, prevented radiation-induced Bax insertion, oligomerization and MOMP. MCRM deconstruction using purified mouse hepatic mitochondria revealed ceramide alone is non-apoptogenic. Rather Bax integrates into MCRMs, oligomerizing therein, conferring 1-2 log enhanced cytochrome c release. Consistent with this mechanism, MCRM Bax isolates as high molecular weight "pore-forming" oligomers, while non-MCRM membrane contains exclusively MOMP-incompatible monomeric Bax.

Conclusions/significance: Our recent studies in the C. elegans germline indicate that mitochondrial ceramide generation is obligate for radiation-induced apoptosis, although a mechanism for ceramide action was not delineated. Here we demonstrate that ceramide, generated in the mitochondrial outer membrane of mammalian cells upon irradiation, forms a platform into which Bax inserts, oligomerizes and functionalizes as a pore. We posit conceptualization of ceramide as a membrane-based stress calibrator, driving membrane macrodomain organization, which in mitochondria regulates intensity of Bax-induced MOMP, and is pharmacologically tractable in vitro and in vivo.

Show MeSH