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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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Ionizing radiation-induced ceramide elevation is confined to mitochondria.(A) Time- and dose-dependent induction of apoptosis in response to ionizing radiation. Morphologic changes of nuclear apoptosis were detected using the DNA-specific fluorochrome bis-benzimide. Data (mean±SEM) are collated from 3 experiments analyzing 500 cells per point. (B) Time-dependent cytochrome c (Cyt. c) release after 10 Gy. Cytosolic fractions of HeLa cells, collected at the indicated times post-irradiation were analyzed by immunoblotting using α-Cyt. c. Data are from 1 representative of 3 studies. (C) Increased CS activity in isolated mitochondria (MT) after 20 Gy. CS activity was measured at 28 h post-irradiation. Data are from 2 experiments. (D) Radiation increases mitochondrial ceramide content. At 33 h post-irradiation, ceramide was quantified by the diacylglycerol kinase assay in mitochondrial-enriched and ER-enriched fractions from HeLa cells. Inset: The ER-enriched fraction was devoid of mitochondrial contamination based on western blotting with anti-COXII (mitochondrial marker), while the mitochondrial fraction was 10.9±1.7% (mean±SE) ER based on anti-Climp63 (ER marker) blotting. Data (mean±SE) are from 4 experiments performed in triplicate. *, p<0.05 vs. control. (E) Ionizing radiation increases CS activity in MAM and ceramide levels in MAM-free mitochondria (MT). HeLa cells were harvested 33 h post-10 Gy and organelles isolated as in Materials and Methods. ER (P100) was fractionated by differential centrifugation and mitochondria and MAM within the heavy membrane fraction (P10) were further separated from each other by 30% Percoll gradient. Upper panel; CS activity was measured in each fraction using sphinganine and palmitoyl-CoA as substrates as in Materials and Methods. Middle panel: purity of ER, Mitochondria and MAM fractions was analyzed by immunoblotting with antibodies to Calnexin (ER marker) and COXII (MT marker). Based on anti-Calnexin blotting, Percoll-purified mitochondria were 3–4% contaminated with ER. Lower panel; Ceramide levels were quantified by diacylglycerol kinase assay as in Materials and Methods. (F) Co-localization of mitochondrial ceramide and COXI after 20 Gy. At 33 h post-irradiation, HeLa cells were stained with anti-ceramide IgM (red) and anti-COXI IgG (green). Images were acquired with a Leica TCS AOBS SP2 confocal microscope equipped with a 63×1.4NA OIL DIC D objective combined with 4× scan zoom, and co-localization (yellow) was analyzed with MetaMorph 7.5 software. Scale bar; 10 µm. Inset images (rectangles) represent 4× magnification of left upper (0 Gy) and left lower (20 Gy) regions for observation of co-localization. Data are from 1 of 5 experiments.
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pone-0019783-g001: Ionizing radiation-induced ceramide elevation is confined to mitochondria.(A) Time- and dose-dependent induction of apoptosis in response to ionizing radiation. Morphologic changes of nuclear apoptosis were detected using the DNA-specific fluorochrome bis-benzimide. Data (mean±SEM) are collated from 3 experiments analyzing 500 cells per point. (B) Time-dependent cytochrome c (Cyt. c) release after 10 Gy. Cytosolic fractions of HeLa cells, collected at the indicated times post-irradiation were analyzed by immunoblotting using α-Cyt. c. Data are from 1 representative of 3 studies. (C) Increased CS activity in isolated mitochondria (MT) after 20 Gy. CS activity was measured at 28 h post-irradiation. Data are from 2 experiments. (D) Radiation increases mitochondrial ceramide content. At 33 h post-irradiation, ceramide was quantified by the diacylglycerol kinase assay in mitochondrial-enriched and ER-enriched fractions from HeLa cells. Inset: The ER-enriched fraction was devoid of mitochondrial contamination based on western blotting with anti-COXII (mitochondrial marker), while the mitochondrial fraction was 10.9±1.7% (mean±SE) ER based on anti-Climp63 (ER marker) blotting. Data (mean±SE) are from 4 experiments performed in triplicate. *, p<0.05 vs. control. (E) Ionizing radiation increases CS activity in MAM and ceramide levels in MAM-free mitochondria (MT). HeLa cells were harvested 33 h post-10 Gy and organelles isolated as in Materials and Methods. ER (P100) was fractionated by differential centrifugation and mitochondria and MAM within the heavy membrane fraction (P10) were further separated from each other by 30% Percoll gradient. Upper panel; CS activity was measured in each fraction using sphinganine and palmitoyl-CoA as substrates as in Materials and Methods. Middle panel: purity of ER, Mitochondria and MAM fractions was analyzed by immunoblotting with antibodies to Calnexin (ER marker) and COXII (MT marker). Based on anti-Calnexin blotting, Percoll-purified mitochondria were 3–4% contaminated with ER. Lower panel; Ceramide levels were quantified by diacylglycerol kinase assay as in Materials and Methods. (F) Co-localization of mitochondrial ceramide and COXI after 20 Gy. At 33 h post-irradiation, HeLa cells were stained with anti-ceramide IgM (red) and anti-COXI IgG (green). Images were acquired with a Leica TCS AOBS SP2 confocal microscope equipped with a 63×1.4NA OIL DIC D objective combined with 4× scan zoom, and co-localization (yellow) was analyzed with MetaMorph 7.5 software. Scale bar; 10 µm. Inset images (rectangles) represent 4× magnification of left upper (0 Gy) and left lower (20 Gy) regions for observation of co-localization. Data are from 1 of 5 experiments.

Mentions: Morphologic evidence of apoptosis was detected in 5–15 Gy irradiated HeLa cells by 36 h and became significant at 48 h (Figure 1A; p<0.001 vs. control). Increased cytosolic effector caspase 3 activity, measured fluorometrically by cleavage of the substrate Z-DEVD-AFC, was similarly detected by 30 h post-irradiation, peaking at 36 h, remaining elevated for at least 72 h after 10 Gy (see Figure 2E for 36 h data). Cytochrome c release from mitochondria into cytosol was detected at 30–32 h after 10 Gy by Western analysis (Figure 1B) and was maximal at 36 h. Consistent with the cytochrome c release profile, Bax insertion into the MOM also began at 30 h, peaking at 34 h after 10 Gy (Figure S1A). In agreement with previous studies [44], Bax insertion occurred without apparent translocation from cytosol (not shown), but rather from a Bax pool loosely attached to unirradiated HeLa mitochondria but not inserted into MOM [37], [45]. This pool, which is readily removed by 100 mM Na2CO3 washing and does not spontaneously induce MOMP [45], [46], represents the large majority of endogenous Bax co-isolated with HeLa mitochondria (estimated at ∼80% in our preparations based on semi-quantitative densitometry normalized for protein recovery; n = 8). At 34 h post-irradiation, the fraction of Bax inserted into the MOM had increased 4–5-fold (Figure S1A, p<0.001 vs. control unirradiated, n = 6). Bax multimerization into low molecular weight (not shown) and higher molecular weight oligomers (Mr 360 KD) occurred concomitant with Bax insertion (Figure S1B). Whereas mitochondrial Bax was primarily monomeric in unirradiated HeLa cells (n = 4), a majority of Bax (estimated at ∼60% of the total by densitometry) redistributed into high molecular weight oligomeric fractions post-irradiation (Figure S1B). In contrast, Bak and VDAC were found in both fractions in unirradiated controls, and as previously reported [37] redistributed minimally upon irradiation (Figure S1B).


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)

Ionizing radiation-induced ceramide elevation is confined to mitochondria.(A) Time- and dose-dependent induction of apoptosis in response to ionizing radiation. Morphologic changes of nuclear apoptosis were detected using the DNA-specific fluorochrome bis-benzimide. Data (mean±SEM) are collated from 3 experiments analyzing 500 cells per point. (B) Time-dependent cytochrome c (Cyt. c) release after 10 Gy. Cytosolic fractions of HeLa cells, collected at the indicated times post-irradiation were analyzed by immunoblotting using α-Cyt. c. Data are from 1 representative of 3 studies. (C) Increased CS activity in isolated mitochondria (MT) after 20 Gy. CS activity was measured at 28 h post-irradiation. Data are from 2 experiments. (D) Radiation increases mitochondrial ceramide content. At 33 h post-irradiation, ceramide was quantified by the diacylglycerol kinase assay in mitochondrial-enriched and ER-enriched fractions from HeLa cells. Inset: The ER-enriched fraction was devoid of mitochondrial contamination based on western blotting with anti-COXII (mitochondrial marker), while the mitochondrial fraction was 10.9±1.7% (mean±SE) ER based on anti-Climp63 (ER marker) blotting. Data (mean±SE) are from 4 experiments performed in triplicate. *, p<0.05 vs. control. (E) Ionizing radiation increases CS activity in MAM and ceramide levels in MAM-free mitochondria (MT). HeLa cells were harvested 33 h post-10 Gy and organelles isolated as in Materials and Methods. ER (P100) was fractionated by differential centrifugation and mitochondria and MAM within the heavy membrane fraction (P10) were further separated from each other by 30% Percoll gradient. Upper panel; CS activity was measured in each fraction using sphinganine and palmitoyl-CoA as substrates as in Materials and Methods. Middle panel: purity of ER, Mitochondria and MAM fractions was analyzed by immunoblotting with antibodies to Calnexin (ER marker) and COXII (MT marker). Based on anti-Calnexin blotting, Percoll-purified mitochondria were 3–4% contaminated with ER. Lower panel; Ceramide levels were quantified by diacylglycerol kinase assay as in Materials and Methods. (F) Co-localization of mitochondrial ceramide and COXI after 20 Gy. At 33 h post-irradiation, HeLa cells were stained with anti-ceramide IgM (red) and anti-COXI IgG (green). Images were acquired with a Leica TCS AOBS SP2 confocal microscope equipped with a 63×1.4NA OIL DIC D objective combined with 4× scan zoom, and co-localization (yellow) was analyzed with MetaMorph 7.5 software. Scale bar; 10 µm. Inset images (rectangles) represent 4× magnification of left upper (0 Gy) and left lower (20 Gy) regions for observation of co-localization. Data are from 1 of 5 experiments.
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pone-0019783-g001: Ionizing radiation-induced ceramide elevation is confined to mitochondria.(A) Time- and dose-dependent induction of apoptosis in response to ionizing radiation. Morphologic changes of nuclear apoptosis were detected using the DNA-specific fluorochrome bis-benzimide. Data (mean±SEM) are collated from 3 experiments analyzing 500 cells per point. (B) Time-dependent cytochrome c (Cyt. c) release after 10 Gy. Cytosolic fractions of HeLa cells, collected at the indicated times post-irradiation were analyzed by immunoblotting using α-Cyt. c. Data are from 1 representative of 3 studies. (C) Increased CS activity in isolated mitochondria (MT) after 20 Gy. CS activity was measured at 28 h post-irradiation. Data are from 2 experiments. (D) Radiation increases mitochondrial ceramide content. At 33 h post-irradiation, ceramide was quantified by the diacylglycerol kinase assay in mitochondrial-enriched and ER-enriched fractions from HeLa cells. Inset: The ER-enriched fraction was devoid of mitochondrial contamination based on western blotting with anti-COXII (mitochondrial marker), while the mitochondrial fraction was 10.9±1.7% (mean±SE) ER based on anti-Climp63 (ER marker) blotting. Data (mean±SE) are from 4 experiments performed in triplicate. *, p<0.05 vs. control. (E) Ionizing radiation increases CS activity in MAM and ceramide levels in MAM-free mitochondria (MT). HeLa cells were harvested 33 h post-10 Gy and organelles isolated as in Materials and Methods. ER (P100) was fractionated by differential centrifugation and mitochondria and MAM within the heavy membrane fraction (P10) were further separated from each other by 30% Percoll gradient. Upper panel; CS activity was measured in each fraction using sphinganine and palmitoyl-CoA as substrates as in Materials and Methods. Middle panel: purity of ER, Mitochondria and MAM fractions was analyzed by immunoblotting with antibodies to Calnexin (ER marker) and COXII (MT marker). Based on anti-Calnexin blotting, Percoll-purified mitochondria were 3–4% contaminated with ER. Lower panel; Ceramide levels were quantified by diacylglycerol kinase assay as in Materials and Methods. (F) Co-localization of mitochondrial ceramide and COXI after 20 Gy. At 33 h post-irradiation, HeLa cells were stained with anti-ceramide IgM (red) and anti-COXI IgG (green). Images were acquired with a Leica TCS AOBS SP2 confocal microscope equipped with a 63×1.4NA OIL DIC D objective combined with 4× scan zoom, and co-localization (yellow) was analyzed with MetaMorph 7.5 software. Scale bar; 10 µm. Inset images (rectangles) represent 4× magnification of left upper (0 Gy) and left lower (20 Gy) regions for observation of co-localization. Data are from 1 of 5 experiments.
Mentions: Morphologic evidence of apoptosis was detected in 5–15 Gy irradiated HeLa cells by 36 h and became significant at 48 h (Figure 1A; p<0.001 vs. control). Increased cytosolic effector caspase 3 activity, measured fluorometrically by cleavage of the substrate Z-DEVD-AFC, was similarly detected by 30 h post-irradiation, peaking at 36 h, remaining elevated for at least 72 h after 10 Gy (see Figure 2E for 36 h data). Cytochrome c release from mitochondria into cytosol was detected at 30–32 h after 10 Gy by Western analysis (Figure 1B) and was maximal at 36 h. Consistent with the cytochrome c release profile, Bax insertion into the MOM also began at 30 h, peaking at 34 h after 10 Gy (Figure S1A). In agreement with previous studies [44], Bax insertion occurred without apparent translocation from cytosol (not shown), but rather from a Bax pool loosely attached to unirradiated HeLa mitochondria but not inserted into MOM [37], [45]. This pool, which is readily removed by 100 mM Na2CO3 washing and does not spontaneously induce MOMP [45], [46], represents the large majority of endogenous Bax co-isolated with HeLa mitochondria (estimated at ∼80% in our preparations based on semi-quantitative densitometry normalized for protein recovery; n = 8). At 34 h post-irradiation, the fraction of Bax inserted into the MOM had increased 4–5-fold (Figure S1A, p<0.001 vs. control unirradiated, n = 6). Bax multimerization into low molecular weight (not shown) and higher molecular weight oligomers (Mr 360 KD) occurred concomitant with Bax insertion (Figure S1B). Whereas mitochondrial Bax was primarily monomeric in unirradiated HeLa cells (n = 4), a majority of Bax (estimated at ∼60% of the total by densitometry) redistributed into high molecular weight oligomeric fractions post-irradiation (Figure S1B). In contrast, Bak and VDAC were found in both fractions in unirradiated controls, and as previously reported [37] redistributed minimally upon irradiation (Figure S1B).

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
Related in: MedlinePlus