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Mitochondrial fusion is increased by the nuclear coactivator PGC-1beta.

Liesa M, Borda-d'Agua B, Medina-Gómez G, Lelliott CJ, Paz JC, Rojo M, Palacín M, Vidal-Puig A, Zorzano A - PLoS ONE (2008)

Bottom Line: Here, we demonstrate that reduced mitochondrial size observed in knock-out mice for the transcriptional regulator PGC-1beta is associated with a selective reduction in Mitofusin 2 (Mfn2) expression, a mitochondrial fusion protein.This PGC-1beta-induced elongation specifically requires Mfn2 as this process is absent in Mfn2-ablated cells.Finally, we show that PGC-1beta increases Mfn2 promoter activity and transcription by coactivating the nuclear receptor Estrogen Related Receptor alpha (ERRalpha).

View Article: PubMed Central - PubMed

Affiliation: Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain.

ABSTRACT

Background: There is no evidence to date on whether transcriptional regulators are able to shift the balance between mitochondrial fusion and fission events through selective control of gene expression.

Methodology/principal findings: Here, we demonstrate that reduced mitochondrial size observed in knock-out mice for the transcriptional regulator PGC-1beta is associated with a selective reduction in Mitofusin 2 (Mfn2) expression, a mitochondrial fusion protein. This decrease in Mfn2 is specific since expression of the remaining components of mitochondrial fusion and fission machinery were not affected. Furthermore, PGC-1beta increases mitochondrial fusion and elongates mitochondrial tubules. This PGC-1beta-induced elongation specifically requires Mfn2 as this process is absent in Mfn2-ablated cells. Finally, we show that PGC-1beta increases Mfn2 promoter activity and transcription by coactivating the nuclear receptor Estrogen Related Receptor alpha (ERRalpha).

Conclusions/significance: Taken together, our data reveal a novel mechanism by which mammalian cells control mitochondrial fusion. In addition, we describe a novel role of PGC-1beta in mitochondrial physiology, namely the control of mitochondrial fusion mainly through Mfn2.

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Related in: MedlinePlus

PGC-1β increases the rate of mitochondrial fusion and changes mitochondrial morphology.(A) Representative Western blot of three independent experiments of C2C12 myoblasts transduced with LacZ or PGC-1β adenovirus at a MOI 200 during 48 h. 40 µg of protein from lysates were loaded and Mfn2, Mfn1, Opa1, Drp1, Fis1 and Porin were detected with specific antibodies. (B) Representative images from a duplicate of one of the same three independent transduction experiments performed in panel (A). Mitochondria were labelled with anti-Cox1 antibody detected with a secondary antibody conjugated to Alexa 594. Scale bar, 10 µm. (C) C2C12 myoblasts quantification and classification attending to mitochondrial tubule length (150–200 cells counted per transduction). Myoblasts with a mean mitochondrial tubule length <4 µm were considered “Short” and >4 µm were considered “Long”. Results are mean±SEM and expressed as percentage of total C2C12 myoblasts counted from three independent transduction experiments with LacZ (white bars) or PGC-1β (black bars) at MOI of 200. *, statistical difference at p<0.01. (D) Electron microscopy images of C2C12 myoblasts transduced with LacZ or PGC-1β adenovirus as in panel (A) were taken at 40.000x magnification . Scale bar 500 nm. (E) 4 hours after PEG, polykaryons of C2C12 myoblasts transduced as in (A) were counted attending to mtGFP (mitochondrial matrix- targeted green fluorescent protein) and mtRFP (mitochondrial matrix-targeted red fluorescent protein) mixing (detected as yellow mitochondria). Polykaryons classified as “Low” displayed <50% of mixed mtGFP and mtRFP (yellow mitochondria). Polykaryons classified as “High” displayed ≥50% of yellow mitochondria. Graphs show mean±SEM of percentage of total polykaryons counted from three independent cell fusion experiments and transduction experiments with LacZ (white bars) or PGC-1β (black bars) at MOI of 200. *, statistical difference at p = 0.02. (F) Representative images from one of three independent transduction and polyethylene glycol-mediated (PEG) cell fusion experiments. C2C12 myoblasts stably expressing mitochondrial matrix-targeted green fluorescent protein (mtGFP) or red fluorescent protein (mtRFP) were transduced with LacZ or PGC-1β adenovirus as in panel (A). Polykaryons were fixed 4 h after 30”–40” PEG treatment. During these 4 h, cells were incubated in growth medium with 100 µg/ml of cycloheximide to inhibit de novo synthesis of mtGFP and mtRFP. Degree of fusion is directly proportional to mixing of mtGFP and mtRFP (yellow mitochondria). Scale bar, 10 µm.
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pone-0003613-g003: PGC-1β increases the rate of mitochondrial fusion and changes mitochondrial morphology.(A) Representative Western blot of three independent experiments of C2C12 myoblasts transduced with LacZ or PGC-1β adenovirus at a MOI 200 during 48 h. 40 µg of protein from lysates were loaded and Mfn2, Mfn1, Opa1, Drp1, Fis1 and Porin were detected with specific antibodies. (B) Representative images from a duplicate of one of the same three independent transduction experiments performed in panel (A). Mitochondria were labelled with anti-Cox1 antibody detected with a secondary antibody conjugated to Alexa 594. Scale bar, 10 µm. (C) C2C12 myoblasts quantification and classification attending to mitochondrial tubule length (150–200 cells counted per transduction). Myoblasts with a mean mitochondrial tubule length <4 µm were considered “Short” and >4 µm were considered “Long”. Results are mean±SEM and expressed as percentage of total C2C12 myoblasts counted from three independent transduction experiments with LacZ (white bars) or PGC-1β (black bars) at MOI of 200. *, statistical difference at p<0.01. (D) Electron microscopy images of C2C12 myoblasts transduced with LacZ or PGC-1β adenovirus as in panel (A) were taken at 40.000x magnification . Scale bar 500 nm. (E) 4 hours after PEG, polykaryons of C2C12 myoblasts transduced as in (A) were counted attending to mtGFP (mitochondrial matrix- targeted green fluorescent protein) and mtRFP (mitochondrial matrix-targeted red fluorescent protein) mixing (detected as yellow mitochondria). Polykaryons classified as “Low” displayed <50% of mixed mtGFP and mtRFP (yellow mitochondria). Polykaryons classified as “High” displayed ≥50% of yellow mitochondria. Graphs show mean±SEM of percentage of total polykaryons counted from three independent cell fusion experiments and transduction experiments with LacZ (white bars) or PGC-1β (black bars) at MOI of 200. *, statistical difference at p = 0.02. (F) Representative images from one of three independent transduction and polyethylene glycol-mediated (PEG) cell fusion experiments. C2C12 myoblasts stably expressing mitochondrial matrix-targeted green fluorescent protein (mtGFP) or red fluorescent protein (mtRFP) were transduced with LacZ or PGC-1β adenovirus as in panel (A). Polykaryons were fixed 4 h after 30”–40” PEG treatment. During these 4 h, cells were incubated in growth medium with 100 µg/ml of cycloheximide to inhibit de novo synthesis of mtGFP and mtRFP. Degree of fusion is directly proportional to mixing of mtGFP and mtRFP (yellow mitochondria). Scale bar, 10 µm.

Mentions: On the basis of the effects of PGC-1β on Mfn2 and Porin expression, we also analyzed whether this coactivator regulates the expression of other proteins involved in mitochondrial dynamics and in the ETC system. PGC-1β-transduced myotubes induced the expression of Mfn1, OPA1, Drp1 and Fis1 (Fig. 2B) in mitochondrial-enriched extracts (1.7-, 2.2-, 1.4-, or 2.1-fold values over basal values, respectively) to a level similar to that detected for Porin (1.45-fold stimulation, Fig. 2C). Mfn2 displayed a significantly higher increase in expression compared to Porin abundance in mitochondrial-enriched extracts (4.3-fold induction, Fig. 2C). When data were expressed as protein levels relative to Porin expression, we only detected significant stimulation of Mfn2 and Fis1 in response to PGC-1β over-expression (Figure S3). Similar results were obtained in total lysates (data not shown). This superior induction of Mfn2 protein was also detected in C2C12 myoblasts (Fig. 3A).


Mitochondrial fusion is increased by the nuclear coactivator PGC-1beta.

Liesa M, Borda-d'Agua B, Medina-Gómez G, Lelliott CJ, Paz JC, Rojo M, Palacín M, Vidal-Puig A, Zorzano A - PLoS ONE (2008)

PGC-1β increases the rate of mitochondrial fusion and changes mitochondrial morphology.(A) Representative Western blot of three independent experiments of C2C12 myoblasts transduced with LacZ or PGC-1β adenovirus at a MOI 200 during 48 h. 40 µg of protein from lysates were loaded and Mfn2, Mfn1, Opa1, Drp1, Fis1 and Porin were detected with specific antibodies. (B) Representative images from a duplicate of one of the same three independent transduction experiments performed in panel (A). Mitochondria were labelled with anti-Cox1 antibody detected with a secondary antibody conjugated to Alexa 594. Scale bar, 10 µm. (C) C2C12 myoblasts quantification and classification attending to mitochondrial tubule length (150–200 cells counted per transduction). Myoblasts with a mean mitochondrial tubule length <4 µm were considered “Short” and >4 µm were considered “Long”. Results are mean±SEM and expressed as percentage of total C2C12 myoblasts counted from three independent transduction experiments with LacZ (white bars) or PGC-1β (black bars) at MOI of 200. *, statistical difference at p<0.01. (D) Electron microscopy images of C2C12 myoblasts transduced with LacZ or PGC-1β adenovirus as in panel (A) were taken at 40.000x magnification . Scale bar 500 nm. (E) 4 hours after PEG, polykaryons of C2C12 myoblasts transduced as in (A) were counted attending to mtGFP (mitochondrial matrix- targeted green fluorescent protein) and mtRFP (mitochondrial matrix-targeted red fluorescent protein) mixing (detected as yellow mitochondria). Polykaryons classified as “Low” displayed <50% of mixed mtGFP and mtRFP (yellow mitochondria). Polykaryons classified as “High” displayed ≥50% of yellow mitochondria. Graphs show mean±SEM of percentage of total polykaryons counted from three independent cell fusion experiments and transduction experiments with LacZ (white bars) or PGC-1β (black bars) at MOI of 200. *, statistical difference at p = 0.02. (F) Representative images from one of three independent transduction and polyethylene glycol-mediated (PEG) cell fusion experiments. C2C12 myoblasts stably expressing mitochondrial matrix-targeted green fluorescent protein (mtGFP) or red fluorescent protein (mtRFP) were transduced with LacZ or PGC-1β adenovirus as in panel (A). Polykaryons were fixed 4 h after 30”–40” PEG treatment. During these 4 h, cells were incubated in growth medium with 100 µg/ml of cycloheximide to inhibit de novo synthesis of mtGFP and mtRFP. Degree of fusion is directly proportional to mixing of mtGFP and mtRFP (yellow mitochondria). Scale bar, 10 µm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2570954&req=5

pone-0003613-g003: PGC-1β increases the rate of mitochondrial fusion and changes mitochondrial morphology.(A) Representative Western blot of three independent experiments of C2C12 myoblasts transduced with LacZ or PGC-1β adenovirus at a MOI 200 during 48 h. 40 µg of protein from lysates were loaded and Mfn2, Mfn1, Opa1, Drp1, Fis1 and Porin were detected with specific antibodies. (B) Representative images from a duplicate of one of the same three independent transduction experiments performed in panel (A). Mitochondria were labelled with anti-Cox1 antibody detected with a secondary antibody conjugated to Alexa 594. Scale bar, 10 µm. (C) C2C12 myoblasts quantification and classification attending to mitochondrial tubule length (150–200 cells counted per transduction). Myoblasts with a mean mitochondrial tubule length <4 µm were considered “Short” and >4 µm were considered “Long”. Results are mean±SEM and expressed as percentage of total C2C12 myoblasts counted from three independent transduction experiments with LacZ (white bars) or PGC-1β (black bars) at MOI of 200. *, statistical difference at p<0.01. (D) Electron microscopy images of C2C12 myoblasts transduced with LacZ or PGC-1β adenovirus as in panel (A) were taken at 40.000x magnification . Scale bar 500 nm. (E) 4 hours after PEG, polykaryons of C2C12 myoblasts transduced as in (A) were counted attending to mtGFP (mitochondrial matrix- targeted green fluorescent protein) and mtRFP (mitochondrial matrix-targeted red fluorescent protein) mixing (detected as yellow mitochondria). Polykaryons classified as “Low” displayed <50% of mixed mtGFP and mtRFP (yellow mitochondria). Polykaryons classified as “High” displayed ≥50% of yellow mitochondria. Graphs show mean±SEM of percentage of total polykaryons counted from three independent cell fusion experiments and transduction experiments with LacZ (white bars) or PGC-1β (black bars) at MOI of 200. *, statistical difference at p = 0.02. (F) Representative images from one of three independent transduction and polyethylene glycol-mediated (PEG) cell fusion experiments. C2C12 myoblasts stably expressing mitochondrial matrix-targeted green fluorescent protein (mtGFP) or red fluorescent protein (mtRFP) were transduced with LacZ or PGC-1β adenovirus as in panel (A). Polykaryons were fixed 4 h after 30”–40” PEG treatment. During these 4 h, cells were incubated in growth medium with 100 µg/ml of cycloheximide to inhibit de novo synthesis of mtGFP and mtRFP. Degree of fusion is directly proportional to mixing of mtGFP and mtRFP (yellow mitochondria). Scale bar, 10 µm.
Mentions: On the basis of the effects of PGC-1β on Mfn2 and Porin expression, we also analyzed whether this coactivator regulates the expression of other proteins involved in mitochondrial dynamics and in the ETC system. PGC-1β-transduced myotubes induced the expression of Mfn1, OPA1, Drp1 and Fis1 (Fig. 2B) in mitochondrial-enriched extracts (1.7-, 2.2-, 1.4-, or 2.1-fold values over basal values, respectively) to a level similar to that detected for Porin (1.45-fold stimulation, Fig. 2C). Mfn2 displayed a significantly higher increase in expression compared to Porin abundance in mitochondrial-enriched extracts (4.3-fold induction, Fig. 2C). When data were expressed as protein levels relative to Porin expression, we only detected significant stimulation of Mfn2 and Fis1 in response to PGC-1β over-expression (Figure S3). Similar results were obtained in total lysates (data not shown). This superior induction of Mfn2 protein was also detected in C2C12 myoblasts (Fig. 3A).

Bottom Line: Here, we demonstrate that reduced mitochondrial size observed in knock-out mice for the transcriptional regulator PGC-1beta is associated with a selective reduction in Mitofusin 2 (Mfn2) expression, a mitochondrial fusion protein.This PGC-1beta-induced elongation specifically requires Mfn2 as this process is absent in Mfn2-ablated cells.Finally, we show that PGC-1beta increases Mfn2 promoter activity and transcription by coactivating the nuclear receptor Estrogen Related Receptor alpha (ERRalpha).

View Article: PubMed Central - PubMed

Affiliation: Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain.

ABSTRACT

Background: There is no evidence to date on whether transcriptional regulators are able to shift the balance between mitochondrial fusion and fission events through selective control of gene expression.

Methodology/principal findings: Here, we demonstrate that reduced mitochondrial size observed in knock-out mice for the transcriptional regulator PGC-1beta is associated with a selective reduction in Mitofusin 2 (Mfn2) expression, a mitochondrial fusion protein. This decrease in Mfn2 is specific since expression of the remaining components of mitochondrial fusion and fission machinery were not affected. Furthermore, PGC-1beta increases mitochondrial fusion and elongates mitochondrial tubules. This PGC-1beta-induced elongation specifically requires Mfn2 as this process is absent in Mfn2-ablated cells. Finally, we show that PGC-1beta increases Mfn2 promoter activity and transcription by coactivating the nuclear receptor Estrogen Related Receptor alpha (ERRalpha).

Conclusions/significance: Taken together, our data reveal a novel mechanism by which mammalian cells control mitochondrial fusion. In addition, we describe a novel role of PGC-1beta in mitochondrial physiology, namely the control of mitochondrial fusion mainly through Mfn2.

Show MeSH
Related in: MedlinePlus