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Mitochondrial fusion provides an ‘initial metabolic complementation’ controlled by mtDNA

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Abstract

Heteroplasmic cells, harboring both mutant and normal mitochondrial DNAs (mtDNAs), must accumulate mutations to a threshold level before respiratory activity is affected. This phenomenon has led to the hypothesis of mtDNA complementation by inter-mitochondrial content mixing. The precise mechanisms of heteroplasmic complementation are unknown, but it depends both on the mtDNA nucleoid dynamics among mitochondria as well as the mitochondrial dynamics as influenced by mtDNA. We tracked nucleoids among the mitochondria in real time to show that they are shared after complete fusion but not ‘kiss-and-run’. Employing a cell hybrid model, we further show that mtDNA-less mitochondria, which have little ATP production and extensive Opa1 proteolytic cleavage, exhibit weak fusion activity among themselves, yet remain competent in fusing with healthy mitochondria in a mitofusin- and OPA1-dependent manner, resulting in restoration of metabolic function. Depletion of mtDNA by overexpression of the matrix-targeted nuclease UL12.5 resulted in heterogeneous mitochondrial membrane potential (ΔΨm) at the organelle level in mitofusin-null cells but not in wild type. In this system, overexpression of mitofusins or application of the fusion-promoting drug M1 could partially rescue the metabolic damage caused by UL12.5. Interestingly, mtDNA transcription/translation is not required for normal mitochondria to restore metabolic function to mtDNA-less mitochondria by fusion. Thus, interplay between mtDNA and fusion capacity governs a novel ‘initial metabolic complementation’.

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Abbreviations

Cap:

Chloramphenicol

DMEM:

Dulbecco’s modified Eagle’s medium

EB:

Ethidium bromide

FBS:

Fetal bovine serum

FI:

Fluorescence intensity

IMM:

Inner mitochondrial membrane

KFP:

Kindling fluorescent protein

MEF:

Mouse embryonic fibroblast

mt:

Mitochondrial

mtFP:

Mitochondrial matrix-targeting fluorescent protein

mtDNAs:

Mitochondrial DNAs

OMM:

Outer mitochondrial membrane

PAGFP:

Photoactivatable green fluorescent protein

PBS:

Phosphate-buffered saline

PEG:

Polyethylene glycol

Q-PCR:

Quantitative polymerase chain reaction. Rho0 cells: cells lacking mtDNA

Tfam:

Mitochondrial transcription factor A

TMRM:

Tetramethylrhodamine methyl ester

VK3:

Vitamin K3

WT:

Wild type

ΔΨm:

Mitochondrial membrane potential

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Acknowledgments

We thank Hongwen Pang for his technical advice and Prof. György Hajnóczky, Prof. Xiaodong Shu, Dr. Juan Du, and Dr. Shen Chen for their expert views on the manuscript. This work was financially supported by the ‘Strategic Priority Research Program’ of the Chinese Academy of Sciences (XDA01020108), the Ministry of Science and Technology 973 program (2013CB967403 and 2012CB721105), the Ministry of Science and Technology 863 Program (2012AA02A708), the National Natural Science Foundation projects of China (31271527), International Cooperation Project of Guangdong Science and Technology Program (2012B050300022), Guangzhou Science and Technology Program (2014Y2-00161), Guangdong Natural Science Foundation for Distinguished Young Scientists (S20120011368), and the “One hundred Talents” Project for Prof. Xingguo Liu from the Chinese Academy of Sciences.

Conflict of interest

The authors declare that they have no conflicts of interest.

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  1. Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China

    Liang Yang, Qi Long, Jinglei Liu, Haite Tang, Yuxing Li, Feixiang Bao, Dajiang Qin, Duanqing Pei & Xingguo Liu

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Fig. S1 mtDNA nucleoids are shared after complete fusion but not ‘kiss-and-run’ in HeLa cells. a Tfam-DsRed co-localizes with anti-mtDNA fluorescence (Scale bar: 2 μm). b Quantitation of mtDNA nucleoid number after overexpressing Tfam-DsRed or mtDsRed in HeLa cells by Anti-DNA immunofluorescence (UT, Untreated). c Time course of mtDNA nucleoid movement during ‘kiss-and-run’ events after PAGFP photoactivation (Scale bar: 1 μm). The graph shows an increase of PAGFP FI in the acceptor mitochondrion (#1). d Time course of a typical mtDNA nucleoids sharing events in HeLa cells by mitochondrial complete fusion as determined by photoactivation (Scale bar: 1 μm). e The percentage of mtDNA nucleoid sharing events that occurred during ‘kiss-and-run’ events per 466.4 s imaging interval (n≥17). #, p < 0.01

Fig. S2 Acquisition of mtDNA nucleoids by mtDNA-less mitochondria by fusion events in Rho0 cells. a b The identification of Rho0 Hela cells. a The protein expression level of Tfam in WT and Rho cells. b Relative levels of Cox1 and Cox2 mtRNA in WT and Rho0 cells. c Time course of a typical mitochondrial fusion event in Rho0 cells (Scale bar: 1 μm). The graph shows an increase of PAGFP FI in the acceptor mitochondrion (#1). d e The heteroplasmic fusion between mtDNA-less and normal mitochondria by PEG-induced cell fusion. d Anti-DNA and anti-Tfam immunofluorescence of Rho0 and WT cells (Scale bar: 10 μm). e Anti-DNA and Anti-Tfam immunofluorescence of WT × Rho0 (mtDsRed) cell hybrids after PEG-mediated cell fusion for 7 h (Scale bar: 10 μm)

Fig. S3 OMM fusion frequency in Rho0 cells. a Labeling of a subpopulation of mitochondria by photoactivation of PAGFP in WT and Rho0 HeLa cells expressing both Omp25-PAGFP and mtDsRed (Scale bar: 10 μm). b OMM fusion was monitored by measuring the dilution of Omp25-PAGFP in subset for 200 s after photoactivation (n=5). c Comparison of OMM fusion events numbers in WT and Rho0 cells (n=5). #, p < 0.01

Fig. S4 Measurements of ΔΨm in WT and Rho0 HeLa cells and the generation of mouse Rho- cell lines by genetic approach. a Measurements of ΔΨm in WT and Rho0 by JC-1 staining (Scale bar: 10 μm). The ratio of red/green FI is used to measure ΔΨm. Rho0 cells had lower ratio, indicating lower ΔΨm than WT. b Measurements of ΔΨm in WT and Rho0 by TMRM staining (Scale bar: 10 μm). c Relative level of ND5 mtDNA in the days following infection with UL12.5-GFP. d Relative level of ND5 mtRNA in the days following infection with UL12.5-GFP. e Measurement of ΔΨm in MEF cells, Mfn1,2-/- MEF cells, and Opa1-/- MEF cells by TMRM staining (Scale bar: 10 μm)

Fig. S5 A single mtDNA-less mitochondrion gains a higher ΔΨm after fusing with a normal mitochondrion by ‘kiss-and-run’ (Scar bar 1 μm)

Fig. S6 Relative levels of Cox1/2 mtDNA in HeLa cells treated with 0.4 μg/mL EB for 48 h. #, p < 0.01

Fig. S7 Overexpression of Mfn1 or Mfn2 does affect neither mtDNA nor mtRNA levels. a Relative levels of ND2 and ND5 mtDNA in MEF cells after overexpression of Mfn1, Mfn2 or Mfn1/2 for 6 days. b Relative levels of ND2 and ND5 mtRNA in MEF cells after overexpression of Mfn1, Mfn2 or Mfn1/2 for 6 days

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Yang, L., Long, Q., Liu, J. et al. Mitochondrial fusion provides an ‘initial metabolic complementation’ controlled by mtDNA. Cell. Mol. Life Sci. 72, 2585–2598 (2015). https://doi.org/10.1007/s00018-015-1863-9

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