In Mitochondria β-Actin Regulates mtDNA Transcription and Is Required for Mitochondrial Quality Control

I stumbled upon a new Cell Press journal called “iScience”. This really sounds like a journal for apple fanboys as defined by the urban dictionary

“…One who believes adding an "i" prefix to anything is automatically superior to anything that does not have an "i" in front of it.” –Urban dictionary

I was yesterday reading a recent article in this journal and I have to say the “I” prefix doesn’t seem to make this journal any better. If something, it seems to be worse than the actual Science journal by publishing even more correlative, unsubstantiated and hyperbolic findings.

The article was about actin within mitochondria (Xie et al. 2018). This publication refers extensively to an older paper suggesting that actin would reside within mitochondria (Reyes et al. 2011), although on my opinion this is probably just an experimental artefact. As it turns out Xie et al. didn’t try to reproduce these findings but took them at face value.

In this article from Xie et al. the authors compared mitochondria in WT mouse embryonic fibroblasts (MEFs) and beta-actin knockout fibroblasts. First they stained mitochondria with MitoTracker Orange and saw that in KO MEFs mitochondria have changed morphology, which is not surprising as mitochondria are known to utilize the actin cytoskeleton to move and dock i.e. actin network is needed for proper mitochondrial dynamics. The authors believe this change in morphology is caused by the beta-actin’s ability to control mitochondrial membrane potential, which could affect also mitochondrial morphology. Somehow I prefer the idea that mitochondrial dynamics is disrupted to altered cytoskeleton.

Next they treated cells with oxidative phosphorylation system (OXPHOS) inhibitors and CCCP, which will uncouple various cellular membrane potentials (Padman et al. 2013). In most conditions the KO MEF mitochondria were more sensitive to these treatments, which is, again, not surprising as the KO MEF mitochondria have altered dynamics. A notable exception was the complex IV inhibitor cyanide (KCN) had a similar effect in both cell types. Also, inhibiting complex V increased mitochondrial membrane potential more in the knockout cells. To me this would suggest that the KO MEFs have less leakage across the mitochondrial inner membrane or that the electron transport system is working at an increased rate. The authors suggest that the KO MEFs have “more unused capacity for proton storage” whatever that means.

Next, the authors measure complex II/III activity and saw that this was decreased in the knockout MEFs (Fig. 2). Quite often, complex II activity is used to normalize the activities of other complexes because complex II is the only complex which all proteins are nuclear encoded. Because the complex II/III activity was decreased in knockout MEFs, this would suggest that the mitochondrial transcription could be increased leading to increased complex III activity. This result is at odds with other experiments in this article but this conflict is not discussed.

They also re-analyzed some published RNA-seq data to observe possible changes in nuclear encoded OXPHOS genes. This, however, is rather meaningless as we previously showed that OXPHOS subunit RNA levels do not correlate well with the protein levels (Kühl et al. 2017).

I think the Figure S3E is one of the most important ones in this paper. The authors tried to restore mitochondrial membrane potential in knockout MEFs by over-expressing actin-GFP. They did not see a rescue effect when expressing actin in the cytosol or nucleus, but importantly the over-expression was less than 1/30 of the amount of endogenous actin. For this reason it is unclear to me why they even show this experiment. Instead, they should have designed an actin over-expression system reaching the WT actin levels.

Figure 3 left me completely unconvinced. The authors show using immunocytochemistry that some actin staining and mitochondrial staining overlap and so they conclude that actin is within mitochondria. It is just like people studying mitochondria-endoplasmic reticulum contacts who stain both networks and see overlapping signals but provide no evidence that these are actual contact sites. Similarly, Xie et el. provide no evidence that this actin is actually within mitochondria.

To artificially target actin into mitochondria the authors added a mitochondrial targeting sequence (MTS) to the N-terminus of actin. Unfortunately, they did not show whether A) actin is functional with this targeting sequence or whether MTS-actin is actually imported into mitochondrial matrix or just co-localizes with mitochondrial staining. Surprisingly, this MTS-actin slightly increased the mitochondrial membrane potential and some mitochondrial transcript levels. I would have liked to see a control where the authors would have targeted some other non-mitochondrial proteins into mitochondria, such as MTS-GFP or MTS-LacZ, to see whether any protein would have had the same effect.

All in all the authors didn’t provide any convincing evidence that actin would be imported into mitochondria. Knocking out actin has clearly various effects on mitochondrial morphology and respiration etc. but this is to be expected because mitochondria move and dock using actin but concluding from this that actin regulates mitochondrial gene expression is just naïve.

References:

Kühl I, Miranda M, Atanassov I, Kuznetsova I, Hinze Y, Mourier A, Filipovska A, Larsson NG. Transcriptomic and proteomic landscape of mitochondrial dysfunction reveals secondary coenzyme Q deficiency in mammals. Elife. 2017. PMID: 29132502

Padman BS, Bach M, Lucarelli G, Prescott M, Ramm G. The protonophore CCCP interferes with lysosomal degradation of autophagic cargo in yeast and mammalian cells. Autophagy. 2013. PMID: 24150213

Reyes A, He J, Mao CC, Bailey LJ, Di Re M, Sembongi H, Kazak L, Dzionek K, Holmes JB, Cluett TJ, Harbour ME, Fearnley IM, Crouch RJ, Conti MA, Adelstein RS, Walker JE, Holt IJ. Actin and myosin contribute to mammalian mitochondrial DNA maintenance. Nucleic Acids Res. 2011. PMID: 21398640

Xie X, Venit T,  Drou N, Percipalle P. In Mitochondria β-Actin Regulates mtDNA Transcription and Is Required for Mitochondrial Quality Control. iScience 2018. PMID: Not available

Endonuclease G promotes mitochondrial genome cleavage and replication

I have always had in mind the textbook model where EndoG is localized in the mitochondrial intermembrane space (IMS) from where it is released upon apoptosis to degrade nuclear DNA. Every now and then though, there are papers suggesting it would have a mitochondrial function such as regulation of mitochondrial DNA (mtDNA) removal in fruit fly sperm (DeLuca et al. 2012, Yu et al. 2017).

In this paper from Wiehe et al. (Wiehe et al. 2018) the authors suggest EndoG has something to do with mtDNA replication. In the introduction the authors cite a classical paper showing the IMS localization of EndoG (Ohsato et al. 2002) but also another paper suggesting EndoG is either directly or indirectly associated with the mitochondrial inner membrane (Uren et al. 2005). It is important to point out that Uren et al. never showed that EndoG would be in the mitochondrial matrix.

There is data both for and against the function of EndoG in mtDNA maintenance although the former is rather weak. The EndoG knockout mice have normal mtDNA copy number (Irvine et al. 2005, David et al. 2006) providing the strongest evidence for the lack of mitochondrial function. There is one study, however, shoving that EndoG knockout mice have decreased mtDNA copy number (McDermott-Roe et al. 2011). Wiehe et al. refer to this same McDermott-Roe et al. study suggesting that EndoG would directly interact with mtDNA. This evidence seems spurious at best, because the authors performed chromatin immunoprecipitation (ChIP) using purified mitochondria (McDermott-Roe et al. 2011). It is expected that if you carry out a pulldown with a DNA binding protein (EndoG) and the only DNA around comes from mitochondria, one will enrich mtDNA. This does not show that EndoG would interact with mtDNA in vivo. It seems there is no strong evidence showing that EndoG would be localized in the mitochondrial matrix.

To visualize mitochondrial DNA replication and transcription in cells, the Wiehe et al. use so called mitochondrial Transcription and Replication Imaging Protocol (mTRIP). This method is based on fluorescence probes binding mtDNA (mREP) or mtRNA (mTRANS). The mtDNA binding probes is located upstream of the replication origin of the heavy strand, between the light- and heavy-strand promoters (LSP and HSP) (Chatre & Ricchetti 2013). It has been suggested that this probe can access mtDNA only during the initiation of mtDNA replication and 7S DNA synthesis. It is unclear to me, why it would not access this region during the initiation of mtDNA transcription from the LSP and how sensitive this probe is to changing mtDNA topology.

Wiehe et al. use this mREP mtDNA binding probe as evidence for initiation for mtDNA replication but it seems to me these probes can measure several indistinguishable things (initiation of transcription, initiation of mtDNA replication, initiation of 7S DNA synthesis, differences in mtDNA topology). The authors also used qPCR to quantify the levels of 7S DNA and mtDNA and it seems that the amount of 7S DNA was decreased as was the mtDNA copy number when measured proximal, but not distal, to the replication origin (Fig. 2). It would have been nice to see a Southern blot of mtDNA to actually see whether there is some paused/abortive replication going on.

Next, Wiehe et al over-expressed WT and catalytically inactive EndoG but it is not shown how strong this over-expression was (Fig. 3). Also, the over-expressed proteins were tagged (Myc-DDK) and it is not addressed whether the tagged proteins are functional. The authors compared the effects of these over-expressions on mREP signal and saw a statistically significant but to me a biologically meaningless increase in mREP signal, which, as explained above, can come from many sources.

Next in figure 4 the authors amplified mtDNA in two large overlapping fragments using long-range PCR and saw that upon EndoG knockdown the amount of amplified product decreases in only one of the fragments, which included the whole minor arc. This long-range PCR could be affected by various factors such as replication stalling, DNA base damage and nicking of DNA so it is unclear what the authors are observing here. Weirdly enough, this effect on long-range PCR amplification was not reproduced in a later experiment (Fig. 5D).

All in all the paper assumes without any evidence that EndoG is in the mitochondrial matrix. The results are largely based on fluorescence probes and long-range PCR, both of which are sensitive to various effectors.

Just to be pedantic I have to correct the first sentence of this manuscript. MtDNA encodes 13 proteins, 11 of which are part of the electron transport system and all 13 are part of the oxidative phosphorylation system.

Stats:

The authors used statistical tests which do not assume normal distribution. Therefore it would have been nice to see etc. box plots instead of bar plots to see the sample distribution. Also, the authors do not seem to correct any statistical methods for multiple comparisons.

References:

Chatre L, Ricchetti M. Prevalent coordination of mitochondrial DNA transcription and initiation of replication with the cell cycle. Nucleic Acids Res. 2013. PMID: 23345615

David KK, Sasaki M, Yu SW, Dawson TM, Dawson VL. EndoG is dispensable in embryogenesis and apoptosis. Cell Death Differ. 2006. PMID: 16239930

DeLuca SZ, O'Farrell PH. Barriers to male transmission of mitochondrial DNA in sperm development. Dev Cell. 2012. PMID: 22421049

Irvine RA, Adachi N, Shibata DK, Cassell GD, Yu K, Karanjawala ZE, Hsieh CL, Lieber MR. Generation and characterization of endonuclease G null mice. Mol Cell Biol. 2005. PMID: 15601850

McDermott-Roe C, Ye J, Ahmed R, Sun XM, Serafín A, Ware J, Bottolo L, Muckett P, Cañas X, Zhang J, Rowe GC, Buchan R, Lu H, Braithwaite A, Mancini M, Hauton D, Martí R, García-Arumí E, Hubner N, Jacob H, Serikawa T, Zidek V, Papousek F, Kolar F, Cardona M, Ruiz-Meana M, García-Dorado D, Comella JX, Felkin LE, Barton PJ, Arany Z, Pravenec M, Petretto E, Sanchis D, Cook SA. Endonuclease G is a novel determinant of cardiac hypertrophy and mitochondrial function. Nature. 2011. PMID: 21979051

Ohsato T, Ishihara N, Muta T, Umeda S, Ikeda S, Mihara K, Hamasaki N, Kang D. Mammalian mitochondrial endonuclease G. Digestion of R-loops and localization in intermembrane space. Eur J Biochem. 2002. PMID: 12444964

Uren RT, Dewson G, Bonzon C, Lithgow T, Newmeyer DD, Kluck RM. Mitochondrial release of pro-apoptotic proteins: electrostatic interactions can hold cytochrome c but not Smac/DIABLO to mitochondrial membranes. J Biol Chem. 2005. PMID: 15537572

Wiehe RS, Gole B, Chatre L, Walther P, Calzia E, Ricchetti M, Wiesmüller L. Endonuclease G promotes mitochondrial genome cleavage and replication. Oncotarget. 2018. PMID: 29719607

Yu Z, O'Farrell PH, Yakubovich N, DeLuca SZ. The Mitochondrial DNA Polymerase Promotes Elimination of Paternal Mitochondrial Genomes. Curr Biol. 2017. PMID: 28318978

Acetylation and phosphorylation of human TFAM regulate TFAM–DNA interactions via contrasting mechanisms

For years now there has been a lot of hype regarding the importance of mitochondrial protein acetylation (Carrico et al. 2018). As covered in our recent review (Kauppila et al. 2017) and also backed up by some more recent research (James et al. 2017), mitochondrial acetylation is non-enzymatic i.e. random. In addition, most acetylations at any given site are present at very low levels (1%) (Weinert et al. 2014, Weinert et al. 2015) making it difficult to see that they would have any biological function.

Now a new paper from King et al. (King et al. 2018) suggests that mitochondrial transcription factor A (TFAM) would be both acetylated and phosphorylated and that this would regulate TFAM-mtDNA interactions. Phosphorylated TFAM was published already few years ago by some of the same authors (Lu et al. 2013). According to the previous study the levels of phosphorylated TFAM in vivo is miniscule perhaps because phosphorylation targets TFAM for degradation. In this newer study by King et al. the authors detect TFAM acetylation in HEK293 cells over-expressing TFAM. However, it seems the authors did not quantify to what extent each lysine residue is acetylated. Based on the low global acetylation stoichiometries in mitochondria, I would guess the TFAM acetylation levels to be very low. Rest of the experiments were done using TFAM with acetyl-lysine mimicking mutations basically reflecting a 100% acetylation.

At this point I stopped reading the manuscript, because one should first show how prevalent these acetylations are in vivo before studying their effects in vitro. In other words, I think the authors might have put a lot of effort studying something that does not exist.

References:

Carrico C, Meyer JG, He W, Gibson BW, Verdin E. The Mitochondrial Acylome Emerges: Proteomics, Regulation by Sirtuins, and Metabolic and Disease Implications. Cell Metab. 2018. PMID: 29514063

James AM, Hoogewijs K, Logan A, Hall AR, Ding S, Fearnley IM, Murphy MP. Non-enzymatic N-acetylation of Lysine Residues by AcetylCoA Often Occurs via a Proximal S-acetylated Thiol Intermediate Sensitive to Glyoxalase II. Cell Rep. 2017. PMID: 28249157

Kauppila TES, Kauppila JHK1, Larsson NG. Mammalian Mitochondria and Aging: An Update. Cell Metab. 2017. PMID: 28094012

King GA, Shabestari MH, Taris KKH, Pandey AK, Venkatesh S, Thilagavathi J, Singh K, Koppisetti RK, Temiakov D, Roos WH, Suzuki CK, Wuite GJL. Nucleic Acids Res. 2018. PMID: Not yet in PUBMED

Lu B, Lee J, Nie X, Li M, Morozov YI, Venkatesh S, Bogenhagen DF, Temiakov D, Suzuki CK. Phosphorylation of human TFAM in mitochondria impairs DNA binding and promotes degradation by the AAA+ Lon protease. Mol Cell. 2013. PMID: 23201127

Weinert BT, Iesmantavicius V, Moustafa T, Schölz C, Wagner SA, Magnes C, Zechner R, Choudhary C. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. Mol Syst Biol. 2014. PMID: 24489116

Weinert BT, Moustafa T, Iesmantavicius V, Zechner R, Choudhary C. Analysis of acetylation stoichiometry suggests that SIRT3 repairs nonenzymatic acetylation lesions. EMBO J. 2015. PMID: 26358839

Replication machinery degrading linear mitochondrial DNA

Linear mitochondrial DNA (mtDNA) molecules with deletions are a curious case. To my knowledge, they are only present at high levels in a few genetic models, including the mtDNA mutator mouse(Trifunovic et al. 2004, Bailey et al. 2009), mtDNA mutator flies (Bratic et al. 2015), patients carrying mutations in mitochondrial genome maintenance exonuclease 1 (MGME1)(Nicholls et al. 2014) and MGME1 knockout mouse (Matic et al. 2018).

In the case of mtDNA mutator mouse, the level of this linear mtDNA with deletion remains stable with time (Kukat et al. 2009). Additionally, it has been shown using mitochondrially targeted restriction enzymes that linear mtDNA is rapidly degraded (Bayona-Bafaluy et al. 2005). Therefore the question remains, who is degrading these mtDNA fragments?

Moretton et al. tried to answer this question by linearizing mtDNA with mitochondrial targeted restriction enzyme followed by systematic knockdown of exonucleases known/suggested to be in mitochondria (Moretton et al. 2017). These exonucleases included ExoG, EndoG, MGME1, DNA2 and FEN1. EXD2 was not tested, but that was later shown to be localized on the mitochondrial outer membrane (Hensen et al. 2018). Perhaps surprisingly, none of the exonucleases studied by Moretton et al. seemed to participate in degradation of linear mtDNA fragments.

There is still at least one exonuclease that Moretton et al. did not study, which is the exonuclease activity of mitochondrial DNA polymerase (POLG). Indeed, in the absence of nucleotides POLG can engage in so called exonuclease mode where it starts degrading the primer in 3’-5’ direction (Bratic et al. 2015). A recent study in fruit flies tried to answer the question what enzyme degrades mtDNA in fly sperm (Yu et al. 2017). Yu et al. concluded that DmPOLG is involved in this degradation but it takes place independent of its exonuclease activity. I was not convinced by this paper because they saw mtDNA replication in punctae without DmPOLG-GFP and more importantly all the models were based on knocking down various proteins of the minimal mitochondrial replisome with variable efficiencies. Also, when I read this paper I was aware of some in-house work taking place in the group of Martin Graef.

Medeiros et al. showed in yeast that under starvation there is a strong mtDNA depletion if autophagy is inhibited and that this depletion can be rescued by increasing nucleoside pools through supplementation or genetically (Medeiros et al. 2018). Therefore, the authors hypothesized that under starvation the autophagy-deficient cells might have changes in nucleotide pools causing MIP1 (yeast POLG) to enter the above mentioned exonuclease mode and degrade mtDNA. Using an exonuclease-deficient POLG the authors showed that indeed it seems to be POLG which is degrading mtDNA.

The most recent paper on the subject comes from Peeva et al. (Peeva et al. 2018). Peeva et al. tested multiple exonucleases (EXOG, APEX2, ENDOG, FEN1, DNA2, MRE11, RBBP8, MGME1 and POLG) to see whether the knockout or knockdown of these enzymes would affect the degradation of linear mtDNA molecules. Somewhat similar to Medeiros et al., Peeva et al. also suggest that POLG together with MGME1 and TWINKLE would be degrading linear mtDNA.

So there it is. Both in yeast and human cells mtDNA seems to be degraded by the exonuclease activity of POLG. It would be interesting to see how the yeast mtDNA actually looks like at the molecular level when degraded as yeasts do not possess MGME1. One could therefore predict that these degraded molecules would not have blunt ends similarly to the results from humans where POLG is suggested to degrade mtDNA to the 3’-5’ direction and MGME1 to the 5’-3’ direction. It is still open question what controls this degradation, if it is controlled to begin with. Both Medeiros et al. and Peeva et al. hypothesize that low nucleotide pools might be a controlling factor. Although this hypothesis would make sense in the case of yeast under starvation and inhibited autophagy, I don’t see how this would work with these human cells under normal culture conditions. Clearly just linearizing mtDNA is sufficient to promote mtDNA degradation without manipulation of the dNTP pools.

Some open questions for the future:

• Is this kind of degradation present also in more in vivo conditions or whether it is only present in somewhat artificial conditions where most of mtDNA is linearized by restriction enzymes.
• What, if any, is controlling the balance between mtDNA synthesis and degradation?
• Is there a specific endonuclease cleaving the yeast mtDNA before POLG can start degrading mtDNA?
• How is mtDNA degraded in organisms without MGME1?

Perhaps this is a stretch but in Peeva et al. study the authors showed how in WT cells linear mtDNA is degraded piece by piece. Where mitophagy selective for damaged mtDNA to exist, which I think it does not (Kauppila et al. 2017), one would not expect to see these slowly degrading molecules but instead a bulk disappearance of linear mtDNA fragments.

References:

Bailey LJ, Cluett TJ, Reyes A, Prolla TA, Poulton J, Leeuwenburgh C, Holt IJ. Mice expressing an error-prone DNA polymerase in mitochondria display elevated replication pausing and chromosomal breakage at fragile sites of mitochondrial DNA. Nucleic Acids Res. 2009. PMID: 19244310

Bayona-Bafaluy MP, Blits B, Battersby BJ, Shoubridge EA, Moraes CT. Rapid directional shift of mitochondrial DNA heteroplasmy in animal tissues by a mitochondrially targeted restriction endonuclease. Proc Natl Acad Sci U S A. 2005. PMID: 16179392

Bratic A, Kauppila TE, Macao B, Grönke S, Siibak T, Stewart JB, Baggio F, Dols J, Partridge L, Falkenberg M, Wredenberg A, Larsson NG. Complementation between polymerase- and exonuclease-deficient mitochondrial DNA polymerase mutants in genomically engineered flies. Nat Commun. 2015. PMID: 26554610

Hensen F, Moretton A, van Esveld S, Farge G, Spelbrink JN. The mitochondrial outer-membrane location of the EXD2 exonuclease contradicts its direct role in nuclear DNA repair. Sci Rep. 2018. PMID: 29599527

Kauppila TES, Kauppila JHK, Larsson NG. Mammalian Mitochondria and Aging: An Update. Cell Metab. 2017. PMID: 28094012

Kukat A, Trifunovic A. Somatic mtDNA mutations and aging--facts and fancies. Exp Gerontol. 2009. PMID: 18585880

Matic S, Jiang M, Nicholls TJ, Uhler JP, Dirksen-Schwanenland C, Polosa PL, Simard ML, Li X, Atanassov I, Rackham O, Filipovska A, Stewart JB, Falkenberg M, Larsson NG, Milenkovic D. Mice lacking the mitochondrial exonuclease MGME1 accumulate mtDNA deletions without developing progeria. Nat Commun. 2018. PMID: 29572490

Medeiros TC, Thomas RL, Ghillebert R, Graef M. Autophagy balances mtDNA synthesis and degradation by DNA polymerase POLG during starvation. J Cell Biol. 2018. PMID: 29519802

Moretton A, Morel F, Macao B, Lachaume P, Ishak L, Lefebvre M, Garreau-Balandier I, Vernet P, Falkenberg M, Farge G. Selective mitochondrial DNA degradation following double-strand breaks. PLoS One. 2017. PMID: 28453550

Nicholls TJ, Zsurka G, Peeva V, Schöler S, Szczesny RJ, Cysewski D, Reyes A, Kornblum C, Sciacco M, Moggio M, Dziembowski A, Kunz WS, Minczuk M. Linear mtDNA fragments and unusual mtDNA rearrangements associated with pathological deficiency of MGME1 exonuclease. Hum Mol Genet. 2014. PMID: 24986917

Peeva V, Blei D, Trombly G, Corsi S, Szukszto MJ, Rebelo-Guiomar P, Gammage PA, Kudin AP, Becker C, Altmüller J, Minczuk M, Zsurka G, Kunz WS. Linear mitochondrial DNA is rapidly degraded by components of the replication machinery. Nat Commun. 2018. PMID: 29712893

Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink JN, Rovio AT, Bruder CE, Bohlooly-Y M, Gidlöf S, Oldfors A, Wibom R, Törnell J, Jacobs HT, Larsson NG. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature. 2004. PMID: 15164064

Yu Z, O'Farrell PH, Yakubovich N, DeLuca SZ. The Mitochondrial DNA Polymerase Promotes Elimination of Paternal Mitochondrial Genomes. Curr Biol. 2017. PMID: 28318978