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