Diet impact on mitochondrial bioenergetics and dynamics

http://journal.frontiersin.org/article/10.3389/fphys.2015.00109/full

Rosalba Putti, Raffaella Sica, Vincenzo Migliaccio and Lillà Lionetti

 

In this study they investigate the effect of two different fat dietary sources (saturated (HL diet) vs polyunsaturated (HFO diet) omega 3 ) on mitochondrial dynamics and function in rat liver and skeletal muscle. The consequences of starvation and caloric restriction (CR) are also discussed.

The high fat diet rich in saturated fatty acid (HL diet) decreases mitochondrial function and increases ROS production. Mitochondria became more fragmented. Mitochondria do become more efficient due to a decrease in proton leak. An increase in energy efficiency reduces energy expenditure and can contribute to obesity development. In several studies it has been suggested that decreases in Mfn2 lead to decreases in proton leak. Mfn2 has also been linked to regulation of in vivo insulin resistance. Increased mitochondrial fragmentation induced by HL diet may be an adaptive cellular response to increase oxidation of surplus dietary fatty acids, which results in higher ROS production.

In contrast to the HL diet, the HFO diet rich in polyunsaturated fatty acids seems to improve mitochondrial function. ROS production is reduced, and increased mitochondrial fusion is seen. HFO diet also leads to a mild mitochondrial uncoupling due to enhanced expression of uncoupling protein 2. Mitochondrial efficiency is thus decreased, which may explain the decrease in ROS and observed increase in fatty acid utilization. There was less weight gain in rats with HFO diet compared to rats with HL diet.

Opposite effects on mitochondrial dynamics are seen for two types of nutrient deficiency, starvation and caloric restriction (CR):

Upon starvation, mitochondria fuse, has been associated with increases in ATP production. The fusion of mitochondria during starvation has been suggested to maximize energy production to sustain the cell during nutrient deprivation.

On the other hand, CR (e.g. mice submitted to 40% CR for 6 months) leads to mitochondrial fission. Mitochondrial biogenesis also increases. The larger number of mitochondria seen was linked to a reduction in oxygen consumption, membrane potential and ROS. Levels of ATP production were no different in CR conditions vs. control. It is like the cell increases the number of mitochondria so that each mitochondrion works less hard, which then decreases ROS. Having mitochondria fragmented also means that dysfunctional mitochondria can be more easily degraded.

What is the function of mitochondrial networks? A theoretical assessment of hypotheses and proposal for future research

 http://onlinelibrary.wiley.com/doi/10.1002/bies.201400188/full

 Hanne Hoitzing, Iain G. Johnston and Nick S. Jones

Mitochondria are dynamic organelles: sometimes they are fragmented, sometimes they form giant fused networks across the cell, and sometimes they take on intermediate shapes. What is the reason for having these different possible morphologies?

In this paper we discuss the function of large fused mitochondrial networks. Some hypotheses existing in the literature are being discussed, and some new ones are proposed. A mathematical perspective is taken. Coarse-grained models, simulations and estimations are used to try to gain insights. To enable a mathematical description of mitochondrial fusion, the terms microfusion, mesofusion, static hyperfusion and dynamic hyperfusion are introduced. Improvements for models are suggested, and future experiments are proposed.

Among the insights found are the possibilities that selective fusion alone leads to an increase in mitochondrial quality control; that increased fusion may have non-linear effects on the diffusion rate of proteins; that the effect on membrane potential of fusion may be more complicated than a simple averaging; and that fusion can act to dampen biochemical fluctuations.

Cardiolipin is a key determinant for mtDNA stability and segregation during mitochondrial stress

http://www.sciencedirect.com/science/article/pii/S0005272815000572#bb0060

Luis Alberto Luévano-Martínez,Maria Fernanda Forni,Valquiria Tiago dos Santos,Nadja C. Souza-Pinto and Alicia J. Kowaltowski

Cardiolipin makes up about 15% of the phospholipid content of the mitochondrial inner membrane. Cardiolipin is known to stabilize ATP synthase dimers and respiratory chain super complexes, and is able to promote cristae-like structures by responding to pH gradients.

In this paper, they investigate the role of cardiolipin in the cellular response to mitochondrial stress in yeast. A genetic model which lacks the cardiolipin synthase gene is used.

Results show that cells lacking cardiolipin have increased levels of PG, the precursor of cardiolipin. In optimal growth conditions, cells without cardiolipin show no defects in respiration, whereas under thermal stress these cells show decreases in both basal and maximal respiratory rates. Thermal stress reduces the amount of respiratory chain complexes, including subunits encoded by mtDNA, this suggests that lack of cardiolipin may lead to mtDNA instability. Further results suggest that mtDNA indeed becomes more prone to stress-induced damage when no cardiolipin is present.

Cardiolipin may be involved in mtDNA segregation during budding.

MtDNA is normally anchored to the inner membrane, and perhaps cardiolipin plays a role in this anchoring. The inner membrane of mitochondria in yeast in composed of phosphatidylcholine (PC, about 38%), phosphatidylethanolamine (PE, 24 %), phosphatidylinositol (16%), cardiolipin (16%), phosphatidylserine (PS, 4%) and phosphoatidic acid (1.5%). In the paper they show that isolated nucleoids bind more to cardiolipin than to its precursor PG. No significant binding is observed to PC. Also when nucleoids isolated from mammalian cell lines were used, affinity for binding to cardiolipin was higher than binding to any of the other phospholipids. Further results suggest that mtDNA can anchor to the inner membrane in the absence of cardiolipin under control conditions, but not under thermal stress.

Asymmetric apportioning of aged mitochondria between daughter cells is required for stemness

http://www.sciencemag.org/content/early/2015/04/01/science.1260384.long

Pekka Katajisto, Julia Döhla, Christine Chaffer, Nalle Pentinmikko, Nemanja Marjanovic, Sharif Iqbal, Roberto Zoncu, Walter Chen, Robert A. Weinberg, David M. Sabatini

The amount of mitochondrial content in a stem cell is thought to influence its tendency to differentiate. In this study, the authors use stemlike cells (SLCs), expressing photoactivatable green fluorescent protein (paGFP), to investigate the effect of protein aging. PaGFP only fluoresces after exposure to UV light. After UV exposure, and allowing the cells to age, old proteins fluoresce whereas new proteins do not. The authors tag a particular mitochondrial protein (Omp25) with paGFP and find that stem cells apportion the >10 hour old proteins asymmetrically between daughters, by a factor of ~5.6. This effect was not found for membrane proteins of other organelles, and not found at all in non-stem cells.

The authors probe this further, by using mitochondrial proteins fused to a Snap-tag. This method allows precise temporal labelling: young proteins appear green and proteins which are 10h old appear red. Interestingly, they find that young proteins are mainly found in the periphery of the cell. Whilst old mitochondria were asymetrically partitioned, young mitochondria were more uniformly distributed between daughters. They found that this asymmetry indicated the formation of two lineages amongst the daughters: daughters with mainly young mitochondria were more stem-like whereas those with old mitochondria tended to differentiate.

Finally, the authors sought to determine the cause of this asymmetry. Membrane potential was investigated, as this is known to correlate with stemness properties. Although they did indeed find that more stem-like cells tended to have higher ΔΨm, alterations of ΔΨm with an uncoupler had no effect on age-selective segregation, so this appears to be an effect rather than a cause of the age asymmetry. However, upon inhibition of Parkin (a pro-mitophagy protein) or Drp1 (pro-fission), daughter cells tended to inherit old and young mitochondria more symmetrically. Thus the authors suggest that perturbations which challenge mitochondrial quality control tend to remove this age-asymmetric partitioning, which maintains stemness properties.

Fatty Acid Trafficking in Starved Cells: Regulation by Lipid Droplet Lipolysis, Autophagy, and Mitochondrial Fusion Dynamics

 http://www.sciencedirect.com/science/article/pii/S1534580715000726

 Angelika S. Rambold, Sarah Cohen and Jennifer Lippincott-Schwartz

Under nutrient starvation, fatty acids (FAs), which are often stored in lipid droplets, move into mitochondria to drive beta oxidation-based metabolism to sustain energy levels. Exactly how FA become mobilized and delivered into mitochondria is unclear. In this paper they investigate which mechanisms are used to release FAs into the cytoplasm and how FAs move into mitochondria.

They find that FAs are mainly released from lipid droplets by lipolysis (as opposed to lipophagy).

In starved MEFs, almost all lipid droplets were closely associated with mitochondria and this allows FAs to move directly from the lipid droplets into mitochondria. Mitochondria were also highly fused in the starved cells, enabling equilibration of FAs throughout the mitochondrial population. In cells with fusion deficiencies (through knockout of Mfn1 or Opa1), many mitochondrial elements were not close to lipid droplets and FAs did not become homogeneously distributed across the mitochondrial population.

In starved wild-type cells, a rapid increase in FA oxidation was seen, and cells could almost maintain total mitochondrial respiration levels over the entire starvation period (24 hours). Mfn1 knockout cells initially showed an increase in FA oxidation but within 24 hours FA oxidation reduced significantly, causing total mitochondrial respiration levels to drop over time.

It is suggested that delivery of FAs to mitochondria occurs at limited sites, only there where lipid droplets are in close proximity to mitochondria. This may explain why lipid droplets and mitochondria are so closely associated. Having too many free FAs in the cytoplasm can cause damage, so efficient movement of FAs from the droplets into mitochondria is beneficial. Having mitochondria all fused up then enables the FAs to distribute themselves homogeneously throughout the mitochondrial population.

Lineage correlations of single cell division time as a probe of cell-cycle dynamics

http://www.nature.com/nature/journal/v519/n7544/full/nature14318.html

Oded Sandler, Sivan Pearl Mizrahi, Noga Weiss, Oded Agam, Itamar Simon & Nathalie Q. Balaban

Heterogeneity amongst populations of cells is a fundamental observation in cell biology. One example of this is cell cycle duration, which can be thought of as being determined stochastically, perhaps by inheritance of mitochondrial content at mitosis. According to this intuition, mathematical models can be constructed to describe the expected correlation between mothers, daughters and cousin cell cycle periods, such as the bifurcating autoregression model. This model predicts that the correlation between cousins is less than that between a mother and daughter cell. 

However, time series which appear stochastic in nature can sometimes be derived from underlying deterministic, chaotic, behaviour. The authors set out to determine whether cell cycle distributions are indeed stochastic, or deterministically chaotic. Using Fucci markers, the authors generated lymphoblasts which fluoresced red at G1 phase, yellow at S phase, green at G2 and no fluorescence during mitosis. Using single-cell microscopy, they found that the cell cycle period between cousins had a greater Spearman's correlation (0.63) than between mothers and daughters (0.04). In other words, this is the reverse of what is to be expected from the bifurcating autoregression model, which they label as the 'cousin-mother inequality'.

The authors go on to suggest that the cousin-mother inequality is evidence for deterministic inheritance. They use the Grassberger-Procaccia algorithm on the cell cycle periods, to examine whether the data is stochastic in nature, or in fact deterministic chaos. This returns a quantity called the 'correlation dimension', which is thought to be low for deterministic systems and high for stochastic ones (although see here for subtleties associated with this). They find a small correlation dimension (~3) for the cell cycle period data (whereas random noise is >10), and use this to conclude that cell cycle times are deterministic. They suggest a dynamical model (containing 6 parameters) to explain their data: the 'kicked cell cycle' model. This states that cell-cycle duration is drawn from a deterministic circadian clock oscillator (see Fig. 3D).

It remains to be seen whether there exist alternative stochastic models to the bifurcating autoregression model, which can explain the heterogeneity in cell cycle times. For instance, can the inheritance of mitochondrial content at cell division also recover the correlation between cousins?

Proportionality: A Valid Alternative to Correlation for Relative Data

http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1004075

David Lovell, Vera Pawlowsky-Glahn, Juan José Egozcue, Samuel Marguerat, Jürg Bähler

When trying to understand when a quantity covaries with another, a standard set of tools which come to mind are correlation coefficients. But consider three statistically independent variables: X, Y and Z which have no correlation. Plotting a large number of samples from X vs Y, Y vs Z or X vs Z will indeed give small correlation coefficients. However, the quantities X/Z and Y/Z must be correlated due to their common divisor, which can mislead us in believing that X is correlated with Y, which we know is untrue (this is clearly shown in Fig. 1A). Thus, if we are interested in relationships between X and Y, searching for correlation between X/Z and Y/Z can be misleading. It should be noted that this is only a concern when Z is a random variable, with a large enough variance. If Z is a constant number across experiments, then our intuition for correlation coefficients is restored. The lesson is 'correlation between relative abundances is meaningless, if we are using different normalisations for each condition'.

This statistical trap is easy to overlook, as it is commonplace to search for correlations in quantities which are normalised (say mRNA of gene 1/total mRNA, i.e. X/Z).  The authors highlight that if X/Z and Y/Z are proportional across each sample, then X must be proportional to Y. They therefore suggest a 'goodness-of-fit to proportionality' as a more appropriate statistic when searching for covariation in relative abundances. This is defined as ϕ = var(log(A/B))/var(log A), where A = X/Z and B = Y/Z. ϕ is zero when A and B are perfectly proportional.

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Update: For enthusiasts!

Let's use some Monte Carlo to test this out! Using the notation Unif(p,q) as a uniform distribution with p as the minimum and q as the maximum. I have generated draws from three uniform random variables: X ~ Unif(1,2), Y ~ Unif(4,8), Z ~ Unif(5, 300). We see that none of the variables correlate with each other. However, when we create new variables A = X/Z and B = Y/Z, we see a striking correlation (0.95). So one cannot claim that X is correlated with Y, just because A is correlated with B.

 

This is a particularly pathogenic example, since Z has a huge variance. This simulation yielded ϕ=0.11. Check out the comments on this post to see some back-and-forth between David and I on this.

Leptin modulates mitochondrial function, dynamics and biogenesis in MCF - 7 cells

http://www.ncbi.nlm.nih.gov/pubmed/25752935

Blanquer-Rosselló MD, Santandreu FM, Oliver J, Roca P, Valle A.

Leptin is a hormone that regulates energy expenditure and suppresses food intake. The concentration of leptin in the blood rises as body weight and fat mass increase. Leptin is also involved in many other processes including sex maturation, lactation, immune response and the development of mammary gland. Leptin can increase cell proliferation and inhibits apoptosis in breast cells.

In this paper they investigate the link between leptin and metabolism in breast cancer cells. Cancer cells must rewire cell metabolism to satisfy demands of growth and proliferation. They analyze the effects of a physiological dose of leptin in several features of cellular and mitochondrial metabolism in MCF-7 breast cancer cells.

They find that cellular ATP levels become more reliant on mitochondria in leptin-treated cells and rates of glycolysis decreased. Mitochondrial oxygen consumption increases, but no changes are seen in mitochondrial volume density, respiratory chain proteins or proton leak. ROS levels were decreased and autophagy was increased.

They conclude that leptin ameliorates oxidative stress and increases mitochondrial ATP production in breast cancer cells, which may benefit growth and survival.

Stable heteroplasmy at the single-cell level is facilitated by intercellular exchange of mtDNA

http://nar.oxfordjournals.org/content/43/4/2177.full.pdf

Anitha D Jayaprakash, Erica Benson, Swapna Gone et al.

Deep sequencing allows the measurement of mtDNA heteroplasmy at a single-cell level. However, regions of mtDNA exist in the nuclear genome (nDNA), called Numts. This is due to ancestral transfer of genetic material from the mitochondria to the nucleus. Numts may have variable sequence and copy number, so failure to separate nDNA from mtDNA can cause inaccuracy in heteroplasmy measurement. Although methods currently exist to deal with this, it is unclear whether they are able to resolve heteroplasmies below 5%.

The authors present a method called Mseek, to enzymatically digest linear nDNA and leave behind circular mtDNA, to a purity >98% (whereas endogenously, mtDNA can form <1% of genetic material). Thus ability to resolve different haplotypes is only limited by sequencing depth.  By inspecting multiple cell lines, the authors found that heteroplasmy is a ubiquitous phenomenon, with most mutations being transitions, indicating replication errors of polymerase-γ.

To determine whether heteroplasmy in cull culture originates from a population of cells homoplasmic for different mutations, or a population of heteroplasmic cells, the authors performed the following experiment. They extracted two single cells from a colony, and passaged them for ~25 generations to derive two new colonies. They then measured single-cell heteroplasmy levels for the two new colonies. They found that the derived colonies were heteroplasmic and had similar haplotype distributions. They show that a simple computational model of random genetic drift would shift the haplotype distribution by a large extent over this many generations. Their observations imply that individual cells are heteroplasmic, and this heteroplasmy is relatively stable over a time scale of ~25 generations. There is therefore a mechanism to counteract random drift.

Exchange of mtDNA between cells could bring the haplotype distribution closer to the average across the population. To test this hypothesis, the authors co-cultured cell lines with distinct haplotype distributions, one of which was GFP-labelled. After 4 weeks of co-culture, one of the cell lines was selected for (either with FACS or antibiotic-resistance), and then cultured for a further 4 weeks. In two of four pairs of cell lines tested, they found transfer of mtDNA from one cell line to the other. In the other two pairs, there was no genetic transfer. Thus mtDNA transfer does appear to occur, but is not universal. Furthermore, it is not a necessary mechanism to counteract genetic drift, but it is perhaps sufficient.

The Mitochondrial-Derived Peptide MOTS-c Promotes Metabolic Homeostasis and Reduces Obesity and Insulin Resistance

http://www.sciencedirect.com/science/article/pii/S1550413115000613

Changhan Lee, Jennifer Zeng, Brian G. Drew, Tamer Sallam, Alejandro Martin-Montalvo, Junxiang Wan, Su-Jeong Kim, Hemal Mehta, Andrea L. Hevener, Rafael de Cabo, Pinchas Cohen

Summary: A short open reading frame (sORF) encoding the signaling peptide humanin was previously identified in mitochondria, and suggested the possible existence of others. The authors identified an sORF in 12S rRNA which encodes 16-aa peptide they name MOTS-c, which regulates insulin sensitivity & metabolic homeostasis. This peptide targets skeletal muscle, inhibiting folate and purine biosynthesis, which activates AMPK.

Retrograde signalling via ROS, calcium and cytochrome C is a well-known phenomenon, however this signalling mechanism, dubbed 'mitokines' by the authors, represents a novel means of mitonuclear signalling. sORFs have recently emerged as an area of interest as techniques to study them have improved. sORFs have been found throughout mRNAs, and may be translated through leaky scanning or transcriptional re-initiation.

The authors used an in silico search to identify potential sORFs in human 12S mitochondrial rRNA, which they named MOTS-c (mitochondrial open reading frame of 12S rRNA type-c). It appears that although MOTS-c is transcribed in the mitochondrion, it must be translated in the cytoplasm. The possibility that MOTS-c has arisen from nuclear mtDNA transfer (NUMT) was ruled out as no NUMT peptides are homologous to MOTS-c. Mitochondrial depletion showed elimination of 12S rRNA and MOTS-c transcripts in HeLa cells. Fasting reduced MOTS-c in skeletal muscle, testes and plasma, but heart and brain tissues showed no change.

MOTS-c expression alters metabolite levels, as well as changing gene expression profiles. The authors assess that its target is the folate-methionine cycle, which also affects de novo purine biosynthesis. MOTS-c action leads to a significant decrease in purines. The changes to the folate cycle also have the effect of activating AMPK and causing fatty acid oxidation, as well as increasing glucose utilisation.

MOTS-c treatment in mice on a normal diet led to body weight, food intake, and blood glucose reductions, as well as a decrease in circulating signalling proteins implicated in pathogenesis of both obesity and insulin resistance. When mice were fed a high-fat diet, however, MOTS-c treatment prevented obesity without reducing caloric intake. Heat generation significantly increased, as did respiratory exchange ratio (RER). An increase in RER is indicative of a shift towards higher glucose utilisation. This suggests that MOTS-c treatment leads to avoidance of obesity through increase of energy expenditure due to heat generation. MOTS-c appears to act in skeletal muscle. Promisingly, treating mice for 7 days restored the insulin sensitivity of 12 month old mice to nearly the level of 3 month old mice.

Changes in mitochondrial morphology and bioenergetics in human lymphoblastoid cells with four novel OPA1 mutations.

 http://www.iovs.org/content/early/2015/03/04/iovs.14-16288.long

 Shu-Huei Kao, May-Yung Yen, An-Guor Wang et al

OPA1 is a GTPase protein required for mitochondrial inner membrane fusion. Long and short isoforms of OPA1 exist, a mixture of both of them seems to be necessary for normal mitochondrial fusion. Dissipation of membrane potential induces OPA1 cleavage into its short isoforms and this causes mitochondrial fragmentation.

In this paper they investigate four different human OPA1 mutations in lymphoblastoid cells, to find out whether they affect mitochondrial morphology and bioenergetics in different ways. Two of their mutants only have OPA1 short isoforms, the other two also have some long isoforms.

Normal control cells showed a balanced mitochondrial network between filamentous and fragmented states. In all of the OPA1 mutated cells, mitochondria became more fragmented. The proportions of filamentous, intermediate and fragmented networks were 37%, 44% and 19% in control cells, and 1%, 22%, 77% in the OPA1 mutant cells. Membrane potential and ATP concentrations were reduced in mutant cells (ATP concentration ranged from 56% to 63% of that of control cells).

Additionally, all the OPA1 mutants showed decreases in oxygen consumption rate, maximal respiratory rate and increases (3-5 fold) in proton leak. Higher levels of ROS and oxidative damaged were also found in the mutant cells (a 2-fold increase in hydrogen peroxide and a 3-4 fold increase in superoxide). Also, a 4-8 fold increase in lipid-peroxidation was observed. OPA1 deficient cells preferred glycolysis rather than OXPHOS.

No significant differrences between the different OPA1 mutations were observed.

Mitochondrial function and lifespan of mice with controlled ubiquinone biosynthesis

http://www.nature.com/ncomms/2015/150306/ncomms7393/full/ncomms7393.html

Ying Wang, Daniella Oxer and Siegfried Hekimi

Ubiquinone (UQ) is a central metabolite of the electron transport chain, accepting electrons from complexes I and II, and passing them to complex III. Using Cre-Lox recombination, the authors were able to knock out the enzyme which generates UQ (MCLK1) in adult mice after allowing them to develop normally, as conventional knock out of MCLK1 is embryonic lethal. Two weeks after the chemically-induced knock out, the mice had almost zero MCLK in all tested tissues, besides the liver which was unaffected.  

One might expect the mice to not survive much beyond the intervention, yet astonishingly the median survival was 276 days. UQ became depleted by as much as 90% over 6 months, in the most heavily affected tissues. Blood lactate levels were elevated, suggesting raised glycolysis. Respiratory rates in isolated tissues were reduced, but by no more that a factor of two. Respiratory enzyme activities were raised, likely in compensation for the low UQ availability.  8 months after treatment, the mice had almost zero UQ in heart tissue, and yet were still able to survive. This is particularly surprising, since the heart is such an energetically demanding tissue. Phenotypically, the animals tended to lose body fat, hair and generate a hunched posture. Further studies will be required to elucidate how such severe depletions in this metabolite can be withstood.

Mitochondrial control by DRP1 in brain tumor initiating cells

http://www.nature.com/neuro/journal/vaop/ncurrent/pdf/nn.3960.pdf

Qi Xie, Qiulian Wu, Craig M Horbinski et al.

Brain tumor initiating cells (BTICs) are capable of self-renewal, they are highly proliferative and show chromosomal abnormalities. BTICs are known to take over the glucose transporter GLUT3 so that they can withstand metabolic stress more easily. All tumor cells have dysregulated metabolic pathways, but the highly proliferative nature of BTICs suggests that these tumor subpopulations have some metabolic features that distinguish them from the tumor bulk. In this paper they look at mitochondrial morphology in the most common primary intrinsic brain tumor, gliblastoma.

They compare mitochondrial morphology from BTICs with non-BTIC tumor cells and found out that mitochondria in BTICs are more fragmented and less tubular : in non-BTIC tumor cells, mitochondria show an elongated tubulated structure whereas in BTICs they are shorter and rounded. This suggest that BTICs have increased mitochondrial fragmentation or decreased fusion. It turns out that BTICs show increased phosphorylation of Drp1 at Ser-616 and decreased phosphorylation at Ser-637, both of these changes enhance fission activity of Drp1.

They then checked whether these changed in Drp1 phosphorylation levels were responsible for increased fission in BTICs. A Drp1 gain-of-function mutant, with increased Ser-616 phosphorylation activity and blocked Ser-637 phosphorylation, was expressed in non-BTIC tumor cells. Mitochondria in non-BTIC tumor cells expressing these mutants indeed became more fragmented and less elongated. It also induced expression of some stem cell regulators and repressed some differentiation markers. Expression of the Drp1 mutant was, however, not sufficient to reprogram non-BTIC tumor cells into BTICs.

They then tried to find out whether the change in Drp1 activity is crucial for BTIC maintenance, because it is observed that differentiation of BTICs reduces the hyperactivation of Drp1. Drp1 was knocked down in BTICs using small hairpin RNA lentiviral constructs (shDrp1) , which significantly decreased the growth of BTICs, whereas Drp1 knockdown had no effect on non-BTIC tumor cells or normal neuronal progenitor cells (which are, just like BTICs, capable of differentation and self-renewal). Targeting Drp1 lead to a fourfold decrease in tumorsphere size. They evaluated the potential anti-tumor effects of Drp1 knockdown in vivo in mice, and found that mice with BTICs expressing shDrp1 had reduced tumor formation and increased survival compared to mice with BTICs expressing non-targeted shRNA.

Mdivi-1 is an inhibitor of the GTPase activity of Drp1, and using Mdivi-1 to block Drp1 activity also lead to decreased growth of BTICs. BTICs were implanted into brains of mice, and the mice where then injected with Mdivi-1 which increased mice survival compared with control.

AMPK is a cellular stress sensor. They found that activation of AMPK decreased BTIC growth, and both Drp1 knockdown and Mdivi-1 treatment lead to an increase in AMPK activation. This suggests that the hyperactivity of Drp1 in BTICs may lead to a decreased AMPK activity. Downregulation of both Drp1 and AMPK activity did not reduce the growth of BTICs (as opposed to only downregulating Drp1) which indeed suggests that Drp1 is a critical node in the response of BTICs to metabolic stress through AMPK regulation.

They go on to find the molecular mechanisms that activate Drp1 in BTICs, it turns out that CDK5 and CAMK2 are important. CAMK2 inhibits Drp1 in non-BTIC tumor cells, and its expression is lower in BTICs. CDK5 activates Drp1 and is preferentially expressed in BTICs.

Identification and functional prediction of mitochondrial complex III and IV mutations associated with glioblastoma

http://neuro-oncology.oxfordjournals.org/content/early/2015/03/02/neuonc.nov020.long

Rhiannon E. Lloyd, Kathleen Keatley, D. Timothy J. Littlewood et al.

The role of mtDNA mutations in cancer is subtle. Across most cancers, it has been suggested that mutations are negatively selected for. In this study, the authors provide contrary evidence for glioblastoma multiforme (GBM). They searched for heteroplasmic mutations of complexes III and IV from a mixture of cell lines and human biopsies in GBM patients. They clustered mutations according to three parameters: prevalence in the GBM population; prevalence in the general population; and heteroplasmy level, using ANOVA to check statistical significance. This split the >200 identified mutations into a group of 9 rare functional GBM mutations, and nonfunctional mutations. 43% of GBM tumours carried at least one of the functional mutations.

They mapped the mutations onto bovine crystal structures of the corresponding enzymes, to classify each mutation into one of the following groups: i) frameshift, ii) active site, iii) binding pocket, iv) protein interaction region, v) non-functional. 3D modelling provided mechanistic insight into the function of these mutations.

Integrity of the yeast mitochondrial genome, but not its distribution and inheritance, relies on mitochondrial fission and fusion

http://www.pnas.org/content/112/9/E947.full

Christof Osman, Thomas R. Noriega, Voytek Okreglak et al.

In this study, the authors develop a new means of imaging mtDNA nucleoids in budding yeast, without using dyes. They do this by introducing genes into the mitochondrial genome, whose protein products fluoresce. They constructed 3D images of mtDNA and mitochondrial content distributions. They found that the total length of the mitochondrial network correlates strongly with the number of nucleoids, with approximately 1 nucleoid per micron of network. They found that i) the inter-nucleoid distance, and ii) the distance between the network tip and the closest nucleoid, are significantly different to randomly distributed nucleoids. Indeed, they found that nucleoids tend to be preferentially distributed at the tips of the network, aiding mtDNA inheritance to daughter cells. 

After investigating the physiological case, the authors investigated the effect of knocking out mitochondrial fission (Dnm1) and fusion (fzo1). Remarkably, none of the above observations changed by a large magnitude, indicating that nucleoid distribution and inheritance in yeast is essentially fusion/fission independent. The authors show that respiratory-deficient cells accumulate in  fusion/fission knockouts. This suggests that the role of fusion and fission in yeast is for complementation of respiratory defects in individual mitochondria, rather than distribution and inheritance of mtDNA.

Extensive tissue-related and allele-related mtDNA heteroplasmy suggests positive selection for somatic mutations

http://www.pnas.org/content/112/8/2491.full

Mingkun Li, Roland Schröder, Shengyu Ni et al.

Currently, there is little evidence to support the role of positive selection for mutations in mtDNA; indeed there is evidence that in proliferative tissue, it is negative selection which is the greater force. In this large-scale study, the authors explore the prevalence of different mutations from post-mortem tissues across 152 individuals. They find that particular heteroplasmies occur at particularly high frequencies, based on their nucleotide position, the tissue and the consensus allele. In agreement with other studies, the authors find that the number of heteroplasmies correlates with age. 

The functional explanation for tissue-related and allele-related selection remains unknown, but the authors suggest that it is possibly the metabolic requirements of each tissue which generates a selective pressure. As a case-study, the authors show that there is a strong positive selection for heteroplasmies in the liver which decrease mitochondrial function. Since many metabolic processes in the liver generate damaging byproducts, mutations which result in reduced function may be a means to avoid further damage, dubbed as 'survival of the slowest'.

 

Sensory Detection of Food Rapidly Modulates Arcuate Feeding Circuit

http://www.sciencedirect.com/science/article/pii/S0092867415000768

Yiming Chen, Yen-Chu Liu, Tzu-Wei Kuo, Zachary A Knight

Summary: AgRP and POMC neurons have been observed via optogenetic methods with sub-second time resolution in actively behaving mice. Neural activity fluctuates in response to food presentation and feeding, and has characteristic dynamics within both meals and bouts. This represents a fast-responding, learning/knowledge-based modulation to the slower-fluctuating endocrine-mediated homeostatic regulation.

In this paper, the authors use optogenetic techniques to image hypothalamic activity during feeding behaviour, specifically activity of AgRP and POMC neurons. These share downstream POMC-activated/AgRP-inhibited melanocortin receptors. The paper addresses a particular knowledge gap: short-term dynamics of AgRP and POMC before, during and immediately following feeding.

AgRP neurons (which stimulate feeding) and POMC (which inhibit it) are both believed to have their activity modulated by endocrine signals encoding information about nutritional state, which is supported by this paper. A challenge with ghrelin (an endocrine indicator of hunger) led to AgRP activity almost doubling and POMC activity dropping to nearly half, and this change was reversed after food was consumed.

Surprisingly, AgRP & POMC can be strongly regulated simply by observing food, rather than longer-term endocrine-mediated homeostatic responses.  In fasted mice both POMC and AgRP neurons show a strong and rapid response to presentation of food, with most of the response having already taken place before the first bite occurs. In mice that were previously fed ad libitum this is not observed. Food removal was found to reverse the effects of food presentation, but on a slower timescale.

Detectable but inaccessible food (either in a cage or hidden from view) led to a much smaller and transient response, with the hidden food evoking a smaller change. The appeal of the food also influences the strength of the neural response, with highly appetising foods (peanut butter and chocolate) causing larger responses.

POMC and AgRP neurons also fluctuate within feeding bouts in a meal, with both AgRP and POMC having consistent fluctuations lasting approximately 30 seconds. These resemble the responses on first presentation of food, but at a much smaller level of variation.

Trans-mitochondrial coordination of cristae at regulated membrane junctions

http://www.nature.com/ncomms/2015/150217/ncomms7259/full/ncomms7259.html

Martin Picard, Meagan J. McManus, György Csordás et al.

The existence of junctions which electrically couple adjacent mitochondria through their outer mitochondrial membranes, called inter-mitochondrial junctions (IMJs), has been known for almost three decades.  IMJs do not necessarily form when mitochondria come into contact, and the phenomenon is distinct from mitochondrial fusion, since the inner/outer mitochondrial membranes remain distinct when an IMJ is formed. Indeed, the authors note that the presence of IMJs tend to correlate with how dependent the cell is on oxidative phosphorylation. They are dynamically regulated, and can form on addition of electron donors to generate membrane potential.

The authors investigated the influence of IMJs on cristae structure, and found that cristae align themselves perpendicularly to IMJs, and at a higher density than other regions. Even after genetic perturbations to interfere with cristae structure, the cristae continued to align with IMJs. They observed that cristae would bend a great deal, so they could participate in IMJs, which suggests biological regulation. The authors present a novel drug-inducible system which can physically tether adjacent mitochondria. They show that both cristae and IMJ formation can be induced with this system, by stable juxtaposition of mitochondria. Their data suggest that such structures exist to promote information transfer inside the cytoplasm of eukaryotic cells.

SET overexpression in HEK293 cells regulates mitochondrial uncoupling proteins levels within a mitochondrial fission/reduced autophagic flux scenario

http://www.sciencedirect.com/science/article/pii/S0006291X15001436

Luciana O. Almeida et al

SET is a protein that is accumulated in Alzheimer's disease and some types of cancer. It is hypothesized here that SET influences mitochondrial mechanisms.

In this paper they find that, in HEK293 cells overexpressing SET, levels of mitochondrial uncoupling proteins UCP2 and UCP3 are increased (uncoupling proteins allow for dissipation of the electrochemical gradient across the inner membrane of the mitochondrion). Cellular ATP content was decreased. Also, levels of mitochondrial fission were increased due to increased levels of the fission proteins Drp1 and Fis1.

Heterogeneity for IGF-II production maintained by public goods dynamics in neuroendocrine pancreatic cancer

http://www.pnas.org/content/112/6/1833.full

Marco Archetti, Daniela A. Ferraro, and Gerhard Christofori

Significant heterogeneity exists in intratumour populations. At first glance, this appears to be at odds with the strong selective pressure for the most proliferative subclones. The authors explore this by considering neuroendocrine pancreatic cancer cells, which produce a growth factor called IGF-II. They mix populations of IGF-II positive (producer) cells and IGF-II knockout (nonproducer) cells, to explore under what circumstances heterogeneity can be supported. 

They find that, when cells are grown in nutrient-high conditions, the fraction of producer cells reduces to zero over time. This is because the nonproducer cells are able to free-ride on the IGF-II produced by the producer cells, in a tragedy of the commons. However, at low nutrient conditions, producer cells dominate over the nonproducer cells (at intermediate concentrations of nutrient there was no clear winner). The authors argue that this can be understood in terms of the nonlinear cost of generating IGF-II. When the cost/benefit ratio for producing IGF-II is low (i.e. in nutrient poor conditions), cells that can endogeneously produce the growth factor have an advantage. However, as nutrient availability increases, there is a diminishing return on generating the growth factor, so cells that are nonproducers have an advantage. The authors show via simulation that when such agents are spatially distributed on a lattice, coexistence between producers and nonproducers can occur due to heterogeneity in nutrient availability.

Breast-cancer-secreted ​miR-122 reprograms ​glucose metabolism in premetastatic niche to promote metastasis

http://www.nature.com/ncb/journal/v17/n2/full/ncb3094.html
  
Miranda Y. Fong, Weiying Zhou, Liang Liu et al.

The authors find that many breast cancer cell lines secrete the microRNA miR-122, in vesicles composed of both protein and microRNA. They show that this factor suppresses glucose metabolism by suppressing pyruvate kinase: the last step in glycolysis which generates pyruvate, which is the substrate for the citric acid cycle. The authors show that these vesicles are easily taken up by untransformed cells, and that glucose metabolism is then inhibited as a result. In vivo models show that mice receiving high levels of miR-122 developed significant metastatic colonisation, despite reduced primary tumour formation. The authors suggest that miR-122 is involved in preparing the pre-metastatic niche for tumour cell arrival, by suppressing nutrient uptake of other cells, to favour themselves.

Replication-transcription switch in human mitochondria

http://www.sciencemag.org/content/347/6221/548.full

Karen Agaronyan, Yaroslav I. Morozov, Michael Anikin and Dmitry Temiakov

Mitochondrial DNA (mtDNA) replication coincides with transcription in both time and space. As such, there is potential for collisions between the separate machineries of these processes to cause detrimental effects. This study elucidates the mechanisms of decision making, between transcription and replication. The authors investigate the effects of human transcription elongation factor (TEFM) binding to mitochondrial RNA polymerase (mtRNAP). The authors suggest that mtRNAP initiates replication of mtDNA when unbound to TEFM,  by transcribing short 120nt replication primers and then falling off of the mtDNA. However, when bound to TEFM, mtRNAP transcribes the entire heavy strand, ready for translation into protein. Thus, TEFM behaves as a molecular switch, toggling between replication and transcription of mtDNA, suggesting they are mutually exclusive processes.

Antibiotics that target mitochondria effectively eradicate cancer stem cells, across multiple tumor types: Treating cancer like an infectious disease

http://www.ncbi.nlm.nih.gov/pubmed/25625193?dopt=Abstract

R. Lamb , B. Ozsvari, C.L. Lisanti et al.

It is becoming increasingly clear that cancer stem cells play an important role in tumorigenesis. A key observation is that these cells are highly dependent on mitochondrial oxidative phosphorylation. In this paper, the authors raise the fact that a number of FDA-approved drugs inhibit mitochondrial biogenesis as one of their side-effects. This is because many antibiotics target bacterial ribosomes, which share many similarities with the mitochondrial ribosome. They show that a number of existing antibiotics can reduce tumour-sphere number in a dose-dependent manner: for instance Doxycycline can eradicate two breast cancer cell lines at doses as low as 50um. This approach is perhaps a step towards mutation-independent cancer therapy.

Mitochondrial dynamics and viral infections: A close nexus

http://www.sciencedirect.com/science/article/pii/S0167488915000099

Mohsin Khan, Gulam Hussain Syed, Seong-Jun Kim and Aleem Siddiqui

This paper discusses how viruses manipulate cellular machinery, in particular mitochondria, for their own good. It makes sense for viruses to target mitochondria because it gives them control over the energy production of the cell: the more energy, the more viruses can be made. Perhaps more importantly, by targeting mitochondria the virus may have some control over the survival of the cell as mitochondria are involved in apoptosis. The virus wants to keep the cell alive for as long as possible in order to produce many copies of itself before bursting out the cell and killing it. The paper focusses on how viruses influence mitochondrial dynamics, and how this may influence cell survival. Several viruses and their effects on mitochondrial dynamics are discussed.

Hepatitus C viruses (HCV) cause ER stress, release of calcium from the ER and subsequent uptake of calcium by mitochondria which then depolarize and become dysfunctional. Proteins of HCV can associate directly with the mitochondria and localize to the outer mitochondria membrane, or the mitochondria-associated-ER membrane (MAM). Once associated with MAM, HCV proteases are able to cleave mitochondria associated antiviral signalling proteins (MAVS). MAVS play an important role in immune signalling, and by cleaving MAVS, the virus may be able to evade an immune response. Other HCV proteins are able to perturb the activity of complex I of the respiratory chain, promote mitochondrial calcium uptake and promote ROS production. The virus also messes with mitochondrial quality control, by altering Drp-1 phosphorylation which causes mitochondria to fragment, and increasing the expression of Parkin and PINK1 which promotes mitophagy. When Parkin and Drp1 were depleted from HCV-infected cells, the virus could not induce fragmentation and mitophagy any more, but suddenly apoptotic signalling and cytochrome c release increased, with apoptosis as a consequence. It may thus be that the HCV induced fission followed by mitophagy boosts mitochondrial quality control and helps to prevent cytochrome c release, so that the cell survives longer.

Besides hepatitus C, they also discuss the hepatitus B virus, the Epstein–Barr virus, Human cytomegalovirus, Pseudorabies virus, Influenza virus, Measles virus, Newcastle disease virus, and the SARS coronavirus. Most of these virusses trigger mitochondrial fission, except for the SARS coronavirus, which causes mitochondria to fuse. Why does this virus work differently? Read the paper, and perhaps the answer will be there..

Inhibition of oxidative metabolism leads to p53 genetic inactivation and transformation in neural stem cells

http://www.pnas.org/content/112/4/1059.full

Stefano Bartesaghi, Vincenzo Graziano, Sara Galavotti et al.

Evasion of cell death is known to be one of the hallmarks of cancer. As we know, mitochondria are central organelles to the execution of cell death, and are observed to be perturbed in cancer. In this study, the authors perturb the mitochondria of neural progenitor stem cells in two ways. Firstly, through knockdown of NDUFA10 (a subunit of complex I), they find a shift to glycolytic metabolism and an increase in cell confluence (i.e. cell growth). They then interfere with the gene TK2, which is involved in mtDNA biosynthesis. Perhaps surprisingly, they find TK2 KO cells have a higher ATP yield that the wild-type, despite their glycolytic shift. These cells also had increased cell diameter, an accumulation of cells in S-phase and resistance to differentiation. They found that these KO cells did not tend to express the pro-apoptotic transcription factor p53, rather a shorter isoform Δp53, and were more susceptible to neoplastic transformation.

Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells

http://www.nature.com/cdd/journal/vaop/ncurrent/full/cdd2014211a.html

X Wang and H-H Gerdes

It has previously been shown that cells have the ability to transfer mitochondria through the formation of tunnelling nanotubes (TNTs), which can extend from one cell to another. The authors use UV light to promote apoptosis in a culture of pheochromocytoma 12 cells, and show that cell death can be rescued through coculture with healthy cells. They show that after cytochrome-c release, but before caspase-3 activation, stressed cells extend microtubules out to healthy cells. The healthy cells then donate healthy mitochondria to the stressed cells, causing a reduction in cell death. Inhibition of TNT formation almost eliminates the ability of healthy cells in coculture to rescue cell death.

Variation in cancer risk among tissues can be explained by the number of stem cell divisions

http://www.sciencemag.org/content/347/6217/78.full

Cristian Tomasetti and Bert Vogelstein

Most cells in tissues are partially or fully differentiated, typically short-lived and unlikely to be able to initiate a tumour. However, stem cells have the capacity to self-renew and can be involved in tumourigenesis. It is well known that environmental factors, such as carcinogens, can increase cancer risk; but also that different tissues are intrinsically more predisposed to neoplastic transformation than others. The authors investigate which of these factors explains lifetime cancer risk.

They find that the total number of stem cell differentiations in a particular tissue, correlates strongly (0.804) with the lifetime risk of cancer in that tissue. They calculate that 65% (39% to 81%; 95% CI) of the variance in cancer risk can be attributed to the total number of stem cell divisions in the tissue. The authors propose classifying cancers into two categories: deterministic tumours (D-tumours) which are heavily influenced by the environment, and replicative tumours (R-tumours) which are driven by mainly stochastic factors. They use unsupervised clustering to find that most are R-tumours.

Evidence for frequent and tissue-specific sequence heteroplasmy in human mitochondrial DNA

http://www.sciencedirect.com/science/article/pii/S1567724914001858

Jana Naue, Steffen Hörer, Timo Sänger et al.

The authors measured heteroplasmy levels across 100 individuals taken during autopsy. They examined a number of tissues: blood (control), buccal cells, liver, brain, muscle, heart, lung, bone and hair, with 883 samples in total across a range of ages. They find that muscle and liver cells are the most susceptible tissues to developing mtDNA mutations (79% and 69% of individuals respectively), with only 12 individuals displaying no mutations whatsoever in the measured tissues. Bone (19.8%), blood (18%), lung (17%) and buccal cells (16.2%) showed the fewest number of individuals with mtDNA mutations. They find a strong correlation between the mean number of heteroplasmies in muscle and age (r=0.746).

Origins and functional consequences of somatic mitochondrial DNA mutations in human cancer

http://elifesciences.org/content/3/e02935

Young Seok Ju, Ludmil B Alexandrov, Moritz Gerstung et al.

Mitochondrial DNA (mtDNA) mutations are often observed in cancer, but their causal significance in tumorigenesis has been called into question. This study extensively explores somatic mtDNA mutation in 1675 tumours, and compare their findings to a null model of random genetic drift. They find that silent/missense mutations occur at a rate predicted by simulations of random genetic drift, due to endogenous copying errors during mtDNA replication. However, severe mutations which result in protein truncation (such as frame shifts) are actually negatively selected for - meaning that tumours are observed to have fewer such mutations than expected by chance. Although low heteroplasmy levels of such mutations are tolerable, cells which cannot generate sufficient energy output have a lower fitness and are selected against.

Explicit Tracking of Uncertainty Increases the Power of Quantitative Rule-of-Thumb Reasoning in Cell Biology

http://www.cell.com/biophysj/abstract/S0006-3495%2814%2901124-2

Biological quantities often come with substantial associated uncertainty, either because experimental measurements have errors, or biological systems are naturally variable (or both). This uncertainty sometimes makes it hard to fully interpret rough calculations in biology, because uncertainties in quantities can combine in non-obvious ways. This (awesome :-) ) paper introduces an approach and web tool designed to perform calculations while explicitly tracking uncertainty, so that a calculation doesn't result in a single number but rather a descriptive interpretable distribution. The web tool is linked to the BioNumbers database of experimental measurements in biology, facilitating quick and easy "back-of-the-envelope" calculations in biology.

As a mitochondrial example, here's a toy calculation of the number of protons in a mitochondrion. BioNumber #100438 gives us a guess at the volume of a mitochondrion; BioNumber #105939 tells us the internal pH (each quantity has an uncertainty). We can use the two values to estimate the number of protons in a mitochondrion:
http://www.caladis.org/compute/?q=10^%28-%24105939%29*6e23*%24100438*1e-15&v=105939%3Alogn%2C7.98%2C0.07%3B100438%3Alogn%2C0.5%2C0.25&x=off&n=m&h=fd&a=rad

Aging: A mitochondrial DNA perspective, critical analysis and an update

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4237642/

Inna N Shokolenko, Glenn L Wilson, and Mikhail F Alexeyev

 

This review discusses the recent criticisms of the mainstream view that a vicious cycle of ROS-induced mtDNA damage induces further ROS production and mtDNA damage. For instance, the authors cite evidence that chronic exposure of cells to rotenone (a complex I inhibitor and ROS generator) and hydrogen peroxide causes no significant increase in mtDNA mutation. Indeed, the superoxide radical inhibits the enzyme aconitase, suppressing the Krebs cycle and reducing the supply of NADH and FADH2, which reduces the electron flow through the ETC. Thus there may exist a negative feedback loop for ROS production in oxidative phosphorylation. The authors suggest that ROS may in fact contribute to adaptive signalling to mitigate the effects of ageing. For instance, naked mole-rats live almost 8 times longer than mice, and yet have a much higher oxidative burden, especially in their mtDNA.

Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS

http://www.nature.com/nature/journal/v515/n7527/full/nature13909.html

Edward T. Chouchani, Victoria R. Pell, Edoardo Gaude et al.

The authors investigate the mechanism of ischaemia-reperfusion (IR), which occurs when the blood supply to a tissue is disrupted and then restored, such as during a heart attack or stroke. It is already well-established that tissue damage occurs once blood supply is restored, as this causes the electron transport chain to run in reverse: using ATP to pump protons into the mitochondrial matrix, generating reactive oxygen species (ROS) and inducing cell death. This study shows that accumulation of succinate is a universal feature of ischaemia, due to complex II reversal at low oxygen concentration. After reperfusion, the large amount of succinate drives conventional electron transport in complex III and IV, whilst driving complex I to run in reverse, to generate ROS. The authors show that inhibition of complex II ameliorates reperfusion injury. This cardiprotection is lost again when dimethyl succinate is added back, indicating that succinate elimination is the key protective feature of complex II inhibition.

Compartmentalization of the protein repair machinery in photosynthetic membranes

http://www.pnas.org/content/111/44/15839.full

Sujith Puthiyaveetil, Onie Tsabari, Troy Lowry, Steven Lenhery, Robert R. Lewis, Ziv Reich and Helmut Kirchoff

Photosynthesis relies on the functioning of the PSII complex, which is embedded in the thylakoid membranes of plant chloroplasts. This complex is large and composed of multiple subunits, of which the D1 subunit bears most of the brunt of the damage from photosynthesis. The complex undergoes a repair cycle which reduces the amount of protein synthesis required by only replacing the D1 subunit through a series of reactions designed to remove and degrade the damaged D1 subunit without destroying the entire complex, before synthesising a new D1 subunit. This repair process occurs in the stroma lamellae (which connect the stacked grana), however PSII is concentrated in the stacked grana. PSII phosphorylation triggers its disassembly before it is transported through the grana margin to the stroma lamellae for D1 replacement. The repair cycle is involved and must happen rapidly - the entire PSII complement of a plant can be turned over in just 1 hour. This requires an efficient process with minimal back-reactions.

The authors investigated the organisation of the PSII repair system through fractionation of thylakoid membranes and electron microscopy (EM) investigation of stacked grana. High-light (HL) treatment, which causes increased damage to PSII, led to growth in grana margins at the expense of grana core and stroma lamellae. By quantifying levels of key proteins involved in PSII repair in the grana core, margins and stroma lamellae, the authors deduced that localising different components in different areas allows the PSII repair system to improve its throughput by minimising back-reactions. In addition, localisation of proteolytic steps also reduces the amount of unnecessary protein degradation, by ensuring that access to proteasomes is restricted.

The authors identify discovering factors that govern the localisation of enzymes to the appropriate compartment as an important future step.

Transcription could be the key to the selection advantage of mitochondrial deletion mutants in aging

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3939916/pdf/pnas.201314970.pdf

Axel Kowald and Thomas B. L. Kirkwood

In this paper they discuss a mechanism that could be responsible for the observed "clonal expansion" of mtDNA mutations. Clonal expansion means that in individual cells, the mitochondrial population is often taken over by a single type of mutant (as opposed to many different mutants). There have been various suggestions as to how clonal expansion occurs but none of these suggestions can account for all the experimental observations. For example, the hypothesis that clonal expansion of a single mutant happens merely by chance (random drift) can explain clonal expansion in long lived, but not short lived species.

In this paper, they propose a new mechanism of clonal expansion. The idea they propose is that normally, mtDNA replication rate decreases if concentrations of some of its products are high (a negative feedback loop). Mutants that miss that part of the genome that encodes for proteins involved in this feedback loop will not be able to suppress mtDNA replication rate through this feedback mechanism. These deletion mutations therefore have a replicative advantage over wild-type mitochondria.

By studying the position in the genome of mtDNA deletions that were observed to be clonally amplified, they found that most deletions overlap a subunit of complex I of the respiratory chain. This means that it could be that this subunit is involved in the negative feedback loop, and missing this subunit gives a replicative advantage that allows for clonal expansion.

They then provide an ODE model describing the dynamics of wildtype and mutant mtDNA as well as ATP concentration. In their model they assume that mutants have a 50% higher replication rate because they lack the negative feedback loop.  Because of this, the mutant population increases exponentially until the cell collapses (collapse is defined by a certain drop in ATP concentration and each extra mutant present lowers the ATP concentration by some amount).  Their model predicts that, if one starts with 1000 wildtype mtDNAs and 1 mutant mtDNA, at the moment of collapse the number of mutants is 9-fold higher than the number of wildtypes. This is in agreement with experimental results.

They continue to make a stochastic version of their ODE model, which can predict clonal expansion of a single type of mutant (rather than accumulated of various mutants) in both long- and short-lived species.

A Mitochondrial ATP Synthase Subunit Interacts with TOR Signaling to Modulate Protein Homeostasis and Lifespan in Drosophila

http://www.cell.com/cell-reports/abstract/S2211-1247(14)00685-8

Xiaoping Sun, Charles T Wheeler, Jason Yolitz, Mara Laslo, Thomas Alberico, Yaning Sun, Qisheng Song, Sige Zou

The authors studied a Drosophila model in which ATP synthase d (ATPsyn-d) was knocked down after reaching adulthood using a drug-inducible gene switch system (simply knocking down ATPsyn-d led to lethality before pupation). ATPsyn-d knockdown after reaching adulthood increased lifespan in female but not male Drosophila with no alteration to food intake, however overexpression did not have any effect. The lifespan extension was diet-dependent, and enhanced lifespan in high protein, low sugar diets but decreased them when the proportions were reversed.

ATPsyn-d knockdown paradoxically led to increased ATP levels through a decrease in UCP expression, but only on a protein-rich diet. It also caused significant transcriptional changes, including proteins involved in a range of protective, proteostatic and energy-generating roles.

The authors also explored the effects of ATPsyn-d knockdown on TOR and MAPK pathways, and found that overexpression of the TOR signalling suppressor Tsc2 cancelled out the longevity-promoting effects of ATPsyn-d knockdown. Feeding with rapamycin had a similar but less pronounced effect, reducing the lifespan extension due to ATPsyn-d knockdown. This is attributed to overlapping pathways of lifespan extension between ATPsyn-d and rapamycin, which are likely to include TOR signalling.

Finally, a decrease in protein polyubiquitination and polyubiquitinated protein aggregates was observed. These are biomarkers for proteostasis, leading the authors to conclude that the lifespan extension due to ATPsyn-d knockdown is at least partially accomplished through improved proteostasis. The authors suggest that ATPsyn-d and other mitochondrial proteins regulate TOR signalling, and that perhaps the beneficial effects exist in high protein, low carbohydrate diets because there TOR levels are high, whereas in low protein, high carbohydrate diets TOR levels are low so there is nothing to knock down.

Kinetic evidence against partitioning of the ubiquinone pool and the catalytic relevance of respiratory-chain supercomplexes

James N. Blaza, Riccardo Serreli, Andrew J. Y. Jones, Khairunnisa Mohammed, and Judy Hirst

http://www.pnas.org/content/111/44/15735.full

The electron transport chain (ETC) consists of protein complexes, which pump protons across a membrane to store electrochemical potential energy. This potential energy is then converted by another element of the ETC into an energy currency: ATP. Recent studies have shown that the complexes of the electron transport chain exist in supercomplexes, where multimers of the ETC components combine, which is thought to increase their efficiency. 

The authors present evidence disputing the importance of these supercomplexes (but not their existence). Ubiquinone (Q) is a protein which ferries electrons through the lipid membrane of the ETC, and exists in a pool between the leaflets of the membrane. It has been proposed that the Q pool is partitioned such that supercomplexes have a separate pool to the TCA-cycle. However, the authors show that altering the levels of substrate for the ETC and TCA causes global changes in the Q pool, which is evidence against partitioning.

Supercomplexes are also widely cited to exist because of the observation that flux through the ETC is almost entirely controlled by both complex I and III of the ETC, suggesting they exist in a complex together. The authors here, however, question this finding because these studies rely on a particular inhibitor, rotenone, to inhibit complex I. Upon replication of previously published protocols, the authors find flux control coefficients of >100%, which is impossible. Using alternative inhibitors, the authors measured more modest control coefficients, which the authors interpret as evidence against the catalytic importance of supercomplexes.

Feedback regulation via AMPK and HIF-1 mediates ROS-dependent longevity in Caenorhabditis elegans

http://www.pnas.org/content/111/42/E4458.short

There's a wealth of stuff in this paper and it's all pretty cool. The idea is to examine the finding that inhibiting mitochondrial respiration extends lifespan. These guys pick apart a feedback mechanism involving HIF-1 and AMPK that responds to, and balances, ROS levels. They find that HIF-1 and AMPK cross-repress and regulate ROS levels in different directions, potentially allowing for fine control over ROS levels. The feedback system is coupled to free iron homeostasis (free iron can lead to the production of ROS) and intriguingly seems to modulate resistance to bacterial pathogens (suggesting that mitochondrial ROS lowers rates of infection).

(By the way, they're using ROS probes DCF-DA and Amplex Red -- not cpYFP, which we have issues with)

A Mitofusin-2 – dependent inactivating cleavage of Opa1 links changes in mitochondria cristae and ER contacts in the postprandial liver

  http://www.pnas.org/content/early/2014/10/28/1408061111.long

Aditi Sood,Danny Vijey Jeyaraju, Julien Prudent, Alexandre Caron, Philippe Lemieux, Heidi May McBride, Mathieu Laplante, Katalin Tóth, and Luca Pellegrini; PNAS

In this paper, they study the liver of two groups of mice: 1) the first group was killed 2 hours after feeding 2) the second group was killed 5h after feeding. Nutrient levels become limited in the 5h group, they then studied the shape of cristae and the amount of ER-mitochondria contact in the 5h group as compared to the 2h group.

Changes in cristae structure

The number of cristae per mitochondrion had decreased in liver cells with limited nutrients. The average length per cristae remained the same.

 
Changes in mitochondria-ER contact
About 1 in 4 mitochondria in both groups was in contact with the ER, but in the nutrient limiting group the mitochondrial surface area in contact with the ER had increased. The ER thus wrapped more extensively around mitochondria when nutrients were limited.

OPA1 cleavage observed which is MFN2 dependent
They then continue to investigate what caused the cristae remodelling. Total OPA1 expression levels were the same in both groups, but two new forms of OPA1 were identified in nutrient-limiting conditions. These two forms were the result of OPA-1 cleavage by an unknown cysteine protease.The sites of cleavage were named C1 and C2. Cleavage at either C1 or C2 is likely to inactive the dynamin-like activity of OPA1, but does not interfere with mitochondrial dynamics. Mfn2 was required for the observed OPA1 cleavage, further suggesting a link between changes in cristae shape and changes in mitochondria-ER contact (because MFN2 is involved in mitochondria-ER tethering).

Conclusions 
 Mitochondria adapt to changes in nutrient availability by remodelling their cristae and changing the amount of mitochondria-ER contact. The cristae remodelling is mediated through MFN2-dependent OPA1 cleavage by a cysteine protease. The study suggests that cristae remodelling and changes in mitochondria-ER contact are coupled during nutrient depletion.

Making Proteins in the Powerhouse

B Martin Hällberg and Nils-Göran Larsson; Cell Metabolism (5 August 2014)

http://www.sciencedirect.com/science/article/pii/S155041311400309X

This review discusses the elements of mitochondrial transcription and translation, and the pathogenic effects of defects.

Mitochondrial DNA contains essential subunits of proteins involved in the oxidative phosphorylation system, as well as the tRNA and rRNA required for translation of proteins inside the mitochondrion. Although the vast majority of mitochondrial proteins are imported, failure to correctly translate the proteins encoded by mtDNA can lead to defects in oxidative phosphorylation, which can lead to significant negative consequences for the organism. Such a failure can occur either through mutation of a gene encoding a protein, or through damage to tRNA or rRNA impairing the mitochondrial translation system as a whole. The authors give the example of two mutations in the 12S rRNA gene of mitochondria which can lead to deafness, as well as referencing a subset of mtDNA mutations found in aging which can impair mitochondrial translation.

mtDNA transcription by mitochondrial RNA polymerase (POLRMT) leads to the creation of two long transcripts, one from each strand. These are punctuated by tRNA which are then cleaved at their 5' and 3' ends to release the mRNA strands held between them. It is currently unclear how mRNAs which are not between two tRNAs are released and processed. Early transcript processing is believed to take place alongside transcription in mitochondrial RNA granules, which may be followed by a second round of processing outside of these granules. Mitochondria possess a specific polyA polymerase (mtPAP) which performs polyadenylation of mitochondrial mRNA; without this polyadenylation mitochondrial mRNA stability is impaired and translation decreases.

Mammalian tRNAs are inherently less stable than other types of tRNA due to structural differences. The authors state that this makes them more susceptible to processing and modification defects, and that over 100 mutations in mitochondrial tRNAs have been observed to be pathogenic. The aminoacyltransferases responsible for charging mitochondrial tRNAs are all encoded in the nucleus.

Mitochondrial ribosome biogenesis requires the co-ordination of both nuclear and mitochondrial processes; 12S and 16S mitochondrial rRNA must be assembled with ribosomal proteins imported from outside of the mitochondrion. Translating mitoribosomes have been reported to be tethered to the inner mitochondrial membrane. The authors also discuss post-transcriptional modifications of 12S and 16S mitochondrial rRNA and the biogenesis of the mitoribosomal subunits.

The mammalian mitoribosome has a mass ratio of RNA to protein of 1:2, whereas bacterial and eukaryotic cytosolic ribosomes have a ratio of 2:1. The authors suggest that this reflects the loss of rRNA components since absorption of the proteobacterium that formed primitive mitochondria, and the replacement of these components with nuclearly encoded proteins.

The authors discuss the fact that at least one tRNA gene is always lost in all pathogenic single large deletion mutations of human mtDNA and that these mutations always lead to heteroplasmy and a require a heteroplasmy of >60% to impair translation. Heteroplasmic tRNA point mutations are also stated to be common causes of mitochondrial disease. Nuclear mutations affecting genes controlling mitochondrial translation also have a wide variety of potential pathological effects.

Finally, the authors discuss the fact the surprising complexity of the mitochondrial translation given its limited remit, and the number of nuclear-encoded genes that must be coordinated with mitochondrial translation in order to permit correct function. They emphasise the importance of studying mitochondrial translation in order to understand both mitochondrial disease and aging.