Accurate concentration control of mitochondria and nucleoids

 Rishi Jajoo, Yoonseok Jung, Dann Huh, Matheus P. Viana, Susanne M. Rafelski, Michael Springer, Johan Paulsson

http://classic.sciencemag.org/content/351/6269/169.long

When a cell divides, all components in the cell need to go into either one of the daughter cells. The cell has developed mechanisms to make sure that some components, for example chromosomes, are correctly segregated. How does this work for mitochondria? Does the cell have active mechanisms to push equal amounts of mitochondria in each daughter cell, or do the mitochondria randomly move into one of the daughters?

In this paper, they try to answer these questions in fission yeast S. Pombe.
It was shown before that mitochondria are pushed to both of the cell poles before cell division, which would suggest that this is a mechanism of segregating the mitochondria.

However, in this paper they show that in the last 15% of the cell cycle, mitochondria spatially re-equilibrate themselves throughout the cell. When the cell divides, the mitochondrial volume in a daughter cell tracks the cytoplasmic volume of that daughter cell. For example, if the two daughters get 60% and 40% of the cytoplasmic volume, they will on average also get 60% and 40% of the mitochondrial volume.
The same is true of mitochondrial nucleoids, which contain the mitochondrial DNA; they too segregate in proportion to the cytoplasmic volume.

However, the errors made in nucleoid segregation are smaller than you would expect from passive mechanisms. Using the example from above, passively one would expect that each nucleoid has a probability of 0.6 of ending up in the larger daughter (the one with 60% of the cytoplasm) and a probability of 0.4 of ending up in the other daughter. This is called binomial partitioning and has a certain error size associated with it. The actual errors that are made in S. Pombe (i.e. the deviations from perfect partitioning) are smaller than binomial errors.

A model that would explain these sub-binomial nucleoid segregation errors is to assume that the nucleoids are regularly spaced out within the mitochondria. It is not known how this regular spacing is accomplished by the cell.

The authors also find that the number of nucleoids that are produced from beginning to end of the cell cycle does not depend on the initial amount that was present. This suggests that S. Pombe does not not use feedback control to produce its nucleoids (or other mitochondrial proteins). Feedback control is energetically expensive. Rather, nucleoids are randomly added throughout the cell cycle, following a Poisson distribution.

TCA Cycle and Mitochondrial Membrane Potential Are Necessary for Diverse Biological Functions

http://www.sciencedirect.com/science/article/pii/S1097276515009363

Inmaculada Martínez-Reyes, Lauren P. Diebold, Hyewon Kong, Michael Schieber, He Huang, Christopher T. Hensley, Manan M. Mehta, Tianyuan Wang, Janine H. Santos, Richard Woychik, Eric Dufour, Johannes N. Spelbrink, Samuel E. Weinberg, Yingming Zhao, Ralph J. DeBerardinis, Navdeep S. Chandel

In this study, the authors investigate the effect of eliminating mtDNA from cells, on metabolism. This is often achieved by incubation with the chemical ethidium bromide, which prevents the protein responsible for mtDNA replication (POLG) from working. However, ethidium bromide is toxic and has potentially off-target effects. The authors engineered cells which, upon exposure to an antibiotic, express a dominant-negative form of POLG (DN-POLG), thereby removing potential off-target effects. Within 6 days of induction of DN-POLG, mtDNA transcription had ceased.

Cells had a greatly reduced proliferation rate (although still viable), despite having access to glucose and pyruvate: metabolites required to drive glycolysis and the TCA cycle respectively. As discussed in [1] (see here) mitochondria oxidise NADH to NAD+, the substrate of glycolysis. The authors introduced two non-mammalian proteins (NDl1 and AOX), which together carry electrons in a similar manner to the electron transport chain, but do not pump protons across the inner mitochondrial membrane. In this way, NAD+ can be restored, without generating mitochondrial ATP from oxidative phosphorylation. The authors show that these proteins are sufficient to drive flux through the TCA cycle, and increase the NAD+/NADH ratio. However, the authors find that these cells still have an impaired proliferation rate (at odds with the findings in [1]?)

The authors investigated an alternative reason for the impaired proliferation of these cells: mitochondrial membrane potential (ΔΨm). Cells without mtDNA cannot pump protons to maintain ΔΨm, but ATP synthase is still able to hydrolyse ATP from glycolysis, to maintain ΔΨm. An endogenous inhibitor of this function is the protein ATPIF1. The authors knocked out this gene, and showed that cells without mtDNA are able to maintain their ΔΨm at wild-type levels. These ATPIF1-KO cells had a, statistically significant, partial recovery in proliferation rate.

Interestingly, treating the ATPIF1-KO cells with an antioxidant, which mops up ROS (canonically considered to be the bad-guy of mitochondrial physiology, see here), reduced the proliferation rate. This shows that ROS, as well as ΔΨm, are necessary for cells to proliferate.

[1] Wiley, Christopher D., et al. "Mitochondrial Dysfunction Induces Senescence with a Distinct Secretory Phenotype." Cell metabolism (2015).

Mitochondrial Dysfunction Induces Senescence with a Distinct Secretory Phenotype

http://www.sciencedirect.com/science/article/pii/S1550413115005781

Wiley CD, Velarde MC, Lecot P, Liu S, Sarnoski EA, Freund A, Shirakawa K, Lim HW, Davis SS, Ramanathan A, Gerencser AA, Verdin E, Campisi J

Cellular senescence is the process by which dividing cells permanently lose their ability to replicate. This phenotype can inhibit the growth of cancerous cells, but it is also thought to occur in normal tissues during aging. It is known that mitochondrial dysfunction can induce senescence, however the mechanisms of this are unclear.

The authors show that several different kinds of mitochondrial dysfunction can induce senescence in the IMR-90 cell line: mtDNA depletion; drugs which inhibit the electron transport chain (rotenone and antimycin A); and inhibition of a particular mitochondrial chaperone (HSPA9) which aids in import of proteins into mitochondria.

Mitochondria oxidise NADH to NAD+ in a number of metabolic reactions involved in generating energy. NAD+ is a substrate of glycolysis, whereas NADH is a product, which can inhibit the pathway if it is not removed. As glycolysis provides pyruvate, the substrate of oxidative phosphorylation, a reduced NAD+/NADH ratio may be expected to slow down glycolysis and therefore oxidative phosphorylation.

The authors provide evidence that the mechanism of mitochondrially-induced senescence is lowered NAD+/NADH ratios, suggesting energetic collapse. This is supported by an increased ADP/ATP ratio in these cells. They find that supplementation of pyruvate to cells can partially rescue the senescent phenotype.

The authors further show the relevance of mitochondrially-induced senescence, by investigating the effect in POLG mutator mice (mice which accumulate mtDNA mutations in time and have a progerioid phenotype). The authors found that the progerioid mice had many more senescent cells in affected tissues, with lowered NAD+/NADH ratios compared to wild-type mice.

Mitochondrial Membrane Potential Identifies Cells with Enhanced Stemness for Cellular Therapy

http://www.sciencedirect.com/science/article/pii/S1550413115005690

Sukumar M, Liu J, Mehta GU, Patel SJ, Roychoudhuri R, Crompton JG, Klebanoff CA, Ji Y, Li P, Yu Z, Whitehill GD, Clever D, Eil RL, Palmer DC, Mitra S, Rao M, Keyvanfar K, Schrump DS, Wang E, Marincola FM, Gattinoni L, Leonard WJ, Muranski P, Finkel T, Restifo NP

Cancer immunotherapy involves using the immune system to target and eradicate tumours. One method is to isolate and transfer immune cells into the patient. There are a number of different kinds of immune cell, one of which is called the T cell. T cells themselves divide into a number of subtypes, two of which are: effector memory (EM) and stem-cell memory (SCM) T cells. It is known that SCM cells are able to persist for longer periods of time, and are more effective in attacking tumour cells than EM cells. It is therefore desirable to be able to enrich for SCM cells, in a mixed population of T cells, to deliver a more potent immunotherapy.

In this study, the authors stain a mixed population of T cells with a chemical which causes cells with a large mitochondrial membrane potential (ΔΨm) to fluoresce more strongly (using TMRM). They find that fractions with low membrane potential are enriched for SCM cells, whereas fractions with high membrane potential are enriched for EM cells. Indeed, the authors show in a variety of cell lines that low ΔΨm is associated with stem-like properties.

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Thoughts:
Curiously, low ΔΨm cells had a lower glycolysis rate, and higher spare respiratory capacity, and lower baseline respiratory rate, compared to high ΔΨm cells. Low ΔΨm cells tend to favour fatty acid oxidation, which provides an alternative substrate to glucose for energy production. However, fatty acid oxidation drives the Krebs cycle, which in turn can drive oxidative phosphorylation. So, given that these cells are not undergoing glycolysis, one might expect ΔΨm low cells to have a higher resting oxygen consumption rate? It would be interesting to know the ATP concentration of the ΔΨm low cells: my guess would be that they have a lower ATP concentration?