Thursday, 30 October 2014

Making Proteins in the Powerhouse

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

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.

Wednesday, 29 October 2014

Myo19 Ensures Symmetric Partitioning of Mitochondria and Coupling of Mitochondrial Segregation to Cell Division

Jennifer L. Rohn, Jigna V. Patel, Beate Neumann, Jutta Bulkescher, Nunu Mchedlishvili, Rachel C. McMullan, Omar A. Quintero, Jan Ellenberg, Buzz Baum

At cell divison mitochondria are segregated between the halves of the cell that will form the daughter cells. In order to ensure both offspring are viable they must both be provided with a sufficient complement of functioning mitochondria. The authors use a small interfering RNA (siRNA) library to target genes known to be associated with cell division errors. The resulting phenotypes were analysed by visual inspection of videos compiled from automated microscopy images to determine which of the candidate genes led to a cell division defect.

In addition to identifying a number of already-known genes, the authors identified the myosin protein Myo19 as being crucial for cell division. Myo19 was determined to be targeted to mitochondria, where it is associated with the outer membrane through a 150 amino acid domain known as MyMOMA (Myo19-specific mitochondria outer membrane association).

Inhibiting mitochondrial fission led to a failure of cell division, which the authors hypothesise may be caused by the division ring being obstructed by an excessively fused mitochondrial network. Treatment with siRNA inhibiton of mitofusin-2 (Mfn2) rescued the Myo19 depletion phentype, lending support to this idea.

Finally, and most remarkably, Myo19 inhibition led to highly asymmetric organisation of mitochondria in anaphase. This demonstrates a role for Myo19 in the correct partitioning of mitochondria at cell division; without it mitochondria are not recruited correctly to the poles of the cell and can sometimes obstruct the division ring, causing cell division to fail. Given that mitochondria are known to move along microtubules the authors suggest that Myo19 acts as a tether, regulating their poleward movement along microtubules during segregation.

Tuesday, 28 October 2014

Rapid rates of newly synthesized mitochondrial protein degradation are significantly affected by the generation of mitochondrial free radicals

A. Basoah, P. M. Matthews, K. J. Morten; FEBS Letters (November 2005)

Proteins are vulnerable to oxidation by reactive oxygen species (ROS), and mitochondrial proteins are especially at risk due to their proximity to ROS production. Nuclear proteins localised to mitochondria are at additional risk due to their partially unfolded state immediately after translocation.

Once proteins have been damaged, they are targeted for degradation by the cell's degradation machinery, however severe ROS damage can lead to proteins becoming harder to degrade. These undegraded proteins can then form protein aggregates which can prove toxic to the cell. Alternatively, if the protein degradation machinery is successful in destroying the damaged protein, this can lead to proteins being removed before they can be fully assembled. This can cause a deficiency of the damaged protein if it happens repeatedly.

The authors study a C2C12 myoblast cell culture and investigate the effects of ROS production on newly synthesised mitochondrial protein turnover. Cells were treated with menadione, which increased ROS production and led to an increase in protein degradation rate, which was observed using labelled methionine. Menadione levels were kept low so as not to affect cell viability (high doses have been shown to trigger cell death), but led to elevated ROS levels.

The changes in protein degradation were not uniform, and some proteins even showed decreased degradation rates. Confusingly, two different subunits of ATP synthase showed opposite effects, with one degradation rate increasing while the other decreased. The experimental timescale was not long enough to determine if these changes in degradation rates led to changes in steady-state abundance of the proteins concerned.

Mitochondrial ROS in cancer: initiators, amplifiers or an Achilles’ heel?

Mitochondrial ROS in cancer: initiators, amplifiers or an Achilles’ heel?

Reactive oxygen species (ROS) are typically generated by the leak of electrons from the electron transport chain (ETC) in mitochondria: the powerhouse of the cell. The function of ROS in both normal and pathological circumstances is subtle, as highlighted by this review. ROS are thought to damage mitochondrial DNA, which may result in the ETC becoming more leaky, to generate more ROS. ROS can switch on a variety of pro-survival signals such as HIF, MAPK-ERK and AMPK in cancer, as well as causing nuclear DNA-damage to increase the chances of oncogenic mutations. However, through controversial mechanisms, ROS can also act as death inducers in cancer, perhaps by spurring on apoptotic signalling. The authors argue that ROS are in fact the initiators, amplifiers and the Achilles' heel of cancer.

Monday, 27 October 2014

Enhancing nucleotide metabolism protects against mitochondrial dysfunction and neurodegeneration in a PINK1 model of Parkinson's disease

Loss of the mitochondrially-localised kinase PINK1 can cause early-onset Parkinson's disease, and a possible cause of this is through mitochondrial dysfunction following mutation of the Pink1 gene. The authors used a combination of transcriptional and metabolic profiling to discover that Pink1 mutant Drosophila have changes in nucleotide metabolism, in addition to the upregulation of the mitochondrial unfolded protein response which has been observed previously.

Following loss of pink1 a number of metabolic pathways were significantly upregulated, including glycine and serine metabolism, as well as nucleotide salvage, purine biosynthesis, and folate metabolism. The authors interpret this as a metabolic stress response induced by the cell to compensate for mitochondrial impairment due to loss of pink1.

The increase in folate metabolism was due to an upregulation of the kinase dNK, which was found to be the rate-limiting enzyme in the nucleotide salvage pathway. Pink1 mutants have upregulated dNK expression, which is shown to lead to organellar biogenesis. The authors find that inducing overexpression of dNK rescues mitochondrial dysfunction in pink1 mutant flies through enhancement of the nucleotide salvage pathway, and that dietary supplementation with deoxynucleosides also improved the suppression of mitochondrial dysfunction.

Thursday, 23 October 2014

Endogenous Drp1 Mediates Mitochondrial Autophagy and Protects the Heart Against Energy Stress

In this paper they investigate the role of Drp1 in mediating mitochondrial autophagy and stress resistance in cardiomyocytes.

Cells with down-regulated Drp1 show increases in the amount of cleaved caspase 3 (which activates the caspase and this can result in apoptosis) suggesting that Drp1 plays a role in protection against apoptosis. Also, autophagic flux was reduced upon Drp1 downregulation. Results suggest that Drp1 is required to stimulate mitochondrial degradation through autophagy.

There were significantly more mitochondria in cells with Drp1 downregulation, suggesting that the reduction in autophagy leads to an accumulation of mitochondria. Expression of PGC-1a (which causes mitochondrial biogenesis) was the same as in control cells. ATP production was lower in Drp1 downregulated cells, membrane potential was lower and mPTP opening was likely to be accelerated (increased mPTP opening could have been the reason why apoptosis was seen more). Maximum respiratory rate was lower, but the amount of proton leak was not significantly different.

Genetics of the Pig Tapeworm in Madagascar Reveal a History of Human Dispersal and Colonization

The pig tapeworm Taenia solium can cause the tropical disease cysticercosis. Humans are the only definitive hosts of the worm and pigs are the principal intermediate hosts.

The tapeworm can be divided into two mtDNA lineages, Asian and Afro-American, with disjunct geographical distributions. Recently it was found that both lineages exist in Madagascar. The first humans settled in Madagascar about 2000 years ago. Linguistic and archeological evidence suggests that people on Madagascar have ancestry from Island South-east Asia and East Africa. Recently, by studying mtDNA, a genetic contribution from India has been suggested.
By studying the genetics of the tapeworm insights for the distributional history of hosts and parasites can be gained.

In this paper, they collected larvae from pigs across five provinces on Madagascar. Their results indicate the importance of Indian influence on the diversity of people and culture in Madagascar, and that the tapeworms were introduced in Madagascar (within the past hundreds of years) multiple times with people and swines from East Africa. They also find evidence for hybridization between tapeworms with different genotypes.

Wednesday, 22 October 2014

Tumor suppressor p53 cooperates with SIRT6 to regulate gluconeogenesis by promoting FoxO1 nuclear exclusion

Tumor suppressor p53 cooperates with SIRT6 to regulate gluconeogenesis by promoting FoxO1 nuclear exclusion

p53 is a commonly mutated transcription factor in cancer, and is one of the most studied tumour suppressors. Here, the authors elucidate the interaction of p53 with the metabolism, specifically gluconeogenesis: the process by which glucose is built from non-carbohydrate amino acids, which could conceivably be essential for tumour growth. The authors show that p53 directly activates SIRT6, which induced translocation of FOXO1 to the cytoplasm, which activates the enzymes PCK1 and G6PC, which encode the rate-limiting enzymes for gluconeogenesis.

Tug-of-war between driver and passenger mutations in cancer and other adaptive processes

Tug-of-war between driver and passenger mutations in cancer and other adaptive processes

The authors generate a model of tumour growth by defining a fitness function, which depends on the number of driver and passenger mutations which have accumulated. The birth rate of cells is set by the fitness, and the total death rate saturates with the number of cells in the population. In the rare event that a driver mutation is generated, the population size increases rapidly. The population then decays due to the accumulation of passenger mutations which are assumed to reduce the fitness by a constant amount, per mutation. The resultant dynamics is a sawtooth, due to a tug-of-war between drivers and passengers. Their model predicts a critical population size, above which tumour growth is exponential, and below results in extinction. 

Monday, 20 October 2014

Hexokinase II integrates energy metabolism and cellular protection: Akting on mitochondria and TORCing to autophagy

Hexokinase II integrates energy metabolism and cellular protection: Akting on mitochondria and TORCing to autophagy

This review discusses the role of hexokinase II (HKII), a protein which is commonly misregulated in cancer. Hexokinases phosphorylate glucose, which is the first rate-limiting step of glycolysis. Glucose-6-phosphate then inhibits the action of hexokinases to form a feedback mechanism. Furthermore, HKII takes a central role in pro-survival signals, through its inhibition of apoptosis and enhancement of mitophagy. Thus, HKII can be viewed as a molecule which bridges the metabolism and cell survival.

Friday, 17 October 2014

Circulating Tumor Cell Clusters Are Oligoclonal Precursors of Breast Cancer Metastasis

Circulating Tumor Cell Clusters Are Oligoclonal Precursors of Breast Cancer Metastasis

Primary tumours spread to other parts of the body by shedding cells into the blood stream, which can seed tumours elsewhere. This process is thought to be mediated by single cells, but the authors here show that cancer cells are also found to exist in clusters when extracted from the blood, but at a very low abundance. The authors identify the factor plakoglobin which is necessary for cluster formation. Knockdown of this factor had little effect on primary tumour size, but had a striking 80% reduction in metastatic node formation in mice. This indicates that these clusters have a stronger contribution to metastases formation, than just by their abundance.

We can only speculate as to the mechanism by which these clusters gain their super-linear metastatic potential. Perhaps cooperation through sharing of a common resource makes them energetically more competent?

Thursday, 16 October 2014

The distribution of mitochondria and endoplasmic reticulum in relation with secretory sites in chromaffin cells

In this paper they investigate I) the distribution of mitochondria and the ER in chromaffin cells, and ii) how this distribution plays a role in exocytosis.

Exocytosis occurs in response to elevations of cytoplasmic calcium levels and requires energy. Mitochondria-ER interactions are important in shaping cellular calcium signals and therefore the distribution of these two organelles in the cell may well influence exocytotic events.

Mitochondria in chromaffin cells exist in two main populations, one of higher density in the cortical region and another one in the perinuclear region. Mitochondria were noticably smaller in size in the cortical region. The two populations showed different mobilities, mitochondria in the perinuclear area were faster and moved through F-actin and microtubular structures.

Distribution of the ER is more uniform (though the density increases gradually from the cortical zone to regions close to the nucleus) and ER elements were also smaller in cortical regions.

They find that the cortical populations of mitochondria and ER themselves consists of two subpopulations, one close to exocytotic sites and one further away. The local cortical subpopulations could be directly involved in the regulation of calcium signals and ATP supply in the immediate vicinity of exocytotic sites.

Wednesday, 15 October 2014

OPA1‐dependent cristae modulation is essential for cellular adaptation to metabolic demand

OPA1 mediates fusion of the inner mitochondrial membrane and is also involved in cristae remodelling. Maintenance of the cristae structure requires oligomerization of OPA1 because disruption of OPA1 oligomers is necessary for cristae opening and cytochrome c release.

In this paper, they observe that OPA1 dynamically regulates cristae shape and that OPA1 is required for resistance against starvation-induced cell death, and that these processes are independent of mitochondrial fusion (a mutant of OPA1 that does oligomerize but has no fusion activity is still able to maintain cristae structure).

They also show that some group of mitochondrial solute carriers (SLC25A) interacts with OPA1. These SLC25A transporters can sense changes in energy substrate availability. So if there are changes in energy substrate levels, SLC25A transporters respond to this and in turn interact with OPA1 which modulates cristae structure (which regulates assembly of ATP synthases) to respond to the change in energy substrate availability. All of this is independent of OPA1's role in fusion.

Why are most organelle genomes transmitted maternally?

Mitochondrial DNA (mtDNA) is maternally inherited which means it does not undergo sexual recombination. But recombination is thought to be required to prevent an accumulation of mutations (a process known as Muller's ratchet, summarized below in red). Why do mitochondria survive then? How did maternal inheritance evolve?

Paternal leakage occurs
There is increasingly more evidence that occasionally sexual recombination does occur (known as paternal leakage) and this may slow down Muller's ratchet (this is only speculated). There is, however, a selection pressure towards the evolution of uniparental transmission.

Hypotheses for uniparental inheritance
Several theoretical models for the occurrence of uniparental organelle inheritance exist, including the following:
  1. Avoiding competition between organelles and avoiding negative interaction between organelle genomes and/or other organelle genomes and the nuclear genome.
  2. The genetic bottleneck makes it possible to get rid of mutations because genetic drift to homoplasmy can occur. Paternal leakage interferes with this process.
  3. Many genes from endosymbionts have been transferred to the nuclear genome of the host cell and a lot of gene products are then re-imported into the endosymbiont. This means tight co-evolution and co-adaptation between endosymbiont and host cell is required. Mathematical models have shown that co-adaptation is enhanced by uniparental inheritance and could thus be the driving force of uniparental inheritance.

However, there are arguments against all of these hypotheses. For example, they fail to explain why , if uniparental transmission occurs, it is almost always maternal (uniparental paternal inheritance is very rare) and why killing of the paternal cytoplasm occurs (which can be costly).

In this paper, a unifying model for organelle inheritance is proposed.
They argue that uniparental inheritance evolved to avoid the spread of faster replicating organelle genomes that are incompatible with the host nucleus. This uniparental inheritance, however, is unstable (because of Muller's ratchet) and this drives a relaxation of strict maternal inheritance by paternal leakage or biparental transmission. Paternal leakage is then again susceptible to the evolution of fast replicating genomes incompatible with the host nucleus, so paternal leakage is lost again. Then it is restored again (because of Muller's ratchet), then lost again etc... They thus claim that uniparental inheritance is an unstable state. The maternal predominance is due to more mutations in the paternal cytotypes.

They then discuss some evidence for their theory.

Muller's ratchet
In asexual reproduction, genomes are inherited as indivisible blocks. If mutations occur, they will be carried over to the next generation. They will keep on accumulating and eventually the population will go extinct. In sexual reproduction, this is prevented because recombination occurs. Recombination allows for the possibility to generate genomes with fewer mutations from genomes that are highly mutated, by putting together mutation free portions of the parental chromosomes.
Note → Having a small genome reduces the probability of mutations to accumulate and therefore the effect of Muller's ratchet can be very small when genomes are small.

Activation of mitochondrial protease OMA1 by Bax and Bak promotes cytochrome c release during apoptosis

When cytochrome c is released from the intermembrane space of mitochondria into the cytoplasm, caspases in the cytoplasm become activated and they initiate apoptosis. Besides cytochrome c, other proteins that normally reside in the intermembrane space are also released into the cytoplasm, one of them is Smac which inhibits inhibitors of apoptosis. Controlling the permeability of the outer membrane of mitochondria is therefore crucial to the survival of the cell. Which event trigger the permeability to increase so that apoptosis follows?

This paper finds that first, Bax and Bak are recruited to the surface of mitochondria. This then activates OMA1, an m-AAA inner membrane protease  that is responsible for cleavage of the long isoforms of OPA1. OPA1 can shape cristae, so when it is cleaved the cristae will remodel and this is presumably what makes it possible for cytochrome c to be released.

To summarize:

  1. Bax/Bak oligomerize on the mitochondrial surface 
  2. This oligomerization somehow changes the inner membrane structure and   activates OMA1, an inner membrane m-AAA protease.  
  3. OMA1 cleaves L-OPA1 
  4. A change in the ratio of long to short isoforms of OPA1 influences cristae structure, so the cristae start remodelling
  5. cytochrome c is released

Tuesday, 14 October 2014

Mitochondrial dynamics and inheritance during cell division, development and disease

This is a nice review on mitochondrial dynamics during cell division, development and disease. A few highlights are mentioned here.

In cells lacking Drp1, mitochondria exist in elongated networks but they are still segregated to daughter cells (but less uniformly), probably because the machinery involved in cytokinesis is strong enough to cleave the mitochondria.

Mitochondria can be non-selectively removed as part of an autophagy response, but mitophagy can be selective too. Mitochondria that are experimentally depolarized attract autophagy machinery and undergo mitophagy.

The mtDNA bottleneck that occurs during oogenesis is discussed as well. A second mtDNA bottleneck occurs during early embryogenesis (there is no mtDNA replication in this stage which reduces mtDNA copy number).

A discussion about the depletion of parental mtDNA is also given, reasons for uniparental inheritance of mtDNA remain unclear..

Mitochondrial Fission Factor Drp1 Maintains Oocyte Quality via Dynamic Rearrangement of Multiple Organelles

Drp1 was knocked out in a transgenic line of female mice which were actively growing oocytes, to examine the effects mitochondrial dynamics on the development and aging of oocytes.

Effects on oocyte formation and growth
The Drp1 KO female mice produced less pups per mating (only 0.63 compared to the 6.7 pups per mating of control mice) despite normal vaginal plug formation. Few or no oocytes could be recovered from the KO mice, they therefore do not ovulate normally Follicle growth was low in KO ovaries and granulosa proliferation was blocked. Defects accumulated with age.

Effects on mitochondrial and ER morphology
Not surprisingly, Drp1 KO in oocytes lead to elongated and aggregated mitochondria. mtDNA nucleoids were clustered.  Most of the ER clustered around mitochondrial clusters. In control mice around 80% of the mitochondria were in contact with the ER, but in KO mice mitochondria tended to cluster with various other membranous structures (e.g. small vesicles). Peroxisomes and secretory vesicles were deformed.

Effects on energetics
There was no difference in membrane potential measured in mitochondria from control and KO oocytes and ATP content was not significantly affected.

Effects on calcium response
There were less calcium oscillations recorded in KO oocytes. The frequency of the oscillations was similar but the amplitude was reduced in KO mice.

Other observations
Oocytes contain a structure called the germinal vesicle, which is a nuclear structure and it is enlarged during oocyte maturation. The germinal vesicle breaks down before polar body formation. In normal mice, mitochondria move from the germinal vesicle to the cytoplasm during breakdown but this does not happen in KO mice. Mitochondrial movement is restricted in KO oocytes and the breakdown of the germinal vesicle is defective.   

OPA1‐dependent cristae modulation is essential for cellular adaptation to metabolic demand

OPA1‐dependent cristae modulation is essential for cellular adaptation to metabolic demand

It is well-established that cristae, the organized invaginations of the mitochondrial inner membrane, remodel themselves upon induction of cell death. In this study, the authors also show that substantial remodelling occurs in response to altered energetic conditions of the cell. These changes are mediated by the protein OPA-1, which is also associated with fusion of the mitochondrial network, but independent of the activity described here. The authors propose that OPA-1 can sense the presence of fuel substrates, which oligomerizes during cell starvation and tightens the cristae. These ultrastructural changes are associated with increased ATP-synthase assembly, and increased efficiency/capacity of energy production.

Monday, 6 October 2014

PGC-1α mediates mitochondrial biogenesis and oxidative phosphorylation in cancer cells to promote metastasis

PGC-1α mediates mitochondrial biogenesis and oxidative phosphorylation in cancer cells to promote metastasis

The canonical explanation for how tumour cells derive their energy is mainly through glycolysis, an anaerobic pathway. However, the work in this paper shows that circulating cancer cells (CCCs) in breast cancer favour mitochondrial oxidative phosphorylation, which can generate more energy and is aerobic. Moreover, the authors find that the protein PGC-1α, which modulates mitochondrial biogenesis, heavily influences CCCs invasiveness and ability to form distant metastases.