AUSTRALIAN FRONTIERS OF SCIENCE, 2008
The Shine Dome, Canberra, 21-22 February
Understanding how mitochondria grow and divide
by Associate Professor Mike Ryan
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Mike Ryan has a PhD from La Trobe University and the University of Adelaide. He received an Alexander von Humboldt Fellowship and worked on protein import into mitochondria in Freiburg, Germany. Mike returned to La Trobe University as a lecturer and later became an associate professor. Mike’s research focuses on mitochondrial biogenesis and disease, with a particular interest in the assembly of proteins into multi-subunit containing complexes. Mike also the heads the Biology Program in the ARC Centre of Excellence for Coherent X-Ray Science, a joint venture with physicists to undertake novel imaging methods to determine biological structures. In 2006, Mike received the Roche medal from the Australian Society for Biochemistry and Molecular Biology. |
I apologise for the very vague title. It was proposed a few months ago, before this symposium was organised.
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As Harvey Millar and Trevor Lithgow have both mentioned, mitochondria are a lovely organelle that once was a bacterium, and during that process the mitochondria transferred their DNA over to the nucleus. That left the mitochondria as basically a double-membraned organelle, with an inner and an outer membrane, and a little bit of remnant DNA that must be inherited when the cells divide.
Most proteins, as Harvey has mentioned, are encoded by the nucleus. That means these proteins need to get into the mitochondria. They grow, they make metabolites, they make lipids, and as the mitochondrion gets bigger and bigger, like the bacterium it once was, it also divides. The division is important, because the mitochondria need to go into daughter cells during mitosis, so we also get a complement of mitochondria within each cell that divides.
As part of this process of inheritance, mitochondria as I will show you in a moment are dynamic structures. They are not just sitting static within the cell; they are players in very important processes.
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So mitochondria are these essential, unique organelles. They are the powerhouse of the cell, producing most of the ATP (adenosine triphosphate) for the body, but, again as Harvey mentioned, they perform lots of other important functions within the cell.
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But in the animal systems, the higher eukaryotes, mitochondria are also the poison cupboard of the cell. One particular molecule that is involved in making ATP is actually involved in killing the cell as well. There are certain factors that actually come and bind to the mitochondria, and punch a hole make a nice pore somehow and release a certain protein called cytochrome c. That is released out into the rest of the cell, and that then triggers a process that is involved in the death of the cell.
This is a very important process that occurs in our bodies all the time. When we are making new cells, we need cells to die as well. If we don't have this process occur, and cells continue to divide, then cancers can arise.
This has been a very hot topic. It was almost thought that most of the processes and fundamental aspects about mitochondria were well known, and only in the last decade or so, when this came by, have mitochondria become hot again.
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Also, mitochondria at this time became a little bit hot by virtue of looking at using new techniques. The particular technique here was the use of green fluorescent protein (GFP), which was identified in a jellyfish and the protein was extracted out. You see here the structure of green fluorescent protein, which has a fluorescent chromophore trapped within a cage, so it is a very stable protein.
The DNA encoding this particular protein can be integrated into a plasmid, and this DNA can be fused to DNA encoding a protein that would normally go into mitochondria, for example, and this DNA can then be introduced into cells grown in culture. The protein that would go into mitochondria would then be taking GFP into the mitochondria for a ride. That means that we could actually see and visualise what mitochondria look like in live cells, as opposed to the electron micrograph which was previously utilised.
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This is what we see of mitochondria within a HeLa cell, a cancer cell that is grown in culture, and you can see that the green fluorescent protein is making these mitochondria nice and bright green these are real colours, not fake and in blue we have the DNA labelled in the nucleus.
This is a snapshot. This is something we see of the mitochondria; you can see threadlike organelles that are running through, and you can almost imagine that they are probably moving around, and twisting and curling.
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In fact, we know that is the case, because we can also do live-cell imaging of these.
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This is another cell, where the nucleus is not labelled but would be within this circle.
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And the mitochondria do indeed move. This is a little bit different it is not green fluorescent protein that we have put into the cells, but instead another fluorescent protein, Dendra, targeted to mitochondria. Dendra is a green fluorescent protein but it has a nice little extra aspect, which you might notice here. Hopefully, you can see some red mitochondria. That is because we can use confocal microscopy to target individual mitochondria, give them a short zap of appropriate wavelength of light, and convert the green fluorescence to a red fluorescence that green fluorescent protein becomes a red fluorescent protein. And so we can monitor what happens within individual mitochondria within one cell, rather than trying to figure out what is going on.
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I will point you to the region near the top of the main image here, to the large mitochondrion in red, and just show you a zoom of what happens in terms of dynamics of mitochondria. Mitochondria undergo fission, so you can see this mitochondrion break up into three different mitochondria. It is actually undergoing a molecular process where molecules are constricting around mitochondria and causing them to break apart.
But, interestingly, mitochondria can also undergo fusion. That means the mitochondria will come together, they kiss, and in this case you will see one particular red mitochondrion fuse with a green mitochondrion, and the fluorescent protein inside the red mitochondrion will be released into the other mitochondrion as well. Then it will leave that particular mitochondrion. You see it go; it has now been fused and there has been mixing. That mixing is important: it maintains the homogeneity of the mitochondria within the cell, so that the mitochondria basically exist as much as possible as an overall single population.
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Mitochondrial dynamics in general are very important, and they are important in many differentiated cells in our bodies.
For example, in the panel at the left of this slide we have a nerve cell, where mitochondria are actually transported up from the cell body right along the axon to the synapses which are involved in the release of neurotransmitter substances to neighbouring neurons or to muscle. We need this triggering and release of neurotransmitter molecules, and that means that the vesicles shown here as green dots which are loaded, need to fuse with the membrane. The mitochondria provide the ATP to perform this process, and they are also involved in buffering the calcium that is part of the triggering.
Mitochondria are also part of actually forming the nerve cells. It has been found that mitochondria sit in spines within nerve cells, and that is involved in the growth of the projections within the nerve as well.
To move to the centre panel of the slide: mitochondria have also been found to be important in pancreatic cells, where they provide a buffering type of maintenance for the whole cell, so that mitochondria prevent the calcium waves that come into these pancreatic cells from going right through the cell. Also, ATP is utilised from the mitochondria directly to result in the release of insulin from the pancreatic cells.
Finally, in the right-hand panel: a more extreme view of mitochondria is in sperm cells, where the mitochondria are, basically, wrapped around the sheath of the tail, the flagellum, of sperm. This is providing the ATP for the motility of the sperm. These mitochondria are really quite changed in their morphology they are fused and wrapped around and the purpose of them is to make these sperm swim. When the sperm actually reaches the egg, the DNA is transferred into the egg but the mitochondria from the sperm is not, it is degraded. The mitochondria that we have in our bodies are inherited only from our mothers. The mitochondria come exclusively from the egg, and that means that the mitochondrial DNA that is present is always present from our mothers. We inherit maternal mitochondrial DNA only; all the boys out there won't be passing on their mitochondrial DNA to their children.
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Mitochondrial dynamics are important, and I will just mention that mitochondria are involved in trafficking along microtubules which are highways within cells. I have got three cells on this slide, and the mitochondria in this case look really quite abnormal because we are actually expressing a different form of a GFP protein, called Milton. This protein is involved in increasing the attachment of mitochondria to microtubules, which are shown here in red with a different fluorescent protein.
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These mitochondria are mobile because the microtubules are there. They hop onto these microtubule highways through adaptor molecules and motor proteins to traffic the mitochondria along.
But if we dissipate or disrupt the microtubule highways, using a chemical, then the red fluorescence will disappear, the mitochondria will clump and they will lose their motility. This movie shows that we have actually added a chemical, the red fluorescence disappears and those mitochondria, which were quite mobile, suddenly start to clump up and are not mobile any more. You see a last one come across and it basically finishes up at the end of a microtubule.
So we can perform a little bit of analysis, looking at these particular processes, with these sorts of tools.
The future in studies looking at mitochondria in terms of morphology is to understand the molecular events, the proteins involved in fission and fusion: how are they actually doing that? We know a number of molecules now, but we don't actually know how they do it, and how they are regulated to do it. We need to know this in a little bit more detail, and of course structural information will be great to have in the future.
We also don't understand enough about the internal dynamics of mitochondria. They contain an outer membrane and an inner membrane, and the inner membrane also has to undergo rearrangements during both fission and fusion processes, but we don't know enough about that either. There are some indications of molecules inside the mitochondria involved in these processes, but they are not well understood. We really need to look inside mitochondria in more detail.
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We can do that using electron microscopy, but in a somewhat different version. The images here were obtained by using tomography, so you can look at thicker sections and you can look at the tilts, angles, of mitochondria. You get to see inside the mitochondria in more detail, seeing these tubules of what are called cristae formed by the inner membrane, causing invaginations in the mitochondria. So we can look at these and compare them in normal cells and cells that have mitochondria that are defective or have changed.
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We will need approaches to look at mitochondria in higher resolution, and of course other molecules in other ways.
As a side point: I am part of the ARC Centre of Excellence for Coherent X-ray Science, which is directed by Keith Nugent. He is at the Department of Physics in the University of Melbourne, and is involved in a number of universities and CSIRO in Melbourne. This is really a joining together of, mainly, physicists in aspects such as detector and beamline development, theory and modelling, experimental methods, and so forth. And then there is an extra component, the biological sciences, which has people like me and Leann Tilley, who works on malaria. We hope to be able to provide some of our expertise toward looking at biological structures, and talking to physicists really, it is learning a new language, with us trying to understand each other's language as well. The mission of this centre is to be the world leader in development of coherent X-ray diffraction for biological imaging.
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This slide offers a nice continuation to look on the inside of mitochondria. As Harvey Millar has already mentioned, mitochondria are the powerhouse of the cell, and inside is the particular powerhouse, the respiratory chain, or the chain involved in oxidative phosphorylation, which is an electron transport chain that in the end causes the production of the ATP. We have been especially interested in looking at these complexes in a biochemical sense. In particular, we are looking at this in a sense of what these complexes are doing and how they are defective in disease.
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Diseases of mitochondrial respiratory chain, or oxidative phosphorylation, occur in about one in 5000 live births. This is a collaboration with David Thorburn, at the Murdoch Children's Research Institute, and he has been collating a number of patients who have been diagnosed there. So far, there are 378 children who have been diagnosed with oxidative phosphorylation disorders, and these children have real energy generation defects. Typically they will die within days to months of life.
A break-up of these patients found that the mitochondrial DNA is defective in about 84 of the patients, and seven genes out of 13 genes that we have (encoding proteins, at least) are defective there are also other genes involved in making these proteins. There are also 74 that have mutations in known genes in the nucleus, other than encoding proteins of the mitochondrial respiratory chain. But another 220 patients have genes which are mutated, or presumably mutated, in the nuclear genes, although the actual genes have not been identified so far.
Through enzymatic analysis, complex I the first complex in the respiratory chain has been identified to be the main cause of these energy generation disorders. We have been performing some work on complex I. It is a rather large complex, containing 45 different subunits, and not only are its defects involved in energy generation but it has also been implicated in Parkinson's. Harvey showed rotenone dust, which is put on tomato plants. If you ingest enough of that you can develop Parkinson's-like symptoms, because you are causing complex I to be defective. And there are various other things, as well, that complex I is involved with.
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We have been trying to look at the assembly of complex I, shown on this slide as a jigsaw puzzle. In humans, 45 different proteins need to come together to make this whole complex. It is a very difficult procedure that mitochondria manage to do. What is even more complicated is that seven protein subunits are actually encoded by that mitochondrial DNA that is still left in our bodies, in our mitochondria, while the other 38 are encoded by nuclear DNA. These proteins need to be imported into the mitochondrion, and they all need then to be assembled together in some manner. Obviously, our mitochondria do this very well, and do it properly, in most situations.
Because the complex I defects are the main cause of oxidative phosphorylation disorders, we (and others) have proposed that the defects that are found in patients might not be in the subunits themselves, but in proteins that are bringing the subunits together: the assembly factors.
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We have looked at patient cell lines that we can grow in culture, even though the patients may have died. We can look at the fibroblasts, the skin cells, from patients grown in culture still and we can then test for the molecular diagnosis of it. And we can look at complex I.
Shown here is a gel of complex I, which in its normal state sits at the top of this gel at a size of 980 kilodaltons. But when we look at these other patient cell lines for complex I, we see that in some cases there is less of complex I; it is depleted in these patients. In some cases there is virtually no complex I. The two cases at the left here are actually a faster-running complex I, and we think that represents a complex I that is defective in its assembly.
In summary, the molecular deficiency is unknown in 50 per cent of patients, so the mutation is not in the complex I subunits. So we thought the mutations might be in the assembly factors.
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Through biochemical analysis we have been looking at the assembly of complex I, as have a number of other groups, and we have a model here. I won't go through the model, but it is basically building blocks or modules of complex I together, and these modules come together at certain stages within the inner membrane, and they are brought together to make the full complex I.
We also have identified an assembly factor, called CIA30, which is involved in early steps of building complex I, and other assembly factors have more recently been identified as well.
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So CIA30 was a potential candidate as an assembly factor that might actually be mutated and cause disease. And so we used antibodies we had raised against CIA30, testing them in our patient cell lines. We found one patient cell line that contained very little CIA30. The other patient cell lines had basically normal CIA30, so it meant that their mutations probably aren't in CIA30 but other genes.
But this particular patient cell line was an interesting one, because this patient had an unknown defect in the nuclear genome, and CIA30 is a gene in the nucleus. The patient has a complex I defect, and presented with cardioencephalomyopathy, which is heart, brain and muscle defects, as is typical of anything that is going to cause defects in making energy. The patient had decreased complex I activity and also reduced levels.
We found subsequently, after we had noticed this at the protein level, that the gene of CIA30 did have mutations in it.
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To close the circle, we put CIA30 back into these patient cell lines in this slide we have expressing CIA30 back again. We found that complex I was then restored. The assembly was restored, as compared with the patient that had virtually no CIA30 (shown here as well).
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The future, though, is difficult. Looking at patient cell lines is not the easiest thing to do. We have got to grow them up. The patient cell lines are making ATP they need ATP for growth but cell lines don't grow very well if they don't make enough ATP. So we can go through other, new methodologies and we can look at different models. One such model is a mouse model.
To take you back to this particular gel, showing that the defect in assembly of complex I within this oval: this defect was found to be actually in a subunit of complex I.
We were lucky enough to uncover a useful mutation, through Hamish Scott, who was at the Walter and Eliza Hall Institute (WEHI) and is now at the IMVS, with Dillon Leong, who is at WEHI, and David Thorburn in collaboration. Hamish and Dillon identified a mouse that, actually through a spontaneous mutation, was defective. It had a defective phenotype.
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Here you see a wildtype mouse running out of the box, very easily, exploring and looking around. And the other mouse has the same defect as that particular patient had, with very poor motility as well as neurological defects typical symptoms of complex I defects and a mitochondrial disease.
This was a fortuitous type of thing to happen, and it was great that it happened so locally, in Melbourne, so that we can now analyse this mouse as a model.
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Importantly, when we looked at the mouse extracts we found complex I in the mouse mutant to also have an assembly defect, consistent with the patient.
So we can use this mouse model in the future to look at pathogenic mechanisms what is the biochemistry underlying these defects, what else happens, and what happens in different specific organs? as well as the potential to devise and trial new treatment strategies, and, finally, to look at whether internal defects, those inside mitochondria, such as defects in complex I, also can change the morphology. We are getting some hints that this might be the case.
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I will finish up now by thanking the people who have been involved, certainly in my laboratory, working on different aspects of mitochondria, but importantly my collaborator David Thorburn, working very closely with complex I biogenesis. And of course I thank the funding bodies, the ARC and the NHMRC.
