New in ISBUC: Eva Kummer

So, what is the principle research question that you're working on at the moment.

I'm interested in mitochondria and in mitochondrial DNA. Even though it constitutes a tiny fraction of the genetic information in the body, it encodes essential subunits of the respiratory chain complexes and is thus vital to energy production in our cells. Specifically, I would like to understand how this DNA is amplified, maintained and expressed. To look at these processes in molecular detail, my group will use a combination of structural and biochemical approaches.

At the moment we are especially interested in the replication machinery. We would particularly like to investigate how the replications factors interact at the replication fork to efficiently synthesize DNA and solve the structures of the factors where no or only very limited 3D information is available.

And this mitochondrial replication machinery, does it look like the bacterial system of the mitochondrial ancestors?

No, actually, it is more related to the bacteriophage systems. Mitochondria represent a very weird mixture of components from bacteria, bacteriophages and the cytoplasm in addition to mitochondria specific proteins. So it is a very odd case.

Moreover, maintenance of mitochondrial DNA entails much more than just replication. We are also interested in addressing how, and if, mitochondrial DNA is repaired. There are several hypotheses in the field and there is actually not that much known about it. Intriguingly, unlike in the nucleus you do not have only two copies of DNA per cell, but you can have hundreds to thousands, which appear to be equally distributed throughout the mitochondrial network. It is still an open question how these DNA copies are distributed in order to ensure that the cell has sufficient mitochondrial DNA everywhere to drive energy production. Here, we are especially interested to understand how the distribution of mitochondrial DNA correlates with its replication.

Another branch that we will pursue is how mitochondrial DNA is expressed because the transcribed RNAs are long polycistronic precursor transcripts that need to undergo maturation before they can be functional. We are very interested in how this maturation takes place.

I suppose they mainly encode membrane proteins? 

Yes, exactly. Membrane proteins, mitochondrial tRNAs and mitoribosomal RNAs.

So, coming back to this DNA distribution in the mitochondrial network: each mitochondrion is not born with DNA?

No, first of all, for mitochondria you would say that there is no de novo formation. You have to envision mitochondria as a dynamic network where mitochondria constantly fuse and divide. In that way, they can change their shape and sometimes parts of the network are degraded because they are no longer functional and other parts expand to generate new mitochondrial mass. But, it's not like you have this bean shaped mitochondrion - as it is usually shown in illustrations - and one bean shaped mitochondria has one DNA molecule. Depending on the needs of the cell the network can adopt different shapes and it is often an elongated network with multiple DNA molecules within each branch.

OK, so DNA distribution is not controlled when they divide?

Actually, our current understanding indicates that it is controlled. For example, it has recently been shown that replication is coupled to localized fission events that help distributing the produced DNA copies in the resulting mitochondria. This is something that we want to have a closer look at. More recently it has also been shown that the location of the fission events on the mitochondria determines the fate of the resulting mitochondria: if the fission event occurs towards the end of the mitochondrion then the produced, small mitochondrial fission product will likely be degraded because its function has declined, while if they divide in the middle they will undergo further rounds of division. And it appears that these ‘healthy’ middle divisions may also be the sites of mitochondrial DNA replication.

You are really venturing into uncharted land here.

Yes, although we know the core proteins involved in the replication process, how, for example, replication and mitochondrial division are locally coupled, that is not so well understood.

So, will you be using any fancy techniques that you believe could be of interest to other ISBUC members?

Because we're just setting up, the fanciest technique I can offer at the moment is probably single particle cryo-EM - even though that might not be too fancy for ISBUC members. In the future, we will expand our palette and will be happy to share new techniques.

OK, and are you looking for any techniques or collaborations?

I've mostly been working with bacterial protein expression so far, so the two new things we wish to establish in the lab are mammalian and insect cell expression systems. We are lucky to have the CPR protein production platform next door with lots of expertise that we can draw on. But of course, especially when it involves more sophisticated approaches like CRISPR/Cas9 genome editing we would like to establish internal expertise, for example by hiring a technician or postdoc who is specifically good at this. But this is indeed something that could also be subject to collaboration.

Another area where I envisage collaborations is in the purification of membrane proteins that may be involved in the distribution of DNA in the mitochondrial network. We would like to explore the possibilities to use nanodiscs or maybe some of the polymers that Henriette Autzen from the Department of Biology is developing.

What courses are you going to teach?

First, I will have to do the paedagogicum and I will contribute to the CPR summer school and the cryo-EM introductory workshop organized by CPR and CFIM. And then, of course, I will also teach at the CPH Bioscience PhD program (https://cphbiosciencephd.org/). But details for further course work remain to be figured out in detail.

At the moment I have accepted to give a talk at the Linderstrøm-Lang symposium taking place in the Biocenter 19 November (https://www.bio.ku.dk/protein).

Do you have a publication that you are most proud of?

Actually, I have two projects that I am very proud of. During my PhD I was working in molecular biology and biochemistry and didn't do structural biology. I think biochemical work is very different to structural biology approaches, because you do a lot of smaller experiments and then all of these experiments in their entirety lead to a working model or a new hypothesis. During my PhD I was working on molecular chaperones and AAA+ proteins. We uncovered how two chaperone systems act together in order to recover aggregated, nonfunctional proteins.  Our major discovery was that one of the chaperones is a very potent protein disaggregase that needs to be kept inactive most of the time to avoid harming or killing the cell because it would otherwise start ‘disaggregating’ correctly folded and functional substrates. So its activity is only unleashed when it sees the right substrate together with the partner chaperone. Once done, it goes back into the inactive mode. Our results have been published back-to-back in two very nice papers:

Oguchi, Y.*, Kummer, E.* et al. A tightly regulated molecular toggle controls AAA+ disaggregase. Nat Struct Mol Biol 19, 1338–1346 (2012). https://doi.org/10.1038/nsmb.2441

Seyffer, F.*, Kummer, E.* et al. Hsp70 proteins bind Hsp100 regulatory M domains to activate AAA+ disaggregase at aggregate surfaces. Nat Struct Mol Biol 19, 1347–1355 (2012).
https://doi.org/10.1038/nsmb.2442

For my postdoc I switched to structural biology and a completely different topic. I started working on mitochondrial ribosomes and had to set up an entirely new system for in vitro reconstitution of mitochondrial translation complexes. I wanted to have the complexes as close to the native ones as possible including the special mitochondrial tRNAs and tRNA synthetases. All of these systems needed to aminoacylate the tRNAs, produce the special leaderless mitochondrial mRNAs, and assemble the complexes hadn't been established. So, essentially, I had to do all the biochemistry before we could study the complexes structurally. I am very proud of the results, some of which were published here:

Kummer, E., Leibundgut, M., Rackham, O. et al. Unique features of mammalian mitochondrial translation initiation revealed by cryo-EM. Nature 560, 263–267 (2018). https://doi.org/10.1038/s41586-018-0373-y 

Impressive! And what are your favorite papers written by others at the moment?

The lab of Suliana Manley at EPFL has published a couple of really nice papers recently. One of them is the one that elucidated how the location of a mitochondrial fission event affects the fate of mitochondria as mentioned earlier. This is one of my favorite papers at the moment:

Kleele, T., Rey, T., Winter, J. et al. Distinct fission signatures predict mitochondrial degradation or biogenesis. Nature 593, 435–439 (2021). https://doi.org/10.1038/s41586-021-03510-6

And from the same lab last year:

Rey, T., Zaganelli, S., Cuillery, E. et al. Mitochondrial RNA granules are fluid condensates positioned by membrane dynamics. Nat Cell Biol 22, 1180–1186 (2020). https://doi.org/10.1038/s41556-020-00584-8

Here, they study for the first time the dynamics of mitochondrial RNA granules. Like in the nucleus and cytoplasm it has been discovered that RNAs can accumulate in RNA deposits, so-called RNA granules, in mitochondria. Mitochondrial RNA granules have been proposed to be the sites of RNA maturation and mitoribosome biogenesis and are therefore very interesting to us.

Do you have any specific expectations to ISBUC?

I would like to get to know a little bit more about the structural biology community in Copenhagen to understand which projects and approaches the different labs pursue and to discover what kinds of collaborations might be possible.

Well, that is exactly what ISBUC is all about, so I sincerely hope that we will be able to meet your expectations.

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