Without kinetochores, cells wouldn't get far in separating their chromosomes during mitosis. This intricate network of around 100 proteins assembles at the centromere of every chromatid and attaches them to the mitotic spindle, aligning them at the center before partitioning them to the poles during anaphase. But kinetochores are much more than a simple link between DNA and microtubules—they coordinate chromosome and spindle dynamics, and are signaling centers that ensure the fidelity of chromosome inheritance.
Iain Cheeseman has been fascinated by the kinetochore since his days as a graduate student with David Drubin and Georjana Barnes at the University of California, Berkeley, where he identified a subgroup of kinetochore proteins called the Dam1 complex that links yeast chromosomes to the mitotic spindle (1–3). Cheeseman switched to C. elegans and human kinetochores for his postdoctoral studies, working with Arshad Desai at the Ludwig Institute for Cancer Research, in San Diego, CA. He purified a set of kinetochore proteins, called the KMN network, which forms the core microtubule-attachment site on metazoan chromosomes (4, 5), and controls kinetochore assembly (6). Cheeseman began his own laboratory at the Whitehead Institute and the Biology Department at MIT in Cambridge, MA, in 2007 and recently characterized another kinetochore subcomplex that helps chromosomes move during anaphase by clinging on to depolymerizing spindle fibers (7).
“It really felt like a compelling structure that I could study for a long time.”
Cheeseman believes that the kinetochore parts list is nearing completion, and he is excited to begin understanding how they all combine to direct chromosome segregation. In a recent interview, he explained his long-standing attachment to kinetochores, and where his research is likely to be pulled next.
What do you think you'd do if you weren't running a laboratory?
My friends from grad school are doing very diverse jobs. Some of them are in academic science, but some are teaching high school. Some of them work at journals or in biotech. I think all of those things are great ways to pursue science. If academia hadn't worked out the way that it has so far, I would be really happy doing any of those things.
What were some of your earliest scientific experiences?
I grew up in a university town—my father's a professor of plant biology—so in summers during high school, as well as mowing lawns and things like that, I worked in laboratories, washing dishes and making solutions. I really liked being around the laboratories, so in college, I worked in a different laboratory nearly every year: plant laboratories, bacteriophage, Chlamydomonas. One summer I worked in a honey bee laboratory on circadian rhythms. We watched the bees 24 hours a day. Since I was the young kid, they gave me the 9 p.m. to 9 a.m. time point! It was fun trying different things. I think it gave me a good idea of what I liked and what I didn't. Often learning what you don't like is an easier way to define the kind of problems that do interest you. Basic research questions about how something worked always excited me so it was an easy choice to go to grad school.
Did you intend to work on kinetochores as a student in the Drubin/Barnes Laboratory?
Georjana's focus had been on microtubules. The Dam1 proteins—at least the two that had been identified—came out of a screen for cytoskeletal components.
For the first year and a half, I focused on the role of these proteins as cytoskeletal elements. But a lot of the data that we found were consistent with a second role, directing chromosome segregation in a way that you wouldn't see for a purely microtubule-binding protein. It took a while to convince people, but it became clear that they were actually at the kinetochore. After that, I really wanted to take more directed approaches to the entire thing—thinking of the diversity of things that the kinetochore does, it really felt like a compelling structure that I could study for a long time. I decided to do a postdoc in a kinetochore laboratory, partly because I came to it a little bit later in my PhD.
You joined Arshad Desai's laboratory in San Diego. What was that like?
I arrived in Arshad's laboratory after he had been there for less than a month. For the first experiments that I did, I had to unpack the gel power supply before I could run the gel. Seeing things from the beginning gave me a nice perspective on how to start my own laboratory someday. I saw the startup process and the decisions that he made, what worked well and occasionally what didn't.
Arshad's laboratory was a really fantastic place to work. He is very creative and thoughtful about how to approach a problem, was a lot of fun to argue with, and was very supportive. I also had the pleasure of working with exceptional laboratory colleagues, including Paul Maddox and Reto Gassman, and amazing people next door in the Oegema and Cleveland laboratories. It made for a really dynamic environment where there was always exciting science and diverse perspectives.
What is it that fascinates you about the kinetochore?
The most exciting thing to me is that the kinetochore has so many different activities that it's almost a model for anything that you could want to study in cell biology. It's such a complex and intricate structure that there are a lot of exciting questions to answer about it.
Even going back 100 years, there was a good understanding of what a kinetochore is and for the next, say, 60 years people were able to learn a huge amount from just watching how the chromosomes behaved. It wasn't until 1987 that the first human kinetochore protein was identified. For the next 10 years, it was like pulling teeth to find other components of the structure. But from about 2000 to 2007 it exploded, going from 10 proteins to 80. We contributed a lot to that by pulling down tagged proteins from yeast, worms, and human cells. However, it's started to level off in the past couple of years, which makes me think that we're getting to the end of this phase.
Consider all the things a kinetochore does: binding to DNA, specifying a position on the chromosome, binding to microtubules, sensing and correcting defects, assembling and disassembling the kinetochore during the cell cycle. You could guess the players most likely to be involved in each of these functions, but we have no idea how they work. Now you have this parts list, however, you can direct your experiments in a really intelligent way. You can understand the individual functions of the proteins and how they integrate to facilitate chromosome segregation. This really is the golden age of the kinetochore.
MAKING THE SEPARATION
What's next for your laboratory?
The last paper I published in grad school looked at kinetochore regulation, and that is something I've always been really excited about. Not only do you have to physically segregate chromosomes, you have to control it in such a way that it never produces errors. The phosphorylation events that alter the behavior or assembly of the kinetochore are almost completely unknown.
We're starting to sort through that. Not only is it a great way to define regulatory paradigms, but we can also learn how an individual protein works mechanistically. For example, Aurora B is a master regulatory kinase in kinetochores that phosphorylates Ndc80 to turn off its microtubule-binding activity. If you didn't know anything about Ndc80 to begin with, it would tell you a lot about how it works, because those phosphorylation sites occur in a very short stretch of the protein's N terminus that binds microtubules.
A lot of your work has focused on the attachment of kinetochores to microtubules…
That's always fascinated me. You have lots of different players at the microtubule interface. There's the KMN network, of which Ndc80 is a part, which really forms the core attachment to microtubules that resists the huge forces generated during mitosis. Then there's the Ska1 complex, which helps hold onto a microtubule as it depolymerizes, harnessing that energy to move the chromosomes. You have motors like CENP-E and dynein that drive translocation along the microtubule polymer and factors that direct microtubule dynamics: CLASP guides polymerization of kinetochore-bound microtubules, while MCAK destabilizes them.
“This really is the golden age of the kinetochore.”
So there are lots of individual activities that you need to define, but ultimately we have to integrate all of them—determine how they fit into this larger structure and work together. We're trying to understand more about how the KMN and Ska1 complexes work. Although they're very different in their properties and cellular phenotypes, I would be hard-pressed to draw a tight line between their functions. They're both amazing machines; we need to figure out how they coordinate with each other.
Do you only work with human cells now?
Arshad was really generous in letting me take the projects that I had set up and a lot of that was in human cells. It was a nice way to go our separate ways, as he continues to focus on worms.
Lots of human kinetochore proteins don't exist in C. elegans and I was curious about how they work. And it would be technically much harder to study kinetochore regulation in C. elegans. It's easier to map the phosphorylation sites and look at their function in human cells. So it really made sense; I'd say 98% of what we do now is in human cells. But there are places where C. elegans or yeast are better suited to a particular experiment, so when we come across those specific questions, we'll move to a different organism.
We don't want to be limited by approach. If it makes sense to do biochemistry, we'll do biochemistry. If it makes sense to do cell biology, we'll do cell biology. The back and forth between approaches is really important. If you have a phenotype or localization that says something about a protein, it really guides your choice of biochemical assays. But once you've done the biochemistry, being able to go back and test whether the things you've learned are really important to the cell is also very useful.
Similarly, if it makes sense to crystallize something or learn how to do single molecule studies, we'll figure out how to go in that direction. The people I get to work with are really smart, they work their butt off, and they do fantastic stuff. I love working with the people in my laboratory.
Text and Interview by Ben Short