Westermann Group Research
Molecular Mechanisms of Kinetochore Function
Eurkaryotic cells have evolved complex and extremely precise chromosome segregation mechanisms, which ensure that genetic information is passed correctly from one generation to the next. In order to understand how the cell moves chromosomes during mitosis, our group is studying the structure, function and regulation of the kinetochore, a complex multi-protein assembly that connects chromosomes to dynamic microtubules. In a reductional biochemical approach we aim to study and assemble functional kinetochores from their constituent parts in vitro. In addition, we use yeast genetics to manipulate and engineer simple chromosome segregation systems in vivo.
Construction of a kinetochore in the test tube
- Figure 1 (Click to view legend)
Understanding kinetochore function requires study of its constituent parts, assembly of these parts into higher-order structures, and ultimately the reconstitution of kinetochore function in vitro. The kinetochore is a complex macromolecular machine that is hierarchically assembled from a set of conserved multi-protein complexes. We have reconstituted a number of these complexes by co-expressing multiple subunits in bacteria and studied their biochemical properties. This analysis has already yielded some important insights: the Dam1 complex, a specialized microtubule-binding component of the budding yeast kinetochore (Figure 1), oligomerizes to form a ring around microtubules in vitro. The ring slides along the microtubule lattice and remains attached to the plus-end even during microtubule disassembly. These properties make the Dam1 ring a very ef ficient force coupler at the kinetochore. A challenge for the future is to understand how the Dam1 ring is connected to the rest of the kinetochore, visualize the structure of the fully assembled interface, and analyze how it is regulated - for instance by mitotic kinases.
Building simplified chromosome segregation systems in the cell
- Figure 2 (Click to view legend)
To define functional modules within the complex kinetochore architecture, we have adopted a reductional approach in designing simple kinetochores in vivo. By artificially recruiting individual kinetochore components to engineered binding sites on circular plasmids and on native yeast chromosomes, we were able to demonstrate that the Dam1 complex is not only necessary but also sufficient to generate an interface that supports chromosome segregation (Figure 2). In the future we will further characterize the protein composition and precise function of these “artificial” kinetochores.
A fur ther challenge for the future is to understand how kinetochore structure and function are modulated throughout the cell cycle. The basic signals that couple cell cycle progression with the regulation of kinetochore function have remained elusive. Combining time-resolved analysis of post-translational modifications with yeast genetics is expected to disclose general principles of regulation.
Analyzing the interaction of kinetochores with dynamic microtubules
- Figure 3 (Click to view legend)
A defining feature of kinetochores is their ability to interact with microtubule plus-ends through multiple rounds of polymerization and depolymerization. How does the kinetochore achieve this remarkable task? What features allow it to follow a polymerizing microtubule end as well as stay connected during disassembly? How does the kinetochore modulate microtubule dynamics? To analyze this process we reconstituted dynamic microtubules in vitro and visualized the interaction of individual kinetochore components using total internal reflection fluorescence (TIRF) microscopy. This technique allows the observation of individual kinetochore complexes and microtubule-binding proteins with single-molecule sensitivity to reveal their mode of interaction with dynamic plus-ends. Our initial analysis has focused on the yeast EB1 protein Bim1p (Figure 3). We showed that this protein uses a microtubule-binding interface composed of a calponin-homology domain and a flexible basic linker to autonomously track growing microtubule ends in vitro. Multi-site phosphorylation of the linker domain by the Aurora kinase Ipl1p steers the interaction of Bim1p with microtubules and critically regulates the quantity of Bim1p on the mitotic spindle in vivo. In the future we aim to reconstitute additional kinetochore plus-end tracking systems to define functional dependencies.