Towards a mechanistic understanding of the chromosome segregation machinery
An understanding of kinetochore function requires the study of its constituent parts, the 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 hierarchically assembles from a set of conserved multi-protein complexes (Figure 1).
Figure 1: Schematic overview of the budding yeast kinetochore.
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: For example, the Dam1 complex, a specialized microtubule-binding component of the budding yeast kinetochore oligomerizes to form a ring around microtubules in vitro (Figure 2).
Figure 2: Negative stain electron microscopy of Dam1 complex decorating microtubules. Microtubules have been pseudocolored in gold. The Dam1 ring complex acts as a force coupler at the yeast kinetochore..
This 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 efficient 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, to visualize the structure of the fully assembled interface and analyze how it is regulated, for example by mitotic kinases. Our investigations into kinetochore assembly have led to the identification of a conserved receptor molecule for the microtubule-binding Ndc80 complex. We have solved the crystal structure of the interface between Ndc80 and the histone-fold protein Cnn1 and we are further investigating how the cell employs different Ndc80 receptors to promote chromosome segregation.
Single molecule analysis of microtubule-associated proteins and motors
Movie 1: Dynamic Microtubules (red) growing in vitro from a Microtubule-organizing center (MTOC). The growing plus-ends of microtubules are decorated with EB1 (green). The movies with taken using TIRF microscopy.
Movie 2: Single Cik1-Kar3 Kinesin-14 motor proteins (red) moving along taxol-stabilized microtubules (blue), visualized by TIRF microscopy. Note unidirectional, processive runs of individual motor molecules along the microtubules.