Kinetochores are megadalton protein structures that assembled on centromeric chromatin to form the microtubule-binding site.  Kinetochores are assembled from conserved protein subcomplexes. Although many models of kinetochore structure have been proposed based on protein-protein interaction experiments and physical studies of individual subcomplexes, little is known about the architecture of native kinetochores and the mechanism by which kinetochores bind to and maintain attachments to dynamic microtubules. In addition, the post-translational modifications required for kinetochore assembly are not known.  To address these issues, we have developed a method to assemble kinetochores de novo and are using this to dissect the assembly process. We are also pursuing kinetochore structure using EM techniques.

Projects

How do kinetochores assemble?

We have developed assays for de novo kinetochore assembly in vitro using a centromeric DNA template and yeast lysate. We are using this assay to identify the post-translational modifications required for assembly, to order the steps of assembly and to monitor kinetochore assembly in real time using a single molecule TIRF microscopy assay. 

What is the structure of a native kinetochore?

We are performing cryoelectron microscopy and cryotomography to elucidate the overall architecture of kinetochores purified from yeast. Our goal is to obtain high resolution images of native kinetochores bound to microtubules.  We would also like to capture assembly intermediates using the de novo assembly assay we developed.