The kinetochore is a network of protein complexes that assembles on centromeric chromatin to act as the connection point between chromosomes and the microtubules that segregate them into daughter cells. During anaphase kinetochores allow chromosomes to track the depolymerizing ends of microtubules, which are the primary site of force generation. Thus, kinetochores not only do not let go of microtubule ends as they breakdown, but are able to harness the energy stored in the microtubule lattice and released during depolymerization to produce processive movement of chromosomes to the spindle poles. In this proposal we aim to probe the molecular mechanisms of directed chromosome motion by examining a set of kinetochore protein complexes that are the site of microtubule attachment. Our approach is to combine activity assays, electron microscopy and image analysis, and bioinformatic methods, in the characterization of fully functional complexes. We are studying the yeast-specific DAM1 complex, which assembles in a microtubule-dependent manner into a ring structure that is able to diffuse on the microtubule surface and to track depolymerizing ends. We are also characterizing the conserved NDC80 complex, and the complete KMN network in yeast, which includes also the MTW1 complex and Spc105. Our ultimate objective is to gain a mechanistic understanding of the molecular interactions governing the dynamic attachment of kinetochores to microtubules. Our structural models would be further tested with our collaborators using the powerful yeast system.
Our overall goal is to gain a mechanistic understanding of the molecular interactions governing the regulated, dynamic attachment of kinetochores to microtubules that underlies the accurate segregation of chromosomes during mitosis. Misregulation of this essential process leads to aneuploidy and tumorigenesis.
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