Chromosome movements during mitosis are linked to depolymerization and growth of microtubule (MT) filaments, the tips of which transmit tension to specialized sites on each chromosome, called kinetochores. Our overall goal is to understand how kinetochores harness MT assembly and disassembly to organize and separate chromosomes during cell division. We focus here on the Dam1 complex, an essential component of kinetochores in yeast. Recent work suggests the Dam1 complex contributes directly to kinetochore-MT attachment, force production, and regulation of attached MTs, perhaps by forming a ring encircling the MT. To test these hypotheses, we have developed an in vitro (cell free) motility assay where Dam 1-coated beads attach to the tips of individual dynamic MTs. Like kinetochores, the beads remain tip-bound and undergo assembly- and disassembly-driven movement. This reconstitution of movement using purified proteins is already a novel result supporting a direct role for the Dam1 complex in kinetochore-MT attachment. Moreover, it allows us to apply advanced optical trapping techniques to assess quantitatively the potential for the complex to contribute to attachment and force production in vivo, and to test key predictions of the ring hypothesis. We propose to (1) determine if the Dam1 complex can form load-bearing attachments to dynamic MT tips, (2) determine if Dam 1-based motility depends on a structure encircling the MT, (3) determine if curling protofilaments at the MT tip physically push against Dam1 to drive its movement, and (4) determine if Dam1 alters MT dynamics in a tension-dependent manner. This work will provide insight into the mechanisms by which kinetochores and other tip-attachment structures harness MT growth and shortening to produce pushing and pulling forces to move organelles. Elucidating the molecular basis for these kinetochore functions is essential for understanding cancer progression because chromosome loss, which occurs frequently in cancer, can result from mutations that weaken kinetochore-MT attachments. Promising new chemotherapeutics are being developed to target components of the mitotic machinery, and these efforts will benefit substantially from a more complete knowledge of the roles and mechanisms of specific kinetochore proteins. ? ? ?
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