PROJECT SUMMARY Accurate chromosome segregation crucially depends on the dynamic attachments between chromosomal kinetochores and spindle microtubules. Recent years brought tremendous progress in identifying molecular components of the kinetochores, but mechanistic studies of how these proteins interact with microtubules and enable chromosome motions are lagging behind. We propose to address this deficiency by using reductionist multiscale approaches and innovative assays that reconstitute physiological aspects of kinetochore-microtubule interactions in vitro. We have molecular tools, equipment and expertise to address in a quantitative and rigorous manner some of the most fundamental questions about mitotic chromosome segregation: (1) how the kinetochores convert their initial microtubule-wall binding into microtubule-end attachment, (2) how they subsequently hang onto the microtubule ends and move in conjunction with tubulin assembly/disassembly, (3) and how these mobile links persist under force. In Aim 1 we will recreate these interactions using purified Ndc80 protein complex and strategically chosen assisting proteins. We recently reconstituted microtubule end conversion by Ndc80, assisted by a plus-end directed kinesin CENP-E. Different Ndc80 variants will be used to uncover the underlying mechanism, and additional kinetochore components will be added to reveal their relative impact. Our findings with purified components will be critically compared with the activity of native kinetochore complexes isolated from extracts of mitotic human cells (Aim 2). We have found that complexes associated with the kinetochore scaffold protein CENP-T can move at the dissembling microtubule ends. This essential achievement lays the groundwork for our functional assays, and subsequent identification and characterization of key kinetochore components for microtubule end coupling in human cells. In Aim 3 we will use advanced laser tweezers techniques to critically compare the ability of purified proteins and complexes to move under pulling force, mimicking tension between sister kinetochores. The results from these studies will help us to construct an integrative view of the mechano-molecular coupling at human kinetochore, define the specific roles of key kinetochore proteins Ndc80, Ska1 and others, and reveal functional difference between kinetochore complexes assembled with different scaffolds. Little is known about functional behavior of human kinetochore proteins, and our research will undoubtedly provide novel insights into the fundamentals of kinetochore-microtubule interactions, and promote new discoveries in the cell division field.