Kinetochores are multiprotein machines that drive mitotic chromosome segregation by harnessing energy released during microtubule tip disassembly to generate force and movement. Kinetochores also ensure the accuracy of mitosis. They sense and release improper microtubule attachments and, when unattached or improperly attached, they also recruit checkpoint proteins that generate `wait' signals, delaying anaphase and giving more time for proper attachments to form. To uncover how these vital functions occur, we are reconstituting kinetochore activities using pure components and applying state-of-the-art biophysical tools for manipulating and tracking individual molecules. Our unique in vitro approach allows long-standing questions about kinetochore function to be answered in direct ways that would be impossible in living cells. Specifically, we will: (1) measure the force-generating capacity of protofilaments as they curl out from a disassembling tip and determine the contribution that this `conformational wave' can make to force production at kinetochores; (2) determine how the conserved microtubule polymerase, Stu2, stabilizes kinetochore-microtubule attachments and how its activity at kinetochores is regulated in a tension-dependent manner; (3) directly observe the association of individual checkpoint proteins with single kinetochores and distinguish whether their binding is directly inhibited by lateral attachment to the side of a microtubule, by end-attachment, or by mechanical tension. Together this work will elucidate how kinetochores generate force to move chromosomes, and how their attachments to spindle microtubules are regulated. Understanding the 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-microtubule attachments or disrupt kinetochore regulation. Promising new chemotherapeutics are also being developed to target kinetochore and spindle checkpoint components, and these efforts will benefit substantially from a more complete knowledge of the roles of specific components and the mechanisms by which they operate.