Equal segregation of the replicated genome during cell division is essential for the development and propagation of all living organisms. Errors in mitotic processes are a hallmark of cancer cells and major chemotherapeutics target the mitotic spindle. One critical function of the kinetochore is to generate the spindle checkpoint signal until each kinetochore has properly attached to microtubules. This signal is initiated by the localization of the MPS1 protein to the calponin homology domain of the Ndc80 complex, which is regulated by Aurora B. However, this domain of Ndc80 protrudes on a long coiled coil far from Aurora kinase, making it unclear how it could be phosphorylated. We have employed a vastly improved super-resolution microscope to visualize human kinetochores. This microscopes unique ability to provide super-resolution in the Z-plane is essential for the study of cellular structure the size of a kinetochore. This visualization of single molecules of Ndc80 in unattached kinetochores has identified a novel pool of the MPS1 binding region of the Ndc80 complex in the central region of spindle checkpoint signaling kinetochores. We hypothesize that this internal pool is a more efficient generator of the spindle checkpoint than the previously appreciated outer pools. We are employing super-resolution microscopy to both test this hypothesis and further map the sub-kinetochore location of key events in generating the spindle checkpoint signals. We will also identify how the spindle checkpoint is turned off by when kinetochores attach to microtubules. We are building upon two important new discoveries about the Ska complex. First, we have identified the steps that enable Ska to be recruited to kinetochores after microtubule attachments. Second, we have shown that Ska binds PP1 providing a mechanism to specifically recruit a phosphatase to properly attached kinetochores. Building upon this strong foundation we will determine how the Ndc80, Ska and PP1 proteins turn off the spindle checkpoint and generate the kinetochore microtubule attachment that allows kinetochores to remain bound to depolymerizing microtubules to move chromosomes.