Mitosis is a critical phase of the cell cycle where chromosome are equally partitioned to daughter cells using the structure and forces from the mitotic spindle apparatus to power this process. The spindle is made of hundreds of dynamic microtubule polymers. Within the spindle, subpopulations of microtubules are tightly controlled by microtubule associated proteins and signaling gradients to achieve distinct properties and functions. Small perturbations to microtubules dynamics or these regulatory players can profoundly affect chromosome segregation and is associated with aneuploidy and genomic instability. Although many mitotic players have been identified, quantitative and mechanistic views of how they are regulated to control microtubule dynamics are missing. I propose to quantitatively define how different mitotic microtubule subpopulations are regulated in a space and time dependent manner. I have identified the kinetochore and centrosome-localized kinase Plk1 as a key factor in modulating microtubule dynamics. Using cell biology, advanced quantitative analysis and biochemistry, we will identify Plk1 substrates that modulate microtubule dynamics throughout mitosis. In addition, we will examine how a Plk1 locally regulates microtubules to create different microtubule populations. These studies will inform how genomic fidelity and chromosome segregation are ensured through maintaining accurate local and global microtubule dynamics.