More than two populations of microtubules comprise the dynamic mitotic spindle.

The microtubules of the mitotic spindle mediate chromosome alignment to the metaphase plate, then sister chromatid segregation to the spindle poles in anaphase. Previous analyses of spindle microtubule kinetics utilizing fluorescence dissipation after photoactivation described two main populations, a slow and a fast turnover population, and these were ascribed as reflecting kinetochore versus non-kinetochore microtubules, respectively. Here, we test this categorization by disrupting kinetochores through depletion of the Ndc80 complex in U2OS cells. In the absence of functional kinetochores, microtubule dynamics still exhibit slow and fast turnover populations, although the proportion of each population and the timings of turnover are altered. Importantly, the data obtained following Hec1 (also known as Ndc80) depletion suggests that other subpopulations, in addition to kinetochore microtubules, contribute to the slow turnover population. Further manipulation of spindle microtubules revealed a complex landscape. For example, although Aurora B kinase functions to destabilize kinetochore bound microtubules it might also stabilize certain slow turnover non-kinetochore microtubules. Dissection of the dynamics of microtubule populations provides a greater understanding of mitotic spindle kinetics and insight into their roles in facilitating chromosome attachment, movement and segregation during mitosis.