Measurement of Microtubule Half-Life and Poleward Flux in the Mitotic Spindle by Photoactivation of Fluorescent Tubulin.

The study of microtubule dynamics is of utmost importance for the understanding of the mechanisms underlying mitotic fidelity. During mitosis, the microtubular cytoskeleton reorganizes to assemble a mitotic spindle necessary for chromosome segregation. Several methods, such as controlled exposure to cold, high pressure, high calcium concentration, or microtubule depolymerizing drugs, have been widely used to evaluate the dynamic properties of specific spindle microtubule populations. However, while these methods offer a qualitative approach that is sufficient to discern differences among specific spindle microtubule populations, they fall short in providing a robust quantitative picture that is sensitive enough to highlight minor differences, for example when comparing spindle microtubule dynamics in different genetic backgrounds. In this chapter we describe a detailed methodology to measure spindle microtubule dynamics using photoactivation of fluorescently tagged tubulin in living cells. This methodology allows the quantitative discrimination of the turnover of specific microtubule populations (e.g., kinetochore vs. non-kinetochore microtubules), as well as determination of microtubule poleward flux rates. These two conspicuous features of metazoan spindles must be tightly regulated to allow, on the one hand, efficient error correction, and on the other hand the satisfaction of the spindle assembly checkpoint that controls mitotic fidelity.