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

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 2101)

Abstract

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.

Key words

Photoactivation Fluorescence microscopy Microtubule dynamics Mitosis Mitotic spindle Flux 
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Notes

Acknowledgments

Work in the laboratory of H.M. is funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. 681443) and FLAD Life Science 2020. H.G. holds a Ph.D. fellowship from Fundação para a Ciência e a Tecnologia (SFRH/BD/141066/2018).

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Authors and Affiliations

  1. 1.Chromosome Instability & Dynamics LaboratoryIBMC - Instituto de Biologia Molecular e Celular, Universidade do PortoPortoPortugal
  2. 2.i3S—Instituto de Investigação e Inovação em Saúde, Universidade do PortoPortoPortugal
  3. 3.Experimental Biology Unit, Faculdade de Medicina, Cell Division Group, Department of BiomedicineUniversidade do PortoPortoPortugal

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