Optimized protocol for live-cell analysis of kinetochore fiber maturation in Indian muntjac cells
Ana Coelho Almeida, Joana Soares-de-Oliveira, Helder Maiato
Microtubules, Kinetochore, Protocol
Here, we take advantage of the low chromosome number (2N=6) and distinctively large kinetochores of female Indian muntjac cells to investigate the molecular mechanism underlying k-fiber maturation. We describe steps for monitoring kinetochore-microtubule dynamics over time. Specifically, we detail the combination of live-cell super-resolution CH-STED microscopy of microtubule growth events within individual k-fibers and a laser-mediated k-fiber injury/repair assay. These tools provide a direct assessment of microtubule amplification mechanisms within k-fibers in metazoans. For complete details on the use and execution of this protocol, please refer to Almeida et al. (2022).