Abstract
Mitotic and meiotic spindles consist primarily of microtubules, which originate from centrosomes and within the vicinity of chromatin. Indirect evidence suggested that microtubules also originate throughout the spindle, but the high microtubule density within the spindle precludes the direct observation of this phenomenon. By using meiotic Xenopus laevis egg extract and employing total internal reflection (TIRF) microscopy, microtubule nucleation from preexisting microtubules could be demonstrated and analyzed. Branching microtubule nucleation is an ideal mechanism to assemble and maintain a mitotic spindle, because microtubule numbers are amplified while preserving their polarity. Here, we describe the assays that made these findings possible and the experiments that helped identify the key molecular players involved.
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Acknowledgements
This work was supported by grants from the NIH/NIGMS (4R00GM100013), the Pew Scholars Program in the Biomedical Sciences, the Sidney Kimmel Foundation, and the David and Lucile Packard Foundation to S.P.
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King, M., Petry, S. (2016). Visualizing and Analyzing Branching Microtubule Nucleation Using Meiotic Xenopus Egg Extracts and TIRF Microscopy. In: Chang, P., Ohi, R. (eds) The Mitotic Spindle. Methods in Molecular Biology, vol 1413. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3542-0_6
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DOI: https://doi.org/10.1007/978-1-4939-3542-0_6
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