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Methods Mol Biol. 2016;1413:77-85. doi: 10.1007/978-1-4939-3542-0_6.

Visualizing and Analyzing Branching Microtubule Nucleation Using Meiotic Xenopus Egg Extracts and TIRF Microscopy.

Author information

1
Department of Molecular Biology, Princeton University, Washington Road, Princeton, NJ, 08544, USA.
2
Department of Molecular Biology, Princeton University, Washington Road, Princeton, NJ, 08544, USA. spetry@princeton.edu.

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.

KEYWORDS:

Cell division; Cytoskeleton; Meiotic spindle; Microtubule; Microtubule nucleation; Mitotic spindle; TIRF microscopy; Xenopus laevis egg extract

PMID:
27193844
PMCID:
PMC5016078
DOI:
10.1007/978-1-4939-3542-0_6
[Indexed for MEDLINE]
Free PMC Article

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