title: Visualizing and Analyzing Branching Microtubule Nucleation Using Meiotic Xenopus Egg Extracts and TIRF Microscopy.
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.
authors.affiliation: Department of Molecular Biology, Princeton University, Washington Road, Princeton, NJ, 08544, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: King
authors.affiliation: Department of Molecular Biology, Princeton University, Washington Road, Princeton, NJ, 08544, USA. spetry@princeton.edu.
authors.forename: Sabine
authors.initials: S
authors.lastname: Petry
mesh_terms: D000818:Animals; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism*; D017930:Genes, Reporter; D008540:Meiosis; D018613:Microscopy, Confocal / Q000379:methods; D008870:Microtubules / Q000378:metabolism*; D008938:Mitosis; D009865:Oocytes / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D008941:Spindle Apparatus / Q000378:metabolism; D013194:Staining and Labeling; D014982:Xenopus laevis
chemical_list: D011993:Recombinant Fusion Proteins
keywords: Cell division; Cytoskeleton; Meiotic spindle; Microtubule; Microtubule nucleation; Mitotic spindle; TIRF microscopy; Xenopus laevis egg extract