利用非洲爪蟾卵母细胞减数分裂提取物和全内反射荧光显微镜观察及分析分支微管成核
Visualizing and Analyzing Branching Microtubule Nucleation Using Meiotic Xenopus Egg Extracts and TIRF Microscopy.
作者信息
King Matthew, Petry Sabine
机构信息
Department of Molecular Biology, Princeton University, Washington Road, Princeton, NJ, 08544, USA.
出版信息
Methods Mol Biol. 2016;1413:77-85. doi: 10.1007/978-1-4939-3542-0_6.
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.
有丝分裂和减数分裂纺锤体主要由微管组成，这些微管起源于中心体以及染色质附近。间接证据表明微管也在整个纺锤体中形成，但是纺锤体内微管的高密度使得这一现象无法直接观察到。通过使用非洲爪蟾卵母细胞减数分裂提取物并采用全内反射（TIRF）显微镜技术，可以证明并分析由已存在的微管形成微管的过程。分支微管形成是组装和维持有丝分裂纺锤体的理想机制，因为微管数量在保持其极性的同时得以增加。在此，我们描述了使这些发现成为可能的实验方法以及有助于确定相关关键分子参与者的实验。
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