Decker Franziska, Brugués Jan
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany; Max Planck Institute for the Physics of Complex Systems, Dresden, Germany.
Methods Cell Biol. 2015;125:61-75. doi: 10.1016/bs.mcb.2014.11.004. Epub 2015 Jan 8.
Here, we describe a detailed protocol, based on laser ablation and fluorescence optical microscopy, to measure the microtubule organization in spindles, including microtubule length distribution, polarity, and plus and minus end densities. The method uses the asymmetry in microtubule depolymerization after a cut, where the newly created microtubule plus ends depolymerize all the way to the minus ends, whereas the newly created minus ends remain stable. The protocol described in this chapter is optimized for spindles, but can be easily applied to any microtubule-based structure. The chapter is divided into two parts. First, we provide the theoretical basis for the method. Second, we describe in detail all steps necessary to reconstruct the microtubule organization of a spindle assembled in Xenopus laevis egg extract. Compared to electron microscopy, which in theory can resolve individual microtubules in spindles and provide similar structural information, our method is fast and simple enough to allow for a full quantitative reconstruction of the microtubule organization of several X. laevis spindles—which have volumes tens of thousands of times larger than spindles whose structures have been previously solved by electron microscopy—in a single experimental session, as well as to explore how the architecture of these structures changes in response to biochemical perturbations.
在此，我们描述了一种基于激光消融和荧光光学显微镜的详细方案，用于测量纺锤体中的微管组织，包括微管长度分布、极性以及正端和负端密度。该方法利用切割后微管解聚的不对称性，即新产生的微管正端一直解聚到负端，而新产生的负端保持稳定。本章所述方案针对纺锤体进行了优化，但可轻松应用于任何基于微管的结构。本章分为两部分。首先，我们提供该方法的理论基础。其次，我们详细描述重建非洲爪蟾卵提取物中组装的纺锤体微管组织所需的所有步骤。与理论上可以分辨纺锤体中单个微管并提供类似结构信息的电子显微镜相比，我们的方法快速且简单，足以在单个实验过程中对多个非洲爪蟾纺锤体的微管组织进行完整的定量重建（这些纺锤体的体积比以前通过电子显微镜解析其结构的纺锤体大数万倍），还能探究这些结构的架构如何响应生化扰动而变化。
