Legent Kevin, Tissot Nicolas, Guichet Antoine
Institut Jacques Monod, UMR 7592 - CNRS, Université Paris Diderot, 15 rue Hélène Brion, Bât Buffon, 75205, Paris, France.
Methods Mol Biol. 2015;1328:99-112. doi: 10.1007/978-1-4939-2851-4_7.
The microtubule cytoskeleton is a plastic network of polarized cables. These polymers of tubulin provide orientated routes for the dynamic transport of cytoplasmic molecules and organelles, through which cell polarity is established and maintained. The role of microtubule-mediated transport in the asymmetric localization of axis polarity determinants, in the Drosophila oocyte, has been the subject of extensive studies in the past years. However, imaging the distribution of microtubule fibers in a large cell, where vitellogenesis ensures the uptake of a thick and hazy yolk, presents a series of technical challenges. This chapter briefly reviews some of these aspects and describes two methods designed to circumvent these difficulties. We provide a detailed protocol for the visualization by immunohistochemistry of the three-dimensional organization of tubulin cables in the oocyte. Additionally, we detail the stepwise procedure for the live imaging of microtubule dynamics and network remodeling, using fluorescently labeled microtubule-associated proteins.
微管细胞骨架是一个由极化纤维组成的可塑性网络。这些微管蛋白聚合物为细胞质分子和细胞器的动态运输提供了定向路径，通过这些路径细胞极性得以建立和维持。在过去几年中，微管介导的运输在果蝇卵母细胞中轴极性决定因子不对称定位中的作用一直是广泛研究的主题。然而，在一个大细胞中对微管纤维分布进行成像存在一系列技术挑战，在这个大细胞中，卵黄发生确保了对浓厚且模糊的卵黄的摄取。本章简要回顾了其中一些方面，并描述了两种旨在克服这些困难的方法。我们提供了一个详细的方案，用于通过免疫组织化学可视化卵母细胞中微管纤维的三维组织。此外，我们详细说明了使用荧光标记的微管相关蛋白对微管动力学和网络重塑进行实时成像的逐步程序。
