Visualizing Microtubule Networks During Drosophila Oogenesis Using Fixed and Live Imaging.
Visualizing Microtubule Networks During Drosophila Oogenesis Using Fixed and Live Imaging.
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DOI：
10.1007/978-1-4939-2851-4_7
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发表时间：
2015-01-01
期刊：
影响因子：
--
通讯作者：
Guichet, Antoine
中科院分区：
文献类型：
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作者：
Legent, Kevin;Tissot, Nicolas;Guichet, Antoine
微管细胞骨架是一个由极化电缆组成的塑料网络。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的途径，通过这些途径建立和维持细胞的极性。在果蝇卵母细胞中，微管介导的转运在轴极性决定因素不对称定位中的作用，在过去的几年里一直是广泛研究的主题。然而，成像微管纤维在大细胞中的分布，其中卵黄发生确保吸收厚重和模糊的卵黄，提出了一系列技术挑战。本章简要回顾了其中的一些方面，并描述了两种旨在规避这些困难的方法。我们提供了一个详细的方案，通过免疫组织化学的方法显示卵母细胞中微管蛋白电缆的三维组织。此外，我们详细介绍了使用荧光标记的微管相关蛋白对微管动力学和网络重建进行实时成像的分步过程。
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.