Pigino Gaia, Cantele Francesca, Vannuccini Elisa, Lanzavecchia Salvatore, Paccagnini Eugenio, Lupetti Pietro
Max-Planck-Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
Methods Enzymol. 2013;524:325-42. doi: 10.1016/B978-0-12-397945-2.00018-4.
Cilia and flagella play very important roles in eukaryotic cells, ranging from cell motility to chemo- and mechanosensation with active involvement in embryonic development and control of cell division. Cilia and flagella are highly dynamic organelles undergoing constant turnover at their tip, where multiprotein precursors synthesized in the cell cytoplasm are assembled, turnover products are released and carried back for recycling. Such bidirectional trafficking is maintained by an ATP-dependent active transport that is carried out by intraflagellar transport (IFT) particles. Despite our knowledge of the cell biology, the genomic, and the biochemistry of IFT, high-resolution 3D models for IFT are still missing. To date, the only information on the 3D structure of IFT come from our analysis of full-length flagella from the biflagellate green alga Chlamydomonas reinhardtii: the model organism where IFT was discovered and first characterized. In this chapter, we describe and discuss the strategy we implemented to produce the first 3D models of in situ IFT trains in flat-embedded flagella. We provide detailed information about the acquisition of tomographic images, the simultaneous alignment of the double-tilt tomographic series, and the analysis of the tomograms by subtomogram averaging for the generation of detailed 3D models of IFT particles.
纤毛和鞭毛在真核细胞中发挥着非常重要的作用，从细胞运动到化学感应和机械感应，积极参与胚胎发育和细胞分裂的控制。纤毛和鞭毛是高度动态的细胞器，其顶端不断更新，在那里，在细胞质中合成的多蛋白前体被组装，更新产物被释放并带回进行循环利用。这种双向运输由鞭毛内运输（IFT）颗粒进行的ATP依赖性主动运输维持。尽管我们对IFT的细胞生物学、基因组学和生物化学有所了解，但仍缺少IFT的高分辨率三维模型。迄今为止，关于IFT三维结构的唯一信息来自我们对双鞭毛绿藻莱茵衣藻全长鞭毛的分析：这是发现并首次表征IFT的模式生物。在本章中，我们描述并讨论了我们为生成扁平包埋鞭毛中原位IFT列车的首个三维模型而实施的策略。我们提供了有关断层图像采集、双倾斜断层扫描系列的同步对齐以及通过子断层扫描平均分析断层图以生成IFT颗粒详细三维模型的详细信息。
