Winey Mark, Stemm-Wolf Alexander J, Giddings Thomas H, Pearson Chad G
MCD Biology, University of Colorado at Boulder, 347 UCB, Boulder, Colorado 80309-0347, USA.
Methods Cell Biol. 2012;109:357-78. doi: 10.1016/B978-0-12-385967-9.00013-X.
Since their first detection in pond water, large ciliates such as Tetrahymena thermophila, have captivated school children and scientists alike with the elegance of their swimming and the beauty of their cortical organization. Indeed, cytology - simply looking at cells - is an important component of most areas of study in cell biology and is particularly intriguing in the large, complex Tetrahymena cell. Cytological analysis of Tetrahymena is critical for the study of the microtubule cytoskeleton, membrane trafficking, complex nuclear movements and interactions, and the cellular remodeling during conjugation, to name a few topics. We briefly review previously reported cytological techniques for both light and electron microscopy, and point the reader to resources to learn about those protocols. We go on to present new and emerging technologies for the study of these marvelous cells. These include the use of fluorescent-protein tagging to localize cellular components in live cells, as well as for tracking the dynamic behavior of proteins using pulse labeling and fluorescence recovery after photobleaching. For electron microscopy, cellular and antigenic preservation has been improved with the use of cryofixation and freeze-substitution. The technologies described here advance Tetrahymena cell biology to the cutting-edge of cytological analysis.
自从嗜热四膜虫等大型纤毛虫首次在池塘水中被发现以来，它们优雅的游动姿态和皮层组织的美妙形态吸引了学生和科学家。实际上，细胞学——仅仅观察细胞——是细胞生物学大多数研究领域的重要组成部分，在大型、复杂的四膜虫细胞中尤其引人入胜。对四膜虫进行细胞学分析对于研究微管细胞骨架、膜运输、复杂的核运动和相互作用以及接合过程中的细胞重塑等诸多课题至关重要。我们简要回顾了先前报道的用于光学显微镜和电子显微镜的细胞学技术，并为读者指出了解这些实验方案的资源。接着，我们介绍用于研究这些奇妙细胞的新兴技术。这些技术包括使用荧光蛋白标签在活细胞中定位细胞成分，以及通过脉冲标记和光漂白后的荧光恢复来追踪蛋白质的动态行为。对于电子显微镜，通过冷冻固定和冷冻置换改进了细胞和抗原的保存。这里描述的技术将四膜虫细胞生物学推进到了细胞学分析的前沿。
