Valentine Megan, Yano Junji, Lodh Sukanya, Nabi Ashikun, Deng Bin, Van Houten Judith
State University of New York, Plattsburgh, NY, United States.
University of Vermont, Burlington, VT, United States.
Methods Cell Biol. 2023;175:177-219. doi: 10.1016/bs.mcb.2022.12.003. Epub 2023 Jan 23.
In this chapter we provide some tools to study the ciliary proteins that make it possible for Paramecium cells to swim by beating their cilia. These proteins include many ion channels, accessory proteins, peripheral proteins, structural proteins, rootlets of cilia, and enzymes. Some of these proteins are also found in the soma membrane, but their distinct and critical functions are in the cilia. Paramecium has 4000 or more cilia per cell, giving it an advantage for biochemical studies over cells that have one primarily cilium per cell. Nonetheless, a challenge for studies of many ciliary proteins in Paramecium is their low abundance. We discuss here several strategies to overcome this challenge and other challenges such as working with very large channel proteins. We also include for completeness other techniques that are critical to the study of swimming behavior, such as genetic crosses, recording of swimming patterns, electrical recordings, expression of very large channel proteins, RNA Interference, among others.
在本章中，我们提供了一些工具来研究纤毛蛋白，正是这些蛋白使得草履虫细胞能够通过摆动纤毛来游动。这些蛋白包括许多离子通道、辅助蛋白、外周蛋白、结构蛋白、纤毛的根丝以及酶。其中一些蛋白也存在于体细胞的膜中，但它们独特且关键的功能存在于纤毛中。每个草履虫细胞有4000根或更多的纤毛，这使其在生化研究方面比每个细胞只有一根初级纤毛的细胞更具优势。然而，研究草履虫中许多纤毛蛋白面临的一个挑战是它们的丰度较低。我们在此讨论几种克服这一挑战以及其他挑战（如处理非常大的通道蛋白）的策略。为了全面起见，我们还介绍了对游泳行为研究至关重要的其他技术，如遗传杂交、游泳模式记录、电记录、非常大的通道蛋白的表达、RNA干扰等。
