Dishinger John F, Kee H Lynn, Verhey Kristen J
Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, USA.
Methods Enzymol. 2013;524:75-89. doi: 10.1016/B978-0-12-397945-2.00005-6.
Cilia and flagella are microtubule-based organelles that play important roles in human health by contributing to cellular motility as well as sensing and responding to environmental cues. Defects in cilia formation and function cause a broad class of human genetic diseases called ciliopathies. To carry out their specialized functions, cilia contain a unique complement of proteins that must be imported into the ciliary compartment. In this chapter, we describe methods to measure the permeability barrier of the ciliary gate by microinjection of fluorescent proteins and dextrans of different sizes into ciliated cells. We also describe a fluorescence recovery after photobleaching assay to measure the entry of ciliary proteins into the ciliary compartment. These assays can be used to determine the molecular mechanisms that regulate the formation and function of cilia in mammalian cells.
纤毛和鞭毛是以微管为基础的细胞器，通过促进细胞运动以及感知和响应环境信号，在人类健康中发挥重要作用。纤毛形成和功能的缺陷会导致一大类被称为纤毛病的人类遗传疾病。为了执行其特定功能，纤毛包含必须导入纤毛区室的独特蛋白质组合。在本章中，我们描述了通过将不同大小的荧光蛋白和葡聚糖显微注射到纤毛细胞中来测量纤毛门通透性屏障的方法。我们还描述了一种光漂白后荧光恢复测定法，以测量纤毛蛋白进入纤毛区室的情况。这些测定法可用于确定调节哺乳动物细胞中纤毛形成和功能的分子机制。
