Lechtreck Karl F
Department of Cellular Biology, University of Georgia, Athens, Georgia, USA.
Methods Enzymol. 2013;524:265-84. doi: 10.1016/B978-0-12-397945-2.00015-9.
Intraflagellar transport (IFT) is a specialized intracellular transport which is required for the assembly and maintenance of cilia and eukaryotic flagella. IFT protein particles move bidirectionally along the flagella in the space between the flagellar membrane and the axonemal doublets. The particles consist of more than 20 different polypeptides and are transported by kinesin-2 from the cell body to the flagellar tip and by cytoplasmic dynein back to the cell body. Chlamydomonas reinhardtii is unique in that IFT can be visualized by two distinct microscopic approaches: differential interference contrast (DIC) and tracking of fluorescently tagged IFT proteins. In vivo imaging of IFT is critical to determine, for example, the role of individual proteins in the IFT pathway and how flagellar proteins are transported by IFT. Here, the microscopic requirements and the procedures for the imaging of IFT by DIC and by total internal reflection fluorescence microscopy will be described. Kymograms, graphical representations of spatial position over time, provide a convenient way to analyze in vivo recordings of IFT. In the future, multicolor in vivo imaging of IFT and its cargoes will be used to understand how flagella are assembled, maintained, and repaired.
鞭毛内运输（IFT）是一种特殊的细胞内运输方式，对于纤毛和真核生物鞭毛的组装与维持至关重要。IFT蛋白颗粒在鞭毛膜与轴丝双联微管之间的空间中沿鞭毛双向移动。这些颗粒由20多种不同的多肽组成，由驱动蛋白-2从细胞体运输至鞭毛尖端，并由胞质动力蛋白运回细胞体。莱茵衣藻的独特之处在于，IFT可以通过两种不同的显微镜方法进行观察：微分干涉相差（DIC）和对荧光标记的IFT蛋白进行追踪。例如，对IFT进行体内成像对于确定单个蛋白在IFT途径中的作用以及鞭毛蛋白如何通过IFT进行运输至关重要。在此，将描述通过DIC和全内反射荧光显微镜对IFT进行成像的显微镜要求和操作步骤。波形图是空间位置随时间的图形表示，为分析IFT的体内记录提供了一种便捷的方法。未来，对IFT及其货物进行多色体内成像将用于了解鞭毛是如何组装、维持和修复的。
