Dentler William, Vanderwaal Kristyn, Porter Mary E
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.
Methods Cell Biol. 2009;93:145-55. doi: 10.1016/S0091-679X(08)93008-9. Epub 2009 Dec 4.
The transport of materials to and from the cell body and tips of eukaryotic flagella and cilia is carried out by a process called intraflagellar transport, or IFT. This process is essential for the assembly and maintenance of cilia and flagella: in the absence of IFT, cilia cannot assemble and, if IFT is arrested in ciliated cells, the cilia disassemble. The major IFT complex proteins and the major motor proteins, kinesin-2 and osm-3 (which transport particles from the cell body to ciliary tips) and cytoplasmic dynein 1b (which transports particles from ciliary tips to the cell body) have been identified. However, we have little understanding of the structure of the IFT particles, the cargo that these particles carry, how cargo is loaded and unloaded from the particles, or how the motor proteins are regulated. The focus of this chapter is to provide methods to observe and quantify the movements of IFT particles in Chlamydomonas flagella. IFT movements can be visualized in paralyzed or partially arrested flagella using either differential interference contrast (IFT) microscopy or, in cells with fluorescently tagged IFT components, with fluorescence microscopy. Methods for recording IFT movements and analyzing movements using kymograms are described.
物质在真核生物鞭毛和纤毛的细胞体与末端之间的运输是通过一种称为鞭毛内运输（IFT）的过程来完成的。这个过程对于纤毛和鞭毛的组装与维持至关重要：在没有IFT的情况下，纤毛无法组装，并且如果在有纤毛的细胞中IFT被阻断，纤毛就会解体。主要的IFT复合蛋白以及主要的驱动蛋白，即驱动蛋白-2和osm-3（它们将颗粒从细胞体运输到纤毛末端）和细胞质动力蛋白1b（它将颗粒从纤毛末端运输到细胞体）已被确定。然而，我们对IFT颗粒的结构、这些颗粒所携带的货物、货物如何从颗粒上装卸，或者驱动蛋白如何被调控了解甚少。本章的重点是提供观察和量化衣藻鞭毛中IFT颗粒运动的方法。IFT运动可以在瘫痪或部分受阻的鞭毛中通过微分干涉相差（IFT）显微镜观察到，或者在具有荧光标记的IFT成分的细胞中通过荧光显微镜观察到。文中描述了记录IFT运动以及使用波形图分析运动的方法。
