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. 2009 Elsevier Inc. All rights reserved.
Bibliographical noteFunding Information:
Studies of IFT in our laboratories have been made possible by support from NIH ( P20 RR016475 to WD) and the National Institutes of Health ( GM55667 to MEP). Kristyn VanderWaal was also supported in part by a predoctoral fellowship from the American Heart Association , Midwest Affiliate ( 0715799Z ), and a Grant-in-Aid ( 20828 ) from the University of Minnesota Graduate School to M.E.P.