Purification and Localization of Intraflagellar Transport Particles and Polypeptides
The growth and maintenance of almost all cilia and flagella are dependent on the proper functioning of the process of intraflagellar transport (IFT). This includes the primary cilia of most human cells that are in the Go phase of the cell cycle. The model system for the study of IFT is the flagella of the biflagellate green alga Chlamydomonas. It is in this organism that IFT was first discovered, and genetic data from a Chlamydomonas mutant first linked the process of IFT to polycystic kidney disease in humans. The information provided in this chapter addresses procedures to purify IFT particles from flagella and localize these particles, and their associated motor proteins, in flagella using light and electron microscopic approaches.
Key wordsFlagella Cilia Intraflagellar transport IFT Motility Immunofluorescence Immunogold EM
Work in the author’s lab is supported by the NSF (MCB 0950402). This support is greatly appreciated.
- 10.Signor D, Wedaman KP, Orozco JT, Dwyer ND, Bargmann CI, Rose LS, Scholey JM (1999) Role of a class DHC1b dynein in retrograde transport of IFT motors and IFT raft particles along cilia, but not dendrites, in chemosensory neurons of living Caenorhabditis elegans. J Cell Biol 147:519–530PubMedCentralCrossRefPubMedGoogle Scholar
- 12.Cole DG, Diener DR, Himelblau AL, Beech PL, Fuster JC, Rosenbaum JL (1998) Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons. J Cell Biol 141:993–1008PubMedCentralCrossRefPubMedGoogle Scholar
- 22.Hutner SH, Provasoli L, Schatz A, Haskins CP (1950) Some approaches to the study of the role of metals in the metabolism of microoganisms. Proc Am Phil Soc 94:152–170Google Scholar