For over 40 years, the humble green alga Chlamydomonas reinhardtii
has carried the banner of flagellar biology. Chlamydomonas
possesses a wealth of flagellar mutants, which have greatly informed our understanding of these organelles. The cell's two flagella (identical to cilia in every way but their name) are readily purified for biochemical studies (King, 1995
) and are amenable to numerous experimental perturbations, including regeneration after amputation (Rosenbaum et al., 1969
; Lefebvre, 1995
). However, one of this little alga's greatest contributions to flagellar biology was the discovery and characterization of intraflagellar transport (IFT).
IFT is indispensable for the assembly and maintenance of eukaryotic flagella. The only way for axonemal precursors to reach the site of flagellar assembly at the flagellar tip is to be carried there by large IFT particles, powered by the anterograde motor heterotrimeric kinesin-2. At the tip, IFT particles are remodeled and loaded with axonemal turnover products for their return trip to the cell body, driven by the retrograde motor cytoplasmic dynein-1b. The IFT machinery is highly evolutionarily conserved and many Chlamydomonas
genes that encode IFT proteins are homologous to human disease genes (Pazour et al., 2000
IFT is a live-cell microscopy-defined phenomenon. Though IFT particles (originally dubbed “rafts”) had been observed in electron micrographs for decades, their function was not understood until IFT was visualized by DIC microscopy (Kozminski et al., 1993
). Subsequent biochemisty studies have revealed much about the composition (Piperno and Mead, 1997
; Cole et al., 1998
) of IFT particles, as well as their interactions with specific axonemal precursors (Qin et al., 2004
; Hou et al., 2007
). In addition, cryo-electron tomography studies have produced insights into the modular 3D architecture of IFT particles (Pigino et al., 2007
). However, since IFT is a description of the movement of proteins, live-cell microscopy remains an invaluable technique for understanding this important cellular behavior. In this paper, we briefly review the history of transmitted light and fluorescence microscopy in Chlamydomonas
and then detail the promising new application of TIRF microscopy to studying IFT and other dynamic flagellar processes.