Analysis of cargo transport by IFT and GFP imaging of IFT in Chlamydomonas

Abstract

Chlamydomonas reinhardtii is the organism in which intraflagellar transport (IFT) was first visualized and in which the composition of IFT particles was originally elucidated. As the universality of IFT among ciliated/flagellated cells was uncovered, the diversity of organisms used to study IFT has grown. Still, because of the ease of isolation of flagella from Chlamydomonas and the battery of temperature-sensitive mutants affecting IFT proteins and motors, this unicellular alga remains the principal model for biochemical studies of IFT motors and cargo; furthermore, the long, exposed flagella of this cell are ideally suited for observing IFT in real time with GFP-tagged components of IFT.

Fig. 1

The vector designed for tagging Chlamydomonas genes with GFP, pHK85, has two copies of the GFP gene separated by two tandem linker sequences. The first or second GFP can be replaced by a cDNA or genomic sequence encoding a protein to be tagged with GFP at either its C- or N-terminus, respectively. Transcription is driven by the PsaD promoter and is terminated by the PsaD 3′ genomic sequence. The plasmid also encodes aphVIII as a selectable marker in Chlamydomonas.

Fig. 2

Visualization of IFT27::GFP in the flagella of Chlamydomonas. (A) IFT27::GFP is visible in IFT trains (arrows) in the two flagella of the cell. A video of the IFT movement of this cell is presented in the supplementary material. (B) Traces of IFT trains are visible as lines on this kymograph of the video shown in the supplement. The downward pointing dotted line shows retrograde IFT and the upward pointed dotted line shows anterograde IFT.