Single-Molecule Observation of IFT Turnover using a Photobleaching Gate

Abstract

Intraflagellar transport (IFT) is required for the assembly and maintenance of eukaryotic cilia and flagella. In Chlamydomonas reinhardtii, IFT trains are transported by kinesin-2 to the flagellar distal tip and by dynein-1b back to the basal body. Remodeling of IFT trains and motor protein regulation are believed to occur at the flagellar tip complex. Due to the complex and crowded nature of the flagellar turnaround zones, IFT train and motor behavior at the tip remains unclear. To investigate the turnover of a single IFT train or a motor cluster, we introduced a high-power laser gate that continuously photobleaches incoming IFT trains and selectively allows a single unbleached train to enter the flagellum. We observed that anterograde IFT trains pause at tip for ∼4 s on average and split into multiple retrograde trains. In contrast, the KAP subunit of kinesin-2 releases from IFT trains at the tip after a shorter pausing period. Different from C. elegans IFT, in which KAP is recycled as a retrograde cargo, KAP in Chlmaydomonas returns to the basal body by diffusion with an average diffusion constant of 1.2 μm2/s. We also observed a concentration gradient for the KAP-GFP background that increases towards the tips of flagella. We anticipate our photobleaching gate assays to be a starting point to address the detailed mechanism of IFT regulation at turnaround zones.