Single-Molecule Studies of Intraflagellar Transport in Chlamydomonas
- Author(s): Shih, Sheng-Min
- Advisor(s): Yildiz, Ahmet
- et al.
Intraflagellar transport (IFT) is a universal process that is required for the assembly and maintenance of all eukaryotic cilia and flagella. Defects in IFT are linked to a wide range of human diseases, including polycystic kidney disease and retinal degeneration. IFT has been implicated in sensory and motile ciliary functions, but the mechanisms of this relationship remain unclear.
We used Chlamydomonas flagellar surface motility (FSM) as a model to test whether IFT provides force for gliding of cells across solid surfaces, and whether IFT transports flagellar membrane glycoproteins (FMGs). We show that IFT trains are coupled to FMGs in a Ca2+-dependent manner. IFT trains transiently pause through surface adhesion of their FMG cargos, and dynein-1b motors pull the cell towards the distal tip of the axoneme. Using the IFT-FMG linkage, we performed optical trapping studies to measure the force produced by single IFT trains. The stall force measurements show that each IFT train is transported by at least four active motors. Opposite-polarity motors are reciprocally coordinated to avoid a tug-of-war. Our results demonstrate the mechanism of Chlamydomonas gliding motility and suggest that IFT plays a major role in adhesion-induced ciliary signaling pathways.
To understand the regulation mechanism of IFT, key information needs to be acquired by monitoring IFT remodeling at the turnaround zones. However, the collective behavior of IFT particles poses unique challenges, because individual IFT trains often cannot be resolved at the flagellar tips and bases. We developed a photobleaching gate assay to track single molecules in dense specimens. The assay controls the number of fluorescent particles in a region of interest by photobleaching its boundary. To test this method, we tracked GFP-labeled receptors at a 100 ms temporal resolution on a live cell membrane, even though the average distance between the receptors is less than 50 nm. We directly observed ligand-induced dimerization of epidermal growth factor receptor (EGFR) on a cell membrane.
We applied the photobleaching gate method to IFT in Chlamydomonas. We tracked individual IFT complexes along the length of a cilium and observed their remodeling at the ciliary tip. After transported by kinesin-2 to the flagellar tip, IFT trains split and recombine, and are transported back to the flagellar base bydynein-1b. Kinesin associated protein (KAP), on the other hand, diffuses back to the base after reaching the tip. On the basis of the results above, we propose new models for mixing and remodeling of IFT particles at the flagellar tip, coordination of IFT motors and flagellar length control.