Single-Molecule Analysis of Intraflagellar Transport in Live Chlamydomonas Cells

Abstract

Eukaryotic cilia and flagella are highly specialized for long-distance cargo transportation. Intraflagellar transport (IFT) in Chlamydomonas flagella requires two proteins: kinesin II motors that transport submembranous protein particles from the flagellar base to the tip by moving along a unipolar array of microtubules, and IFT dynein motors that recycle these particles back to the cell body. To investigate the interactions between microtubule motors and individual IFT cargos, we have attached fluorescent polystyrene beads coated with an antibody for FMG1, a transmembrane glycoprotein. Multicolor tracking of FMG1-beads and GFP-tagged IFT cargos showed that FMG1 is transported by the IFT machinery. The movement of the FMG1-beads was tracked with 1 nm precision at 400 microsecond temporal resolution. We observed the beads taking 8 nm steps in both the retrograde and anterograde directions. Analysis of bead motion also revealed that IFT cargos move unidirectionally from one end of the flagella to the other with infrequent backward steps. Using an optical trap, we have measured the forces that are exerted on the bead by the motor proteins to estimate the number of engaged motors on single IFT cargos. Beads have stalled near 20-30 pN forces in each direction, indicating that there are at least 4-5 active motors involved in the transportation of IFT cargos. Force measurements in a strain that carries a temperature-sensitive mutant of kinesin II showed that heat inactivation of kinesin II did not alter forces in the retrograde direction. Our results suggest that kinesin and dynein motors do not undergo tug-of-war competition with each other in IFT. We favor a model where the regulation of motors is restricted to the base and the tip of the flagellum to provide a continuous stream of IFT cargos along the flagellum.