Dynein Dynamics in Vivo: A Single-Molecule View on its Function in Intraflagellar Transport

Abstract

IFT-dynein (cytoplasmic dynein 2) co-operates with kinesin-2 motors to assemble and maintain cilia in a process called intraflagellar transport (IFT). Relatively little is known about this dynein, while the related cytoplasmic dynein 1 has received a lot of attention of late. Here, we study the in vivo IFT-dynein dynamics at the ensemble and single-molecule level.To this end, we use fluorescence microscopy to visualize labeled IFT-dynein motors, expressed at endogenous levels, in the chemosensory cilia of living C. elegans. Fluorescence movies were processed to kymographs, from which location-dependent velocities and motor numbers were obtained using custom kymograph-analysis software. To obtain insight into the behavior of individual motors, we employed photoactivation of PA-GFP-labeled IFT-dynein, which allowed, for the first time, to track individual IFT-dynein motors in vivo.The data revealed that IFT-dynein moves in trains consisting tens of motor proteins. Retrograde trains are smaller but more frequent than anterograde (kinesin-driven) trains. Anterograde and retrograde IFT-dynein flux are equal along cilia, indicating that the cilium is a closed system for dynein. While the kinesin composition of a train varies along the track, the amount of dynein per train remains relatively constant. Remarkably, this does not result in directionality changes along the track, like in reported ‘tug-of-war’ motor systems, suggesting that retrograde and anterograde motor activities are carefully orchestrated in IFT.Single IFT-dynein measurements support the ensemble findings. Trajectories show distinct motility behavior: diffusion at the ciliary base, pauses, turns and directed motion. Pauses in retrograde or anterograde trajectories are never followed by a directional switch. Moreover, retrograde-to-anterograde turn events are rare.This combined ensemble and single-molecule approach has provided new insights into IFT-dynein transport dynamics in living organisms, shedding light on dynein function in general.