Quantification of IFT-Dynein Dynamics in C.elegans

Abstract

Cytoplasmic dyneins are the main drivers of microtubule-based retrograde transport in eukaryotic cells. Cytoplasmic dynein 1 plays a role in retrograde intracellular transport and cell division, whereas cytoplasmic dynein 2, also known as IFT-dynein, co-operates with kinesin motors to assemble and maintain cilia in a process called intraflagellar transport (IFT). While cytoplasmic dynein 1 has been the subject of many recent studies, relatively little is known about IFT-dynein. Here, we focus on the mechanism and dynamics of IFT-dynein: how does it behave in vivo at the ensemble and single-molecule level?To this end, we use fluorescence microscopy to visualize labeled IFT-dynein motors in the chemosensory cilia of living C. elegans. Transgene worms were generated using the Mos1-mediated single copy insertion (MosSCI) method to ensure endogenous motor expression levels. Time-lapse fluorescence movies showed that IFT-dynein moves in trains consisting of tens of motor proteins. The movies were processed to kymographs, from which location-dependent velocities and motor numbers were obtained using in-house developed kymograph-analysis software. This analysis revealed that IFT-dynein train velocities and motor numbers are dynamic, changing along the cilium. Double-labeled constructs allowed us to look more closely into motor co-operation, determining the dynein:kinesin ratio at different positions in the cilium.To obtain insight into the behavior of individual motors, we employed photoactivation of PA-GFP-labeled IFT-dynein, which allowed, for the first time, the tracking of individual IFT-dynein motors in vivo. Single-motor trajectories revealed distinct features of IFT-dynein motility: diffusive behavior at the ciliary base, pauses, turns, directed motion and switches between these behaviors. This combined ensemble and single-molecule approach has provided novel quantitative insight into IFT-dynein dynamics in living organisms, shedding light on the complex functioning of dynein motors in general.