Abstract
Cytoplasmic dyneins drive microtubule-based, minus-end directed transport in eukaryotic cells. Whereas cytoplasmic dynein 1 has been widely studied, IFT dynein has received far less attention. Here, we use fluorescence microscopy of labelled motors in living Caenorhabditis elegans to investigate IFT-dynein motility at the ensemble and single-molecule level. We find that while the kinesin composition of motor ensembles varies along the track, the amount of dynein remains relatively constant. Remarkably, this does not result in directionality changes of cargo along the track, as has been reported for other opposite-polarity, tug-of-war motility systems. At the single-molecule level, IFT-dynein trajectories reveal unexpected dynamics, including diffusion at the base, and pausing and directional switches along the cilium. Stochastic simulations show that the ensemble IFT-dynein distribution depends upon the probability of single-motor directional switches. Our results provide quantitative insight into IFT-dynein dynamics in vivo, shedding light on the complex functioning of dynein motors in general.
Highlights
Cytoplasmic dyneins drive microtubule-based, minus-end directed transport in eukaryotic cells
To image in vivo intraflagellar transport (IFT)-dynein motor dynamics, we generated C. elegans strains with enhanced green fluorescent protein (EGFP) attached to the IFT-dynein light intermediate chain XBX-1
We find that retrograde and anterograde XBX-1 fluxes are very similar (Fig. 1g), implying that the amount of IFT dynein motors in the cilium is constant in time and that there is no substantial loss of IFT dynein at the ciliary tip
Summary
Cytoplasmic dyneins drive microtubule-based, minus-end directed transport in eukaryotic cells. We use fluorescence microscopy of labelled motors in living Caenorhabditis elegans to investigate IFT-dynein motility at the ensemble and single-molecule level. At the single-molecule level, IFT-dynein trajectories reveal unexpected dynamics, including diffusion at the base, and pausing and directional switches along the cilium. Primary cilia project from the surface of most eukaryotic cells and have important roles in sensory perception and signalling[16] They are built and maintained by the cooperative action of IFT-dynein and kinesin-2 motors in a bidirectional transport process along the axoneme called IFT17–19. Single-motor trajectories reveal unexpected, distinct features of IFT-dynein motility: diffusive behaviour at the ciliary base, pauses, turns, directed motion and switches between these behaviours. Stochastic simulations allow linking this singlemolecule behaviour to the ensemble IFT-dynein distribution along the cilium, revealing that this distribution crucially depends upon the probability of single-motor directional switches
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