Ensemble and single-molecule dynamics of IFT dynein in Caenorhabditis elegans cilia

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.

Figure 1. Characterization of IFT-dynein ensemble dynamics.

(a) Representative XBX-1::EGFP kymograph showing retrograde (green) and anterograde (magenta) motility. Time: vertical; scale bar, 2 s. Position: horizontal; scale bar, 2 μm. (b) Train-averaged retrograde IFT-dynein velocity profile. (c) Train-averaged anterograde IFT-dynein velocity profile. (d) Train-averaged retrograde (green) and anterograde (magenta) XBX-1 train intensity. (e,f) Representative intensity versus time profiles of anterograde (e) and retrograde (f) motility components in (a) (location indicated by dotted vertical line). Black numbers are peaks identified using automated peak detection. (g) Motor flux is calculated by multiplying the number of motors (area under the curve of the position-dependent fluorescence intensity over time) by the position-dependent velocity. Shown is XBX-1 retrograde (green) and anterograde (magenta) flux in the cilium, n= 30 worms, (b–d) averaged over 529 trains obtained from 30 worms (retrograde) and 425 trains from 30 worms (anterograde). Line thickness represents s.e.m.; dotted line represents s.d. See also Supplementary Fig. 1.

Figure 2. Directionality is not modulated by the dynein/kinesin-2 ratio.

(a) Representative Fourier-filtered anterograde kymographs of XBX-1::EGFP (channel 1, M1), OSM-3::mCherry (channel 2, M2) and a randomly chosen control kymograph used for Manders' colocalization analysis. Time: vertical; scale bar, 2 s. Position: horizontal; scale bar, 2 μm. (b) Time-averaged fluorescence images of dual-colour strains XBX-1::EGFP OSM-3::mCherry, XBX-1::EGFP KAP-1::mCherry and XBX-1::EGFP OSM-3::mCherry kap-1. Scale bar, 2 μm. (c) The dynein/kinesin-2 ratio on trains was generated using Fourier-filtered kymographs from dual-colour constructs for each direction of movement (see also Supplementary Fig. 2). A line is drawn along the same train of interest in both channels and corrected for background, photobleaching, bleed-through and excitation intensity. Division of the two corrected position-dependent intensities gives the XBX-1/OSM-3 ratio (n=95 trains, 24 worms), XBX-1/KAP-1 ratio (n=67 trains from 20 worms) and XBX-1/OSM-3 kap-1 ratio (n=56 trains from 14 worms). The XBX-1/OSM-3 ratio was taken in the distal segment region (4.0–7.5 μm) and the XBX-1/KAP-1 ratio in the proximal segment region (0–3.5 μm). Error is s.d. NS, no significant difference, unpaired t-test. See also Supplementary Table 3 and Supplementary Figs 2 and 3.

Figure 3. Single-molecule quantification reveals novel IFT-dynein behaviour.

(a) Quantification of single-motor IFT-dynein trajectories: 37% anterograde motion (magenta), of which 14% with pausing (light magenta); 27% retrograde motion (green), of which 13% with pausing (light green); 16% at the base (blue); 12% stationary (yellow); 8% turnarounds (purple); n=494 trajectories from 7 worms. Only trajectories of 600 ms or longer are taken into account. (b–f) Representative single-motor IFT-dynein traces showing: pausing (light green) in unidirectional trajectories (b), deceleration upon reaching the ciliary base (c), diffusive behaviour at the ciliary base (d) and A-to-R (anterograde to retrograde) directional switches at different positions with corresponding images and kymograph (e,f). (e,f) Time: vertical; scale bar, 2 s. Position: horizontal; scale bar, 2 μm. Arrow indicates turnaround position. The movie corresponding to (f) is shown in Supplementary Movie 2. (g,h) Histograms of IFT-dynein A-to-R turnarounds (n=34) showing their location and pausing time. R-to-A not shown (only four recorded trajectories).

Figure 4. Stochastic modelling connects dynamics of individual IFT dyneins to ensemble behaviour.

(a, left) Cartoon highlighting stochastic simulations of single IFT dyneins walking and turning around in the cilium. The input parameters of the simulations are indicated. The probability densities of an IFT dynein A-to-R turn (P_AR, 0.14 μm⁻¹) and an R-to-A turn (P_RA, 0.07 μm⁻¹), were obtained experimentally. Pausing time for A-to-R turns (τ_AR, no pause) and R-to-A turns at the base (τ_RA, 500 ms) were estimated. (a, right) Measured (black line) and simulated (dotted line) IFT dynein steady-state distribution in the cilium. In the simulations, we assumed that all IFT-dynein motors in the tip region (shown in grey; not in focus in experimental measurements) turn. (b) Simulated numbers and positions of A-to-R (dotted, black) and R-to-A (dotted, purple) turns. (c) The effect of P_AR and P_RA on IFT-dynein motor distribution in stochastic simulations. Motor distribution in black was obtained using probability parameters calculated from the single-molecule experiments (P_AR=0.14 μm⁻¹, P_RA=0.07 μm⁻¹) and most closely corresponds to the IFT-dynein distribution from ensemble measurements (same as solid line in (a)). Green (P_AR=0.14 μm⁻¹, P_RA=0.14 μm⁻¹), blue (P_AR=0.07 μm⁻¹, P_RA=0.14 μm⁻¹) and red (P_AR=0.28 μm⁻¹, P_RA=0.07 μm⁻¹) are simulated distributions of altered turnaround probabilities. To account for the overlap of two phasmid cilia in the distal segment, intensities and histograms were multiplied by 2−(1/(e^{(x−3,500)/200}+1)). See also Supplementary Fig. 5.