Fig. 2. Reconstitution of dynein-dependent transport of AgDD aggregates in Xenopus laevis egg extract (XE).

A Schematic and wide-field images of the aggregate transport assay. Recombinant AgDD (green) was induced to aggregate in XE and 1:20 diluted into a working XE containing Alexa647-labeled microtubule asters (magenta) and imaged in a 1 cm × 1 cm × 20 μm customized chamber once per minute for 30 min at 18 °C. Experiments were performed 10 times independently with similar results. B Example trajectories of AgDD aggregates. Measurement was performed as in (A), with or without 40 μg/mL CC1 to inhibit dynein-dynactin in XE. Aggregates were randomly selected and colored by diameter. The MTOC was marked with an empty red circle at the center. Among the 100 trajectories from the untreated group, Trajectories of the 10 largest and the 10 smallest aggregates are plotted in (C) (mean diameter ± standard deviation in legend), with the averaged trajectories shown as thick lines. D The relationship between the time-averaged transport velocity and diameter of individual aggregates. The average velocity of individual aggregates was calculated as the total travel distance towards the MTOC divided by the trajectory’s duration (illustrated in (E)). The velocities of individual aggregates were grouped by their diameter in the violin plot. Right: a zoom-in plot showing the mean ± SEM within each group. A total of 1152 (n) aggregates around 16 microtubule asters from 2 batches of XE were included in the analysis. Source data are provided as a Source data file. E An example trajectory of AgDD aggregate to illustrate the result of trajectory segmentation. “Pause” and “Transport Engaged (TE)” segments were colored in pink and green respectively. F The relationship between aggregate diameter and lengths of the pause and the TE segments, shown as mean ± SEM within each size group. 851 (n) trajectories from the experiment in (D) that are longer than 15 min were selected and segmented as illustrated in (E). TE (+): transport-engaged segment when the aggregate moves towards the MTOC; TE (-): transport-engaged segment when the aggregate moves away from the MTOC. G The relationship between the aggregate diameter and the proportion of the pause segment, determined using the data in (F). The plot shows the mean proportion ± SEM within each size group. H Aggregates’ diffusion constants during pauses, with or without 40 μg/mL CC1 in XE. Indicated numbers (n) of aggregates that could be tracked for at least 15 min were selected and grouped by their diameters. Diffusion constants during pause were calculated as described in the methods and plotted against the inverse of the mean diameter within each size group. The dashed line represents the prediction by the Stokes-Einstein law as $k_{B}T/\left(3\pi \eta d\right)$, where d is the aggregate diameter, $\eta$ is the dynamic viscosity of XE (0.01 Pa·s), $k_B$ is the Boltzman constant, and $T$ is 291 Kelvin. Data are presented as mean values ± SEM.