Figure 2. Characterisation of the motile properties of h^WT RNPs.

(A) Mean square displacement (MSD) of h^WT RNP trajectories as a function of time plotted in a log–log format. Mean slopes (± SEM) were calculated from a linear fit to the data. Plot of MSD vs time on non-logarithmic axes is shown in Figure 2—figure supplement 1A,B. ATP, 2.5 mM ATP; ATP-vanadate, 2.5 mM ATP plus 100 μM vanadate. N, number of RNPs analysed. See Figure 2—figure supplement 1C for slopes of log–log MSD(t) for individual RNPs in each population. (B–E) Correlation analysis of mean run length and run velocity for the bidirectional subset of h^WT RNPs. Strong correlations exist for individual RNPs between (B) mean minus end and mean plus end run length, (C) mean minus end and mean plus end velocity, (D) mean minus end run length and mean minus end velocity, and (E) mean plus end run length and mean plus end velocity. Only bidirectional RNPs with ≥20 runs in total were used for these analyses (note that no such cut-off was applied for the analysis in H and I). R², correlation coefficient; N, number of RNPs analysed; m, slope. There is no significant bias in minus end vs plus end motile properties in B and C (red line represents slope expected for no bias). (F) Mean number of h^WT RNPs bound per μm of microtubule per movie in the presence of 2.5 mM ATP, 20 U·ml⁻¹ apyrase and 2.5 mM ATP plus 100 μM vanadate (ATP-vanadate). Means were calculate from values for 12 microtubules selected at random in at least three imaging chambers. (G) Mean percentage of microtubule-associated h^WT RNPs that were unidirectional, bidirectional, or stationary. Means were calculated from 12 microtubules as in F. (H and I) Mean run lengths (H) and velocities (I) of individual runs of bidirectional h^WT RNPs. N, number of individual runs of RNPs (number of RNPs from which the individual runs were extracted was 40 for ATP and 20 for ATP-vanadate). See Figure 2—figure supplement 2C,D for distribution of run lengths and velocities. Errors represent SEM in all panels. In F–I, ***p<0.001; **p<0.01; *p<0.05, compared to the ATP condition for the same parameter (Mann–Whitney non-parametric t test). Images for all analyses in the figure were acquired at 15 fps.

DOI: http://dx.doi.org/10.7554/eLife.01596.007

Figure 2—figure supplement 1. Supplementary MSD analysis of h^WT RNP trajectories.

(A and B) Plots of average mean square displacement (MSD) vs time for h^WT RNPs. See Figure 2A for data plotted on logarithmic axes. (A) For unidirectional RNPs there is a quadratic relationship of MSD with time, indicating a deterministic transport process. (B) For bidirectional RNPs there is a linear relationship of MSD with time, indicative of a diffusive process. The linear relationship held over all time scales analysed; for example, MSD (t) also appeared linear between 0 and 0.3 s (data not shown). MSD (t) is similar in the presence of both ATP and ATP-vanadate. D, diffusion coefficients of bidirectional RNPs on the microtubule (calculated from the slopes of the linear fits in B using MSD = 2Dt). In A and B, MSD traces were obtained by internal averaging, with the time interval corresponding to one quarter of the total duration of the shortest-lived RNP trajectory analysed (Saxton, 1997). N, number of RNPs analysed. (C) Scatter plot showing the slope of the log–log plot of MSD vs time for individual RNPs obtained by internal averaging, with the time interval for each RNP corresponding to one quarter of the total duration of its trajectory (Saxton, 1997). Long and short horizontal red lines demarcate the mean and SEM, respectively. Slopes of ∼1.0 or ∼2.0 are expected for diffusive motion and active transport, respectively. We do not find a significant subset of bidirectional RNPs with a slope of ∼1.5 (such a value has been interpreted as indicative of diffusive motion interspersed with bouts of processive movement [Hendricks et al., 2010; Sanchez et al., 2012]). Images for all MSD analyses were acquired at 15 fps.

DOI: http://dx.doi.org/10.7554/eLife.01596.008

Figure 2—figure supplement 2. Distributions of run lengths and velocities of unidirectional and bidirectional h^WT RNPs.

(A and B) Distribution of lengths (A) and velocities (B) of individual runs of unidirectional minus end-directed h^WT RNPs. Note that no plus end-directed unidirectional runs were observed. N, number of runs (from 25 RNPs [many RNPs have more than one run due to interruptions of bouts of minus end-directed motility by short-lived pauses]). (C and D) Distribution of lengths (C) and velocities (D) of individual runs of bidirectional h^WT RNPs in the minus end or plus end direction in the presence of 2.5 mM ATP or 2.5 mM ATP plus 100 μM vanadate (ATP-vanadate). N, number of runs (from 40 RNPs for ATP and 25 RNPs for ATP-vanadate). Note that due to the relative paucity of long or fast runs, those runs >500 nm or >2000 nm·s⁻¹ were binned together in these plots. The maximum run lengths were: minus end, 2190 nm; plus end, 2008 nm. The maximum run velocities were: minus end, 5270 nm·s⁻¹; plus end, 5800 nm·s⁻¹. There was a general tendency for individual runs that were long to also be fast (data not shown).

DOI: http://dx.doi.org/10.7554/eLife.01596.009