Figure 4. The HLE alone promotes unidirectional motion.

(A) Stepwise GFP photobleaching analysis of RNPs assembled on h^WT or HLE RNAs; inset, schematic of HLE RNA that was fused to the aptamer sequence. Note the significant decrease in the relative copy number of GFP::Dlic on the HLE compared to h^WT. N, number of photobleaching traces analysed. See Figure 4—figure supplement 1A for distribution of values. (B) Mean proportion of motile h^WT or HLE RNPs that are unidirectional per imaging chamber. N, number of chambers analysed; n, total number of RNPs. (C) Mean run length per RNP of motile h^WT or HLE RNPs. N, number of RNPs analysed. (D and E) Mean length (D) and velocity (E) of individual runs of unidirectional RNPs. N, number of individual runs of RNPs (from 25 RNPs each for h^WT and HLE). (F and G) Mean length (F) and velocity (G) of individual runs of bidirectional RNPs (from 40 and 20 RNPs for h^WT and HLE, respectively). Images for all analyses in the figure were acquired at a lower frame rate of 4.2 fps; this is because the small number of Cy3 dyes that could be incorporated into the short HLE RNA necessitated imaging with a higher exposure time. Note that the measured mean run lengths of unidirectional and bidirectional RNPs of the same species are higher at 4.2 fps than at 15 fps (Figure 3F,H), presumably due to short pauses and frequent reversals (in the case of bidirectional RNPs) being missed at the lower frame rate. Velocity of measured runs of bidirectional RNPs of the same species is lower at 4.2 fps than at 15 fps, presumably for the same reason, whereas velocity of unidirectional runs is not significantly affected by the different frame rate as short pauses have little affect on the velocity measured for such long runs (Figure 3G,I). ***p<0.001; **p<0.01; *p<0.05 (Mann Whitney non-parametric t test), compared to h^WT values for the same parameter; error bars represent SEM.

DOI: http://dx.doi.org/10.7554/eLife.01596.013

Figure 4—figure supplement 1. Supplemental data on the HLE’s recruitment of dynein and motile properties.

(A) Distributions and means (± SEM) of number of GFP decay steps for GFP::Dlic associated with HLE RNPs, compared to data for h^WT RNPs that were acquired in experiments performed in parallel. ***p<0.001, compared to h^WT value (Mann–Whitney non-parametric t test). (B) Table illustrating the calculations used to estimate dynein copy number per RNP based on stepwise photobleaching. (C and D) Distribution of lengths (C) and velocities (D) of individual runs of unidirectional minus end-directed HLE RNPs, compared to those of h^WT RNPs imaged at the same frame rate. 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]). (E and F) Distribution of lengths (E) and velocities (F) of individual runs of bidirectional HLE RNPs in the minus end or plus end direction compared to h^WT RNPs imaged at the same frame rate. N, number of runs (from 40 and 20 RNPs for h^WT and HLE, respectively). Note that due to the relative paucity of long or fast runs, those >800 nm or >1000 nm·s⁻¹ were binned together in these plots. Data were acquired at 4.2 fps for these and other experiments involving the HLE, which alters the measured run lengths and velocities for h^WT RNPs compared to those obtained from 15 fps imaging (see Figure 4 legends for further details).

DOI: http://dx.doi.org/10.7554/eLife.01596.014