Egl and BicD are required for formation of transport-competent dynein-RNA complexes in vitro. (A) Example kymographs (time-distance plots) showing behavior of dynein and oskar RNA in the presence and absence of Egl and BicD; in each condition, dynactin is also present but not fluorescently labeled (filled and empty circles represent the presence and absence of indicated proteins, respectively). Egl and BicD were co-expressed and co-purified (see Materials and methods). (B and C) Charts showing the total number of microtubule (MT) binding events for oskar RNA (B) and number of processive dynein complexes on microtubules under conditions shown in A. In B, values were corrected for non-specific background binding of oskar RNA to the imaging surface as described in Materials and methods. Plots show the mean ± SD of values from 10 individual microtubules (represented by black circles) derived from 183 to 918 single RNA particles (B) or from 384 to 1,725 single dynein particles (C) per condition. Statistical significance and P-values were determined with Mann–Whitney tests. (D) Example kymographs (time-distance plots) showing the behavior of dynein activated by oskar RNA, Egl/BicD, and dynactin or by BicD2N and dynactin in the presence (filled circle) and absence (open circle) of Staufen. Quantification of these data is presented in Fig. 2, H and I.