Cadherin-2 particles include stationary and highly motile pools (related to Fig. 6 ). (A) Mean speeds of all Cadherin-2–positive particles indicate that a large pool is nonmotile, moving either antero- or retrogradely at <0.2 µm/s, with the remaining particles moving at higher speeds in both directions. Interfering with either vesicle formation or MT motors reduces the fast-moving pool and increases the stationary fraction. n = 682 tracks for drug control, n = 711 tracks for Brefeldin A, n = 287 tracks for Dynasore, n = 416 tracks for injection control, n = 308 tracks for p50-expressing embryos, n = 593 tracks for Kif5C-expressing embryos, n = 441 tracks Kif5C T94N-expressing embryos, n = 373 tracks for Rab11a-expressing embryos, and n = 152 tracks for Rab11a S25N-expressing embryos. (B) Similarly, maximal velocities of Cadherin-2 particles are decreased when motor transport is impaired. For full statistical testing, see Table S5 and Table S6. Anterograde: n = 658 tracks for drug control, n = 690 tracks for Brefeldin A, n = 283 tracks for Dynasore, n = 399 tracks for injection control, n = 295 tracks for p50-expressing embryos, n = 579 tracks for Kif5C-expressing embryos, n = 425 tracks for Kif5C T94N-expressing embryos, n = 367 tracks for Rab11a-expressing embryos, n = 150 tracks for Rab11a S25N-expressing embryos. Retrograde: n = 656 tracks for drug control, n = 696 tracks for Brefeldin A, n = 280 tracks for Dynasore, n = 411 tracks for injection control, n = 299 tracks for p50-expressing embryos, n = 580 tracks for Kif5C-expressing embryos, n = 432 tracks for Kif5C T94N-expressing embryos, n = 364 tracks for Rab11a-expressing embryos, and n = 148 tracks for Rab11a S25N-expressing embryos. (C) The modified PST-1P activation protocol is able to deplete MTs. White box indicates PST-1P activation area, which is magnified in the lower row. MHB, solid line; URL, dotted line. Elapsed time in seconds. Scale bar, 10 µm. (D) Kymographs demonstrate that the motility of Cadherin-2 reporter particles is reduced when MTs are depleted. Horizontal scale bars, 5 µm; vertical bars, 20 s. (E) Plotting mean speeds against the respective maximal velocities for each track reveals that, upon MT depletion, many tracks cluster in the low mean-low maximal velocity area. Area of 0.8 density given in the top left corner and number of tracks in the bottom left corner. (F and G) Mean velocities (n = 315 tracks for red light only, control region PST-1P OFF and n = 191 tracks for UV-activated, MT-reduced region PST-1P ON; F) and maximal velocities of Cadherin-2 particles are reduced in the anterograde direction upon MT depletion (G). (anterograde: n = 306 tracks for PST-1P OFF, n = 187 tracks for PST-1P ON, P = 3.51 × 10−4; retrograde: n = 310 tracks for PST-1P OFF, n = 189 tracks for PST-1P ON). (H and I) Pause times (H; n = 315 tracks for PST-1P OFF, n = 191 tracks for PST-1P ON) and instantaneous velocities (I; anterograde: n = 1,751 values for PST-1P OFF, n = 1,000 values for PST-1P ON, P = 0.004; retrograde: n = 1,677 values for PST-1P OFF, n = 1,015 values for PST-1P ON, P = 0.006) show a trend toward lower speed motility/longer stationary times when MTs are depleted. Boxes in graphs represent 25–75% of all values and whiskers 1.5 times the quartile. Median is shown as a horizontal bar and mean as a square box. Significance level in Kruskal–Wallis ANOVA: **, P < 0.01; ***, P < 0.001; ns, not significant. All data shown in A, B, and E–I represent n = 13 embryos for drug control, n = 19 embryos for Brefeldin A, n = 8 embryos for Dynasore, n = 13 embryos for injection control, n = 14 p50-expressing embryos, n = 11 Kif5C-expressing embryos, n = 16 Kif5C T94N-expressing embryos, n = 16 Rab11a-expressing embryos, n = 7 Rab11a S25N-expressing embryos, n = 13 for PST-1P nonactivated (OFF) embryos, and n = 12 for PST-1P activated (ON) embryos.
