Supplementary Figure 5: Retrograde trafficking is required for persistent cell migration.

(a) Schematic representation of path persistence: ratio of effective maximum displacement, d, to actual trajectory length, D. (b) Speed distribution of RPE-1 cells migrating at least 50 μm in 2D random migration on fibronectin-coated coverslips (pooled cells from independent experiments are represented in the graph. Number of independent experiments: ctrl siRNA = 6 (n = 456 cells), synt16 siRNA = 3 (n = 58 cells), Rab6 siRNA = 5 (n = 361 cells), and Rab11 siRNA = 4 (n = 447 cells), or in 1D migration on fibronectin-coated micropatterned lines (pooled cells from independent experiments are represented in the graph. Number of independent experiments: ctrl siRNA = 5 (n = 836 cells), synt16 siRNA = 3 (n = 552 cells), Rab6 siRNA = 3 (n = 218 cells), and Rab11 siRNA = 2 (n = 507 cells). 10 representative trajectories for the 1D migration condition are shown (17 h of migration). Note the loss of track linearity for the syntaxin 16 and Rab6 depletion conditions. (c) RPE-1 cells were plated on fibronectin-coated non-micropatterned surface (culture dishes), or on fibronectin-coated line micropatterns before be fixed, permeabilized and immunostained with mAb13. Insets show the Golgi compartment. Scale bar = 10 μm. (d) Transmission microscopy images representing the edge progression in wound healing assays with RPE-1 cells Scale bar = 40 μm. Wound edge velocity is quantified. 1 representative of 3 independent experiments for ctrl siRNA and Rab6 siRNA and 2 independent experiments for synt16 siRNA are shown. ×10 objective fields of 2 wounds were quantified: ctrl siRNA n = 18 fields, synt16 siRNA n = 12 fields, Rab6-siRNA n = 13 fields, mean ± s.d.). (e) Box-and-Whiskers plots representing the migratory persistence of edge cells in wound healing assays. 1 representative of 3 independent experiments for ctrl siRNA, and 2 independent experiments for synt16 siRNA and Rab6 siRNA is shown. (ctrl siRNA n = 47 cells, synt16 siRNA n = 49 cells, Rab6 siRNA n = 50 cells). 10 representative trajectories are shown to the right. (f) Golgi polarization towards the wound, derived as the angle between the wound migration direction (defined as a line perpendicular to the wound, passing via the center of the nucleus) and the position of the Golgi, visualized by GFP-Rab1. 1 experiment. (g) Non-ligand-bound (mAb13) and ligand-bound (12G10) β₁ integrin labeling of wound edge cells. For (d), data represent means ± s.d., for (b, e), two-tailed Mann-Whitney and for (d) unpaired t-test. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001. The ends of the Whiskers are set at 1.5× Interquartile Range above the third quartile, and 1.5× Interquartile Range below the first quartile.