IFs control collective astrocyte migration by regulating traction forces. (A) Phase-contrast images of astrocyte (shown in Video 1) wound healing after 24 h of migration. Black lines represent the initial position (0 h), and white lines show the final position (24 h), of the leading edge. (B) Simplified method for calculating persistence and directionality of migration of a cell with nuclear tracking. For more detailed formulas, see Materials and methods. (C) Graphs of cell velocity, directionality, and persistence measured by manual nuclear tracking of leader cells after 24 h of migration. (D) Simplified protocol for plating cells into PDMS rectangular stamps onto hydrogels. The black dotted arrows show the main directions of migration. The central image shows a monolayer of cells migrating on a hydrogel embedded with fluorescent beads (green dots) with representative tractions (T, blue arrows). The last image represents schematically the components of tractions (T_x and T_y) analyzed with TFM. (E) Phase-contrast images of astrocytes migrating on a 9-kPa collagen-coated polyacrylamide hydrogel at different time points. The white dotted line represents the leading edge, and the arrows show the direction of migration. See also Video 2. (F) Tractions in the x direction (T_x) at indicated time points. See also Video 3. (G) Representative kymographs of total tractions (|T|). (H) Graphs of tractions in the x direction (T_x), mean values (left) and values at the edge of the monolayer (middle) and at the center (right). (I) Graph showing the ratio of the tractions at the edge over tractions at the center, plotted as a function of time of migration. Data are from n = 3 independent experiments. The sample size for each repeat is: si ctl 50, 50, 50; si triple IF 50, 50, 50; si GFAP 50, 50, 50; si vimentin 50, 50, 49; si nestin 50, 50, 50 cells for C; two, one, and four videos of si ctl and two, three, and three videos of si triple IF for E–I. Graphs show mean ± SEM. *, P < 0.05; **, P < 0.01; and ***, P < 0.001. Bars, 50 µm.