IFs and plectin regulate FA localization and dynamics. (A) Fluorescence SIM-3D images of a migrating astrocytes transfected with GFP-vimentin and paxillin-Orange shown in Video 6 (see also Video 7). The orthogonal projections show that vimentin and paxillin are found in the same focal plane. (B) Immunofluorescence images of migrating astrocytes stained for nuclei (cyan) and paxillin (green). The white dotted lines indicate the position of the wound. Insets show enlarged images of FAs at the leading edge and in a central region of the cell. (C) The top left graph shows the mean number of FAs per cell, and the bottom left graph, the mean area of FAs. Adhesions were projected on the nucleus-tip axis (see schematics). The central top graph shows the normalized distance to the nucleus center of each FA. The central bottom graph shows the distribution of adhesions along the nucleus-tip axis. (D) Lifetime of GFP-paxillin–positive adhesions in migrating leader astrocytes (top). Duration time of assembly and disassembly of these adhesions (bottom). (E) Lifetime of GFP-paxillin positive adhesions in migrating astrocytes (left) of the second and third rows. Duration time of assembly and disassembly of these adhesions (right). (F) Immunofluorescence images of astrocytes in the migrating monolayer stained for nuclei (cyan) and paxillin (green). Data are from n = 3 independent experiments. The sample size for each repeat: si ctl 49, 50, 50, si triple IF 50, 50, 50, and si plectin 50, 50, 55 for C; si ctl 26, 40, 40 and si triple IF 26, 40, 40 for D; si ctl 16, 16, 35 and si triple IF 8, 36, 37 for E. n.s., P > 0.05; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. Bars: (orthogonal projections of A) 1 µm; (A and insets of B) 5 µm; (B top and F) 10 µm.