CDC42u, but not CDC42b, controls cell polarization and directed and persistent migration of astrocytes. (A) Representative trajectories of astrocytes transfected with siRNAs against the ubiquitous (si-u1), the brain (si-b1), or both (si-both) CDC42 isoforms and migrating in an in vitro wound healing assay for 16 h. (B and C) Directionality (B) and directional persistence (C) of astrocytes transfected with the indicated siRNA and migrating in a scratch wound assay. (D) Immunofluorescence images of wound-edge astrocytes transfected with the indicated siRNA and fixed 8 h after wounding. Images show microtubules (anti-tubulin, white), cis-Golgi (anti-GM130, green), centrosome (anti-pericentrine, red), and the nucleus (DAPI, blue). (E) Quantification of Golgi orientation in astrocytes transfected with the indicated siRNA, 8 h after wounding. The red line indicates the values expected for random positioning of the Golgi apparatus. (F) Fluorescence images showing PKCζ localization in wound-edge astrocytes transfected with the indicated siRNA. (G) Percentage of wound-edge cells showing PKCζ accumulation at the cell front. (H) Quantification of Golgi reorientation in a rescue experiment using astrocytes transfected with control siRNA or with a siRNA targeting both CDC42 isoforms together with the indicated control (GFP) or GFP-tagged CDC42 constructs. Graphs show data presented as means ± SEM of 5 (C), 4 (E and G), and 3 (H) independent experiments. At least 250 cells (A–C) and 150 cells (D–H) were analyzed per condition. Ctl: Control cells transfected with non-relevant siRNA. P values were calculated using two-sided unpaired Student’s t test. Scale bars: 10 µm.