Arf6 colocalizes with Cdc42 and regulates Cdc42 dynamics and localization. (A) Colocalization of GFP-Cdc42 (green) and CFP-Arf6 (red) in a migrating astrocyte. Higher magnification images of boxed areas 1 and 2 are shown on the left and right, respectively. CFP-Arf6 and GFP-Cdc42 colocalized (arrowheads) in discrete areas at the leading edge plasma membrane (box 1) and on intracellular vesicles (box 2). (B) Fluorescence intensities of GFP-Cdc42 (green) and CFP-Arf6 (red) measured along the line shown in A (box 1). (C) GFP-Cdc42 recruitment to intracytoplasmic vesicles in astrocytes microinjected with the indicated constructs or nucleofected with the indicated siRNA. (D) Maximum projection of Video 4. (bottom) A higher magnification of the boxed area is shown. (E) Recruitment of GFP-Cdc42 to the leading edge of migrating astrocytes after microinjection with the indicated constructs or nucleofection with the indicated siRNA. (F) GFP-Cdc42 fluorescence images in cells microinjected with the indicated constructs or nucleofected with the indicated siRNA. (right) Higher magnification images of the leading edges (boxed areas) are shown. Dotted lines indicate the direction of the wound. Data are shown as mean ± SEM of three to six independent experiments totalizing >200 cells. Bars, 10 µm.