GFP-Cdc42 localizes to plasma membrane and intracellular vesicles of migrating cells. (A) GFP-Cdc42 (top left) and GFP (top right) fluorescence in migrating primary astrocytes. (bottom left) Higher magnification view of the boxed region shows accumulation of GFP-Cdc42 at the leading edge. (bottom right) Fluorescence intensity of GFP-Cdc42 (blue) measured along three lines (as indicated) drawn across the leading edge (blue) or side edge (green) is shown. Fluorescence intensity of GFP measured along three lines drawn across the leading edge of the GFP-expressing cell is shown in red. Cell–cell contact areas with increased Cdc42 intensity have been disregarded. (B) GFP-Cdc42 fluorescence in a nonmigrating confluent astrocyte (left) and image showing the maximum projection of Video 3 (right). (C) Images showing the maximum projection of Video 2. A higher magnification of the boxed area is shown on the right. Directed movement of small Cdc42-positive vesicles appears as white lines along the length of the protrusion. Large immobile Cdc42-positive vesicles are indicated with asterisks. (D) Still images from Video 2 showing the retrograde (black arrow) and anterograde movement (white arrows) of Cdc42-positive vesicles in the cell protrusion. (E) GM130 staining (red) in GFP-Cdc42 (green)–expressing cells. (F) EEA1 staining (red) in GFP-Cdc42 (green)–expressing cells showing the endosomal EEA1 marker on Cdc42-positive vesicles (arrowheads). Images are representative of at least 100 cells from three independent experiments. The dashed line indicates the direction of the wound. Bars, 10 µm.