Fig. 3. Cdc42 and Arp3 localize to mTuba-stimulated dorsal ruffles. (A) A single frame taken from a 10 minute movie of B16 cells co-transfected with Cherry-mTuba and GFP-Cdc42 shows co-localization of mTuba and Cdc42 at puncta. Box indicates region of movie used for kymograph construction. Average speed of puncta is 1.52±0.34 microns/minute (n=5). (B) Percentage of total area of mTuba (green) and Cdc42 (red) that coexist during ruffle formation in A. 10% of mTuba co-localizes with Cdc42, and 5-10% of the population of Cdc42 co-localizes with mTuba. (C) B16 cells were co-transfected with Cherry-mTuba and GFP-N17Cdc42. N17Cdc42 generates greater numbers of mTuba puncta relative to cells transfected with Cherry-mTuba alone (arrow indicates formation of ruffle). Boxes indicate regions of movie used for kymograph construction. Note limited mobility of mTuba puncta when co-expressed with GFP-N17Cdc42. (D) A single frame taken from a movie of B16 cells co-transfected with Cherry-mTuba and GFP-Arp3 shows co-localization of Arp2/3 and mTuba at puncta. Box indicates region of movie used for kymograph construction. Speed of puncta is 1.01±0.15 microns/minute (n=6). (E) Percentage of total area of mTuba (green) and Arp3 (red) that coexist during ruffle formation in (D). Approximately 90% of mTuba co-localizes with Arp3, and 85% of the population of Arp3 co-localizes with mTuba. Bars: A,D, 5 µm; C, 15 µm.