Fig. 2. Inhibition of actin polymerization and deletion of mTuba domains disrupts dorsal ruffle formation. (A) Co-expression of Cherry-SH3 and GFP-actin in B16 cells. Deletion of the actin-regulatory protein-binding domain of mTuba results in cytoplasmic distribution of mTuba with no visible puncta formation. Actin organization is also disrupted. Box indicates region of movie used to generate kymographs. Kymographs confirm cytoplasmic distribution of the mutant. (B) Co-expression of Cherry-GEF and GFP-actin in B16 cells. Deletion of the GEF domain of mTuba results in puncta formation but no ruffling. Box indicates region of movie used to generate kymographs. Kymographs show near-stationary puncta. (C) Co-expression of Cherry-BAR and GFP-actin in B16 cells. Deletion of the BAR domain of mTuba causes extensive peripheral ruffling but no dorsal ruffling. Box indicates region of movie used to generate kymographs. Kymographs show rapid peripheral ruffling activity. (D) B16-F1 cells co-transfected with GFP-mTuba and mRFP-actin and treated with 50 nM cytochalasin D (CD). Image shown is following CD treatment. Boxed area indicates region used to generate kymographs. Average speed of puncta following CD treatment is 0.03±0.19 microns/minute (n=6). Actin polymerization is inhibited, but mTuba remains associated with the actin cytoskeleton at `patches'. Kymographs are derived from the full movie with CD addition at five minutes. Dotted line indicates time of CD addition. Note that, following CD treatment, mTuba puncta fail to move. (E) Percentage of total area of mTuba (green) and actin (red) that coexist during ruffle formation in (D) (see Materials and Methods). Plots show that the percentage of actin overlapping with mTuba puncta increases following CD treatment at actin `patches'. Bars, 5 µm.