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Fig. 1. mTuba overexpression stimulates dorsal ruffling. (A) Tuba isoforms. Domain structure of full-length Tuba (Tuba) and mini-Tuba (mTuba) are indicated. Tuba possesses four tandem N-terminal SH3 domains (gray boxes), which bind directly to dynamin. The C-terminal SH3 domain binds N-WASP. Schematic representations of mTuba mutants are indicated. (B) Overexpression of GFP-mTuba. B16 cells were transfected with GFP-mTuba and cell lysates were probed for GFP and Tuba using anti-GFP antibody (
-GFP) and affinity-purified anti-Tuba antibody (AP -Tuba), respectively. (C) GFP-mTuba expression wave front tracking. The front of GFP-mTuba puncta was tracked through 30 frames of a time-lapse movie and shows apparent random movement. (D) A single image from a time-lapse movie of B16 cells co-transfected with Cherry-mTuba and GFP. The lower panel represents kymographs constructed from the boxed region from each image. Note movement of mTuba puncta but not GFP. (E) Series of time-lapse images of a B16-F1 cell co-transfected with GFP-mTuba and mRFP-actin (Movie 1, supplementary material). Tuba puncta localize to the trailing edge of the actin ruffle and radiate outwards concomitant with the passage of the ruffle. The rectangle indicates the region of the movie used to generate the kymographs. Average speed of ruffles is 1.32±0.38 microns/minute (n=29). Arrows indicate the leading edge of the ruffle. Arrowheads indicate localization of mTuba puncta to the trailing edge of the ruffle. (F) Percentage of total area of Tuba (green) and actin (red) from (E) that overlap during ruffle formation over time (see Materials and Methods). Plot indicates that approximately 10% of the total Tuba population overlaps actin directly, and that approximately 10% of the actin population overlaps Tuba puncta directly. Bars: D, 5 µm; E, 3 µm.
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