FIGURE 2:

MYO10 can be harnessed to traffic bait–prey complexes in live cells. Visualization of bait and prey molecule trafficking in live HeLa cells by time-lapse TIRFM. Imaging was performed at 37°C. Fluorescence images have been uniformly inverted. (A) In HeLa cells expressing MYO10-MYO7A(TAIL)^BAIT (left) and MYRIP^PREY (right), both molecules accumulate at filopodial tips. A single frame from the time-lapse is shown. Filopodia selected for kymographs are indicated by red arrows. (B) Kymographs from A reveal molecular trafficking along filopodia. Bait and prey molecules accumulate at the filopodial tips, which are visible as a dark vertical line. Dim puncta of MYO10-MYO7A(TAIL)^BAIT and MYRIP^PREY move synchronously toward the filopodial tip (anterograde, arrowheads). Retrograde flow of bait and prey molecules (toward the cell body) is also observed. (C) MYO10^{NO BAIT} accumulates at filopodial tips but does not traffic MYRIP^PREY molecules. This control experiment demonstrates that trafficking of MYRIP^PREY is critically dependent on the MYO7A(TAIL) moiety being fused to the myosin motor. (D) Kymographs from C confirm that MYRIP^PREY molecules are not trafficked by MYO10^{NO BAIT}. Anterograde (arrowheads) trafficking of MYO10^{NO BAIT} is observed. Filopodia 3 and 4 are indicated by arrows in C. Horizontal scale bars: 10 µm (A and C); 500 nm (B and D); vertical scale bars: 30 s (B and D), with the bottom of each kymograph representing 0 s.