FIGURE 7.

Intrafilopodial movements of myosin-X and the effect of FERM domain deletion at the single molecule level. A, fluorescence images at high fluorescence sensitivity. Yellow lines indicate the regions of interest for kymograph. Scale bars are 1 μm. B, sequential images (upper left) and kymograph (upper right and lower) obtained from the yellow line on the left image in A. Yellow arrowheads in the upper left panel indicate the fluorescent spot moving toward the filopodia tip. The lower panel is a magnified image of the region indicated with a red rectangle on the upper panel. The time resolution was 0.1 s. The scale bars in the upper panels are 2 μm. C, photobleaching of GFP-M10-FULL in the filopodia. The left panel shows the typical photobleaching of fluorescent spot in filopodia. Red is two-step bleaching, and blue is one-step bleaching. The right panel shows the histogram of fluorescence intensities averaged for 1 s just after appearing of individual GFP spots (n = 91). The red line in the right panel is the fitting result with f(x) = a₁·exp[−{(x − x₀)/σ}²/2] + a₂·exp[−{(x − 2·x₀)/σ}²/2] (a₁ = 15.1, a₂ = 20.9, x₀ = 58.1, and σ = 22.4). D, kymograph obtained from the yellow line in the right image in A before (upper) and after (lower) the GFP clusters of backward movements were photobleached. The middle panel is a magnified image of the region indicated with a red rectangle on the upper panel. The color of the rectangles corresponds with the color of the arrowheads of the right image in A. The time resolution is 0.3 s. The scale bars in the upper panels are 2 μm. Yellow rectangles in B and D indicate the movement of GFP-M10-FULL toward the filopodial tip. E, histograms of the forward velocity of myosin-X obtained by the kymographs for M10-FULL (left) and M10-ΔFERM (right). Lines indicate fittings of a single Gaussian function with peaks at 578 nm/s for M10-FULL (n = 112) and 583 nm/s for M10-ΔFERM (n = 94) (p = 0.67 by paired t test).