Figure 2: F-actin is nucleated in two separate regions within the leading edge.

In all images, cold colours indicate low fluorescence intensities, and hot colours denote high actin fluorescence intensities. White indicates saturation in the chosen colour scale but does not reflect saturation during acquisition. Scale bars=5 μm. (a) FRAP experiment for a region including the free membrane at the front of a migrating cell expressing GFP-actin. The white box at t=0 s indicates the photobleached region. In all panels, the white dashed line indicates the position of the free front membrane at time t=0 s. (b) Time course of fluorescence recovery in the photobleached region and a region following the free front membrane. Fluorescence recovery was monitored in the bleached region, reflecting actin turnover (red box in a for t=21 s; red line, ‘bleached region’). For comparison, fluorescence increase arising from polymerization against the free membrane was measured in a region of identical size that followed the cell front over time (final position shown by yellow box in a, t=21 s; yellow line, ‘free membrane’). Fluorescence loss due to image acquisition was estimated in a ROI away from the bleach zone (grey line, ‘control’). (c) Polymerization velocity v_x(y) at the free membrane at the cell front along the y axis for the cell shown in a. See Supplementary Fig. S4A–C for details. (d) FRAP experiment for a region at the cell-wall interface in the leading edge of a migrating cell expressing GFP-actin. The white box at t=0 s indicates the photobleached region. (e) Time course of fluorescence recovery at the cell-wall interface for the cell in panel d. Fluorescence was measured in a region tracking the bleached zone as it moved medially reporting on actin turnover (red box in d for t=17 s; red line, ‘bleached region’) or in a region that stayed stationary at the cell-wall interface that reflected new polymerization (yellow box in d for t=17 s; yellow line, ‘cell-wall interface’). Fluorescence loss due to image acquisition was estimated in a ROI away from the bleach zone (grey line, ‘control’). (f) Polymerization velocity v_y(x) at the cell-wall interface along the x axis calculated from iFRAP data. The front of the cell was taken as the origin of the x axis. See Supplementary Fig. S4D–F for details.