Figure 2.

Bleached and unbleached GFP-actin are incorporated into F-actin structures at the leading edge of spillover protrusions. (A) Still images and associated kymographs from a time lapse series of micrographs of JR20s expressing GFP-actin that were plated as detailed in Fig. 1A, focused on regions between the edge of a Fn attachment site and the edge of a cell protrusion as different regions of the protrusion are photobleached. Yellow lines show the regions the kymographs below were generated from, and red arrows signify where GFP-actin was bleached and then incorporated into the treadmilling actin filaments at the leading edge. Scale bars, 5 μm. (B) Graph of the retrograde flow rate of GFP-actin in spillover protrusions measured from kymographs like those shown in (A). Bars represent the mean ± standard deviation (n = 33 protrusions from 3 independent experiments). (C) Still images from a time lapse series of micrographs of JR20s expressing GFP-actin that were plated as detailed in Fig. 1A and bleached throughout the entire protrusive area (left column) or cell body over the Fn attachment site (right column) every 60 frames. Yellow regions indicate areas measured for intensity traces shown in (D and E). Scale bar, 10 μm. (D) Trace of the normalized GFP-actin intensity over time in the region indicated by the yellow dotted outline in the bottom-left panel of (C). Red arrows mark photobleaching events of the entire protrusive spillover region of the cell. (E) Trace of the normalized GFP-actin intensity over time in the region indicated by the yellow dotted outline in the bottom-right panel of (C). Red arrows mark photobleaching events of the entire region of the cell that sits over the square Fn attachment cell-adhesive pattern. To see this figure in color, go online.