Fig. 1. Cells expressing constitutively activate R-Ras spread by continuously extending lamellipodia with few retraction events. (A) Total area of spread MCF10A cells comparing cells expressing vector alone with those expressing R-Ras^38V. Bar graph represents the average total spread area of 23 R-Ras cells or 16 control cells ± s.e.m. Note that R-Ras-expressing cells are more than twice as spread as control cells. (B) TIRF images of an individual control and R-Ras^38V-expressing cell during spreading on 10 µg/ml fibronectin. Brighter regions represent areas of greater contact with the ECM. (See Movies 1 and 2, in supplementary material.) (C) Analysis of protrusion and retraction events for control and R-Ras^38V-expressing cells. Images such as those shown in B were analyzed as described (Giannone et al., 2004) to characterize cell-edge dynamics. Top traces show the increase in total cell area over time for a representative control and R-Ras^38V cell. Radial edge velocity maps of the entire membrane periphery plotted as a function of time and arc length are shown below, where arc length denotes the separation between two polar coordinates along the periphery of the cell. Membrane activity was determined and expressed as the velocity of protrusion events (warm colors) or retraction events (cool colors). Dotted lines delineate the phases in cell spreading.