Fig. 6. The effect of various mutations on the ability of the ß1 and ß3 cytoplasmic domains to regulate cell spreading. (A,B) Human fibroblasts were transiently transfected with the control receptor, or tac chimeras containing wild-type or mutant ß1 and ß3 cytoplasmic domains as indicated. Cells adherent to fibronectin for 1 hour were analyzed for cell-surface expression of tac and cell area as described in Materials and Methods. The cell area for 100 randomly sampled positively transfected cells is plotted as a function of tac expression. The x axis is a linear scale of cell area from 0 to 4000 µm²; the y axis is a linear scale of arbitrary FITC fluorescence (tac expression) units defined by Image Pro-Plus from 0 to 1.0x10⁵ (A) and from 0 to 4.0x10⁴ (B). We found in using this assay that round cells that have not begun to spread have cell areas less than 600 µm² (Berrier et al., 2000). The percentage of positively transfected cells that had areas less than 600 µm² was calculated from three experiments and graphed as the mean ± s.e.m. (C) The stable CHO cell lines A5 and ETC12 were transfected with the control receptor or tac chimeras containing either the wild-type ß1 or the ß1-787 (V/A) mutant cytoplasmic domain as indicated. A5 cells adherent to fibrinogen (15 µg/ml; Fg) for 30 minutes or ETC12 cells adherent to fibronectin (10 µg/ml; Fn) for 2 hours were analyzed for cell-surface expression of tac and cell area as described above. The cell area for 100 randomly sampled positively transfected cells is plotted as a function of tac expression. The x axis is a linear scale of cell area from 0 to 2000 µm²; the y axis is a linear scale of FITC fluorescence (tac expression) from 0 to 5.0x10⁴. A vertical line positioned at 600 µm² indicates the separation of spread (right) and not spread (left) cells. The percentage of positively transfected cells that had areas less than 600 µm² was calculated from three experiments and graphed as the mean ± s.e.m.