Figure 1.

Effect of Y477F ezrin on cell motility in AC2M2 cells: Panel A) AC2M2 cells transfected with an empty pCB6 vector, or expressing Y477F ezrin, clones (A43 and C13) were lysed in Laemmli buffer, and equal protein amounts were subjected to SDS-PAGE and western blotting with antibodies against VSVG, ezrin, pY477 ezrin, pTERM and γ-tubulin. In the ezrin blot, the ~81 kDa band in pCB6 represents endogenous ezrin, while the bands in A43 and C13 represent both Y477F mutant and endogenous ezrin. Optical density (OD) ratios of ezrin vs γ-tubulin bands show a 2.6-fold and 3.5-fold increase in total ezrin expression of the A43 and C13 clones, respectively, normalized to the pCB6 clone. Ezr, ezrin; moe, moesin. Panel B) AC2M2 clones expressing either pCB6 empty vector or Y477F ezrin were grown to confluence in 10% FBS/DMEM. Confluent cells were wounded by scoring and medium was immediately replaced. Spontaneous wound closure at each of four marked wound sites for each cell clone was monitored for up to 24 h by phase contrast microscopy. The histogram shows relative cell motility for each clone calculated as the area of wound closure at 18 h and 24 h compared to T = 0 h. Values represent the mean +/- SD of 4 wound sites per clone in each of two experiments. There was significant reduction in cell motility in Y477F ezrin expressing clones (A43 and C13) at both 18 h and 24 h, as determined by a one-way ANOVA (p < 0.001). Panel C) Representative fields photographed using phase contrast microscopy (10× objective) at 0 h, 18 h and 24 h are shown. Boxed areas indicate wound area measured at each time point.