Fig. 2. mDia2 forms a stable pool of cortical actin in the lamella. (A) Example of a TIR-FRAP experiment; X-Rhodamine actin TIRF images taken from a time-lapse movie of a PtK1 control cell prior to and after fluorescence photobleaching of 150 nm of the ventral cell cortex, which occurs at t=0. Lamellipodium measurements were taken at the extreme cell edge (red ovals). Green ovals indicate regions where fluorescence intensity measurements were taken in the lamella. (B) t_1/2 of fluorescence recovery of F-actin in the lamellipodium of control (n=5) and mDia2 antibody (anti-mDia2, n=5) -injected cells. (C) Example of actin fluorescence recovery data (diamonds) fit to a single term exponential (squares) in the lamellipodium of a control PtK1 cell. (D) Percentage of fluorescence recovery after photobleaching of F-actin in the lamella of control (n=5) and mDia2 inhibited cells (n=5, ± s.d.). mDia2 antibody inhibition mobilizes a stable, non-recovering pool of fluorescent actin. (E,F) Examples of fluorescence recovery data (diamonds) fit to a single term exponential (black line) obtained from the lamella of (E) a control PtK1 cell and (F) an mDia2 antibody-injected cell. (G) Data from F (diamonds) fit to a two-term exponential (black line), revealing that F-actin in the lamella of mDia2 antibody-injected cells has a different mechanism of fluorescence recovery than controls, in which the data was fit by a single exponential.