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Fig. 3. mDia2 is necessary for the segregation of distinct dynamic F-actin behavior of the lamellipodium and lamella. Phase-contrast (A) and FSM (B) images of X-Rhodamine actin in control and mDia2 antibody-injected (anti-mDia2) cells. (C) Kymographs taken along the axis of F-actin flow, indicated by the lines in B. Three kymographs were taken from each of five cells per treatment for the analyses shown in G and I. In C, lines indicate the F-actin speckle flow rate in the lamellipodium (LP) and lamella (LA); arrowheads indicate lack of the rapid retrograde flow typical of the LP. (D) qFSM maps of F-actin polymerization (red) and depolymerization (green) rates in control and mDia2 antibody-injected (anti-mDia2) cells. Brightness indicates the relative magnitude of the rate. The arrow indicates a wide region of rapid F-actin polymerization along the cell edge (LP), and the arrowhead indicates a region of no rapid F-actin polymerization at the cell edge (no LP). (E) qFSM maps of the speed of F-actin flow in control and mDia2 antibody-injected (anti-mDia2) cells; the arrow indicates a shallow speed gradient. The arrowhead indicates a region of no rapid retrograde flow at the cell edge. (F) qFSM speckle velocity from regions indicated by boxes in E. (G) Average rates of F-actin retrograde (–) or anterograde flow (+) in the LP and LA, determined from kymographs of FSM movies (± s.e.m.). (H) Rate of F-actin flow as a function of distance from the cell edge in control and anti-mDia2 antibody injected cells. F-actin flow speed at all points in the cell, as determined by qFSM, was averaged parallel to the cell edge in 1 µm intervals behind the cell edge for five control and anti-mDia2 antibody-injected cells. (± s.e.m.). (I) Percentage of time in which fast flow of lamellipodium was present, as analyzed from kymographs (± s.e.m.). The asterisk in I indicates statistical significance (P<0.05) between control and mDia2 antibody-injected cells. (J,K) qFSM maps of (J) the speed of F-actin flow and (K) of F-actin polymerization (red) and depolymerization (green) rates in a PtK1 cell expressing a dominant negative FH2 ( ${delta}$ FH2) construct.

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