Under control conditions, Arp2/3 complex inhibition increases peripheral retrograde actin flow but has little effect on actin turnover. (A) Representative G-actin FSM images (top) and corresponding flow maps (bottom) from a growth cone before, 20 min after treatment in 50 µM CK666, and 15 min after washout of CK666. Arrows in FSM images mark the edge of the actin veil. On the flow maps, colors encode speed (see color bar) and vectors indicate flow direction. (B–E) Summary of relative changes in retrograde actin flow rates after manipulations. (B) CK666 reversibility: before, during 50 µM CK666 treatment (15–30 min), and after washout (10–30 min). (C and D) Concentration dependence: treatment for 15–30 min with various concentrations of (C) CK666 or (D) CK689. (E) Treatment for 15–30 min with 50 µM CK869 or CK312. n, growth cones measured. (F) Map of time-averaged assembly (red) and disassembly (green) events detected near the leading edge of a growth cone before and after CK666 (50 µM, 20 min). Images were sampled from a region similar to the dotted blue box in A. Colors indicate relative assembly or disassembly rates (see color bars). Green arrow, polymerization sites on filopodia. (G) Plot of changes in integrated fluorescent intensity within the flow-displaced regions (see inset) tracked by ROI-Based Turnover Analysis before and after CK666 (50 µM, 20 min). n, 9 growth cones, 3–5 ROIs per growth cone. Images acquired every 5 s with 2 min elapsed recording time. Bars, 5 µm.