Figure 5.

Under conditions of low myosin II activity, Arp2/3 complex inhibition decreases peripheral retrograde actin flow rates. (A) Representative G-actin FSM images (top) and corresponding flow maps (bottom) from a growth cone before and after CK666 (50 µM, 20 min) in the presence of blebbistatin (60 µM, 10 min pretreatment). Arrows in FSM images mark the edge of the actin veil. Insets, control actin and flow map recorded before blebbistatin addition. (B) Summary of relative changes in retrograde actin flow rates in response to increasing concentrations of CK666 (15–30 min) in the presence of blebbistatin. Flow rate decreases: 14.8 ± 1.2% with 25 µM, 24.5 ± 2.3% with 50 µM, and 43.0 ± 2.6% with 100 µM CK666. n, growth cones measured. (C) Representative phalloidin-FSM images (top) and corresponding flow maps (bottom) from a growth cone before and after CK666 (50 µM, 20 min) in the presence of Y27632 (10 µM, 20 min pretreatment). Average flow rate before: 4.75 µm/min; CK666: 6.08 µm/min. Insets: control actin and flow map recorded before Y27632 addition, average flow rate 4.73 µm/min. (D) Retrograde actin flow rates still increased by 19.6 ± 2.6% in response to CK666 (50 µM, 15–30 min) in the presence of Y27632 (10 µM, 20 min pretreatment). Flow rates in response to the same concentration of CK666 under control conditions from Fig. 4 C are shown for comparison. (E) Changes in peripheral retrograde actin flow rates in response to blebbistatin after Arp2/3 complex inhibition. Growth cones were pretreated with CK666 or inactive analogue CK689 (50 µM, 15–30 min), then treated with blebbistatin (60 µM, 10–30 min) in the continued presence of either CK666 or CK689. Flow rates were assessed before drug addition, during CK pretreatment, and during cotreatment with CK and blebbistatin. Population averages are shown. CK666 treatment increased the retrograde flow by 19.3 ± 3.2%. CK689 did not alter retrograde flow. In a CK666 background, blebbistatin decreased the flow by 65.6 ± 3.6%. In a CK689 background, blebbistatin decreased the flow by 20.1 ± 2.0%. Numbers in parenthesis: 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) in the presence of blebbistatin. Images were sampled from a region similar to the dotted blue box in A. Colors indicate relative assembly or disassembly rates (see color bars). (G) Plot of change in integrated fluorescent intensity within the flow-displaced regions before and after CK666 (50 µM, 20 min) in the presence of blebbistatin. n, 6 growth cones, 3–5 ROIs per growth cone. *, P < 0.01 with two-tailed paired t test. Images acquired every 5 s with 2 min elapsed recording time. Bars, 5 µm.