Retrograde Flow Requires Actin Depolymerization Figure 5. Myosin II inhibition prevents growth cone collapse but not retrograde flow attenuation by Jasplakinolide. (A) Actin network as visualized by SDC-FSM using Alexa-568 actin after 5 minutes in vehicle (0.75% DMSO, panel 1), 10 minutes in 70 mM blebbistatin (panel 2) and 3, 6, and 9 minutes in 70 mM blebbistatin and 500 nM jasplakinolide (panels 3–5 respectively). The peripheral domain, transition zone and central domains are indicated by P, T, and C respectively. The solid red line in the control indicates where the kymograph in (B) was taken. (B) A kymograph of the peripheral domain and transition zone taken along the solid red line in (A). The time period shown includes 5 minutes in blebbistatin alone and 10 minutes in blebbistatin and jasplakinolide. The dashed red line indicates the time of jasplakinolide addition; the red dots indicate 3, 6, and 9 minutes post addition. Notice that no retraction of the leading edge is seen in A or B. (B) As previously reported, blebbistatin pretreatment inhibited retrograde flow. (C) Quantification of retrograde flow rates during the time-course of treatment (n = 12: 7 actin and 5 phalloidin). Retrograde flow rates were evaluated within 1.5 minute intervals, normalized verses control and averaged across growth cones. Normalization and averaging is justified by the strong correlation between initial and final flow rates (0.798, calculated in Excel). Flow rates were 73.4+/213.3 percent of control after 10 minutes in blebbistatin, 33.2+/211.1 percent of control at 3 minutes, and 12.8+/25.2 percent of control after 9 minutes in blebbistatin and jasplakinolide. Error bars are standard deviations. Scale bar = 10 um. doi:10.1371/journal.pone.0030959.g005 and FSM data presented in Fig. 1–3 that show a decrease in veil actin filament density near the leading edge after jasplakinolide treatment. Our results suggest that the pool of polymerization- competent barbed ends is sustained by ongoing actin turnover. Actin turnover elevates actin monomer concentration in the peripheral domain The hypothesis that monomer concentration limits polymeri- zation at the leading edge is supported by theoretical models, Figure 6. Decay of flow rate with time following turnover inhibition is independent of myosin II. (A) Retrograde flow rates for vehicle control and blebbistatin pretreated growth cones normalized to rates assessed just before jasplakinolide addition. Bar graph shows average flow rates assessed by kymography. Error bars are standard deviations. Solid blue and orange lines are the curves fit to Equation 1. The adjusted R2 values of the fit are 0.9982 and 0.9991 for vehicle and blebbistatin pretreated data respectively. (B) Theoretical fit of flow decay timecourse separating the exponential decay from the offset. Solid lines indicate the curve according to the best fit parameter; dashed lines were calculated with the 95% confidence intervals which were between 2.66 and 3.40 min for vehicle and 2.82 and 3.34 min for blebbistatin pretreatment. Blue lines: control data; orange lines: blebbistatin pretreatment data. doi:10.1371/journal.pone.0030959.g006 PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e30959