Retrograde Flow Requires Actin Depolymerization Figure 3. Retrograde flow attenuation accompanies network retraction from the leading edge. Actin images acquired as in Figure 2. (A) Images of the growth cone before, 3, 6, and 9 minutes after bath addition of 500 nM jasplakinolide. The peripheral domain, transition zone, and central domains are marked with P, T, and C respectively. Degree of retraction is indicated by the red brackets in the 6 and 9 minute time-points. The inner border of the peripheral domain and transition zone is delineated by the dashed yellow line. (B) Kymograph taken along the red line in A. The dashed red line indicates the time-point of drug application. The red dots indicate the 3, 6 and 9 minute time points. The red brackets and dashed yellow lines are as in A. The angle of the solid red lines can be used to determine the averaged flow rates over periods of time. (C) Quantification of retrograde flow rates during the time-course of 500 nM jasplakinolide treatment (n = 5: 4 actin and 1 phalloidin). Retrograde flow rates were evaluated within 1.5 minute intervals, normalized verses control and averaged across growth cones. Error bars are standard deviations. Flow rates were 52.4+/212.7% of control at 3 minutes, and 30.3+/216.5% at 9 minutes post-treatment. Error bars are standard deviations. Scale bar = 10 um. doi:10.1371/journal.pone.0030959.g003 fit of the averaged jasplakinolide alone data was 2.99 minutes. The decay constant derived from the blebbistatin pretreatment data set was 3.01 minutes (Fig. 6B). The nearly identical decay time constants measured in the presence or absence of blebbistatin suggests jasplakinolide attenuates retrograde flow by a mechanism that is independent of myosin II activity. In contrast, the retrograde flow offset was significantly decreased by blebbistatin treatment- consistent with myosin II activity setting the baseline retrograde flow rate as previously reported [3]. Jasplakinolide effects on retrograde flow cannot be attributed to displacement of myosin II from the actin network since myosin II localization in jasplakinolide treated growth cones was similar to that observed in controls (Fig. S1). Turnover inhibition reduces the density of peripheral barbed ends Jasplakinolide treatment decreased the rate of retrograde network flow in the P domain (Figs. 3A–B); but despite the slower rate of actin filament transport away from the leading edge, actin filament density here was dramatically reduced (Fig. 1C). These observations suggest jasplakinolide was reducing the rate of actin assembly which depends on the concentration of polymerization competent actin monomers and the number of uncapped barbed ends [24]. Barbed ends can be generated both through de novo filament nucleation and severing of existing actin filaments. To further investigate which processes were involved we designed ex- periments to assess changes in barbed end density and measure actin monomer concentration. To measure jasplakinolide effects on barbed end density, cells were permeabilized and existing actin networks stabilized TRITC- phalloidin. Cells were then incubated with Alexa-488-G-actin just above the critical concentration for assembly in the presence of ATP to allow barbed end visualization [25]. Under control conditions barbed ends were concentrated in a ,2–3 um band at the leading edge and proximally in the transition zone (Fig. 7a and 7b) where they appeared to be associated with intrapodia (red arrowheads; [26]). Cytochalasin D, a small molecule that inhibits actin polymerization by capping barbed ends, prevented monomer incorporation confirming the specificity of this labeling protocol (Fig. 7C, D, and K). Studies support a role for cofilin-mediated severing of actin filaments and subsequent disassembly in maintaining a pool of G- actin in cells required for stimulus induced actin nucleation at the leading edge [27,28]. Local severing to create free barbed ends may also regulate assembly at the leading edge [29,30,31]. Since jasplakinolide inhibits cofilin-mediated severing, we used the G- actin incorporation assay described above to investigate jasplakino- lide effects on barbed end density near the growth cone leading edge. Barbed end density (Fig. 7F, H, J and L) as well as phalloidin labeling at the leading edge (Fig. 7E, G, I and L) progressively decreased over time with exposure to 500 nM jasplakinolide. Jasplakinolide competes with phalloidin binding [32] so fluorescent phalloidin quantification may underestimate F-actin density. However, the presence of phalloidin in the extraction buffer should not affect the density of pre-existing barbed ends being assayed. The above observations are also in agreement with the ultrastructural PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e30959