roots of filopodia [3] which are comprised of 10–15 actin filaments in polarized bundles [15]. To investigate effects of actin turnover inhibition on growth cone ultrastructure, EM was performed on cells treated with vehicle (Fig. 1B) or 500 nM jasplakinolide, a concentration known to inhibit actin filament depolymerization in a pyrene-actin assay [5], for six minutes (Fig. 1C). A striking result was buildup of buckled filopodial actin bundles (Fig. 1B&C, yellow arrowhead) in an expanded transition zone (demarcated by dashed yellow lines). A second major effect was loss of veil actin networks situated between filopodia near the leading edge (Fig. 1, compare control leading edge (B) with area near dashed white line (C)). These ul- trastructural changes are consistent with images obtained by fluorescent light microscopy (Fig. S1 A, C). Growth cones treated for 10 minutes with 70 mM blebbistatin, a specific myosin II inhibitor [16,17,18], contained longer, unbuck- led filopodia (Fig. 1D) whose proximal roots projected deep into the central domain as predicted from previous light microscopy investigations [3]. Absence of filopodium root buckling in ble- bbistatin made it difficult to identify the transition zone as a structurally distinct domain. Loss of veil actin did not occur after inhibition of myosin II activity alone (Fig. 1, compare C and E). Interestingly, growth cones pre-treated with blebbistatin and then exposed to jasplakinolide with continuous myosin II inhibition showed little depletion of veil actin from the leading edge; however, these growth cones still had a distinct buildup of actin filaments in more central regions when compared to growth cones treated with blebbistatin alone (Fig. 1, compare D&E). Together these results suggest actin turnover plays a role in regulating actin network density in both peripheral and central regions of the growth cone and evidence of these processes can be observed in the absence of myosin II activity. We next investigated the actin filament dynamics underlying these structural changes. Deceleration of retrograde flow and leading edge retraction follow Jasplakinolide treatment Clearance of actin from the leading edge and increased actin filament density in the transition zone following jasplakinolide treatment suggested the continued retrograde transport of a po- pulation of stabilized actin filaments. To quantitatively address jasplakinolide effects on actin filament dynamics we used fluorescent speckle microscopy [19]. Aplysia californica BCNs were injected with a probe for actin, either fluorescently tagged phalloidin or actin monomer, and imaged for ,5 minutes prior to bath application of jasplakinolide as above. Growth cones were then imaged for 6 or more minutes following jasplakinolide addition depending on the experiment. Within ,90 sec of jasplakinolide addition a decrease in actin speckle density was evident near the leading edge (time montage 2A, lower) and was striking after 6 minutes (Fig. 2A, upper). EM replicas obtained at the same time points indicated a dramatic decrease in the density of actin veil networks and marked kinking of remaining filopodial actin bundles (Fig. 2B green arrow heads). Neurite retraction has been reported in response to jasplakinolide [9], however, our use of highly adhesive poly-L-lysine sub strates tended to prevent the membrane leading edge from de-adhering from the substrate. The result was typically a depletion of peripheral actin networks from the adherent membrane edge over time (as in Fig. 2). In addition, central domains of jasplakinolide-treated growth cones appeared to contract and undergo compression. This is shown in the time series in Fig. 3A and supplementary Video S1 (dashed yellow lines approximate central domain area). Kymographs sampled across peripheral and central domains suggested that build up of material in the central domain resulted from influx of peripheral domain actin structures (Fig. 3B, dashed yellow line; cf. Fig. 1C). Retrograde Flow Requires Actin Depolymerization Jasplakinolide treatment dramatically reduced peripheral re- trograde flow rates (Fig. 3B&C, 4A&B). Rates were assessed by kymography using 1.5 minute sampling windows for a total of nine minutes following jasplakinolide addition. An example kymograph sampled along the red line in panel A is shown in Fig. 3B (dashed red line indicates time of drug application). For population analysis, flow rates (red line slopes) were normalized to the flow rate measured just before drug treatment. Within 3 minutes of jasplakinolide addition retrograde flow had decreased by ,50% and by 9 minutes flow had further decreased to ,30% of control levels (Fig. 3C). Jasplakinolide treatment inhibits actin depolymerization and reduces network convergence independent of myosin II activity Our results suggested jasplakinolide was simultaneously reducing actin density in the peripheral domain and increasing actin density in the central domain. To address the underlying mechanism, we developed a custom algorithm to measure changes in actin network density and estimate net actin turnover dynamics in defined regions of interest over time (see Material and Methods and Fig. S2 for details). Briefly, the user specifies a polygonal region of interest (ROI) where actin network density changes will be assessed, then actin particle trajectories [20] are used to displace the ROI over time. Assuming spatially smooth flow fields, changes in the integrated actin intensity within the region reflect the net contribution of polymerization and depolymerization. If polymer- ization and depolymerization were perfectly balanced, no change in integrated intensity would be expected regardless of change in area. Flow fields from single particle tracking show centripetal mo- vement of actin filaments, with all vectors pointing towards the center of the growth cone (Fig. 4A). Consistent with previous reports, we observed faster flow in the periphery and slower flow in the transition zone and central domain, under control conditions [21,22]. Following jasplakinolide treatment P domain retrograde flow rate decreased (Fig. 4B), consistent with the kymograph results. The region tracking algorithm described above was then used to measure changes in actin filament density and network con- vergence before and after jasplakinolide treatment. Examples of tracked ROIs are shown in Fig. 4C and Video S2. Changes in ROI area and intensity are plotted in Fig. 4D. Over the five minutes the control ROI was tracked, the integrated intensity steadily decreased to ,40% of its initial value (blue lines). The near constant slope of this curve indicates that depolymerization occurs throughout the peripheral domain. In marked contrast, the intensity within the tracked ROI remained high, retaining 80% of its initial value after 8 minutes in jasplakinolide (500 nM). The same trends are evident in pooled data (Fig. 4E, see legend for statistical details). Inhibition of actin filament turnover also had a dramatic effect on flow convergence (Fig. 4, C–E, Video S2). In the example shown (Fig. 4C), the control ROI area decreased by 64.7% over 5 minutes, whereas, ROI area decreased by only 38.5% after 8 minutes in jasplakinolide (Fig. 4D, top). The average decrease in area for all ROIs tracked under control conditions was 59.7+/ 222.2% of initial levels after 3 minutes; the decrease in area after 3 minutes in jasplakinolide was only 8.3+/216.7% (Fig. 4E). We previously reported a role for non-muscle myosin II in severing of actin bundles associated with filopodium roots in growth cones [3] and myosin II has recently been implicated in promoting actin network turnover in migrating keratinocytes [23]. Surprising- ly, we found that myosin inhibition with blebbistatin, a specific non- muscle myosin II inhibitor [16,17,18] we have characterized in our system [3], had no significant effect on either turnover rates or convergence of actin networks in the P domain (Fig. 4D–E). PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e30959