actin assembly rates and 2) continued central network compression by myosin II dependent forces account for the leading edge retraction we observed following jasplakinolide application. What is the role of myosin II in actin network turnover? Recent studies suggest a role for myosin II in actin network turnover [23,43,44,45]. Here we have analyzed actin movement and turnover in the presence and absence of myosin II activity and found that myosin has little effect on bulk turnover in the peripheral domain. This is shown explicitly in Figure 4 where tracked regions in blebbistatin treated growth cones had similar fluorescence decay profiles to control. The kinetics of retrograde flow decay after turnover inhibition were also little affected by myosin activity. In fact, the effects of blebbistatin and jasplakino- lide were mathematically separable with myosin activity contrib- uting a constant offset to the exponential decay of flow (Fig. 6). Turnover inhibition with jasplakinolide immediately attenuates leading edge polymerization. In contrast, Aplysia growth cones can sustain a steady state network treadmill after myosin inhibition with persistent retrograde flow at 50–75% of control rates, as previously reported [3]. This suggests that leading edge actin polymerization can drive the peripheral network treadmill inde- pendent of myosin II activity. We previously reported a specific role for myosin II in filopodium actin bundle severing and recycling in the growth cone transition zone [3]. This effect, which can result in filopodial actin bundle elongation to ,200% of control lengths, is evident in Fig. 1B vs. D. We therefore suggest that myosin II affects re- trograde flow rate and turnover of filopodia, but has little influence on the bulk turnover of the peripheral domain necessary for maintenance of leading edge polymerization. How does turnover inhibition affect leading edge polymerization? Contrary to previous predictions [33], we found higher levels of G-actin monomer in the peripheral domain than in more central regions. In many growth cones a clear inward facing negative concentration gradient of actin monomer was observed (Fig. 8, Fig. S4). Our findings suggest the presence of an active process [46] to maintain the concentration gradient in the face of constant actin filament assembly and consequent monomer depletion near the leading edge. A potential solution would be an anterograde cytoplasmic fluid flow capable of carrying soluble proteins from central regions toward the leading edge similar to what has recently been described in motile epithelial cells [47]. Other reports, both experimental and theoretical, hint that turnover may be required to maintain the monomer pool at the leading edge [8,33,34]. In agreement, we observed that the mo- nomer gradient rapidly collapsed following turnover inhibition with jasplakinolide. Our findings suggest that actin filament turnover occurs throughout the peripheral domain (Fig. 4C–D). This process creates a distributed source of actin monomers that in concert with forward fluid flow could account for the high steady state G-actin concentrations we observe in the peripheral domain. In addition to de novo nucleation, actin filament severing generates uncapped barbed ends that can serve as sites for monomer addition [30,33]. In agreement, we found the highest density of exposed barbed ends near the leading edge (Fig. 7B). Significantly, turnover inhibition rapidly reduced free barbed end density here, possibly as a result of competition between jasplakinolide and cofilin for binding to actin filaments. We noted that following jasplakinolide treatment actin veil density was diminished relative to actin bundles comprising filopodia (Figs. 1C, 2, and 3A). It has been previously reported Retrograde Flow Requires Actin Depolymerization that filopodia have a longer half-life than veils [4] and therefore may be more stable structures. We speculate that changes in G- actin concentration could differentially affect veil versus filopodial actin assembly. In particular filopodia may be able to continue to grow processively in jasplakinolide to the extent that they persist after the veil has retracted. What is the general cell biological relevance of the above? The actin polymerization rate is proportional to the concentra- tions of uncapped filament ends and polymerization competent actin monomer. Barbed ends are generated by de novo actin nucleation and severing of existing filaments, while actin mo- nomers are generated by severing and depolymerization. Both these processes have been implicated in regulation of cell motility. For example, photoactivation of cofilin in cancer cells induces crawling in the direction of activation [30]; cofilin is also required for EGF-stimulated lamellipodium extension [25,48]. EGF sti- mulation increases barbed end density and leading edge actin polymerization in carcinoma cells [49]. Similar results were re- ported in response to NGF stimulation of neuronal growth cones [10]. Exogenous G-actin monomer can also compensate for cofilin deficiency [28] suggesting that ADF/Cofilin activity maintains a pool of actin monomer for feeding barbed end growth. This extensive body of literature and our current results point to the importance of actin turnover mechanisms in regulation of actin- based structural dynamics and related motility processes. Relevance of Actin Turnover for Neuronal Growth Cone Behavior Previous studies suggest signaling pathways that regulate the stability of actin filaments can have significant effects on growth cone motility and guidance [10,11,50,51], however, mechanisms underlying these effects are not well understood at the level of cytoskeletal dynamics. To maintain the growth cone’s actin network treadmill at steady state, net filament assembly and disassembly must be balanced. The current study suggests that modulation of leading edge actin assembly rates via changes in barbed end density and/or actin monomer concentration are powerful mechanisms by which turnover pathways affect actin based growth cone motility and axon guidance. Under these conditions, changes in any parameter will have immediate and global effects on actin structure. Myosin II has been implicated as a downstream effector in many axon guidance pathways however the mechanistic role non- muscle myosin II and its various isoforms play in these processes is quite complex [52,53]. In agreement with previous studies, our results show that myosin II activity is required for jasplakinolide- induced actin network retraction, which has been implicated in growth cone retraction responses [9]. We note however, that the related issue of how actin turnover affects development of traction forces involved in turning and substrate dependent growth [54,55] remains largely unexplored. Our finding that the kinetic effects of inhibition of actin turnover are independent of myosin II activity (Fig. 3) has mechanistic implications for understand- ing these processes and highlights the importance of quantitative analysis of the action of drugs commonly used to perturb cyto- skeletal processes involved in axon growth. Materials and Methods Cell Culture and Imaging Media Primary cultures of A. californica bag cell neurons on poly-D- lysine coated coverslips were prepared as described by Forscher and Smith [12], with some modification. Briefly, abdominal ganglia were incubated at 22uC for ,18 hours in L-15 artificial PLoS ONE | www.plosone.org 11 February 2012 | Volume 7 | Issue 2 | e30959