Retrograde Flow Requires Actin Depolymerization Figure 1. Jasplakinolide treatment results in a myosin II independent accumulation of actin filaments in the transition zone and a myosin II dependent depletion of veil actin in the periphery. (A) Electron micrograph of an untreated A. californica BCN growth cone showing domain organization. Growth cone electron micrographs of the peripheral domain and transition zones (typical ROI shown in A) of growth cones treated with vehicle for 6 minutes (B), 500 nM jasplakinolide for 6 minutes (C), 70 mM blebbistatin for 10 minutes (D), or 70 mM blebbistatin for 10 minutes followed by 70 mM blebbistatin and 500 nM jasplakinolide for 10 minutes (E). Following drug treatment, cells were live extracted and processed for platinum/palladium replica electron microscopy. Dashed yellow lines in panels A and B delineate the transition zone, where filopodia buckling occurs (examples marked with yellow arrowheads). Dashed white line in (C) denotes position of leading edge. The scale bar in D is 2 mm and applies to all panels. doi:10.1371/journal.pone.0030959.g001 lizes actin filaments by: 1) decreasing the monomer off rate at filament ends and 2) competitively inhibiting cofilin binding and severing activity, resulting in a decrease in the number of uncapped filament ends [5,6,7]. Jasplakinolide also increases polymerization rates and promotes nucleation, although this can be largely accounted for by its effect on monomer off rate [5]. Jasplakinolide is commonly used in cell biological experiments to perturb actin dynamics because it is membrane-permeable and has been shown to inhibit protrusion of lamellae, slow cell migration, and induce neurite retraction [8,9]. Recently, both attractive [10] and repulsive [11] axon guidance cues have been shown to promote actin turnover through activation of ADF/ cofilin, however, the cytoskeletal mechanisms underlying these effects are not well understood. Here we report that inhibiting turnover by stabilizing actin filaments stops actin assembly at the leading edge. Simultaneously myosin II contractility moves and compresses the stabilized actin filaments into the central domain. These observations provide a quantitative mechanistic framework for understanding the role of actin turnover in controlling axon growth and guidance. Results Turnover inhibition results in actin filament accumulation in the transition zone and actin veil depletion at the leading edge Neuronal growth cones are spatially segregated into two domains: 1) a peripheral (P) domain characterized by dense actin filament networks and occasional microtubules and 2) a central (C) domain densely populated by microtubules and organelles [12,13] and visualized here by platinum/palladium replica electron microscopy (EM) (Fig. 1A). The peripheral actin is organized into filopodium and actin veil structures, which form the basis of an actin assembly-driven protrusion module [14]. A transition zone (T) exists at the interface between the peripheral and central domains, characterized by bending and buckling of the proximal PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e30959