The Role of Actin Turnover in Retrograde Actin Network Flow in Neuronal Growth Cones David Van Goor, Callen Hyland, Andrew W. Schaefer, Paul Forscher* Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, United States of America Abstract The balance of actin filament polymerization and depolymerization maintains a steady state network treadmill in neuronal growth cones essential for motility and guidance. Here we have investigated the connection between depolymerization and treadmilling dynamics. We show that polymerization-competent barbed ends are concentrated at the leading edge and depolymerization is distributed throughout the peripheral domain. We found a high-to-low G-actin gradient between peripheral and central domains. Inhibiting turnover with jasplakinolide collapsed this gradient and lowered leading edge barbed end density. Ultrastructural analysis showed dramatic reduction of leading edge actin filament density and filament accumulation in central regions. Live cell imaging revealed that the leading edge retracted even as retrograde actin flow rate decreased exponentially. Inhibition of myosin II activity before jasplakinolide treatment lowered baseline retrograde flow rates and prevented leading edge retraction. Myosin II activity preferentially affected filopodial bundle disassembly distinct from the global effects of jasplakinolide on network turnover. We propose that growth cone retraction following turnover inhibition resulted from the persistence of myosin II contractility even as leading edge assembly rates decreased. The buildup of actin filaments in central regions combined with monomer depletion and reduced polymerization from barbed ends suggests a mechanism for the observed exponential decay in actin retrograde flow. Our results show that growth cone motility is critically dependent on continuous disassembly of the peripheral actin network. Citation: Van Goor D, Hyland C, Schaefer AW, Forscher P (2012) The Role of Actin Turnover in Retrograde Actin Network Flow in Neuronal Growth Cones. PLoS ONE 7(2): e30959. doi:10.1371/journal.pone.0030959 Editor: Cara Gottardi, Northwestern University Feinberg School of Medicine, United States of America Received October 7, 2011; Accepted December 28, 2011; Published February 16, 2012 Copyright: 2012 Van Goor et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health grants RO1-NS28695 and RO1 NS51786 to P.F. and the Nikon Partners-in-Research Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Paul Forscher was a ‘‘Nikon Partner in Research’’ during some of the period this research was conducted. This program gave his group access to loaned imaging equipment for the experiments. There was no personal compensation involved nor did the agreement in any way infringe on the freedom to share and publish the authors’ scientific results. Moreover, this agreement does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: paul.forscher@yale.edu Introduction Growth cone structure and motility emerge from the balance of actin filament polymerization, depolymerization, transport, and cross-linking into various network architectures. Motile actin structures continuously consume energy and cycle their compo- nents. As structure is continuously turned over, the system can respond rapidly to extracellular guidance cues; small changes affe- cting actin structure may lead to rapid changes in growth cone morphology and behavior. In the absence of changes in substrate or soluble guidance cues, actin filament polymerization, depoly- merization, and transport maintain a steady-state balance where there is little change in position and growth cone morphology despite continuous retrograde actin flux. In such a system, perturbations of a single process can reveal information on the interdependency of the other processes in maintaining the steady- state balance. Actin assembly at the leading edge produces filopodia and veil structures which are transported centripetally by a process called retrograde flow. Both leading edge polymerization and myosin II contractility contribute to retrograde actin movement. Addition of actin subunits between the polymerized actin array and the leading edge membrane pushes the entire network centripetally [1]. According to the dynamic network contraction model [2], myosin II localized in the transition zone (Fig. 1 and Fig. S1D–F) generates contractile force, which propagates through the me- chanical continuum of the actin array and displaces the entire network. Retrograde flow rate is affected by myosin II activity. Flow rates decrease up to 50% in growth cones exposed to the myosin II ATPase inhibitor blebbistatin and residual retrograde flow can largely be accounted for by the force of leading edge polymerization [3]. Filopodia and veil actin are continuously assembled and tran- sported rearward by retrograde flow. The half-life of growth cone veil actin has been estimated to be 1–3 min whereas filopodial actin bundles are considerably more stable [4] and their recycling requires non-muscle myosin II [3]. To maintain the growth cone periphery in a steady state both of these actin structures must be continuously recycled. Despite the potential mechanistic impor- tance of filament recycling in regulation of growth cone motility, very little is known about the kinetics or spatial characteristics of this processes. In the current study we use jasplakinolide, a cyclic depsipeptide from the marine sponge Jaspis johnstoni, to inhibit network turnover and investigate acute effects on actin filament structure and dynamic behavior. Jasplakinolide cooperatively binds and stabi- PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e30959