The Role of Actin Turnover in Retrograde Actin Network Flow in Neuronal Growth Cones

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.

Two-tiered coupling between flowing actin and immobilized N -cadherin/catenin complexes in neuronal growth cones

“…Using pharmacological compounds, we showed that both actin polymerization forces and myosin II tension contributed to flow generation. Actin flow was high in the peripheral region, where actin monomers incorporate into new filaments, and slowed down toward the central region, where actin filaments disassemble, consistent with fluorescence speckle microscopy data (2,18,37) and the universal actin cycling model (38,39). An important parameter is the time that a given actin monomer spends in the retrograde flow, which seems to be independent of the flow rate itself.…”

“…The actin-capping drug cytochalasin D, the monomeric actin-sequestering drug latrunculin A, and the actinstabilizing agent jasplakinolide all dramatically diminished the fraction of rearward-moving actin-mEos2 molecules (21%, 7%, and 2%, respectively) ( Fig. S5), consistent with a major role of actin tread-milling in generating the flow (2,3,5). Myosin-light chain (MLC)-GFP was localized in the intermediary region of the growth cone, and MLC-Eos2 trajectories were mostly confined in this area (Fig.…”

“…Growth cones translate extracellular signals into directional migration through a coordinated regulation of cytoskeleton, adhesion, and membrane processes (1). At the cytoskeletal level, motility is generated by polarized actin treadmilling, which, together with myosin contraction, generates a continuous retrograde actin flow from the periphery to the base of growth cones (2)(3)(4)(5)(6)(7). At the membrane level, adhesion proteins form dynamic bonds with immobilized extracellular ligands, allowing step-by-step locomotion (8).…”

Coordinating Neuronal Actin–Microtubule Dynamics

“…The post-translational modification of F-actin adds an additional level of actin network regulation (reviewed in [69]). The unusually large growth cones of Aplysia bag cell neurons have provided the principal model system for the study of growth cone structure and dynamics [15,[70][71][72][73][74][75][76]. The simultaneous assembly of actin filaments at the growth cone edge and disassembly from pointed filament ends throughout the P-domain generate a continuous actin turnover refered to as 'treadmilling' (Figures 1A and 3A) [3,15].…”

“…F-actin disassembly is mediated by a collection of actin-binding proteins that can facilitate actinfilament depolymerisation or severing, such as those belonging to the Mical and actin-depolymerising factor (ADF)/cofilin families [78,79]. The actin monomers subsequently generated can then be translocated, either by diffusion or active transport, towards the cell edge and re-incorporated into polymerising filaments, thus completing the actin treadmill [16,65,76]. Polymerising F-actin filaments at the edge of the growth cone, which can be modelled as elastic Brownian ratchets, produce a protrusive force upon the cell membrane [80].…”