The activation of ezrin–radixin–moesin proteins is regulated by netrin-1 through Src kinase and RhoA/Rho kinase activities and mediates netrin-1–induced axon outgrowth

Department of Anatomy and Cell Biology, McGill University, Montreal H3A 2B2, PQ, Canada.
Molecular biology of the cell (Impact Factor: 4.47). 08/2011; 22(19):3734-46. DOI: 10.1091/mbc.E10-11-0917
Source: PubMed


The receptor Deleted in Colorectal Cancer (DCC) mediates the attractive response of axons to the guidance cue netrin-1 during development. On netrin-1 stimulation, DCC is phosphorylated and induces the assembly of signaling complexes within the growth cone, leading to activation of cytoskeleton regulators, namely the GTPases Rac1 and Cdc42. The molecular mechanisms that link netrin-1/DCC to the actin machinery remain unclear. In this study we seek to demonstrate that the actin-binding proteins ezrin-radixin-moesin (ERM) are effectors of netrin-1/DCC signaling in embryonic cortical neurons. We show that ezrin associates with DCC in a netrin-1-dependent manner. We demonstrate that netrin-1/DCC induces ERM phosphorylation and activation and that the phosphorylation of DCC is required in that context. Moreover, Src kinases and RhoA/Rho kinase activities mediate netrin-1-induced ERM phosphorylation in neurons. We also observed that phosphorylated ERM proteins accumulate in growth cone filopodia, where they colocalize with DCC upon netrin-1 stimulation. Finally, we show that loss of ezrin expression in cortical neurons significantly decreases axon outgrowth induced by netrin-1. Together, our findings demonstrate that netrin-1 induces the formation of an activated ERM/DCC complex in growth cone filopodia, which is required for netrin-1-dependent cortical axon outgrowth.

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    • "Among the ERM proteins, radixin and moesin were enriched in growth cone structure and associated with neurite extensions in the cultured hippocampal neurons [12]. Ezrin was associated with axon outgrowth induced by netrin-1 stimulation [13], however, expression of ezrin was mainly detected in cell body [12]. Therefore, the role of ezrin in the neuronal morphogenesis has remained unclear. "
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    • "Proper synaptic development depends on pathfinding by axonal growth cones (Lowery and Van Vactor, 2009; Kolodkin and Tessier-Lavigne, 2011). Growth cones detect extrinsic cues and respond by actin filament reorganization that drives growth cone motility (Huber et al., 2003; Gallo and Letourneau, 2004; Geraldo and Gordon-Weeks, 2009; Lowery and Van Vactor, 2009). "
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    ABSTRACT: The development of a functioning neural network relies on responses of axonal growth cones to molecular guidance cues that are encountered en route to their target tissue. Nerve growth factor (NGF) and neurotrophin-3 serve as attractive cues for chick embryo sensory growth cones in vitro and in vivo, but little is known about the actin-binding proteins necessary to mediate this response. The evolutionarily conserved ezrin/radixin/moesin (ERM) family of proteins can tether actin filaments to the cell membrane when phosphorylated at a conserved threonine residue. Here we show that acute neurotrophin stimulation rapidly increases active phospho-ERM levels in chick sensory neuron growth cone filopodia, coincident with an increase in filopodial L1 and β-integrin. Disrupting ERM function with a dominant-negative construct (DN-ERM) results in smaller and less motile growth cones with disorganized actin filaments. Previously, we found that NGF treatment increases actin-depolymerizing factor (ADF)/cofilin activity and growth cone F-actin (Marsick et al., 2010). Here, we show this F-actin increase, as well as attractive turning to NGF, is blocked when ERM function is disrupted despite normal activation of ADF/cofilin. We further show that DN-ERM expression disrupts leading edge localization of active ADF/cofilin and free F-actin barbed ends. Moreover, filopodial phospho-ERM levels are increased by incorporation of active ADF/cofilin and reduced by knockdown of L1CAM.Together, these data suggest that ERM proteins organize actin filaments in sensory neuron growth cones and are crucial for neurotrophin-induced remodeling of F-actin and redistribution of adhesion receptors.
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