Netrin-1 Attracts Axons through FAK-Dependent Mechanotransduction

Abstract

The mechanism by which extracellular cues influence intracellular biochemical cascades that guide axons is important, yet poorly understood. Because of the mechanical nature of axon extension, we explored whether the physical interactions of growth cones with their guidance cues might be involved. In the context of mouse spinal commissural neuron axon attraction to netrin-1, we found that mechanical attachment of netrin-1 to the substrate was required for axon outgrowth, growth cone expansion, axon attraction and phosphorylation of focal adhesion kinase (FAK) and Crk-associated substrate (CAS). Myosin II activity was necessary for traction forces >30 pN on netrin-1. Interestingly, while these myosin II-dependent forces on netrin-1 substrates or beads were needed to increase the kinase activity and phosphorylation of FAK, they were not necessary for netrin-1 to increase CAS phosphorylation. When FAK kinase activity was inhibited, the growth cone’s ability to recruit additional adhesions and to generate forces >60 pN on netrin-1 was disrupted. Together, these findings demonstrate an important role for mechanotransduction during chemoattraction to netrin-1 and that mechanical activation of FAK reinforces interactions with netrin-1 allowing greater forces to be exerted.

Formin-2 regulates stabilization of filopodial tip adhesions in growth cones and affects neuronal outgrowth and pathfinding in vivo

“…Filopodial tip adhesions regulate filopodial contractility and the generation of traction forces (Moore et al, 2009(Moore et al, , 2012Romero et al, 2013). We find that Fmn2-depleted filopodia are severely compromised in their ability to generate traction forces, possibly owing to impaired engagement of the cytoskeleton with the ECM.…”

Formin 2 regulates the stabilization of filopodial tip adhesions in growth cones and affects neuronal outgrowth and pathfinding in vivo

“…FAK autophosphorylation at Y397 is central to assembly and mechanotransduction at integrin-based adhesion sites. In growth cones, FAK activity mediates filopodial dynamics, development of adhesions and traction forces and pathfinding (Chacón et al, 2012;Moore et al, 2012;Robles and Gomez, 2006). Fmn2 depletion reduced pFAK (Y397) at the filopodia tips, suggesting impaired adhesive stability, in concurrence with the decreased filopodial lifetimes observed.…”

“…Integrin-dependent signaling, analogous to that seen in nonneuronal cells, has been reported in a few studies (Gomez and Letourneau, 1994;Gomez et al, 1996;Myers and Gomez, 2011) and mechanochemical signaling through focal adhesion kinase (FAK) has been shown to stabilize the early adhesions (Moore et al, 2009(Moore et al, , 2012Robles and Gomez, 2006).…”

The mechanical control of nervous system development

“…Talin and vinculin, which link actin filaments to integrins and which are involved in mechanotransduction (Margadant et al, 2011), are likely to be part of such clutches. During axon outgrowth, FAK is mechanically activated, which reinforces interactions between growth cones and the guidance cue netrin 1 (Moore et al, 2012). Netrin 1, in turn, positively regulates traction forces via Pak1-mediated shootin1 phosphorylation, thus promoting actin-substrate coupling, force generation and axon outgrowth (Toriyama et al, 2013).…”