Rho family GTPases have been implicated in neuronal growth cone guidance; however, the underlying cytoskeletal mechanisms are unclear. We have used multimode fluorescent speckle microscopy (FSM) to directly address this problem. We report that actin arcs that form in the transition zone are incorporated into central actin bundles in the C domain. These actin structures are Rho/Rho Kinase (ROCK) effectors. Specifically, LPA mediates growth cone retraction by ROCK-dependent increases in actin arc and central actin bundle contractility and stability. In addition, these treatments had marked effects on MT organization as a consequence of strong MT-actin arc interactions. In contrast, LPA or constitutively active Rho had no effect on P domain retrograde actin flow or filopodium bundle number. This study reveals a novel mechanism for domain-specific spatial control of actin-based motility in the growth cone with implications for understanding chemorepellant growth cone responses and nerve regeneration.

Actin veil networks assembled by the Arp2/3 complex constrain myosin II–dependent contractility in neuronal growth cones with consequent effects on peripheral retrograde actin flow rates.

5-HT promotes neurite growth via IP3-dependent Ca2+ release in neuronal growth cones. Outgrowth depends on increased rates of actin array treadmilling mediated by Ca2+-calcineurin–dependent cofilin activation. This mode of growth contrasts with substrate-dependent responses, for which retrograde actin flow and advance rates have been inversely correlated.

The small G protein Rac regulates cytoskeletal protein dynamics in neuronal growth cones and has been implicated in axon growth, guidance, and branching. Intracellular Ca(2+) is another well known regulator of growth cone function; however, effects of Rac activity on intracellular Ca(2+) metabolism have not been well characterized. Here, we investigate how Rac1 activity affects release of Ca(2+) from intracellular endoplasmic reticulum (ER) stores stimulated by application of serotonin (5-hydroxytriptamine). We also address how Rac1 effects on microtubule assembly dynamics affect distribution of Ca(2+) release sites. Multimode fluorescent microscopy was used to correlate microtubule and ER behavior, and ratiometric imaging was used to assess intracellular Ca(2+) dynamics. We report that Rac1 activity both promotes Ca(2+) release and affects its spatial distribution in neuronal growth cones. The underlying mechanism involves synergistic Rac1 effects on microtubule assembly and reactive oxygen species (ROS) production. Rac1 activity modulates Ca(2+) by 1) enhancing microtubule assembly which in turn promotes spread of the ER-based Ca(2+) release machinery into the growth cone periphery, and 2) by increasing ROS production which facilitated inositol 1,4,5-trisphosphate-dependent Ca(2+) release. These results cast Rac1 as a key modulator of intracellular Ca(2+) function in the neuronal growth cone.

PKC activation enhances myosin II contractility in the central growth cone domain while decreasing actin density and increasing actin network flow rates in the peripheral domain. This dual mode of action has mechanistic implications for interpreting reported effects of PKC on growth cone guidance and neuronal regeneration.