Rac1 Modulates Stimulus-evoked Ca2+ Release in Neuronal Growth Cones via Parallel Effects on Microtubule/Endoplasmic Reticulum Dynamics and Reactive Oxygen Species Production

The small G protein Rac regulates cytoskeletal protein dynamics in neuronal growth cones and has been implicated in axon growth, guidance, and branching. Intracellular Ca²⁺ is another well known regulator of growth cone function; however, effects of Rac activity on intracellular Ca²⁺ metabolism have not been well characterized. Here, we investigate how Rac1 activity affects release of Ca²⁺ 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 Ca²⁺ release sites. Multimode fluorescent microscopy was used to correlate microtubule and ER behavior, and ratiometric imaging was used to assess intracellular Ca²⁺ dynamics. We report that Rac1 activity both promotes Ca²⁺ 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 Ca²⁺ by 1) enhancing microtubule assembly which in turn promotes spread of the ER-based Ca²⁺ release machinery into the growth cone periphery, and 2) by increasing ROS production which facilitated inositol 1,4,5-trisphosphate-dependent Ca²⁺ release. These results cast Rac1 as a key modulator of intracellular Ca²⁺ function in the neuronal growth cone.