While Trio is expressed in axons that run on longitudinal tracts and those that cross the midline, enrichment of this protein is evident in the longitudinal fascicles. Trio is largely localized near the membrane, while cytoplasmic Spg and Sos can be recruited to the membrane by their association with Folinic acid calcium salt pentahydrate N-Cadherin and Robo, respectively. It is not yet clear if membrane recruitment is sufficient to promote Rac activation, or if conserved mechanisms exist to activate GEFs where their activity may be needed. For example, by binding to RhoG, ELMO can target DOCK180 to the membrane. In addition, ELMO binding to DOCK180 relieves a steric inhibition by exposing the DHR-2 domain of DOCK180 that binds Rac. This remains to be shown for other DOCK family members. Next, it is possible that each distinct step of neuronal pathfinding requires a unique set of proteins that allow upstream receptors to signal to downstream proteins for a specific biological output. For example, Trio cooperates with the Abelson tyrosine kinase to promote Rac-dependent actin cytoskeletal dynamics in Frazzled-mediated commissure formation. In the separate process of longitudinal fascicle formation, a trimeric complex of Robo-DOCK-Sos activates Rac to promote axon repulsion. Separately, N-cadherin is suggested to be required for fasciculation and directional growth cone migration. Thus, the Ncad-DOCK-ELMO complex may be responsible for this latter aspect of axonal pathfinding, while other steps may be mediated by individual receptor-GEF complexes. However, additional evidence suggests this regulation may be more complex. Preliminary data from our laboratory Diperodon demonstrates that Ncad may genetically interact with other Rac GEFs to affect earlier CNS development Ncad mutants cannot be rescued by expression of RacWT alone in the CNS. DOCK180 binds the vertebrate receptor Deleted in Colorectal Cancer. In addition, inhibition of DOCK180 activity decreased the activation of Rac1 by Netrin. Another study suggests that Robo is required for multiple, parallel pathways in axon guidance and activated Robo function inactivates Ncadherin-mediated adhesion. Current models suggest activated Robo binds to Abl and N-cadherin, thus providing a mechanism to weaken adhesive interactions during fasciculation to allow for mediolateral positioning of axons along the ventral nerve cord. The association of either Mbc or Spg proteins in the Netrin signaling pathway has not been examined. So far, we have not observed significant differences in genetic combinations that remove either robo or slit in elmo mutants. Furthermore, no significant increases in midline guidance errors were observed in Ncad, elmo mutants, suggesting that Ncad and Spg may function in this process independent of ELMO function. It is clear that additional analysis of Robo and N-Cadherin dynamics are needed in the wellestablished CNS fly model to determine their in vivo relevance. Finally, the physical interactions of GEF proteins with specific membrane receptors may allow the GEFs to be in a unique subcellular localization for post-translational modifications that regulate activity. As mentioned above, DOCK180 is capable of binding and activating Rac when sterically relieved upon ELMO binding. In addition, the presence of ELMO1 inhibits the ubiquitination of DOCK180, thus stabilizing the amount of GEF available to activate Rac. Finally, although the significance is unclear, DOCK180 is phosphorylated upon Integrin binding to the extracellular matrix. Trio has also been shown to be tyrosine phosphorylated upon co-expression with Abl, suggesting this may be a common mechanism for GEF regulation. ELMO is also phosphorylated on tyrosine residues, providing another level of GEF regulation. Further experimentation must be done to determine whether these modifications of GEFs also lead to regulation of Rac activity. Proteins are not static entities, but exist as a dynamic ensemble of inter-converting conformations. These ensembles exhibit a wide range of spatial and temporal scales of internal motions.