AN AXON'S JOURNEY TO FIND ITS PATH: IN VIVO CHARACTERIZATION OF THE MODULATORS AND EFFECTORS OF THE RAC GTPASE SIGNALING PATHWAY INVOLVED IN AXON GUIDANCE

Abstract

The molecular mechanisms leading to axonal guidance are vital for the proper wiring of the nervous system. Many psychiatric disorders may arise from the improper development of the brain. If an axon does not form, or is extended to a place where it is not supposed to be, proper synapses will not form and communication between neurons and neighbor cells will be affected. An axon is extended after the migration of the growth cone. Filopodia and lamellipodia are extended from the growth cone in order to sense the environment for guidance cues. Once signaled, the growth cone migrates to its final target and the axon is developed. A class of proteins called the Rac GTPases is essential for the process of axon pathfinding. In the model nematode Caenorhabditis elegans, MIG-2/RhoG and CED-10/Rac1 are the two redundant Rac GTPases involved in growth cone migration. The main role of Rac GTPases in axon guidance is due to its control over the actin cytoskeleton. The actin-binding UNC-115/abLIM acts downstream of CED-10/Rac1 in axon pathfinding. In Chapters II and III, I describe how Rac signaling activates UNC-115/abLIM. I propose that the scaffolding protein RACK-1 recruits UNC-115/abLIM to the plasma membrane and brings other molecules, such as PKC, for its modulation. Rac GTPases switch between an inactive, GDP-bound form, to an active, GTP-bound form. Activation can be aided by guanine-nucleotide exchange factors (GEFs), and inactivation is enhanced by GTPase activating proteins (GAPs). UNC-73/Trio is a GEF that acts with both MIG-2/RhoG and CED-10/Rac1 in axon guidance. Nevertheless, mutations that disrupt the Rac GEF activity of UNC-73/Trio do not affect axon pathfinding to the same levels as the abolishment of Rac activity seen in the double mutant of mig-2 and ced-10. Along with other evidence, this suggested that there were other molecules involved in the control of Rac GTPases in this process. I performed a candidate-based genetic screen in the C. elegans genome in order to find other DH-containing GEFs that could potentially interact with MIG-2/RhoG and CED-10/Rac1. In Chapter IV, I show that the Rac GEF TIAM-1 acts upstream of MIG-2/RhoG and CED-10/Rac1 as a linker between these GTPases and CDC-42, another member of the Rho subfamily of small GTPases. Moreover, previous studies have implicated Rac GTPases, but not UNC-73/Trio, in the UNC-6/Netrin attractive signaling system. I show that TIAM-1 is the GEF recruited for this system. In Chapter V I show that other GEFs are also involved in this process. The Rac GEF PIX-1/βPIX, and two CDC-42 GEFs, UIG-1/Clg and EXC-5/FGD1, are also involved in the control of Rac GTPases during axon development. In summary, my work has shown how Rac GTPases control the activity of the actin-binding protein UNC-115/abLIM, and how Rac GTPases themselves are controlled during the process of axon guidance.