Alan 2013

Characterization of atypical small GTPase CHW-1 in C. elegans nervous system development

Jamie K. Alan and Erik A. Lundquist

Department of Molecular Biosciences, University of Kansas, Lawrence, KS, 66045

During development of the nervous system, neurons must first migrate to their final positions and then extend axons in order to establish synaptic connections.  The leading edges of migrating cells and the growth cones of axons undergo dynamic changes in their actin cytoskeletons that mediate migration.  Many studies have shown that Rho GTPases are required for actin cytoskeleton rearrangement and subsequent neuronal migration and axon extension. Rho family proteins are Ras-related small GTPases that regulate cytoskeletal organization and dynamics, cell adhesion, motility, trafficking, proliferation and survival.  Misregulation of Rho proteins can result in defects in cell morphology and cell migration.  These proteins function as tightly regulated molecular switches, cycling between an active GTP-bound state and an inactive GDP-bound state.  Despite  extensive knowledge about regulation and function of the canonical Rho proteins (RhoA/Rac1/Cdc-42) little is known about the contribution of the atypical Cdc-42-like proteins (e.g. Wrch-1 and Chp) to axon pathfinding and neuronal migration.  In C. elegans, CHW-1 resembles human Chp (Wrch-2/RhoV) and Wrch-1 (RhoU), and CRP-1 resembles TC10 and TCL.  My preliminary studies suggest that both CHW-1 and CRP-1 contribute to axon pathfinding and neuronal cell migration.  To test the roles of CHW-1 and CRP-1 in axon pathfinding and cell migration, we assayed the PDE axons and the migrations of the AQR and PQR neurons in chw-1(ok697) and crp-1(ok685) mutants.  We found that loss of chw-1 or crp-1 alone resulted in only modest pathfinding defects in PDE neurons, similar to cdc-42(gk388).  However loss of both cdc-42 and chw-1 or chw-1 and crp-1 resulted in synergistic increases in PDE axon pathfinding defects.  These results suggest that, similar to the Rac-like GTPases CED-10 and MIG-2, the Cdc-42-family GTPases CDC-42, CHW-1, and CRP-1 act redundantly in axon pathfinding.  Preliminary studies indicate that loss of either chw-1 or crp-1 results in modest defects in AQR migration, suggesting that they might redundantly control cell migration as well.  Unlike other Rho GTPases, little is known about the spectrum of regulators, effectors and cellular/biological functions of CHW-1 and CRP-1.  In Aim 1, I will continue to dissect the redundant roles of CDC-42, CHW-1 and CRP-1 in axon pathfinding and cell migration and their effects on growth cones.  In Aim 2, I will identify molecules associated with CHW-1 and regulatory proteins that engage CHW-1. Then in Aim 3, I will determine if proteins that associate with CHW-1 act genetically in the CHW-1 pathway in axon pathfinding and neuronal migration. These studies will provide a greater understanding of the role of C. elegans atypical Cdc-42-like GTPases in neuronal development and migration, and the regulatory mechanisms and signaling pathways involving these proteins.  Pathways identified in these studies will likely be conserved in human neural development.

Department of Molecular Biosciences, University of Kansas, Lawrence, KS, 66045

During development of the nervous system, neurons must first migrate to their final positions and then extend axons in order to establish synaptic connections.  The leading edges of migrating cells and the growth cones of axons undergo dynamic changes in their actin cytoskeletons that mediate migration.  Many studies have shown that Rho GTPases are required for actin cytoskeleton rearrangement and subsequent neuronal migration and axon extension. Rho family proteins are Ras-related small GTPases that regulate cytoskeletal organization and dynamics, cell adhesion, motility, trafficking, proliferation and survival.  Misregulation of Rho proteins can result in defects in cell morphology and cell migration.  These proteins function as tightly regulated molecular switches, cycling between an active GTP-bound state and an inactive GDP-bound state.  Despite  extensive knowledge about regulation and function of the canonical Rho proteins (RhoA/Rac1/Cdc-42) little is known about the contribution of the atypical Cdc-42-like proteins (e.g. Wrch-1 and Chp) to axon pathfinding and neuronal migration.  In C. elegans, CHW-1 resembles human Chp (Wrch-2/RhoV) and Wrch-1 (RhoU), and CRP-1 resembles TC10 and TCL.  My preliminary studies suggest that both CHW-1 and CRP-1 contribute to axon pathfinding and neuronal cell migration.  To test the roles of CHW-1 and CRP-1 in axon pathfinding and cell migration, we assayed the PDE axons and the migrations of the AQR and PQR neurons in chw-1(ok697) and crp-1(ok685) mutants.  We found that loss of chw-1 or crp-1 alone resulted in only modest pathfinding defects in PDE neurons, similar to cdc-42(gk388).  However loss of both cdc-42 and chw-1 or chw-1 and crp-1 resulted in synergistic increases in PDE axon pathfinding defects.  These results suggest that, similar to the Rac-like GTPases CED-10 and MIG-2, the Cdc-42-family GTPases CDC-42, CHW-1, and CRP-1 act redundantly in axon pathfinding.  Preliminary studies indicate that loss of either chw-1 or crp-1 results in modest defects in AQR migration, suggesting that they might redundantly control cell migration as well.  Unlike other Rho GTPases, little is known about the spectrum of regulators, effectors and cellular/biological functions of CHW-1 and CRP-1.  In Aim 1, I will continue to dissect the redundant roles of CDC-42, CHW-1 and CRP-1 in axon pathfinding and cell migration and their effects on growth cones.  In Aim 2, I will identify molecules associated with CHW-1 and regulatory proteins that engage CHW-1. Then in Aim 3, I will determine if proteins that associate with CHW-1 act genetically in the CHW-1 pathway in axon pathfinding and neuronal migration. These studies will provide a greater understanding of the role of C. elegans atypical Cdc-42-like GTPases in neuronal development and migration, and the regulatory mechanisms and signaling pathways involving these proteins.  Pathways identified in these studies will likely be conserved in human neural development.