Table of Contents
Acknowledgments
Table of Contents
List of Tables
List of Figures
List of Abbreviations
1 Introduction
1.1 Rho GTPase signaling in the nervous system
1.2 RhoGEF proteins regulate Rho GTPase signaling as activators and scaffold proteins
1.3 UNC-73 is a C. elegans RhoGEF with two functional domains
1.4 The model organism Caenorhabditis elegans
1.5 C. elegans locomotory regulation
1.6 Neurotransmission
1.7 Heterotrimeric G protein pathways and neurotransmission
1.8 RhoA/RHO-1 Signaling in neurotransmission
1.9 Overall objectives:
2 Materials And Methods
2.1 Worm strains
2.2 Transgenic lines
2.3 DNA constructs
2.3.1 DNA constructs in C. elegans
2.3.2 Construction of plasmids for protein expression in E. coli
2.3.3 Construction of plasmids for protein expression in HEK293T cells
2.4 Live animal assays
2.4.1 Locomotion assays
2.4.2 Reversal assay
2.4.3 Drug sensitivity assays
2.4.4 PMA treatment
2.4.5 Coelomocyte endocytosis assay
2.4.6 Body length measurement
2.5 Cell specific RNA interference
2.6 Immunoblotting
2.7 Protein preparation and pull-down assays
2.7.1 Protein preparation from E. coli
2.7.2 Protein preparation from HEK293T cells
2.7.3 Pull-down assay
2.8 Microscopy and imaging analysis
3 Results
3.1 UNC-73 RhoGEF2 isoforms are neuronal Rho GEFs required for locomotion
3.1.1 Cell-specific rescue of unc-73 mutant locomotory defects
3.1.2 RHO-1 functions downstream of UNC-73 RhoGEF2 activity in vivo
3.2 UNC-73 RhoGEF2 isoforms modulate cholinergic signaling.
3.3 unc-73 mutants have neuropeptide processing defects
3.4 Activated Gαs pathway mutants suppress unc-73 RhoGEF2 mutant defects
3.4.1 The activated Gαs pathway is required in both muscles and nervous system to fully rescue the locomotory phenotype of unc-73 mutants
3.4.2 Double mutants of unc-73 RhoGEF2 and the activated Gαs pathway are hypersensitive to aldicarb and levamisole
3.4.3 Gαs pathway activation alters neuropeptide levels
3.5 The relationship between UNC-73 and other molecules in the DCV-mediated signaling pathway
3.5.1 rab-2,unc-31 and unc-73 RhoGEF2 mutants have similar slow movement phenotypes that are rescued by Gαs pathway activation
3.5.2 rab-2, unc-31 and unc-73 RhoGEF2 mutants have similar responses to levamisole
3.5.3 unc-73 RhoGEF2, rab-2 and unc-31 mutants respond similarly to PMA treatment
3.5.4 Analysis of unc-73 RhoGEF2 double mutants with egl-3 and tom-1.
3.5.5 gsa-1(gf) and unc-73(lf) mutants have a shorter body length than wildtype
3.6 An examination of the relationship between the UNC-73 RhoGEF2 pathway and glutamate receptor signaling
3.6.1 UNC-73 overexpression increases reversal frequency
3.6.2 GLR-1 is mislocalized in unc-73 mutants
3.7 The UNC-73 RhoGEF2 domain is most similar to mammalian Kalirin, Trio and p63RhoGEF RhoGEF domains
4 Discussion
4.1 UNC-73 RhoGEF2 isoforms and the Gαq pathway
4.2 UNC-73 RhoGEF2 isoforms and the Gαs pathway
4.3 UNC-73 is involved in DCV-mediated secretion pathways
4.4 unc-73 RhoGEF2 mutant aldicarb and levamisole sensitivities
4.5 UNC-73 RhoGEF2 isoform function and GLR-1 localization
4.6 A model of UNC-73/Rho signaling in the regulation of neurotransmission
References
Appendix A UNC-73C1 interacting proteins
Appendix B Cell specific RNAi
