A Dissertation entitled Rho GTPase Signaling Modulates Neurotransmission in Caenorhabditis elegans by Shuang Hu Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology Dr. Robert Steven, Committee Chair Dr. Richard Komuniecki, Committee Member Dr. Bruce Bamber, Committee Member Dr. Song-Tao Liu, Committee Member Dr. William Messer, Committee Member Dr. Scott Molitor, Committee Member Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo May 2013 An Abstract of Rho GTPase Signaling Modulates Neurotransmission in Caenorhabditis elegans by Shuang Hu Submitted to the Graduate Faculty as Partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology The University of Toledo May 2013 Rho family GTPases act as molecular switches in the regulation of diverse cellular functions, including cell division, gene transcription and neurotransmission. In the model organism Caenorhabditis elegans, the Kalirin and Trio ortholog UNC-73 contains two RhoGEF domains that specifically activate either Rac or Rho GTPases, respectively. The RhoGEF1 domain and the Rac pathway are required for axon guidance in neuronal development, while the RhoGEF2 domain is involved in the control of locomotion, possibly through the modulation of neurotransmission. The unc-73 gene is expressed as multiple isoforms that contain either one or both RhoGEF domains. This study focuses on defining the function of the UNC-73 RhoGEF2 containing isoforms and the Rho signaling pathway in the adult nervous system of C. elegans. Animals with mutations in the unc-73 RhoGEF2 domain move more slowly than wild-type animals and this locomotory defect can be rescued by pan-neuronal expression of the UNC-73E isoform, which contains only the RhoGEF2 domain. In addition, unc-73 RhoGEF2 mutants are resistant to the cholinesterase inhibitor aldicarb and are hypersensitive to the cholinergic agonist levamisole, without any obvious changes in synaptic structure based on the localization of fluorescence-tagged synaptobrevin. These iii results suggest that the UNC-73 RhoGEF2 isoforms may modulate synaptic strength and cholinergic signaling at the level of the neuromuscular junction. Neuropeptide signaling is also affected in UNC-73 RhoGEF2 mutants. RhoGEF2 mutants expressing the YFP- tagged neuropeptide NLP-21 in cholinergic motorneurons exhibit decreased axonal, but not somal, YFP fluorescence compared to wild-type animals, suggesting NLP-21::YFP packaging into vesicles or transport to the axons is inhibited. YFP fluorescence in the coelomocytes, an indicator of neuropeptide release, is also decreased in these animals. These results suggest the UNC-73 RhoGEF2 isoforms may play a neuromodulatory role in the regulation of locomotion, perhaps by regulating dense core vesicle (DCV)- mediated neuropeptide signaling at the level of axonal transport or neuropeptide packaging. The lethargic locomotory and aldicarb resistant phenotypes of unc-73 RhoGEF2 mutants were bypassed in animals with constitutively active (CA) Gαs signaling. Gαs(CA) expression is required in both muscles and neurons to rescue unc-73 locomotory phenotypes in RhoGEF2 mutants, as the expression in either muscle or neurons alone failed to yield substantial rescue. unc-73 RhoGEF2 mutants exhibited phenotypes similar to rab-2 and unc-31 mutants, both of which encode proteins that are involved in DCV- mediated signaling. Gαs(CA) signaling also rescues unc-31 and rab-2 locomotory defects, supporting our hypothesis that UNC-73 RhoGEF2 signaling may modulate DCV- mediated neurotransmission. Together, these results indicate UNC-73 RhoGEF2 isoforms may regulate cholinergic signaling indirectly by modulating neuropeptide signaling and that the Gαs pathway acts downstream of, or in parallel to, the neuropeptide signaling pathway. iv Acknowledgments First I thank my advisor and mentor Dr. Robert Steven for his help and guidance throughout my graduate career. He gave me a precious opportunity for my Ph.D. study, and taught me many lab skills hands on. He is always willing to give a hand whenever I need help. Thanks him being gentle and patient to me. Second I would like to thank my husband Qing for his constantly support. I would not finish my Ph.D. without his encouragement and help. He also provided professional consultant on statistic problems. I also thank all the lab members in Dr. Steven’s lab, especially Todd Cramer, Thuy Tran, and John Farver for their companions and technical helps. We spent many long hours working together in the lab. I thank my committee members, Dr. Richard Komuniecki, Dr. Bruce Bamber, Dr. Song-Tao Liu, Dr. William Messer, and Dr. Scott Molitor, Committee Member. Dr. Komuniecki gave me many advices on my project, and his joint lab meeting broadened my view during my study. I thank other students in the biological department, Vera Hapiak, Gareth Harris, Yu Zhan, Haiying Li, Aaron Tipton, and Leah Rider who were there to offer me suggestions. Dr. Bruce Bamber kindly let me use the software Velocity for fluorescent signal analysis. Dr. Maria Diakonova provided TR- BSA for my coelomocyte experiment. Dr. Song-Tao Liu and Aaron Tipton shared their vectors and protocols to me for pull-down assay. v Table of Contents Acknowledgments............................................................................................................... v Table of Contents ............................................................................................................... vi List of Tables ...................................................................................................................... x List of Figures .................................................................................................................... xi List of Abbreviations .......................................................................................................... 1 1 Introduction ................................................................................................................... 2 1.1 Rho GTPase signaling in the nervous system ......................................................... 2 1.2 RhoGEF proteins regulate Rho GTPase signaling as activators and scaffold proteins .................................................................................................................... 3 1.3 UNC-73 is a C. elegans RhoGEF with two functional domains ............................ 5 1.4 The model organism Caenorhabditis elegans ...................................................... 10 1.5 C. elegans locomotory regulation ......................................................................... 12 1.6 Neurotransmission ................................................................................................ 14 1.7 Heterotrimeric G protein pathways and neurotransmission .................................. 16 1.8 RhoA/RHO-1 Signaling in neurotransmission ..................................................... 17 1.9 Overall objectives: ................................................................................................ 19 vi 2 Materials And Methods............................................................................................... 20 2.1 Worm strains ......................................................................................................... 20 2.2 Transgenic lines .................................................................................................... 23 2.3 DNA constructs ..................................................................................................... 24 2.3.1 DNA constructs in C. elegans ...................................................................... 24 2.3.2 Construction of plasmids for protein expression in E. coli .......................... 27 2.3.3 Construction of plasmids for protein expression in HEK293T cells ........... 29 2.4 Live animal assays ................................................................................................ 29 2.4.1 Locomotion assays ....................................................................................... 29 2.4.2 Reversal assay .............................................................................................. 29 2.4.3 Drug sensitivity assays ................................................................................. 30 2.4.4 PMA treatment ............................................................................................. 31 2.4.5 Coelomocyte endocytosis assay ................................................................... 31 2.4.6 Body length measurement............................................................................ 32 2.5 Cell specific RNA interference ............................................................................. 32 2.6 Immunoblotting..................................................................................................... 34 2.7 Protein preparation and pull-down assays ............................................................ 34 2.7.1 Protein preparation from E. coli................................................................... 34 2.7.2 Protein preparation from HEK293T cells .................................................... 35 2.7.3 Pull-down assay ........................................................................................... 35 vii 2.8 Microscopy and imaging analysis ......................................................................... 36 3 Results