Rho GTPase signaling modulates neurotransmission in Caenorhabditis elegans

Hu, Shuang, "Rho GTPase signaling modulates neurotransmission in Caenorhabditis elegans" (2013). Theses and Dissertations. Paper 100. 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 iv 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) Gα s signaling. Gα s (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. Gα s …