Identification of proteins that interact with the SAX-3/Robo receptor in Caenorhabditis elegans

Axon guidance is a specialized cell movement in which only the growth cone of the neuronal cell navigates to its target in response to various attractive and repulsive cues. Among different types of cellular receptors that respond to these cues, SAX-3 is a C. elegans neuronal receptor belonging to the conserved Roundabout family of proteins that mainly mediates repulsion. The proposed ligand for SAX-3 is SLT 1/Slit, and both sax-3 and slt-1 mutants exhibit axon guidance defects. Genetic and/or biochemical assays have indicated that VAB-1/Eph, UNC-40/DCC, and SDN-1/Syndecan can form co-receptors with SAX-3. However, the downstream signaling pathway is largely unknown, i.e., what signaling cascade does SAX-3 trigger upon binding to its ligand either through putative co-receptor complexes or alone? The only identified downstream effector so far is the actin regulator, UNC-34/Enabled, and also known are two upstream regulators, VAB-8L and UNC-73/Trio. To search for more potential SAX-3 binding partners, I employed several rounds of yeast two hybrid screens. Three baits of different intracellular regions of SAX-3 were used. After sequencing 55 positive isolates, few of them have been isolated more than two times, implying that they are too risky to pursue.; Protein-protein interactions (PPIs) play a pivotal role in cell signaling. Numerous assays have been developed to study regular PPIs. However, there are only a few assays for studying membrane PPIs and each of them have some limitations that hamper their applications. To help solve this problem, I designed a novel strategy to show membrane protein-protein interactions. Having experienced some difficulty in mammalian cell culture and C. elegans, I successfully implemented this protein-protein interaction assay in the yeast Saccharomyces cerevisiae.