Netrin Glycosylation Influences Chemotaxis and Haptotaxis

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PROJECT SUMMARY Fidelity in directional motility is critical for development. This proposal addresses an outstanding question that remains a major impasse in the field of developmental neurobiology: How does a single extracellular morphogen, the secreted glycoprotein netrin-1, function as both a both a long-range diffusive cue vs a local, adhesive cue, and both an attractive cue and a repulsive cue. Netrin-1 is critical in development of many organ systems and branching morphogenesis from invertebrates to humans, where it promotes both attractive and repulsive motility. However, whether netrin-1 functions as a soluble, chemotactic cue and/or as an adhesive, haptotactic cue remains under debate. This is significant, as chemotactic cues can guide over long distances, whereas haptotactic cues guide locally, and thus they achieve appropriate development via fundamentally distinct means. We recently established protocols to separate and purify two distinctly glycosylated forms of netrin-1; we found that one of these forms functions as an attractive haptotactic cue; the other acts as a chemotactic guidance cue that elicits concentration-dependent attractive or repulsive responses. Here we will molecularly characterize these different forms of netrin-1 with glycoanalytics. We will examine how differentially glycosylated forms of netrin-1 evoke diverse responses during haptotaxis and chemotaxis, with the ultimate goal of developing novel mouse models to manipulation netrin glycosylation and thus solubility in vivo. We use biochemical assays and develop a novel tension sensor in the netrin receptor DCC to investigate how different presentations of netrin-1 alter signal transduction and mechanotransduction, respectively.