Netrin Glycosylation Influences Chemotaxis and Haptotaxis

项目摘要 方向运动的准确性对发育至关重要。这一建议解决了一个悬而未决的问题， 仍然是发育神经生物学领域的一个主要僵局：一个单一的细胞外形态原， 分泌的糖蛋白netrin-1，既作为长距离扩散性线索又作为局部粘附性线索， 既有吸引力又有排斥力Netrin-1在许多器官系统的发育中至关重要， 从无脊椎动物到人类的分支形态发生，在那里它促进吸引和排斥运动。 然而，netrin-1是否作为可溶性趋化性线索和/或作为粘附性趋触性线索起作用， 仍在讨论中。这是重要的，因为趋化性线索可以长距离引导，而 触觉线索引导局部，因此它们通过根本不同的手段实现适当的发展。 我们最近建立了分离和纯化netrin-1的两种不同糖基化形式的方案;我们发现， 其中一种形式作为一种吸引力的触趋性线索;另一种作为一种趋化性引导线索 激发依赖于浓度的吸引或排斥反应。在这里，我们将分子表征 这些不同形式的netrin-1与糖分析。我们将研究不同糖基化形式的 netrin-1在趋触性和趋化性过程中引起不同的反应，其最终目标是开发新的 小鼠模型来操纵netrin糖基化和因此在体内的溶解度。我们使用生化分析， 在netrin受体DCC中开发一种新的张力传感器，以研究netrin-1的不同表达方式 分别改变信号转导和机械转导。

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.