Netrin Glycosylation Influences Chemotaxis and Haptotaxis

项目总结 定向运动的保真度对发展至关重要。这项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：单一的细胞外形态原如何， 分泌的糖蛋白Netrin-1既起到远程扩散线索的作用，又起到局部粘连线索的作用， 既是一个吸引人的线索，也是一个令人厌恶的线索。Netrin-1在许多器官系统的发育中起关键作用 从无脊椎动物到人类的分支形态发生，在那里它促进了吸引力和排斥性的运动。 然而，无论netrin-1是否作为一种可溶的、趋化的线索和/或作为一种黏附的、趋化的线索发挥作用， 仍在争论之中。这一点意义重大，因为趋化线索可以在很长的距离内引导，而 触觉线索引导着当地，因此它们通过根本不同的方式实现了适当的发展。 我们最近建立了分离和纯化两种不同糖基化形式的netrin-1的方案；我们发现 这些形式中的一种起着吸引人的触觉线索的作用；另一种起着趋化作用的指导作用 这会引起浓度相关的吸引或排斥反应。在这里，我们将从分子上刻画 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在触觉趋化和趋化作用过程中引起不同的反应，最终目标是开发新的 用小鼠模型来操纵网织蛋白糖基化，从而在体内溶解。我们使用生化分析和 在Netrin受体DCC中开发一种新的张力传感器来研究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.