Molecular Mechanisms Regulating Axon Guidance Receptor Activity

描述（由申请人提供）：随着神经系统的发展，轴突被引导沿着特定的路径找到它们的目标并建立功能连接。精确的连接模式对神经系统的正常功能至关重要。轴突的迁移是由细胞外环境的引导信号引导的。在迁移的轴突表面，受体检测到信号并产生控制细胞骨架动力学的信号，从而指导轴突向何处延伸。已经确定了几种类型的引导信号和受体，包括UNC-6/netrin信号和UNC-40/DCC受体。当一个受体被一个引导线索连接时，决定定向反应的分子机制还不是很清楚。为了探索轴突反应的分子基础，我们在秀丽隐杆线虫中进行了基因筛选，寻找抑制由特定的unc-6错义等位基因rh46引起的轴突引导缺陷的突变。我们的研究结果表明，当UNC-40被UNC-6连接时，我们发现了增强UNC-40信号传导的突变。一个突变是c-38的功能缺失等位基因，它编码一种具有跨膜和细胞外c型凝集素样结构域的蛋白质。我们的初步结果表明，在几种不同的神经元中，ecc -38负调控UNC-40信号。在一种情况下，clec- 38功能的丧失导致神经元细胞体迁移失败，并导致其轴突过早形成依赖于unc -6的细胞。我们还在unc-40外结构域序列中发现了一个错义突变，该突变与unc-6（rh46）结合导致新的轴突迁移模式。当unc-6（rh46）被unc-6（rh46ur282）或unc-6（rh46ur301）替代时，这种反应被抑制，这些等位基因具有抑制原始rh46突变的第二位点突变。这些基因内突变也从筛选中恢复。这些观察结果表明，体内UNC-6和UNC-40外畴之间存在相互作用，并表明受体的结扎确认影响轴突反应的性质。我们建议进一步研究UNC-40信号调控的分子机制。我们计划进一步表征CLEC-38，确定调节UNC-40信号传导的其他成分，从遗传学上剖析控制UNC-40轴突反应的信号传导，并扩展我们的遗传筛选。

DESCRIPTION (provided by applicant): As the nervous system develops, axons are guided along specific pathways to find their targets and make functional connections. The precise pattern of connections is essential for proper nervous system function. Axon migrations are directed by guidance cues in the extracellular environment. On the surface of migrating axons, receptors detect the cues and produce signals that control cytoskeletal dynamics to direct where an axon extends. Several classes of guidance cues and receptors have been identified, including the UNC-6/netrin cue and the UNC-40/DCC receptor. The molecular mechanisms that determine the directional response when a receptor is ligated by a guidance cue are not well understood. To explore the molecular basis of axon responses, we preformed a genetic screen in C. elegans for mutations that suppress axon guidance defects caused by a specific unc-6 missense allele, rh46. Our results indicate that we uncovered mutations that enhance UNC-40 signaling when UNC-40 is ligated by UNC-6. One mutation is a loss-of-function allele of clec-38, which encodes a protein with a transmembrane and extracellular C-type lectin-like domains. Our preliminary results indicate that clec-38 negatively regulates UNC-40 signaling in several different neurons. In one case, loss of clec- 38 function causes the failure of a neuron cell body to migrate and the precocious UNC-6-dependent formation of its axon. We also uncovered a missense mutation within the unc-40 ectodomain sequence that in combination with unc-6(rh46) causes new axons migration patterns. This response is suppressed when unc- 6(rh46) is replaced with unc-6(rh46ur282) or unc-6(rh46ur301), alleles that have second site mutations that suppress the original rh46 mutation. These intragenic mutations were also recovered from the screen. These observations indicate an interaction between UNC-6 and the UNC-40 ectodomain in vivo and they suggest that the ligated confirmation of the receptor influences the nature of the axon response. We propose to further study the molecular mechanisms through which the UNC-40 signals are regulated. We plan to further characterize CLEC-38, determine other components that regulate UNC- 40 signaling, genetically dissect the signaling which controls the UNC-40 axon response, and extend our genetic screening.