Defining the Role of RACK1 in Ephrin Reverse Signaling

发育中的神经细胞必须经常通过大脑迁移，形成控制行为的电路，但这个过程是如何控制的还不清楚。有一组对神经细胞迁移很重要的分子(称为肾上腺素)在哺乳动物中以多种形式存在，这使得研究它们的详细功能变得困难。利用昆虫(包括这里使用的烟草角虫Manduca)产生一种单一类型的EPhin蛋白质这一事实，已经确定了一种在神经细胞发育过程中与Ephin主要相互作用的分子(RACK1)。该项目将确定RACK1如何与EPhin一起调节神经细胞迁移和电路发育。在这个项目中的新发现将提供关于在包括人脑在内的更复杂的动物的神经系统发育过程中电子肾上腺素如何发挥作用的有价值的信息。该项目还将为本科生提供独特的机会，学习研究特定蛋白质如何调控神经细胞生长的高级方法。学生们将在Manduca进行实验，以测试EPhin和RACK1在这一过程中的作用，并确定EPhin功能所需的其他蛋白质。该项目产生的数据和资源将在重要的科学会议上发表，在科学期刊上发表，并发布在其他科学家和公众免费获得的网站上。此外，这个项目的发现将在俄勒冈健康与科学大学脑博览会上向更多的普通观众展示，这是一个每年在俄勒冈科学与工业博物馆举行的互动教育机会。该项目将研究A型肾上腺素(通过GPI脂质锚连接到细胞上)如何调节Src家族中细胞内酪氨酸激酶的活性，以及这一信号通路如何控制发育中神经元的迁移行为。初步研究表明，适配蛋白RACK1(活化蛋白激酶C受体)的功能是一个分子变阻器，它限制了Src信号，直到MsEPhin信号被启动。研究将使用培养的Manduca胚胎中神经元迁移的良好特征分析来进行。对基因表达的有针对性的操作将测试RACK1是否对于A型肾上腺素激活特定的Src激酶(Src42)是必不可少的。对Src活性和Src-RACK1相互作用的生化分析将测试RACK1在这一过程中是否同时作为Src42的调节因子和底物。延时成像和神经元迁移的定量分析将被用来研究这一信号通路如何影响发育中神经系统中神经元行为的特定方面。类似的方法将测试Toll家族蛋白(Toll-7)是否在这一途径中作为A型肾上腺素的新的共同受体发挥作用。这些实验还将允许在相关项目中识别A型肾上腺素的下游效应器，这些项目将由选定的本科生参与者进行。

Developing nerve cells must often migrate through the brain to form the circuits that control behavior, but how this process is controlled is not understood. One group of molecules that are important for nerve cell migration (called Ephrins) exist as multiple types in mammals, making their detailed functions difficult to study. Taking advantage of the fact that insects (including the tobacco hornworm Manduca used here) produce a single type of Ephrin protein, a molecule (RACK1) that Ephrin interacts with in a major way during nerve cell development has been identified. This project will determine how RACK1 works together with Ephrin to regulate nerve cell migration and circuit development. The new discoveries made during this project will provide valuable information about how Ephrins function during nervous system development in more complex animals, including the human brain. The project will also provide unique opportunities for undergraduate students to learn advanced methods for investigating how specific proteins regulate nerve cell growth. Students will conduct experiments in Manduca to test the role of Ephrin and RACK1 in this process, and to identify other proteins that are required for Ephrin function. The data and resources produced by this project will be presented at major scientific conferences, published in scientific journals, and be posted on web sites that are freely available to other scientists and to the public. In addition, discoveries from this project will be presented to more general audiences at the Oregon Health and Science University Brain Fair, an interactive educational opportunity that is held annually at the Oregon Museum of Science and Industry.This project will investigate how type-A Ephrins (which are attached to cells by GPI lipid anchors) can regulate the activity of intracellular tyrosine kinases in the Src family, and how this signaling pathway controls the migratory behavior of developing neurons. Preliminary studies indicate that the adapter protein RACK1 (Receptor of Activated Protein Kinase C) functions as a molecular rheostat that restricts Src signaling until MsEphrin signaling is initiated. Studies will be carried out using a well-characterized assay of neuronal migration in cultured Manduca embryos. Targeted manipulations of gene expression will test whether RACK1 is essential for the activation of a specific Src kinase (Src42) by type-A Ephrins. Biochemical assays of Src activity and Src-RACK1 interactions will test whether RACK1 functions as both a regulator and substrate for Src42 in this process. Time-lapse imaging and quantitative assays of neuronal migration will be used to investigate how this signaling pathway affects specific aspects of neuronal behavior in the developing nervous system. Similar methods will test whether a toll family protein (Toll-7) functions as a novel co-receptor for type-A Ephrins in this pathway. These experiments will also permit the downstream effectors of type-A Ephrins to be identified in related projects that will be conducted by selected undergraduate participants.