Defining the Role of RACK1 in Ephrin Reverse Signaling

发育中的神经细胞必须经常在大脑中迁移，以形成控制行为的回路，但这一过程是如何控制的还不清楚。一组对神经细胞迁移很重要的分子（称为Ephrin）在哺乳动物中以多种类型存在，使得它们的详细功能难以研究。利用昆虫（包括这里使用的烟草天蛾Manduca）产生单一类型的Ephrin蛋白的事实，已经确定了Ephrin在神经细胞发育期间以主要方式相互作用的分子（RACK 1）。该项目将确定RACK 1如何与Ephrin一起调节神经细胞迁移和回路发育。该项目的新发现将提供有关Ephrin在更复杂的动物（包括人类大脑）神经系统发育过程中如何发挥作用的宝贵信息。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节神经细胞生长的先进方法。学生将在Manduca进行实验，以测试Ephrin和RACK 1在这一过程中的作用，并确定Ephrin功能所需的其他蛋白质。该项目产生的数据和资源将在主要科学会议上介绍，在科学期刊上发表，并张贴在网站上，供其他科学家和公众免费使用。此外，该项目的发现将在俄勒冈州健康与科学大学大脑博览会上向更多的普通观众展示，这是一个互动的教育机会，每年在俄勒冈州科学与工业博物馆举行。（其通过GPI脂质锚附着于细胞）可以调节Src家族中的细胞内酪氨酸激酶的活性，以及这种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，衔接蛋白RACK 1（活化蛋白激酶C受体）作为一个分子变阻器，限制Src信号，直到MsEphrin信号被启动。将使用培养的Manduca胚胎中神经元迁移的充分表征的测定进行研究。基因表达的靶向操作将测试RACK 1是否是A型肝配蛋白激活特定Src激酶（Src 42）所必需的。Src活性和Src-RACK 1相互作用的生物化学测定将测试RACK 1在该过程中是否同时作为Src 42的调节剂和底物发挥作用。神经元迁移的延时成像和定量分析将用于研究这种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）是否在该途径中作为A型肝配蛋白的新型共受体发挥作用。这些实验还将允许A型肝配蛋白的下游效应子在相关项目中被识别，这些项目将由选定的本科参与者进行。

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.