The role of MRL adaptor protein MIG-10 in neuronal migration in C. elegans

描述(申请人提供)：神经元及其轴突迁移和建立精确连接的能力在神经系统的发育、学习和记忆形成以及修复损伤过程中非常重要。然而，细胞质信号系统允许迁移的神经元对细胞环境中的指导线索做出反应而改变方向的机制还没有被很好地理解。我们实验室的目标是详细了解一种重要的细胞质蛋白--MRL蛋白--如何帮助将细胞表面的指导信号与细胞迁移反应联系起来。在神经元中，MRL蛋白被认为是通过不对称地定位参与肌动蛋白聚合的细胞质蛋白来对指导信号做出反应，从而使轴突沿着所需的方向生长。然而，在T细胞中，MRL蛋白通过激活细胞表面的整合素受体来发挥作用，因此T细胞正在影响而不是简单地对环境做出反应(所谓的“内向外”途径)。我们在线虫的模型系统中有证据表明，MRL蛋白MIG-10不仅可能在神经元中发挥功能，而且在为神经元迁移提供底物的表皮细胞中也具有类似的“内向外”途径。我们在这项研究中的目标是更好地了解MIG-10如何在神经元和表皮细胞中发挥作用，指导线虫的多次迁移。在特定的目标1中，我们建议确定三种MIG-10蛋白亚型中的每一种都在哪些细胞类型中表达，以及它们的表达是功能所必需的。我们将利用含有基因组结构的转基因动物，这些基因组结构包含正确表达和功能异构体所需的所有序列，并使用细胞特异性表达的cDNA结构来挽救功能，使用RNAi结构来破坏功能。为了更好地了解MRL蛋白质的功能途径，我们已经开始在酵母双杂交系统中鉴定与MIG-10物理相互作用的蛋白质。我们关注的是其中一种相互作用的蛋白质，ABI-1，它是肌动蛋白聚合机制的一部分。在特定的目标2中，我们将准确地确定ABI-1与MIG-10共享哪些迁移功能，并使用类似于AIM 1中用于MIG-10的细胞特异性救援/击倒方法来确定ABI-1在哪些细胞类型中起作用。在特定的目标3中，我们将在ABI-1和MIG-10中进行有针对性的缺失，并确定在体外(使用酵母双杂交和细胞培养系统)和在体内(通过创建表达缺失蛋白的转基因并确定它们拯救蠕虫迁移的能力)这两种蛋白质之间需要哪些蛋白质结构域。这里提出的研究将阐明MRL蛋白在神经系统中发挥作用的机制，特别是它们如何与ABI-1相互作用来调节肌动蛋白聚合。 公共卫生相关性：随着神经系统的形成，神经细胞迁移到正确的位置，并相互建立特定的联系，这对纠正神经系统功能至关重要。MiG-10是一种对这些迁移很重要的蛋白质，最近被证明是损伤后神经细胞再生所必需的，而一种密切相关的蛋白质参与了阿尔茨海默病的信号通路。因此，我们对MIG-10蛋白功能的研究将为更好地理解和最终治疗这些疾病提供必要的基本知识。

DESCRIPTION (provided by applicant): The ability of neurons and their axons to migrate and make precise connections is important during development, learning and memory formation, and repair of damage to the nervous system. However, the cytoplasmic signaling systems that allow migrating neurons to change direction in response to guidance cues in the cellular environment are not well understood. The goal of our laboratory is to understand in detail how one important type of cytoplasmic proteins, the MRL proteins, help to connect guidance signals at the cell surface with cellular migratory responses. In neurons, the MRL proteins are thought to respond to guidance cues by asymetrically localizing cytoplasmic proteins involved in actin polymerization, so that the axon will grow out in a desired direction. However, in T cells, the MRL proteins work instead by activating integrin receptors on the cell surface, so that the T cells is affecting rather than simply responding to the environment (the so-called 'inside out' pathway). We have evidence in the model system C. elegans that the MRL protein MIG-10 may function not only in neurons, but also in a similar 'inside out' pathway in the epidermal cells that provide the substrate on which neurons migrate. Our goal in this proposal is to better understand how MIG-10 functions in both neurons and epidermal cells to direct multiple migrations in C. elegans. In Specific Aim 1, we propose to determine in which cell types each of the three MIG-10 protein isoforms is expressed, and also where their expression is required for function. We will utilize transgenic animals containing genomic constructs that contain all the sequences required for correct expression and function of the isoforms, as well as using cell-specific expression of cDNA constructs to rescue function, and RNAi constructs to knock down function. To better understand the pathway in which the MRL proteins function, we have begun to identify proteins that interact physically with MIG-10 in a yeast two hybrid system. We are focusing on one of these interacting proteins, ABI-1, which is known to be part of the actin polymerization machinery. In Specific Aim 2, we will characterize exactly which migration functions ABI-1 shares with MIG-10, and determine in which cell types ABI-1 is functioning, using a cell-specific rescue/knock down approach similar to that used for MIG-10 in Aim 1. In Specific Aim 3, we will make targeted deletions in both ABI-1 and MIG-10, and determine what protein domains are needed for binding between these two proteins in vitro (using the yeast two hybrid and a cell culture system) and in vivo (by creating transgenes expressing deleted proteins and determining their ability to rescue migrations in the worm.) The studies proposed here will clarify the mechanisms by which MRL proteins work in the nervous system, and in particular how they interact with ABI-1 to regulate actin polymerization. PUBLIC HEALTH RELEVANCE: As the nervous system forms, nerve cells migrate to reach their correct locations and make the specific connections with each other that are vital to correct nervous system function. MIG-10, a protein important to these migrations, was recently shown to be required for nerve cell regeneration following damage, while a closely related protein is in a signaling pathway involved in Alzheimer's Disease. Thus, our study of how the MIG-10 protein functions will provide basic knowledge necessary to better understand and eventually treat these conditions.