CHEMOREPULSION MEDIATED NETRIN RECEPTORS UNC5H AND DCC

神经生物学的一个中心问题是确定神经元连接的复杂网络是如何建立的，这些网络是神经系统功能的基础。 当神经元细胞体和它们的轴突在扩散性趋化性线索的引导下迁移到它们的目标时，至少部分地产生了这种复杂的模式。 我的实验室的长期目标是了解细胞如何在梯度的引导线索中检测和定位自己，并将这些信息在细胞内转化为形状和运动的变化，从而导致定向迁移。 Netrin-1是这样的一个指导线索的例子，和指定的两个家庭的受体的引诱和排斥反应netrin-1已被确定。 细胞或轴突对netrin-1的吸引是由DCC（结直肠癌中的轴突）介导的。 在C. elegans证明了远离netrin-1的排斥是由UNC 5受体指定的，并且UNC 5与DCC一起起作用以介导这种排斥。在初步数据中，显示UNC 5 H和DCC物理相互作用以形成驱避蛋白复合物。 基于这些研究，拨款提案的主要假设是，脊椎动物同源物的UNC 5（UNC 5 H），连同DCC，介导netrin-1依赖的化学排斥在脊椎动物体内。为了解决这一假设，本提案的具体目标是双重的。 I）确定UNC 5 H/DCC复合物在指导netrin-1介导的排斥中的体内作用。 研究netrin-1在引导小脑颗粒细胞迁移中的作用。 这些研究检查netrin- 1在体内功能丧失和功能获得的后果，以及小脑颗粒细胞在体外的迁移。 II）为了理解指定排斥相对于吸引的分子机制，将使用酵母双杂交和亲和色谱方法鉴定结合至UNC 5 H并抑制排斥信号的分子。 将在轴突引导或细胞迁移测定中测试这些相互作用蛋白在介导响应于netrin-1的排斥中的功能。 这些实验为理解细胞如何将netrin-1信号转化为定向迁移提供了基础。

A central problem in neurobiology is to determine how the complex networks of neuronal connections, that underlie the functioning of the nervous system, are established. This intricate pattern is generated, at least in part, when neuronal cell bodies and their axons migrate to their targets, guided by diffusible chemotropic cues. The long term goal for my laboratory is to understand how cells detect and orient themselves in gradients of guidance cues, and transduce this information intracellularly into changes in shape and motility that lead to directed migrations. Netrin-1 is an example of such a guidance cue, and two families of receptors that specify attractant and repellent responses to netrin-1 have been identified. The attraction of a cell or axon toward netrin-1 is mediated by DCC (Deleted in Colorectal Carcinoma). Studies in C. elegans demonstrate that repulsion away from netrin-1 is specified by UNC5 receptors, and that UNC5 functions together with DCC to mediate this repulsion. In the preliminary data, it is shown that UNC5H and DCC physically interact to form a repellent protein complex. Based on these studies, the major hypothesis of the grant proposal is that vertebrate homologs of UNC5 (UNC5Hs), together with DCC, mediate netrin-1 dependent chemorepulsion in vertebrates in vivo. To address the hypothesis, the specific aims of this proposal are two-fold. I) To define the in vivo role of the UNC5H/DCC complex in directing netrin-1 mediated repulsion. Studies are proposed on the role of netrin-1 in guiding the migration of cerebellar granule cells. These studies examine the consequences of netrin- 1 loss-of-function and gain-of-function in vivo, and the migration of cerebellar granule cells in vitro. II) To understand the molecular mechanisms that specify repulsion versus attraction, the molecules that bind to UNC5Hs and transduce a repellent signal will be identified using yeast two-hybrid and affinity chromatography approaches. The function(s) of these interacting proteins in mediating repulsion in response to netrin-1 will be tested in axon guidance or cell migration assays. These experiments provide the foundation for understanding how cells translate a netrin-1 signal into directed migrations.