SIGNAL TRANSDUCTION IN THE NEURONAL GROWTH CONE

发育中的神经系统的主要功能是建立 适当的突触连接。为此， 产生了一个专门的结构，生长锥，其任务是 区分细胞外引导线索。因此 了解生长锥内的分子机制 将这些信号转化为细胞内反应， 轴突生长一个特别重要的结构是膜骨架 介导细胞骨架和血浆之间的相互作用 膜负责改变生长锥形态。我们有 假设膜骨架作为整合中心 在生长锥中， 转化为更简单的细胞内反应。此外，委员会认为， 很可能生长锥膜骨架的关键成分 是可以受细胞外调节的神经系统特异性蛋白质 引导线索其中一种成分是生长相关蛋白GAPA 3。 GAPA 3的调节涉及几种钙依赖性酶， 介导其磷酸化、去磷酸化和蛋白水解。我们有 提出，了解GAPA 3的这种调节如何影响膜 骨架函数将提供有关 轴突发生和再生过程中生长锥的行为。我们将 因此，研究GAPA 3之间的相互作用的三个方面， 并调节生长锥的膜骨架：第一：Ca 2 +- 依赖钙蛋白酶的蛋白水解。钙蛋白酶是一种重要的调节因子， 膜动力学在许多细胞类型，但它的作用，在生长锥， 未知我们将使用完整的孤立的生长锥和1度 研究钙蛋白酶蛋白水解如何影响相互作用 GAPA 3与膜骨架的关系，以及对生长锥的影响， 膜动力学第二：与肌动蛋白和底物的结合- 相关质膜：我们将使用亲和色谱法， 共沉淀技术表征GAPA 3与肌动蛋白的相互作用 和质膜，然后确定它们是如何被Ca 2 +- 依赖酶第三：在生长过程中GAP-43磷酸化的调节 轴突发生和再生。为了确定GAPA 3磷酸化是否 可以被不同种类的细胞外信号刺激， 使用三叉神经节共培养，其中轴突发生和 在不同条件下均可诱导再生。 这些实验将增加我们对GAP功能的理解， 43在膜骨架内，并将服务于我们的最终目标， 设计策略来规避某些神经元的故障（如 哺乳动物CNS）成功再生。

The major function of the developing nervous system is the establishment of appropriate synaptic connections.Toward this end, the growing axon produces a specialized structure, the growth cone, whose task is to discriminate between extracellular guidance cues. It is therefore necessary to understand the molecular mechanisms within the growth cone that transduce those signals into the intracellular responses that direct axon growth. One particularly important structure is the membrane skeleton that mediates the interactions between the cytoskeleton and plasma membrane responsible for altering growth cone morphology. We have hypothesized that the membrane skeleton acts as an integrative center within growth cones where a multiplicity of diverse guidance cues are transduced into a simpler range of intracellular responses. Furthermore, it is likely that key components of the growth cone membrane skeleton will be nervous-system specific proteins that can be regulated by extracellular guidance cues. One such component is the growth-associated protein GAPA3. Regulation of GAPA3 involves several calcium-dependant enzymes that mediate its phosphorylation, dephosphorylation and proteolysis. We have proposed that understanding how this regulation of GAPA3 affects membrane skeleton functions will provide important information regarding the behavior of the growth cone during axonogenesis and regeneration. We will therefore investigate three aspects of how the interaction between GAPA3 and the membrane skeleton of growth cones is regulated: First: Ca2+- dependent proteolysis by calpain. Calpain is an important regulator of membrane dynamics in many cell types, however its role in growth cones is unknown. We will use both intact isolated growth cones and 1 degree cultures to investigate how calpain proteolysis affects the interactions of GAPA3 with the membrane skeleton, and the consequences on growth cone membrane dynamics. Second: Association with actin and the substrate- associated plasma membrane: We will use affinity chromatography and cosedimentation techniques to characterize GAPA3 interactions with actin and the plasma membrane and then determine how they are regulated by Ca2+- dependent enzymes. Third: Regulation of GAP-43 phosphorylation during axonogenesis and regeneration.To determine whether GAPA3 phosphorylation can be stimulated by different classes of extracellular signals we will use trigeminal ganglion cocultures in which both axonogenesis and regeneration can be induced under different conditions.The results of these experiments will increase our understanding of the function of GAP- 43 within the membrane skeleton, and will serve our eventual goal of designing strategies to circumvent the failure of some neurons (such as mammalian CNS) to regenerate successfully.