Regulation of Axon Guidance by Second Messengers

描述(申请人提供)：这项研究的长期目标是更好地了解信号转导级联如何导致细胞骨架重排，这是引导轴突生长所必需的。通过细胞外配体与生长锥表面受体的特定相互作用，生长锥导向其靶标。虽然已经确定了一大类可扩散的、细胞表面和细胞外基质(ECM)结合的配体及其受体，但对受体-配体相互作用下游发生的细胞内信号级联和细胞骨架重排知之甚少。特别是，关于生长锥如何整合通过与多个配体相互作用产生的信号的了解很少，这可能发生在发育中的神经系统中分子梯度、指路柱和边界的动态组合中。细胞内信号中间体如钙、cAMP、蛋白酪氨酸激酶和Rho家族GTP酶之间的相互作用可能在调控通路决策中发挥重要作用。这些信号转而组织细胞骨架元件，调节肌动蛋白/微管聚合、膜突起、焦点复合体形成、底物黏附和去黏附，这些都是适当控制生长锥运动的必要过程。我们最近的工作发现，Src家族的激酶是轴突生长的重要介质，而轴突生长受钙激活的钙蛋白的负调控。在这一应用中，我们建议进一步研究Src激酶作为轴突引导信号和细胞骨架动力学效应因子(包括Rho GTP酶及其下游靶点)之间的关键中介的作用。具体来说，我们建议： 1)在活的生长锥体中研究趋化轴突引导信号和经典的第二信使如何调节依赖于Src的酪氨酸磷酸化。 2)检测生长锥体中Src蛋白和CDC42对N-WASP和mDia2的功能调节。 3)确定含磷酸酪氨酸点接触在控制体外生长锥体转动中的作用。更好地了解生长锥体中的信号转导将有助于深入了解发育和神经疾病的分子基础。

DESCRIPTION (provided by applicant): The long-range goal of this research is to better understand how signal transduction cascades lead to cytoskeletal rearrangements that are necessary for guided axon outgrowth. Guidance of growth cones to their targets occurs through specific interactions of extracellular ligands with receptors on the surface of growth cones. While large families of diffusible, cell surface and extracellular matrix (ECM) bound ligands and their receptors have been identified, less is known of the intracellular signaling cascades and cytoskeletal rearrangements that occur downstream of receptor-ligand interactions. In particular, very little is understood about how growth cones integrate signals generated through interactions with multiple ligands, which likely occur in dynamic combinations of molecular gradients, guideposts and boundaries in the developing nervous system. Interactions among intracellular signaling intermediaries such as calcium, cAMP, protein tyrosine kinases and Rho family GTPases are likely to play important roles in regulating pathfinding decisions. These signals in turn organize cytoskeletal elements that regulate actin/microtubule polymerization, membrane protrusion, focal complex formation, substrata adhesion, and de-adhesion, which are all essential processes for proper control over growth cone motility. Our recent work identified Src family kinases as important mediators of axon outgrowth, which are negatively regulated by calcium-activated calpain. In this application we propose to further investigate the role of Src kinases as key intermediaries between axon guidance cues and effectors of cytoskeletal dynamics, which include Rho GTPases and their downstream targets. Specifically, we propose to: 1) Examine in live growth cones how chemotropic axon guidance cues and classic second messengers modulate Src-dependent tyrosine phosphorylation. 2) Test the functional regulation of N-WASP and mDia2 by Src kinases and Cdc42 in growth cones. 3) Determine the role of phosphotyrosine-containing point contacts in the control of growth cone turning in vitro. A better understanding of signal transduction in growth cones will provide insight into the molecular basis of developmental and neurological disorders.