Regulation of Axon Guidance by Second Messengers

描述（由申请人提供）：本研究的长期目标是更好地理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。生长锥通过细胞外配体与生长锥表面受体的特异性相互作用来引导其到达目标。虽然已经发现了大量可扩散的、细胞表面和细胞外基质（ECM）结合的配体及其受体家族，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，对于生长锥如何整合通过与多个配体相互作用产生的信号，人们知之甚少，这些信号可能发生在发育中的神经系统中分子梯度、路标和边界的动态组合中。细胞内信号介质如钙、cAMP、蛋白酪氨酸激酶和Rho家族gtpase之间的相互作用可能在调节寻路决策中发挥重要作用。这些信号反过来组织细胞骨架元件，调节肌动蛋白/微管聚合、膜突出、病灶复合体形成、基质粘附和去粘附，这些都是适当控制生长锥运动的基本过程。我们最近的工作发现Src家族激酶是轴突生长的重要介质，这是由钙激活的钙蛋白酶负调控的。在本申请中，我们建议进一步研究Src激酶作为轴突引导线索和细胞骨架动力学效应器（包括Rho GTPases及其下游靶点）之间的关键中介的作用。具体而言，我们建议：

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.