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了解信号转导级联如何导致引导轴突生长所必需的细胞骨架重排。通过细胞外配体与生长锥表面受体的特异性相互作用，将生长锥引导至其靶标。虽然已鉴定出大量可扩散的、细胞表面和细胞外基质 (ECM) 结合的配体及其受体，但人们对受体-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，人们对生长锥如何整合通过与多个配体相互作用产生的信号知之甚少，这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。细胞内信号传导中介如钙、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、焦点复合物形成、基质粘附和脱粘附，这些都是正确控制生长锥运动的重要过程。我们最近的工作发现 Src 家族激酶是轴突生长的重要介质，受到钙激活钙蛋白酶的负调节。在此应用中，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 及其下游靶标）之间关键中介的作用。具体来说，我们建议： 1) 检查活体生长锥中趋化性轴突引导信号和经典第二信使如何调节 Src 依赖性酪氨酸磷酸化。 2)测试生长锥中Src激酶和Cdc42对N-WASP和mDia2的功能调节。 3)确定含磷酸酪氨酸的点接触在体外控制生长锥转动中的作用。更好地了解生长锥中的信号转导将有助于深入了解发育和神经系统疾病的分子基础。

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.