Role of msps and tacc during axon guidance

描述（由申请人提供）：准确的轴突寻路是神经系统发育过程中必不可少但又非常复杂的过程。轴突形成复杂功能神经网络的机制仍然是一个主要的谜题，它与理解神经元发育异常如何产生以及神经再生治疗有关。我的长期目标是定义在轴突寻径过程中，在细胞骨架动力学控制水平上整合引导信息的逻辑。作为解决这一问题的起点，我将研究Msps和TACC的作用，这两种微管相关蛋白最近被确定为Abl酪氨酸激酶效应蛋白Orbit的抑制因子，该蛋白调节生长锥中的微管动力学并介导果蝇神经系统中线轴突排斥。我将使用遗传、生化、蛋白质组学和细胞生物学分析来研究生长锥中的Msps、TACC和Orbit功能，以定义轴突引导过程中协调正、负微管动力学的相互作用网络。具体来说，我将：1)定义Msps、TACC和Abl激酶途径之间相互作用的潜在遗传途径（例如Slit、Robo、Orbit），利用果蝇胚胎神经系统中的轴突寻路表型作为实验，验证Msps和TACC与Orbit功能相反的假设，并区分可能的遗传模型；2)利用生物化学和蛋白质组学分析确定Msps/TACC、Orbit和Abl之间是否存在直接的物理相互作用，并扩展和定义果蝇细胞培养系和神经元中参与调节微管动力学的Msps和TACC相互作用网络；3)利用爪蟾生长锥的高分辨率实时成像技术，明确Msps和TACC的细胞作用机制。特别是，我将确定Msps和TACC是否通过促进MT向生长锥前缘延伸而对Orbit发挥功能拮抗作用，或者它们是否对爪蟾生长锥中的MT动力学具有不同的影响。轴突引导异常与多种遗传性神经系统疾病有关，因此这项工作可能揭示这些缺陷是如何产生的以及可能如何预防它们。此外，参与轴突引导的机制被认为会影响神经损伤后轴突的再生能力，因此我们可能能够利用这些信息来设计治疗方法，以允许未来的再生。最后，这里研究的蛋白质在某些癌症中也存在失调。因此，本文提出的研究具有广泛的生物医学意义。

DESCRIPTION (provided by applicant): Accurate axon pathfinding is an essential yet highly complicated process during nervous system development. The mechanisms by which axons form complex functional neuronal networks are still a major puzzle and are relevant to understanding how abnormalities in neuronal development arise and also to nerve regeneration therapeutics. My long-term goal is to define the logic by which guidance information is integrated at the level of cytoskeletal dynamics control during axon pathfinding. As a starting point to address this issue, I will study the role of Msps and TACC, two microtubule-associated proteins which have been recently identified as suppressors of the Abl tyrosine kinase effector protein Orbit which regulates microtubule dynamics in the growth cone and mediates midline axon repulsion in the Drosophila nervous system. I will use genetic, biochemical, proteomic, and cell biological assays to investigate Msps, TACC and Orbit function in the growth cone to define the network of interactions which coordinate positive and negative microtubule dynamics during axon guidance. Specifically, I will: 1) define potential genetic pathways of interaction between Msps, TACC, and the Abl Kinase pathway (e.g. Slit, Robo, Orbit), using axonal pathfinding phenotypes in the Drosophila embryonic nervous system as an assay, testing the hypothesis that Msps and TACC function opposite of Orbit and distinguishing between possible genetic models; 2) use biochemical and proteomic analysis to determine if there are direct physical interactions between Msps/TACC, Orbit, and Abl, as well as to expand and define the Msps and TACC interaction networks involved in regulating microtubule dynamics in a Drosophila cell culture line and in neurons; and 3) define the cellular mechanisms of action of Msps and TACC, using highresolution live imaging in Xenopus growth cones. In particular, I will determine if Msps and TACC play a functionally antagonistic role to Orbit, by promoting MT extension towards the growth cone leading edge, or whether they have a different effect on MT dynamics in Xenopus growth cones. Abnormalities in axon guidance have been associated with multiple hereditary neurological disorders and thus this work may shed light on how these defects arise and possibly how to prevent them. Furthermore, mechanisms involved in axon guidance are thought to influence the ability of axons to regenerate after neural injury and so we may be able to use this information to design treatments to allow regeneration in the future. Finally, the proteins studied here are also misregulated in certain cancers. Thus, the research proposed here is of broad biomedical significance.