Axon motility and guidance in three dimensional matrices

描述(由申请人提供)：对控制轴突生长和指导的过程的详细了解对于了解神经系统的发育以及在脊髓损伤后成功地设计切断的神经元的再生和连接是必不可少的。许多负责促进或抑制生长以及具有吸引力和排斥性的信号通路已被确定。然而，我们对轴突运动机制的绝大多数知识都来自于对生长锥体在平坦、僵硬的底物上导航的研究。有相当多的证据表明，细胞在复杂、柔韧、三维(3D)的细胞外环境中的运动性可能与镀膜玻璃盖上的截然不同。这一探索性研究计划的目标是开发必要的工具，以探索3D胶原基质中生长锥体的细胞骨架动力学，并启动3D细胞外引导线索对细胞骨架响应的研究。我们的具体目标是：(1)测量3D胶原凝胶中生长锥细胞骨架的动力学。(2)量化引导因子梯度对三维生长锥体细胞骨架动力学的影响。这些研究3D凝胶中引导的细胞骨架动力学的技术的发展，将为解决有关轴突运动基础的生物物理和生化机制的基本问题奠定基础，并将为以可预测的方式影响轴突运动的受控环境操纵的发展奠定基础。我们将能够生成发育过程中或胶质瘢痕中遇到的抑制性线索的模型，分析这些线索影响轴突运动的机制，然后系统地操纵环境和信号转导途径，以验证关于轴突引导机制的假说，或测试控制轴突生长的干预措施。

DESCRIPTION (provided by applicant): A detailed understanding of the processes that control axon growth and guidance is essential for understanding the development of the nervous system and for engineering successful regrowth and reconnection of severed neurons following spinal cord injury. Many of the signaling pathways responsible for enhancing or inhibiting growth and for attractive and repulsive guidance have been identified. However, the vast majority of our knowledge of the mechanisms of axon motility come from studies of growth cones navigating on flat, stiff substrates. There is considerable evidence that cell motility in complex, pliable, three dimensional (3D) extracellular environments can be very different than on a coated glass coverslip. The goal of this exploratory research proposal is to develop the tools necessary to probe the cytoskeletal dynamics of growth cones in 3D collagen matrices and to initiate studies of the cytoskeletal response to extracellular guidance cues in 3D. Our specific aims are to: (1) Measure growth cone cytoskeletal dynamics in 3D collagen gels. (2) Quantify the effect of gradients of guidance factors on growth cone cytoskeletal dynamics in 3D. The development of these techniques to investigate the cytoskeletal dynamics of guidance in 3D gels will lay the groundwork for addressing fundamental questions about the biophysical and biochemical mechanisms that underlie axon motility, and will lay the groundwork for the development of controlled manipulations of the environment that impact axon motility in predictable ways. We will be in a position to generate models of inhibitory cues encountered during development or in the glial scar, analyze the mechanisms by which those cues affect axon motility, and then systematically manipulate the environment and the signal transduction pathways to validate hypothesis about the mechanisms of axon guidance or to test interventions to control axon growth.