Axon motility and guidance in three dimensional matrices

描述（由申请人提供）：详细了解控制轴突生长和引导的过程对于理解神经系统的发育以及脊髓损伤后切断的神经元的工程成功再生和重新连接至关重要。 许多负责增强或抑制生长以及吸引和排斥指导的信号通路已经被确定。 然而，我们对轴突运动机制的绝大多数了解来自于对生长锥在平坦、坚硬基底上导航的研究。 有相当多的证据表明，在复杂的，柔韧的，三维（3D）的细胞外环境中的细胞运动可能是非常不同的涂层玻璃盖玻片。 这项探索性研究提案的目标是开发必要的工具来探测三维胶原基质中生长锥的细胞骨架动力学，并启动三维细胞骨架对细胞外指导线索的反应研究。 我们的具体目标是：（1）测量三维胶原凝胶中生长锥细胞骨架动力学。 (2)量化导向因子梯度对三维生长锥细胞骨架动力学的影响。 这些技术的发展，以调查细胞骨架动力学的指导，在3D凝胶将奠定基础，为解决基本问题的生物物理和生化机制，轴突运动的基础，并将奠定基础的发展控制操纵的环境，影响轴突运动的可预测的方式。 我们将能够生成在发育过程中或在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通路，以验证有关轴突指导机制的假设，或测试干预措施以控制轴突生长。

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.