Mechanisms of axonal pathfinding 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和脊髓损伤后切断神经元的工程成功再生至关重要。虽然已经确定了许多负责促进或抑制生长以及吸引和排斥引导的分子，但我们对轴突运动机制的大部分知识来自对平坦，无特征，坚硬底物的研究。然而，越来越多的证据表明，细胞运动对结构和机械环境是敏感的。相反，在体外和体内复杂环境中，在创造控制轴突生长的结构特征方面已经取得了重大进展，但在大多数情况下，负责调节运动的机制尚不清楚。该研究的目标是利用成像和分析工具在严格控制的机械和结构环境中研究轴突引导，这些工具将以前所未有的细节揭示亚细胞形态和动力学。这些研究将阐明丝状足、局点粘连、机械力和引导因子在受控环境中的作用，并为在体内以可预测的方式影响轴突运动的细胞外环境操作的发展，以及为促进损伤后神经再生的植入材料的工程设计提供重要信息。我们的具体目标是：(1)确定丝状足-胶原纤维相互作用促进轴突运动和3D引导的机制；(2)评估基质成分对生长锥形态、轴突生长和轴突在3D胶原- i基质中的引导的影响；(3)验证轴突被限制在狭窄通道（模拟纤维限制）中的假设，即轴突对非定向引导信号的反应较弱。本研究将利用PI实验室开发的专用高速、高灵敏度旋转盘共聚焦显微镜系统，以及定制的图像分析软件。这项研究是一项跨学科的合作努力，涉及具有细胞生物学、物理学、材料科学和工程学专业知识的研究人员。这一努力将对轴突运动产生一个全新的视角，并将显著提高我们在复杂环境中理解和控制轴突生长的能力。

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 of severed neurons following spinal cord injury. While many of the molecules responsible for enhancing or inhibiting growth and for attractive and repulsive guidance have been identified, the majority of our knowledge of the mechanisms of axon motility comes from studies on flat, featureless, stiff substrates. There is accumulating evidence, however, that cell motility is sensitive to the structural and mechanical environ- ment. Conversely, there has been significant progress in creating structural features that control axon growth in complex environments in vitro and in vivo, but in most cases the mechanisms responsible for the modulation of motility are not known. The goal of the proposed research is to investigate axonal guidance in rigorously controlled mechanical and structural environments using imaging and analysis tools that will reveal subcellular morphology and dynamics with unprecedented detail. These studies will elucidate the roles of filopodia, focal adhesions, mechanical forces and guidance factors in controlled environments, and provides crucial information for the development of manipulations of the extracellular environment in vivo that impact axon motility in predictable ways, and for the engineering of implant materials to promote nerve regeneration after injury. Our specific aims are to: (1) Determine the mechanisms by which filopodia-collagen fibril interactions promote axon motility and guidance in 3D (2) Assess the effects of matrix composition on growth cone morphology, axon outgrowth, and axon guidance in 3D collagen-I matri- ces, and (3) Test the hypothesis that axons confined to narrow lanes, which mimic fibrillar confinement, are less responsive to non-directed guidance cues. This research will utilize a specialized high-speed, high sensitivity spinning disk confocal microscope system developed in the laboratory of the PI, as well as customized image analysis software. The research is an interdisciplinary collaborative effort involving re- searchers with expertise in cell biology, physics, materials science, and engineering. This effort will produce an entirely new perspective on axon motility and should significantly advance our ability to understand and control axon growth in complex environments.