Single Molecule Imaging of Guided Axonal Development using Plasmon Nanorulers

描述(申请人提供)：神经生长因子(NGF)和中性营养素等神经营养生长因子通过配体诱导的神经特异性膜受体-酪氨酸激酶(NsmRTK)的二聚体刺激神经细胞(NCS)的前神经性信号，并调节神经细胞中的几个关键过程，包括轴突规范和延长。然而，目前还不清楚这些生物分子调控因子如何协调高度复杂的形态发生来形成神经回路网络。为了理解具有高度时空复杂性的轴突发育的分子机制，需要在具有多变量分析能力的单分子水平上对神经细胞进行实时监测。在这里，我们提出了一种创新的方法，利用原位表面等离子体暗视野显微镜在单分子水平上研究轴突发育的分子机制。与传统显微镜只提供细胞状态的静态评估不同，所提出的表面等离子体显微镜允许我们在轴突发生期间以及形态发生过程中连续地可视化信号分子的单分子事件。利用这项新技术，我将具体挑战以下主题：1.TrkA的原位监测 单个神经细胞的二聚化及其与不同的形态发生(轴突规范和延长)的相关性。2.单个细胞的集中突起激活及其与周围细胞的联系。3.验证人工磁性镊子系统用于引导轴突发育的可行性。

DESCRIPTION (provided by applicant): Neurotropic growth factors such as nerve growth factor (NGF) and neutrophins stimulate proneuritic signals of neural cells (NCs) via ligand-induced dimerizations of neural specific membrane receptor- tyrosine kinases (nsmRTKs) and modulate several key processes in neural cells including axon specification and elongation. However, it is still unclear how these biomolecular regulators coordinate highly complex morphogenesis for the neural circuit network formation. To understand molecular mechanisms of axonal development with high spatio-temporal complexity, it is required to monitor neural cells in real-time at the single molecule level with multivariate analytical capability. Here, we propos an innovative approach to investigate molecular mechanisms of axonal development at the single molecule level using in situ surface plasmon dark field microscopy. Unlike conventional microscopy that only provides a static assessment of cell status, the proposed surface plasmon microscopy allows us to visualize single molecule events of the signaling molecules continuously over the axon generation period as well as morphogenetic development. With this new technique, specifically, I will challenge the following topics: 1. In situ monitoring of TrKA dimerization of individual nerual cells and its correlation with distinct morphogenesis (axon specification and elongation). 2. Focused neurite activation of a single cell and its communication with surrounding cells. 3. Test the feasibility of an artificial magnetic tweezer system for the guided axonal development.