Single Molecule Imaging of Guided Axonal Development using Plasmon Nanorulers

描述（由申请人提供）：神经生长因子（如神经生长因子（NGF）和中性粒细胞）通过配体诱导的神经特异性膜受体酪氨酸激酶（nsmRTKs）的二聚化刺激神经细胞（NCs）的前营养性信号，并调节神经细胞的几个关键过程，包括轴突的特化和伸长。然而，目前尚不清楚这些生物分子调节因子如何协调神经回路网络形成的高度复杂的形态发生。为了了解具有高度时空复杂性的轴突发育的分子机制，需要在单分子水平上实时监测神经细胞，并具备多变量分析能力。在这里，我们提出了一种创新的方法，在单分子水平上使用原位表面等离子体暗场显微镜来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使我们能够在轴突产生期间连续地观察信号分子的单分子事件以及形态发生发育。用这个新技术，具体来说，我将挑战以下主题：1。TrKA的原位监测

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.