Spatial Mutation of Membrane Protein Assembly Dynamics Using Nano-Actuators

描述(申请人提供)：血管生成素通过配体诱导的Tie2受体的组装和转位刺激内皮细胞(ECs)的促血管生成信号，并调节ECs中的几个关键过程，包括增殖、迁移和组装。然而，目前尚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，如血管萌发、迁移、分支和网络形成。为了理解具有高度时空复杂性的血管生成的分子机制，必须实时、高分辨率地监测Tie2的时空动力学。在这里，我提出了一种创新的方法，利用一种新的磁等离子体纳米驱动系统来研究TiE_2的动态组装和空间分布变化。与传统显微镜只提供细胞状态的静态评估不同，所提出的系统允许我们在整个血管形成时期以及整个形态发生发展过程中连续可视化信号分子的生物分子事件。此外，通过利用纳米驱动系统的磁钳能力，控制TiE2的空间分布 这使得研究空间极化的Tie2分布如何影响细胞的行为和命运成为可能。具体地说，利用这项新技术，我将挑战以下问题： 1.不同配体如血管生成素刺激后，Tie2的组装状态和分布有何变化？2.Tie2的组装状态是否与不同的亚细胞位置有关？3.Tie2的空间极化分布如何影响细胞的行为？

DESCRIPTION (provided by applicant): Angiopoietins stimulate proangiogenic signals of endothelial cells (ECs) via ligand-induced assembly and translocation of Tie2 receptors and modulate several key processes in ECs including proliferation, migration, and assembly. However, it is still unclear how these biomolecular regulators coordinate highly complex morphogenesis such as vascular sprouting, migration, branching, and network formation. To understand molecular mechanisms of angiogenesis with high spatio-temporal complexity, the spatiotemporal dynamics of Tie2 must be monitored in real-time with high resolution. Here, I propose an innovative approach to investigate dynamic assembly and spatial distribution changes of Tie2 using a new magneto-plasmonic nano-actuation system. Unlike conventional microscopy that only provides a static assessment of cell status, the proposed system allows us to visualize biomolecular events of the signaling molecules continuously over the entire angiogenesis period as well as throughout morphogenetic development. Additionally, by using magnetic tweezing capability of the nano-actuation system, control of Tie2 spatial distributions is possible, enabling the study of how spatially polarized Tie2 distribution influences the cell's behavior and fate. With this new technique, specifically, I will challenge the following questions: 1. How do the assembly state and distribution of Tie2 change after stimulation with various ligands such as angiopoietins? 2. Are the assembly states of Tie2 related to the different subcellular locations such as cell periphery, cell-to-cell contacts, and cell-to-substratum contacts? 3. How do spatially polarized distributions of Tie2 influence a cell's behavior?