Improving Environment Sensitive Dyes for Live Cell Single Molecule Imaging

项目摘要 本研究培训计划的目的是利用单分子荧光成像技术 与结合了新的环境敏感染料的染料基生物传感器结合以研究GT3信号传导 在吞噬过程中。吞噬作用是我们免疫反应的重要组成部分， 并且诸如细菌和真菌的外来颗粒被巨噬细胞、嗜中性粒细胞或 树突状细胞这导致细胞间信号传导的激活，从而使免疫系统适应性地消除 一个局部的威胁无法包裹某些颗粒（吞噬作用受阻），如石棉和 二氧化硅，已显示导致毒性反应性物质的积累，其可最终导致肺 疾病和癌症。从目标识别到成功或不成功的包裹，吞噬 涉及几个不同的步骤，每一个都调用独特而复杂的信号通路来控制细胞的生长。 必要的细胞骨架重组。Rho GTP酶Cdc 42和Rac 1是Fcγ受体所必需的 介导的吞噬作用，但调节其瞬时定位和激活的机制尚不清楚。 我们建议表征Cdc 42和Rac 1信号传导在受抑过程中的时空动态。 使用两种单分子成像技术的吞噬作用：随机光学分辨率显微镜（STORM） 和单粒子跟踪光活化定位显微镜（sptPALM）。因为这些的分辨率 技术取决于探头的亮度，因为我们希望同时监测定位和 构象的单分子，我们建议利用溶剂敏感的mercury（Mero）染料开发 基于染料的生物传感器，将用于活细胞超分辨率显微镜。我们提出了几个新的 用于改进Mero染料以使其能够用于STORM和sptPALM成像的合成方法。也就是说， 将发生分子内亚胺反应以诱导染料可逆地和自发地闪烁， 实现直接，无添加剂的STORM成像;以及，一种简单的方法，用于提高Mero的光稳定性 (and花青）染料将被开发，其利用高价碘亲电基团转移试剂， 在染料的多烯链上安装保护性吸电子基团。我们将利用非自然的 氨基酸诱变以位点选择性地将优化的Mero染料掺入到结合结构域上， 选择性识别Cdc 42或Rac 1的活性状态，或直接识别GTP酶的位置， 染料在活化时由于构象变化而经历环境变化。这些生物传感器将 可用于研究Cdc 42和Rac 1在IgG识别过程中的定位和激活， 使用STORM和sptPALM的吞噬作用，提供将用于开发新的GTdR模型的数据 在这个重要的免疫过程中。

Project Summary The aim of this research training plan is to utilize single molecule fluorescence imaging techniques coupled with dye based biosensors incorporating novel environment sensitive dyes to study GTPase signaling during phagocytosis. Phagocytosis is a crucial component of our immune response, whereby apoptotic cells and foreign particles such as bacteria and fungi are consumed and degraded by macrophage, neutrophil, or dendritic cells. This results in the activation of cell-to-cell signaling that adapts the immune system to eliminate a localized threat. The inability to encapsulate certain particles (frustrated phagocytosis), such as asbestos and silica, has been shown to result in accumulation of toxic reactive species that can ultimately lead to lung disease and cancer. From target recognition to successful or unsuccessful encapsulation, phagocytosis involves several distinct steps that each invoke unique and complex signaling pathways to control the necessary cytoskeletal restructuring. The Rho GTPases Cdc42 and Rac1 are necessary for Fcγ receptor mediated phagocytosis, but the mechanisms that regulate their transient localization and activation are unclear. We propose to characterize the spatio-temporal dynamics of Cdc42 and Rac1 signaling during frustrated phagocytosis using two single molecule imaging techniques: stochastic optical resolution microscopy (STORM) and single particle tracking photoactived localization microscopy (sptPALM). Because the resolution of these techniques depends on the brightness of the probe, and because we wish to monitor both the localization and conformation of single molecules, we propose to utilize solvent sensitive merocyanine (Mero) dyes to develop dye-based biosensors that will be used in live cell super resolution microscopy. We propose several novel synthetic approaches for improving Mero dyes to enable their use for STORM and sptPALM imaging. Namely, an intramolecular imine reaction will be developed to induce the dyes to reversibly and spontaneously blink, enabling direct, additive-free STORM imaging; and, a simple approach for improving the photostability of Mero (and cyanine) dyes will be developed that utilizes hypervalent iodine electrophilic group transfer reagents to install protective electron withdrawing groups onto the polyene chain of the dyes. We will utilize unnatural amino acid mutagenesis to site-selectively incorporate the optimized Mero dyes onto binding domains that selectively recognize the active states of Cdc42 or Rac1, or directly onto positions of the GTPases where the dyes experience environmental changes due to conformational changes upon activation. These biosensors will be used to study the localization and activation of Cdc42 and Rac1 during IgG recognition and frustrated phagocytosis using STORM and sptPALM, providing data that will be used to develop new models for GTPase activity during this important immune process.