Fluorescent single molecule orientation imaging in living cells

描述（由申请人提供）：我们将提出一种基于荧光单分子的取向成像来检测分子内结构或分子间组织变化的方法。我们将开发用于偏振荧光成像的强大仪器，展示监测与感兴趣的蛋白质紧密连接的单个荧光团的3D角度变化所需的速度和灵敏度。在开发光学排列和所需的采集和处理算法的同时，我们将使用该系统监测丝状真菌Ashbya gossypii和出芽酵母中septin分子的组织。使用这些模型系统是因为它们非常适合成像，分子遗传和生化操作，并且这些细胞中的septin具有很好的特征。septin是细胞骨架中高度保守的组成部分，对细胞分裂和细胞内区隔化至关重要。使用偏振荧光成像方法获得了关于septin稳态组织的重要见解，但从未在单分子水平上。作为目标1，我们将开发仪器和探针设计成像的荧光单分子的三维定向成像。我们将在体外和活细胞中分析septin组装和重组的机制。在Aim 3中，我们将揭示septin在细胞膜中扩散屏障功能的机制。在实践中，我们期望在这些方法开发和生物应用之间不断地来回转换。

DESCRIPTION (provided by applicant): We will propose a method to detect changes in intramolecular structure or intermolecular organization based on orientation imaging of fluorescent single molecules. We will develop robust instrumentation for polarized fluorescence imaging exhibiting the speed and sensitivity required to monitor 3D angular changes of individual fluorophores that are rigidly connected to proteins of interest. While developing the optical arrangement and required acquisition and processing algorithms, we will use the system to monitor the organization of septin molecules in a filamentous fungus, Ashbya gossypii and in budding yeast. These model systems are used because they are highly amenable to imaging, molecular genetic and biochemical manipulations and the septins in these cells are well characterized. The septins are a highly conserved component of the cytoskeleton that are critical for cytokinesis and intracellular compartmentalization. Important insights have been gained about the steady state organization of septins using polarized fluorescence imaging approaches but never at the single molecule level. As Aim 1 we will develop instrumentation and probe design for imaging the 3D orientation imaging of fluorescent single molecules. For Aim 2 we will analyze the mechanisms of septin assembly and reorganization in vitro and in living cells. For Aim 3, we will reveal the mechanisms of diffusion barrier function of septins in cellular membrane. In practice, we anticipate continuous back-and-forth transitions between those methods development and biological applications.