Fluorescent single molecule orientation imaging in living cells

活细胞中的荧光单分子定向成像

• 批准号: 8371144
• 负责人: Tomomi Tani
• 金额: $ 40.24万
• 依托单位: MARINE BIOLOGICAL LABORATORY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-20 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8371144
• 关键词: Algorithms; Alzheimer' s Disease; Architecture; Area; Autistic Disorder; Back; Biochemical; Biological; Biological Models; Cells; Cellular Membrane; Cilia; Coupled; Cytokinesis; Cytoskeleton; DNA Sequence Rearrangement; Development; Diffusion; Disease; Distal; Exhibits; Exocytosis; Filament; Fluorescence; Fluorescence Anisotropy; Fluorescence Polarization; Fluorescent Dyes; GTP-Binding Proteins; Huntington Disease; Image; Image Analysis; In Vitro; Individual; Label; Lateral; Learning; Life; Light; Lighting; Link; Lipids; Malignant Neoplasms; Measures; Membrane; Membrane Fluidity; Membrane Proteins; Methods; Molds; Molecular; Molecular Genetics; Molecular Structure; Monitor; Mothers; Neuropathy; Optics; Parkinson Disease; Pheromone Receptors; Phosphatidylinositol 4,5-Diphosphate; Polymers; Procedures; Process; Proteins; Rotation; Saccharomycetales; Sampling; Scaffolding Protein; Signaling Protein; Speed; Structure; System; Systems Analysis; Time; Value Orientations; Vesicle; Work; Yeasts; base; daughter cell; design; fluorescence imaging; fluorophore; imaging probe; in vivo; insight; instrumentation; interest; light microscopy; method development; millisecond; molecular assembly/self assembly; monolayer; mutant; nano; nanometer; research study; scaffold; single molecule; target SNARE proteins; tool

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. PUBLIC HEALTH RELEVANCE: Primary objective of the development of our fluorescent single molecule imaging is to reveal the mechanisms of molecular assembly and functions of septins in living cells. Septin functions are diverse and range from acting as scaffolds to concentrate signaling proteins to forming diffusion barriers that segregate membranes into discrete domains. Misregulation of septins has been implicated in a wide range of neuropathies including Alzheimer's, Parkinsons and Huntington's disease, autism as well as in various cancers. Understanding normal septin organization and assembly is essential to understand how their misfunction promotes disease.

描述(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，以显示监控与感兴趣蛋白质刚性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获取和处理算法的同时，我们将使用该系统监测丝状真菌Ashbya Cotsypii和萌芽酵母中Septin分子的组织。这些模型系统之所以被使用，是因为它们对成像、分子遗传和生化操作具有高度的适应性，并且这些细胞中的隔膜蛋白具有良好的特征。间隔蛋白是细胞骨架中一种高度保守的成分，对胞质分裂和细胞内的区域化至关重要。使用偏振荧光成像方法已经获得了关于隔膜的稳态组织的重要见解，但从未在单分子水平上获得过。作为目标1，我们将开发用于荧光单分子三维取向成像的仪器和探针设计。对于目标2，我们将在体外和活细胞中分析Septin组装和重组的机制。对于目标3，我们将揭示细胞膜上Septins的扩散屏障功能的机制。在实践中，我们预计这些方法的开发和生物应用之间会不断地来回转换。 与公众健康相关：我们开发荧光单分子成像的主要目标是揭示活细胞中间隔蛋白的分子组装和功能机制。Septin的功能是多种多样的，从充当支架到集中信号蛋白，再到形成扩散屏障将膜分离成不同的结构域。广泛的神经疾病包括阿尔茨海默氏症、帕金森氏症、亨廷顿氏症、自闭症以及各种癌症，都涉及到对间隔蛋白的错误调控。了解正常的间隔蛋白的组织和组装对于了解它们的功能障碍如何促进疾病是至关重要的。