Application of Novel Optical Methods to Cell Dynamics

新型光学方法在细胞动力学中的应用

• 批准号: 8827336
• 负责人: ALAN S VERKMAN
• 金额: $ 41.64万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8827336
• 关键词: Address; Biochemistry; Biological; Biology; Brain Neoplasms; Cell membrane; Cells; Color; Crowding; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Development; Diffusion; Drug Delivery Systems; Electrons; Extracellular Matrix; Extracellular Space; Fluorescence; Goals; Homeostasis; In Vitro; Individual; Ion Channel; Ions; Life; Measurement; Measures; Membrane; Membrane Proteins; Membrane Transport Proteins; Methodology; Methods; Microscopy; Modeling; Molecular; Motion; Nutrient; Optical Methods; Pharmaceutical Preparations; Photobleaching; Property; Protein Dynamics; Proteins; Quantum Dots; Regulation; Research; Resolution; Rotation; Signal Transduction; Spectrum Analysis; Tissues; Tumor Tissue; Water; Work; aquaporin 4; aqueous; base; biophysical techniques; brain tissue; cell motility; follow-up; in vivo; insight; macromolecule; millisecond; neurotransmission; novel; particle; protein protein interaction; quantum; retinal rods; single molecule; solute; tumor; water channel

PROJECT SUMMARY/ABSTRACT This MERIT extension remains focused on the development and application of novel fluorescence methods to measure solute mobility and interactions in live cells and tissues. The aims of the research are a direct extension of ongoing work to understand the determinants of extracellular space (ECS) diffusion and volume (Aim 1), plasma membrane protein diffusion (Aim 2) and protein-protein interactions (Aim 3). In Aim 1, novel optical methods will be applied to study regulated ECS diffusion, volume and ionic homeostasis in brain and tumor. We will apply microfiberoptic methods, polarization correlation microscopy, and K'^-sensing fluorescent indicators to study ECS regulation in brain and tumor, addressing questions regarding the microviscosity of the extracellular matrix, the dynamic changes in ECS volume and [K*] during neural signal transduction, and the influence of cellular crowding on macromolecule diffusion in the ECS. In Aim 2, single-molecule fiuorescence methods will be applied to investigate the determinants of membrane protein translational and rotational diffusion. Building on quantum dot-single particle tracking (SPT) studies of aquaporin-4 (AQP4) water channels, we will use multi-color SPT and quantum rod polarization correlation microscopy to investigate the determinants of AQP4 translational and rotation diffusion in live cells, and to study AQP diffusion in lamellipodia of migrating cells and the determinants of AQP4 supramolecular assembly. In Aim 3, single-molecule fluorescence methods will be applied to investigate protein-protein interactions of membrane water and ion transporters. We have implemented methodology to quantify protein-protein interactions in live cells, including multicolor SPT, two-color FCS, and super-resolution microscopy. Building on our recent studies of AQP water channels and CFTR Cl" channels, we will investigate the size and dynamics of AQP4 supramolecular assembly, and the cytoskeletal and other interacting proteins of AQP4 and CFTR. These data will provide molecular-level information about the mechanisms and biological consequences of AQP4 and CFTR protein-protein interactions. In addition, the proposed studies will establish novel and widely applicable methodology to measure protein dynamics and interactions in cell plasma membrane and in the extracellular space.

项目摘要/摘要 该优点扩展仍然集中在新型荧光的开发和应用上 测量活细胞和组织中溶液迁移率和相互作用的方法。研究的目的 是正在进行的工作的直接扩展，以了解细胞外空间的决定因素（EC） 扩散和体积（AIM 1），质膜蛋白扩散（AIM 2）和蛋白质蛋白 互动（目标3）。在AIM 1中，新型的光学方法将应用于研究调节的EC扩散， 脑和肿瘤中的体积和离子稳态。我们将采用微纤维化方法，极化 相关显微镜和K'^ - 感测荧光指标，以研究大脑和 肿瘤，解决有关细胞外基质微度的问题，动态 神经信号转导期间ECS体积和[K*]的变化以及细胞的影响 在EC中的大分子扩散上拥挤。在AIM 2中，将单分子的杂种方法将 应用于研究膜蛋白翻译和旋转扩散的决定因素。 基于Aquaporin-4（AQP4）水通道的量子点点粒子跟踪（SPT）研究 我们将使用多色SPT和量子杆极化相关显微镜研究 活细胞中AQP4转化和旋转扩散的决定因素，并研究AQP扩散 迁移细胞和AQP4超分子组件的决定因素的薄片。在AIM 3中， 单分子荧光方法将应用于研究 膜水和离子转运蛋白。我们已经实施了量化蛋白质蛋白质的方法 活细胞中的相互作用，包括多色SPT，两色FC和超分辨率显微镜。 基于我们最近对AQP水道和CFTR CL“通道的研究，我们将研究 AQP4超分子组件的大小和动力学，以及细胞骨架和其他相互作用 AQP4和CFTR的蛋白质。这些数据将提供有关分子级的信息 AQP4和CFTR蛋白 - 蛋白质相互作用的机理和生物学后果。此外， 拟议的研究将建立新颖且广泛适用的方法来测量蛋白质 细胞质膜和细胞外空间中的动力学和相互作用。