Application of Novel Optical Methods to Cell Dynamics

项目摘要/摘要 这一优点的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质流动性和相互作用的方法。研究的目的 是正在进行的了解细胞外空间(ECS)决定因素的工作的直接延伸 扩散和体积(Aim 1)、质膜蛋白扩散(Aim 2)和蛋白质-蛋白质 互动(目标3)。在目标1中，将应用新的光学方法来研究受调节的ECS扩散， 脑和肿瘤的体积和离子动态平衡。我们将应用微纤维光学方法，偏振 相关显微镜和钾敏感荧光指示剂研究脑和脑内ECS的调节 肿瘤，解决有关细胞外基质的微粘度、动力学 神经信号转导过程中内皮细胞体积和[K*]的变化及细胞因子的影响 ECS中大分子扩散的拥挤现象。在目标2中，单分子荧光方法将 用于研究膜蛋白平移和旋转扩散的决定因素。 建立在水通道蛋白4(AQP4)水通道的量子点-单粒子跟踪(SPT)研究的基础上， 我们将使用多色SPT和量子棒偏振相关显微镜来研究 AQP4在活细胞中平移和旋转扩散的决定因素，并研究AQP在活细胞中的扩散 迁移细胞的片状脂膜和AQP4超分子组装的决定因素。在《目标3》中， 单分子荧光方法将用于研究蛋白质与蛋白质之间的相互作用。 膜上的水和离子转运体。我们已经实施了蛋白质-蛋白质量化的方法学 活细胞内的相互作用，包括多色SPT、双色FCS和超分辨率显微镜。 基于我们最近对AQP水通道和CFTRCl“通道的研究，我们将研究 AQP4超分子组装的大小和动力学，以及细胞骨架和其他相互作用 AQP4和CFTR的蛋白。这些数据将提供有关分子水平的信息 AQP4和CFTR蛋白-蛋白质相互作用的机制和生物学后果。此外, 建议的研究将建立新的和广泛适用的蛋白质测量方法。 细胞膜和胞外空间的动力学和相互作用。

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.