Quantitative Fluorescent Speckle Microscopy

描述(申请人提供)：我们建议继续发展定量荧光散斑显微镜(QFSM)。QFSM是荧光显微镜的一种变体，用于分析活细胞中大分子结构中亚单位的动态变化。在第一轮资助中，我们开发了聚焦稳定的TIRF有限状态机和旋转圆盘共聚焦成像肌动蛋白细胞骨架和组织培养细胞的焦点黏附动力学，并开发了图像分析软件，将随机散斑图像信号转换为肌动蛋白细丝组装、拆卸和运输的高分辨率地图。我们利用这项技术提供的定量信息来研究迁移中的上皮细胞中肌动蛋白细胞骨架的调节，从而建立了细胞如何构建特定的基于肌动蛋白的结构来驱动细胞定向迁移的新范式。在这里，我们试图朝着建立QFSM的长期目标迈出关键的一步，QFSM是一种全面分析活细胞中多个大分子结构的时空动力学和相互作用的方法。我们的具体目标是：目标1：将QFSM扩展到测量聚合物的绝对周转速度，这将使我们能够在活细胞中进行肌动蛋白动力学的原位生物化学。目的2：将QFSM扩展到微管和中间丝细胞骨架的分析。目的3：建立相关的定量有限状态机，探索活细胞中两个或多个大分子结构之间的动态相互作用。这些发展是由我们实验室对细胞骨架功能在细胞迁移中的基本机制进行的假设驱动的细胞生物学研究推动的，但我们的技术是通用的，着眼于它在生物医学研究界的广泛使用。我们建议进一步发展定量荧光散斑显微镜技术，以分析大分子组装的动力学和相互作用。我们的研究将集中于肌动蛋白细丝、微管和中间丝细胞骨架系统动力学的空间和时间整合。这些相互作用集中地牵涉到广泛的细胞功能。因此，拟议的发展将服务于细胞生物学社区，以研究细胞生理学和病理行为的基本方面。

DESCRIPTION (provided by applicant): We propose to continue the development of quantitative Fluorescent Speckle Microscopy (qFSM). qFSM is a variant of fluorescence microscopy to analyze the dynamics of subunits within macromolecular structures in living cells. In the first round of funding, we developed focus-stabilized TIRF FSM and spinning disk confocal imaging of actin cytoskeleton and focal adhesion dynamics in tissue culture cells, and developed image analysis software that converts the stochastic speckle image signal into high- resolution maps of the assembly, disassembly, and transport of the actin filaments. We exploited the quantitative information delivered by this technology to study the regulation of the actin cytoskeleton in migrating epithelial cells and thus established a new paradigm for how the cell builds specific actin-based structures to drive directed cell migration. Here, we seek to make the next critical steps towards our long term goal of establishing qFSM as a method for the comprehensive analysis of spatiotemporal dynamics and interaction of multiple macromolecular structures in living cells. Our specific aims are: Aim 1: To extend qFSM to the measurement of absolute rates of polymer turnover which will allow us to perform in situ biochemistry of actin dynamics in living cells. Aim 2: To extend qFSM to the analysis of microtubule and intermediate filament cytoskeletons. Aim 3: To establish correlational qFSM to probe the dynamic interaction between two or more macromolecular structures in living cells. These developments are motivated by the hypothesis-driven cell biological research in our labs on the fundamental mechanisms of cytoskeletal function in cell migration, but our technology is implemented generically with an eye towards its broad use by the biomedical research community. We propose to further develop the technique of quantitative Fluorescent Speckle Microscopy for the analysis of the dynamics and interaction of macromolecular assemblies. Our studies will focus on the spatial and temporal integration of the dynamics of the actin filament, microtubule, and intermediate filament cytoskeleton systems. These interactions are centrally implicated in a wide array of cell functions. Thus, the proposed developments will serve the cell biology community to investigate fundamental aspects of cell physiology and pathological behaviors.