Quantitative Fluorescent Speckle Microscopy

描述（由申请方提供）：我们建议继续开发定量荧光散斑显微镜（qFSM）。qFSM是荧光显微镜的变体，用于分析活细胞中大分子结构内亚基的动态。在第一轮资金中，我们开发了组织培养细胞中肌动蛋白细胞骨架和粘着斑动力学的焦点稳定TIRF FSM和旋转盘共聚焦成像，并开发了图像分析软件，该软件将随机散斑图像信号转换为肌动蛋白丝组装、拆卸和运输的高分辨率图。我们利用该技术提供的定量信息来研究迁移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肌动蛋白结构以驱动定向细胞迁移的新范式。在这里，我们寻求使我们的长期目标，建立qFSM作为一种方法，在活细胞中的多个大分子结构的时空动力学和相互作用的综合分析的下一个关键步骤。我们的具体目标是：目标1：将qFSM扩展到测量聚合物周转的绝对速率，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qFSM模型，研究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中关于细胞迁移中细胞骨架功能的基本机制的假设驱动的细胞生物学研究推动的，但我们的技术是在生物医学研究界广泛使用的情况下实现的。我们建议进一步发展定量荧光散斑显微镜技术，用于分析大分子组装体的动力学和相互作用。我们的研究将集中在肌动蛋白丝，微管和中间丝细胞骨架系统的动态的空间和时间的整合。这些相互作用集中地涉及广泛的细胞功能。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.