Functional linkages between endocytosis and actin

描述（由申请方提供）：网格蛋白包被的小凹和囊泡介导质膜受体及其配体摄取到细胞中的主要途径，以调节信号转导、免疫应答以及细胞-细胞和细胞底物相互作用。最近的进展使可视化的动态行为的荧光标记的网格蛋白包被的结构（CCS）的质膜上和胞吞作用期间，虽然这种运动的驱动力是未知的。质膜在结构上由下面的皮质肌动蛋白丝细胞骨架支撑，其动态重塑维持并改变细胞的形状。很明显，胞吞囊泡形成过程中质膜的变形一定需要皮层肌动蛋白细胞骨架的局部调节。事实上，我们最近表明，肌动蛋白的组装和拆卸是必不可少的CCS动力学的多个不同方面。网格蛋白介导的内吞作用的复杂动力学是如何与皮质肌动蛋白的组装和拆卸相结合的？这两个高度动态和复杂的细胞机制之间的功能联系是什么？这些不同的，肌动蛋白依赖的CCS动态行为的生理意义是什么？为了解决这些问题，我们已经建立了一个紧密的合作努力，汇集了内吞作用专家与肌动蛋白动力学专家，并结合了我们在细胞生物化学，定量荧光显微镜，计算图像分析和数学建模的技术专长。在目标1中，我们建议开发新的成像检测，轨迹分析和统计指标的方法来分类网格蛋白包被的结构在细胞表面上的不同的运动学，在胞吞作用和新生囊泡形成后。在目标2中，使用全内反射显微镜和定量荧光散斑显微镜，我们将产生第一个高分辨率，互相关，时空地图皮质肌动蛋白和网格蛋白包被的结构动力学在每个运动类，定量研究功能的后果扰动肌动蛋白动态内吞作用和探测的生理意义的动态异质性在CCS。在目标3中，我们将建立协调皮质肌动蛋白动力学与网格蛋白介导的内吞作用的分子机制。该项目将推进我们对我们的长期目标，开发一个生化网络模型的功能之间的联系，内吞作用和肌动蛋白细胞骨架的基础上的网格蛋白涂层结构和皮质肌动蛋白的综合动力学的大型数据库。

DESCRIPTION (provided by applicant): Clathrin coated pits and vesicles mediate the major pathway for uptake of plasma membrane receptors and their ligands into the cell to regulate signal transduction, the immune response and cell-cell and cell substrate interactions. Recent advances have enabled visualization of the dynamic behavior of fluorescently-labeled clathrin coated structures (CCSs) on the plasma membrane and during endocytosis, although the driving forces for this motility are unknown. The plasma membrane is structurally supported by an underlying cortical actin filament cytoskeleton whose dynamic remodeling maintains and changes the shape of the cell. Clearly the deformation of the plasma membrane during endocytic vesicle formation must somehow require concomitant local regulation of the underlying cortical actin cytoskeleton. Indeed, we have recently shown that actin assembly and disassembly are essential for multiple diverse aspects of CCS dynamics. How are the complex dynamics of clathrin-mediated endocytosis integrated with cortical actin assembly and disassembly? What are the functional linkages between these two highly dynamic and complex cellular machineries? What is the physiological significance of these diverse, actin-dependent CCS dynamic behaviors? To address these issues, we have established an intense collaborative effort that brings together experts on endocytosis with experts on actin dynamics and combines our technical expertise in cellular biochemistry, quantitative fluorescence microscopy, computational image analysis and mathematical modeling. In Aim 1, we propose to develop new imaging assays, methods of trajectory analysis and statistical metrics to classify the diverse kinematics of clathrin-coated structures on the cell surface, during endocytosis and after nascent vesicle formation. In Aim 2, using total internal reflection microscopy and quantitative fluorescent speckle microscopy, we will generate the first high-resolution, cross-correlation, spatio-temporal map of cortical actin and clathrin-coated structure dynamics in each kinematic class, to quantitatively examine the functional consequences of perturbing actin dynamics on endocytosis and to probe the physiological significance of the dynamic heterogeneity in CCSs. In Aim 3, we will establish the molecular mechanisms that coordinate cortical actin dynamics with clathrin-mediated endocytosis. The project will advance us towards our long-term objective of developing a biochemical network model of the functional linkage between endocytosis and the actin cytoskeleton based on a large data repository of integrated dynamics of clathrin coated structures and cortical actin.