Functional linkages between endocytosis and actin

网格蛋白包被的小凹和囊泡介导质膜受体摄取的主要途径， 它们的配体进入细胞，以调节信号转导、免疫应答以及细胞-细胞和细胞- 基质相互作用最新的进展已经使可视化的动态行为， 荧光标记的网格蛋白包被结构（CCS）在质膜上和胞吞作用期间， 尽管这种运动性的驱动力是未知的。质膜在结构上由 一种潜在的皮质肌动蛋白丝细胞骨架，其动态重塑维持和改变了细胞骨架的结构。 细胞的形状。很明显，胞吞囊泡形成过程中质膜的变形 在某种程度上需要伴随的潜在皮层肌动蛋白细胞骨架的局部调节。我确已 最近表明，肌动蛋白的组装和拆卸是必不可少的多个不同方面的CCS 动力学网格蛋白介导的内吞作用的复杂动力学如何与皮质肌动蛋白整合 组装和拆卸？这两个高度活跃和 复杂的细胞机制这些多样的、肌动蛋白依赖的 CCS动态行为？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和肌动蛋白动力学专家， 在细胞生物化学，定量荧光显微镜，计算图像分析和 数学建模在目标1中，我们提出开发新的成像检测方法， 对细胞上网格蛋白包被结构的不同运动学进行分类的分析和统计度量 表面，在胞吞作用和新生囊泡形成后。在目标2中，使用全内反射 显微镜和定量荧光斑点显微镜，我们将产生第一个高分辨率， 交叉相关，皮质肌动蛋白和网格蛋白包被的结构动态的时空图，在每个 运动学类，定量研究扰动肌动蛋白动力学对 内吞作用，并探讨CCS的动态异质性的生理意义。在目标3中， 将建立协调皮质肌动蛋白动力学与网格蛋白介导的分子机制， 内吞作用该项目将推动我们实现开发生物化学的长期目标。 网络模型之间的功能联系的内吞作用和肌动蛋白细胞骨架的基础上，一个大的 网格蛋白包被结构和皮层肌动蛋白的综合动力学数据库。

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.