Functional linkages between endocytosis and actin

描述（由申请人提供）：网格蛋白包被的凹坑和囊泡介导质膜受体及其配体进入细胞的主要途径，以调节信号转导、免疫反应以及细胞-细胞和细胞底物的相互作用。最近的进展使荧光标记的网格蛋白包覆结构（CCSs）在质膜上和内吞过程中的动态行为可视化成为可能，尽管这种运动的驱动力尚不清楚。质膜在结构上由底层皮质肌动蛋白丝细胞骨架支撑，细胞骨架的动态重塑维持和改变细胞的形状。很明显，在内吞囊泡形成过程中，质膜的变形一定在某种程度上需要底层皮质肌动蛋白细胞骨架的局部调节。事实上，我们最近已经表明，肌动蛋白的组装和拆卸对于CCS动力学的多个不同方面至关重要。网格蛋白介导的胞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结合在一起的？这两个高度动态和复杂的细胞机器之间的功能联系是什么？这些不同的、依赖于肌动蛋白的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和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方面的技术专长。在目标1中，我们建议开发新的成像分析方法、轨迹分析方法和统计指标，以对细胞表面、内吞过程和新生囊泡形成过程中网格蛋白包被结构的不同运动学进行分类。在目标2中，我们将使用全内反射显微镜和定量荧光散斑显微镜，生成每个运动类中皮质肌动蛋白和网格蛋白包被结构动力学的第一个高分辨率、相互关联的时空图，以定量检查扰动肌动蛋白动力学对内吞作用的功能后果，并探索CCSs中动态异质性的生理意义。在Aim 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。该项目将推进我们的长期目标，即基于网格蛋白包被结构和皮质肌动蛋白的综合动力学的大型数据库，开发胞吞作用和肌动蛋白细胞骨架之间功能联系的生化网络模型。

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.