Functional linkages between endocytosis and actin

笼状蛋白包被的小凹和囊泡介导了质膜受体和 它们的配体进入细胞，调节信号转导、免疫反应和细胞- 底物相互作用。最近的进展使可视化的动态行为成为可能 在质膜上和在内吞过程中荧光标记的笼状蛋白涂层结构(CCSS)， 尽管这种运动的驱动力尚不清楚。质膜在结构上由 一种潜在的皮质肌动蛋白细丝细胞骨架，其动态重塑维持和改变 单元格的形状。显然，质膜在内吞囊泡形成过程中的变形必须 不知何故，需要伴随着对底层皮质肌动蛋白细胞骨架的局部调节。事实上，我们有 最近发现肌动蛋白的组装和拆解对CCS的多个不同方面是必不可少的 动力学。网状蛋白介导的内吞作用如何与皮质肌动蛋白相结合 组装和拆卸？这两个高度动态的和 复杂的细胞机械？这些不同的、依赖肌动蛋白的细胞的生理意义是什么？ CCS动态行为？为了解决这些问题，我们建立了紧张的合作努力， 汇聚了内吞作用专家和肌动蛋白动力学专家，并结合了我们的技术专长 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，我们建议开发新的成像分析方法，弹道的方法 用分析和统计方法对细胞上笼状蛋白涂层结构的不同运动学进行分类 表面、内吞作用期间和新生囊泡形成后。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， 皮质肌动蛋白和笼蛋白包裹的结构动力学的相互关系、时空图 运动学类，以定量研究摄动肌动蛋白动力学在 并探讨CCSS动态异质性的生理学意义。在目标3中，我们 将建立协调皮质肌动蛋白动力学的分子机制与网状蛋白介导的 内吞作用。该项目将推动我们朝着开发生物化学的长期目标前进 细胞内吞作用与肌动蛋白细胞骨架功能联系的网络模型 笼状蛋白涂层结构和皮质肌动蛋白综合动力学的数据库。

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.