Regulation and Function of Intermediate Filaments in Cell Mechanics

项目(摘要-项目(1((高盛)) 在这个项目中，我们正在测试这样一个假设，即 由波形蛋白组成的III型中间丝网是细胞骨架的重要组成部分。 调节细胞的微观机械性能以响应机械应力和信号转导 以及细胞的运动性。波形蛋白是间充质细胞表达的主要中间丝蛋白，是 对细胞迁移、伤口愈合、正常皮肤上皮-间充质转化至关重要 在发育和癌症中，以及细胞对机械应力的反应中。我们建议使用几个 显微技术包括3D结构照明、全内反射和冷冻电子断层扫描 目的：研究细胞内波形蛋白的结构。我们将使用伤口愈合模型来确定过程中的步骤 在成纤维细胞对机械应力的反应中波形蛋白网络的组装/拆解，包括 波形蛋白合成的调控。这些研究还将包括确定和表征 波形蛋白、微管和肌动蛋白应激纤维之间的相互作用，以及 伴局灶性粘连的波形蛋白。不同波形蛋白网络组装状态对整个细胞及细胞周期的影响 亚细胞的力学性能也将通过微观流变学方法和原子力进行检测。 显微镜。最后，波形蛋白磷酸化在网络组装、伤口愈合、与 其他细胞骨架组件和细胞结构，以及细胞力学也将使用 全细胞和生化方法。

Project(Summary—Project(1((Goldman) In this project, we are testing the hypothesis that the assembly states and mechanical properties of the type III intermediate filament network composed of vimentin is an important component of the cytoskeleton in regulating the micromechanical properties of cells in response to mechanical stress and in signal transduction and cell motility. Vimentin is the major intermediate filament protein expressed in mesenchymal cells and is critically important for cell migration, wound healing, the epithelial-mesenchymal-transition in normal development and in cancer, and in the response of cells to mechanical stress. We propose to use several microscopy methods including 3D-structured illumination, total internal reflection, and cryo-electron tomography to investigate the structure of vimentin in cells. We will use a wound healing model to identify steps in the process of vimentin network assembly/disassembly in the response of fibroblasts to mechanical stress, including the regulation of vimentin synthesis. These studies will also include the identification and characterization of interactions among vimentin, microtubules, and actin stress fibers, as well as the details of the interactions of vimentin with focal adhesions. The effects of different vimentin network assembly states on whole cell and subcellular mechanical properties will also be examined by micro-rheological methods and atomic force microscopy. Finally, the role of vimentin phosphorylation on network assembly, wound healing, interactions with other cytoskeletal components and cellular structures, and cellular mechanics will also be examined using both whole cell and biochemical approaches.