Regulation and Function of Intermediate Filaments in Cell Mechanics

描述（由申请人提供）：在本计划项目资助中，我们的首要假设是细胞骨架IF（特别是由波形蛋白（VIF）组成的III型IF）的组装状态和机械特性的改变在调节细胞响应机械和化学信号的微机械特性方面发挥重要作用。这些研究是至关重要的，因为IF是哺乳动物细胞骨架系统的主要成分，但它们在细胞运动中的具体功能仍然未知。6位课题负责人及其研究目标如下：R.西北大学的Goldman将确定波形蛋白IF（VIF）系统如何响应机械和化学信号而变化，并将纯化和表征VIF组装和分解时形成的波形蛋白前体。V. Gelfand将确定VIF与微管和肌动蛋白丝之间的关系，确定负责易位VIF前体的分子马达，并研究VIF如何影响细胞骨架动力学和微管（MT）动力学。G. Danuser，哈佛大学，将测试的假设，即VIF网络的形成涉及一个空间分布的装配线，这一过程涉及分子马达，以及VIF网络组装调制组装的MT和微丝（MF）。D. Weitz，哈佛大学，将确定在体外组装的VIF网络从纯化的波形蛋白，天然VIF网络分离的细胞和活细胞制备的微观力学性能。宾夕法尼亚大学的P. Janmey将确定VIF网络如何对活细胞的微机械特性做出贡献，以及这种特性如何响应基底刚度和外力而变化。康涅狄格大学的P. Burkhard将使用生物物理技术和X射线晶体学来确定波形蛋白二聚体的结构和生物物理特性，并研究VIF组装中间体的形成。在所有这些研究中，将检查不同的翻译后磷酸化事件的调节功能。 公共卫生相关性：中间丝（IF）是参与决定细胞形状，运动和机械完整性的结构蛋白。然而，它们的具体功能仍然未知。我们提出的研究旨在了解他们的具体功能的运动和机械性能的细胞。这些基础研究将提供对它们正常功能的深入了解，并将有助于解释它们在许多疾病中的功能缺陷，这些疾病归因于编码不同IF蛋白的基因突变。

DESCRIPTION (Provided by applicant): In this Program Project Grant our overarching hypothesis is that alterations in the assembly states and mechanical properties of cytoskeletal IF, specifically the type III IF composed of vimentin (VIF), play important roles in regulating the micromechanical properties of cells in response to mechano- and chemo- signaling. These studies are critically important as IF are major elements of the cytoskeletal system of mammalian cells and yet their specific functions in cell motility remain unknown. The 6 Project Leaders, and their research aims are as follows: R. Goldman, Northwestern University, will determine how the vimentin IF (VIF) system changes in response to mechanical and chemical signals, and will purify and characterize the vimentin precursors that form as VIF assemble and disassemble. V. Gelfand, Northwestern University will determine the relationship between VIF with microtubules and actin filaments, identify the molecular motors responsible for translocating VIF precursors, and investigate how VIF affect cytoskeletal dynamics and microtubule (MT) dynamics. G. Danuser, Harvard University, will test the hypotheses that VIF network formation involves a spatially distributed assembly line, that this process involves molecular motors, and that VIF network assembly modulates assembly of MT and microfilaments (MF). D. Weitz, Harvard University, will determine the micromechanical properties of in vitro assembled VIF networks prepared from purified vimentin, native VIF networks isolated from cells and in living cells. P. Janmey, University of Pennsylvania, will determine how the VIF network contributes to the micromechanical properties of living cells and how this changes in response to substrate stiffness and external forces. P. Burkhard, University of Connecticut, will use biophysical techniques and X-ray crystallography to determine the structure and biophysical properties of the vimentin dimer and study the formation of VIF assembly intermediates. In all of these studies, the regulatory function of different posttranslational phosphorylation events will be examined. PUBLIC HEALTH RELEVANCE: Intermediate Filaments (IF) are structural proteins involved in determining cell shape, movement and mechanical integrity. However, their specific functions remain unknown. We propose studies aimed at understanding their specific functions in the movement and mechanical properties of cells. These basic studies will provide insights into their normal functions and will help explain their defective functions in the many diseases attributed to mutations in the genes encoding the different IF proteins.