Regulation and Function of Intermediate Filaments in Cell Mechanics

描述（由申请人提供）：在本项目中，我们的主要假设是细胞骨架IF的组装状态和力学特性的改变，特别是由vimentin （VIF）组成的III型IF，在响应机械和化学信号的细胞微力学特性调节中发挥重要作用。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元素，但它们在细胞运动中的特定功能尚不清楚。6位项目负责人的研究目标如下：美国西北大学的R. Goldman将确定vientin 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组装和拆卸形成的vientin前体。西北大学的V. Gelfand将确定VIF与微管和肌动蛋白丝之间的关系，确定负责VIF前体易位的分子马达，并研究VIF如何影响细胞骨架动力学和微管（MT）动力学。哈佛大学的G. Danuser将测试以下假设：VIF网络的形成涉及空间分布的装配线，这一过程涉及分子马达，以及VIF网络的组装调节MT和微丝（MF）的组装。dr . Weitz，哈佛大学，将确定体外组装的VIF网络的微观力学特性，这些VIF网络由纯化的vimentin、从细胞中分离的原生VIF网络和活细胞中制备。宾夕法尼亚大学的P. Janmey将研究VIF网络如何影响活细胞的微观力学特性，以及这种特性如何随着基质刚度和外力的变化而变化。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vimentin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，不同的翻译后磷酸化事件的调节功能将被检查。

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.