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.