The overall hypothesis of this P01 Grant is that the unique properties of vimentin intermediate filaments (VIF) play a key role in regulating cytoskeletal organization and modulate the micromechanical properties of cells as well as a diverse set of cellular activities, including cell polarization, cell migration, or tissue morphogenesis. The Project Investigators and Core Leaders are all leaders in the field of cell biology and cell mechanics. Over the past funding period collaborative studies have established unique cell reagents and assays leading to key insights into the properties and functions of VIF. These insights provide a strong foundation for this renewal application. In preparation of this application further preliminary results have been collected supporting the feasibility of each of the projects. The projects are interactive conceptually, technically and programmatically, making the aggregate of the projects much greater than the sum of its parts. In Project #1, Dr. Goldman, Northwestern University, will determine the structural interactions among VIF, microtubules (MT) and actin microfilaments (MF) using high resolution microscopic techniques; determine the role of the assembly and disassembly of VIF in wound healing and motility assays; and determine the role of VIF phosphorylation in cellular signal transduction. In Project #2, Dr. Gelfand, Northwestern University will determine the dynamic mechanisms regulating VIF-MT interactions; determine the mechanisms responsible for the dynamic interactions between VIF and MF; and determine how VIF modulate the transport and distribution of membrane-bound organelles. In Project #3, Dr. Danuser, UTSW Dallas, will examine mechanisms by which VIF control MT organization and cell polarity; investigate mechanisms by which VIF control cell traction; and examine mechanisms by which VIF respond to cell-external guidance cues. In Project #4, Dr. Weitz, Harvard University, will determine the properties of reconstituted networks of VIF as well as composite networks comprised either of VIF, MF and myosin motors, or VIF, MT and their associated motors; study the micromechanical properties of VIF networks in living cells in 3D settings and in reconstituted networks derived from these cells. In Project #5, Dr. Janmey, University of Pennsylvania, will determine the mechanisms that regulate force-dependent VIF assembly in cells; study the mechanics of VIF networks under compression in vitro; and determine how VIF regulate the response of cells and tissues to compression loading. Interactions among members of all projects and data sharing will allow for integration of physical characterizations made by different groups using methods unique to their labs that cover a wide range of time and length scales. These efforts will be supported by the Cell and Tissue Core which, under the guidance of Dr. Ridge, Northwestern University, will support all PIs by maintaining the required WT and vimentin null mouse models; by engineering tissue and cell type-specific vimentin knockout animals; by isolating primary cells from various tissues of these animals; and by analyzing gene expression patterns and providing purified proteins as required.

Narrative Intermediate Filaments (IF) are structural proteins involved in determining cell shape, movement and mechanical integrity. Our proposed studies are aimed at understanding their specific functions in the mechanical properties and movement of cells. These basic studies will provide insights into the normal functions of IF and will help explain their defective functions in the many diseases attributed to mutations in the genes encoding the different IF proteins.