Project 2: Gelfand; Regulation and Function of Intermediate Filaments in Cell Mechanics

Project 1. Regulation and Function of Intermediate Filaments in Cell Mechanics (Goldman). We are working with the Danuser laboratory to finalize a detailed analysis of vimentin IF-microtubule interactions using 3D-SIM and computer vision analyses with the goal of understanding the templating of MTs by vimentin IF. In collaboration with the Gelfand laboratory we have demonstrated that kinesin-1 via its heavy chain is directly involved in the transport of vimentin IF and we are now attempting to determine its interacting site on vimentin. This study will yield the mechanism of vimentin cargo loading on to this MT-based motor and thereby will permit us to determine the mechanism of targeting of the various assembly forms of vimentin to regulate the local mechanical properties of the cytoplasm. Together with the Janmey laboratory we are carrying out experiments on the role of the juxtanuclear cage of vimentin IF in regulating the mechanical properties and positioning of nuclei. With Dave Weitz and Mark Johnson ( Biomedical Engineering at Northwestern) we are employing combined sharp tip/blunt tip probe atomic force microscopy (AFM), traction force microscopy (TFM), 3D-SIM and optical magnetic twisting cytometry (OMTC) to demonstrate that vimentin IF, through interactions with actin, regulate the stiffness of the cell cortex. In collaboration with Ohad Medalia (University of Zurich) we are finalizing a cryo-EM tomography study on the sub-nanometer structure of native vimentin IF.