ABSTRACT Cell migration is essential to many fundamental processes, including embryonic development, wound repair, and cancer metastasis. Central to this process is the cellular cytoskeleton comprised of three polymeric networks: F-actin, microtubules, and intermediate filaments (IFs). Vimentin is an IF protein that is essential to the mechanical resilience of cells and regulates cross-talk amongst the cytoskeleton, but its role in how cells squeeze through small pores in tissues is poorly understood. We have shown that loss of vimentin enhances cell motility through three-dimensional 3D micro-fluidic channels and protects the nucleus from damage during migration. Vimentin is thought to play a distinct role in the transfer of forces from cell surface matrix adhesions to the nuclear surface, but new evidence suggests that vimentin may also play a more active role in persistent cell motility via its interactions with actin stress fiber formation and microtubule positioning. Still, the specific mechanisms by which vimentin enables 3D migration through dense tissue remains unclear. This project addresses the overarching question: how does vimentin influence cytoskeletal functions, adhesions with the extracellular matrix, and cell-cell interactions to coordinate 3D cell migration? To address this question, we are pursuing three sub-projects. First, we are determining the effects of vimentin in cytoskeletal- mediated 3D cell motility. Second, we are identifying vimentin’s role in integrin expression and focal adhesion activation, and third, we are identifying the mechanisms by which vimentin mediates collective cell migration through extracellular matrix networks. To achieve our project goals, we request supplemental funding to acquire a PRIMO photopatterning system to support our studies of vimentin’s role in cell migration through 3D tissue-like environments. We are currently traveling one-way 1-hr from Syracuse to Cornell Nanotechnology Facility in Ithaca NY to use microfabrication equipment, placing undue cost and time barriers for this project. For this project to be successful, we need more access to microfabrication technology to rapidly test and develop designs for cell migration studies, which the PRIMO instrument will provide. We expected that these projects will determine new functional roles of vimentin in cell migration, which has important implications for understanding healthy tissue maintenance and diseases that progress by the migration of cells through tissues.