The vimentin intermediate filament cytoskeleton can quickly sense and respond to stress in the environment, and under certain conditions become altered through post-translational modifications (PTMs) to alter its function and disassemble into soluble oligomers for extracellular release. Extracellular vimentin (eVim) has emerged as a regulator of both physiological and pathological processes, however we lack an in-depth understanding about the molecular mechanisms that regulate eVim function. Using an ex vivo post-cataract surgery model that recapitulates the major features of the lens fibrotic disease, posterior capsule opacification, our studies revealed a role for extracellular, cell-surface vimentin in promoting wound healing and fibrosis. In response to cataract surgery wounding, eVim became released into the extracellular environment, bound to the cell-surface of myofibroblast progenitor cells, linked to causing fibrosis and promoted a fibrotic response to lens injury. The objective of this proposal is to decipher the molecular mechanisms by which eVim drives a pathological fibrotic outcome to lens injury. The central hypothesis is that lens wounding stimulates alterations to the cellular environment leading to the activation of factors that cause changes in intracellular vimentin leading to the release of eVim into the extracellular space, and in the process vimentin has acquired a distinct structural form and molecular characteristics that shape eVim pro-fibrotic function, including the ability of eVim to bind to the cell surface of myofibroblast progenitor cells and signal a pathological outcome to lens injury. The rationale fundamental to this proposal is that completion of these studies will provide important new insight into the molecular regulation of eVim function in driving fibrosis that can be applied to understanding eVim function in other pathogenic processes. The proposed work will also identify key aspects of eVim regulation that may be targeted to prevent a fibrotic outcome to lens injury. The central hypothesis will be tested in the following three specific Aims: 1) Investigate mechanisms mediating vimentin release into the extracellular space and cell- surface binding post-lens wounding, 2) Investigate the mechanisms by which extracellular vimentin signals fibrosis in the lens and 3) Investigate the state of extracellular vimentin linked to driving lens fibrosis post- injury. We will pursue these aims using an innovative combination of molecular and biochemical techniques, including vimentin-linked polyacrylamide gels, a novel tool developed to study how eVim interacts with the surface of cells. The proposed research is significant, because there is a lack of comprehensive understanding about the conditions that regulate vimentin release by cells, binding of eVim to the surface of cells, and the eVim-dependent signaling intermediaries that promote fibrosis. It is also significant because the specific oligomeric form(s) and m...