Enzyme-Dependent Initiation of Fibronectin Fibrillogenesis Drives Cell Migration and Mechanosensing

Abstract

Fibronectin (FN) fibrillogenesis is considered to be driven by cytoskeletal forces applied through integrin-FN adhesions. However, FN fibrils are observed within early embryos that are too soft to support high cellular forces, suggesting that an initiation step is required prior to force-induced fibrillogenesis. Previous studies show that Lysyl oxidase-Like-3 (LOXL3) binds to and oxidizes FN, and that loss of LOXL3 leads to improper FN assembly in vivo. Therefore, we set out to test an alternative hypothesis where initiation of FN fibrillogenesis is dependent on the enzymatic activity of Lysyl oxidases rather than on force. To do so we first used cell-free assays to test FN fibrillogenesis in the presence of LOXL3 or LOXL2, followed by multiple cell-based assays to test integrin activation, cell adhesion, migration, and mechanosensing. We find that LOXL2/3 oxidize FN on specific lysines in regions involved in fibrillogenesis. Moreover, HPLC and super-resolution microscopy showed larger FN clusters following FN treatment. Accordingly, cells seeded on treated FN had significantly more mature and organized adhesions compared to control cells and were also significantly more motile. Further, LOXL2/3 were located at the cell leading edge, suggesting that localized secretion of these enzymes might drive persistent migration. We then proceeded to test whether LOXL3 treatment can affect mechanosensing. Indeed, on very soft matrices (0.25kPa), treated FN led to significantly larger cell area and formation of mature adhesions compared to control, and transmission of forces through nascent integrin adhesions was significantly higher. Collectively, these results demonstrate enzyme-dependent force-independent initiation of FN fibrillogenesis, which is translated downstream to force-dependent processes, including adhesion maturation, mechanosensing, and cell migration.