Generation of novel Fn1-fluorescent fusion proteins in vivo for the investigation of cell_ECM dynamics in real time.

Project Summary ECM components play integral roles in regulating cell fates and response to injury; therefore, elucidating the dynamic nature of cell-extracellular matrix (ECM) interactions is of fundamental importance to understand how changes in the composition and architecture of ECM regulate physiological and pathological processes. However, our views of ECM architecture, remodeling and interactions with cells are largely based on static pictures of fixed tissues. Moreover, we don?t understand how changes in ECM composition, architecture and dynamics are interrogated by cells, and how ECM is modified in response to cellular activities. To address these challenges, we generated novel tools to visualize ECM synthesis, assembly, remodeling, and dynamics using CRISPR-mediated genetic engineering, and developed a novel approach to modify the genomic locus of the fibronectin (Fn1) gene to generate a variety of Fn1-fluorescent protein (FP) fusion proteins. This approach has allowed us to visualize Fn1 secretion and fibril assembly while retaining the native regulation of Fn1 expression and splicing in model primary mouse cells, mouse embryonic fibroblasts (MEFs). This R21 application is being submitted in response to the PA-16-141, Development of Animal Models and Related Biological Materials for Research, and outlines our proposal to generate knock-in mice expressing fluorescently tagged Fn1. Our preliminary data show that we have succeeded in modifying the Fn1 genomic locus to generate Fn1-FP fusions proteins, and demonstrate our advanced imaging capabilities to study ECM dynamics and remodeling. Completion of experiments proposed in this grant, will allow to visualize ECM dynamics in real time in live animals, and analyze dynamic changes in ECM in development and disease.