INTERACTIONS BETWEEN HEPARAN SULFATE AND ITS BINDING DOMAIN ON FIBRONECTIN ARE REQUIRED FOR ASSEMBLY OF A FIBRILLAR EXTRACELLULAR MATRIX

Abstract

The extracellular matrix (ECM) is a network of proteins that provides physical scaffolding for cells as well as biochemical and biophysical cues that guide processes such as differentiation and migration. Fibronectin (FN), an essential component of the ECM, is assembled via a cell-mediated process in which integrin receptors bind secreted FN and mediate its polymerization into fibrils that extend between cells, ultimately forming an insoluble matrix. The glycosaminoglycan heparan sulfate (HS) and its binding to FN are essential for the formation of insoluble FN fibrils. In the first part of this study, I investigated the contributions of HS to the early stages of the assembly process using knockdown of exostosin-1 (EXT1), a glycosyltransferase required for HS chain synthesis. NIH 3T3 fibroblasts with decreased EXT1 expression exhibited a significant reduction in both FN and type I collagen fibrils in the insoluble matrix. FN fibril formation is initiated at matrix assembly sites and, while these sites were formed by cells with EXT1 knockdown, their growth was stunted compared to wild type cells. This defect was rescued by the addition of exogenous soluble heparin chains long enough to simultaneously bind multiple FN molecules. The primary heparin-binding site in FN resides in the type III13 module. To determine the requirement for this module during ECM assembly, I constructed an in-frame deletion of III13 in mouse fibroblasts using CRISPR/Cas9 genome editing. Cells homozygous for deletion of III13 assembled a significantly reduced FN matrix. Cells with partial deletion of III13 assembled a FN matrix similar to wildtype fibroblasts. This dosage effect of III13 indicates that HS-binding activity is not required in every subunit of FN but that the absence of binding is detrimental to FN fibrillogenesis. Based on results, I propose a model where heparin/HS binding to III13 is necessary early in assembly for localizing FN molecules at assembly sites, facilitating FN-FN interactions, and eventually forming insoluble fibrils.