Search

Fibronectin (FN), a critical component of the extracellular matrix, is assembled into fibrils through a cell-mediated process. Heparan sulfate (HS) binds to the III(13) module of FN, and fibroblasts lacking this glycosaminoglycan exhibit reduced FN fibril assembly. To determine if HS depends on III(13) to control FN assembly, we deleted both III(13) alleles in NIH 3T3 cells using the CRISPR-Cas9 system. ΔIII(13) cells assembled fewer FN matrix fibrils and less DOC-insoluble FN matrix than wildtype cells. Little if any mutant FN matrix was assembled when purified ΔIII(13) FN was provided to Chinese hamster ovary (CHO) cells, showing that lack of III(13) caused the deficiency in assembly by ΔIII(13) cells. Addition of heparin promoted the assembly of wildtype FN by CHO cells, but it had no effect on the assembly of ΔIII(13) FN. Furthermore, heparin binding stabilized the folded conformation of III(13) and prevented it from self-associating with increasing temperature suggesting that stabilization by HS/heparin binding might regulate interactions between III(13) and other FN modules. This effect would be particularly important at matrix assembly sites where our data show that ΔIII(13) cells require both exogenous wildtype FN and heparin in the culture medium to maximize assembly site formation. Our results show that heparin-promoted growth of fibril nucleation sites is dependent on III(13). We conclude that HS/heparin binds to III(13) to promote and control the nucleation and development of FN fibrils.

Fibronectin (FN), an essential component of the extracellular matrix (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. Our previous work using mutant Chinese hamster ovary (CHO) cells identified the glycosaminoglycan heparan sulfate (HS) and its binding to FN as essential for the formation of insoluble FN fibrils. In this study, we investigated the contributions of HS at an early stage of the assembly process using knockdown of exostosin-1 (EXT1), one of the glycosyltransferases required for HS chain synthesis. NIH 3T3 fibroblasts with decreased EXT1 expression exhibited a significant reduction in both FN and type I collagen in the insoluble matrix. We show that 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 with wild-type cells. The most severe defect observed was in the polymerization of nascent FN fibrils, which was reduced 2.5-fold upon EXT1 knockdown. This defect was rescued by the addition of exogenous soluble heparin chains long enough to simultaneously bind multiple FN molecules. The activity of soluble heparin in this process indicates that nascent fibril formation depends on HS more so than on the protein component of a specific HS proteoglycan. Together, our results suggest that heparin or HS is necessary for concentrating and localizing FN molecules at sites of early fibril assembly.