Nucleation of fibronectin fibril assembly requires binding between heparin and the 13th type III module of fibronectin

Fibronectin (FN), a critical component of the extracellular matrix, is assembled into fibrils through a cell-mediated process. Heparan sulfate (HS) binds to the III₁₃ module of FN, and fibroblasts lacking this glycosaminoglycan exhibit reduced FN fibril assembly. To determine if HS depends on III₁₃ to control FN assembly, we deleted both III₁₃ alleles in NIH 3T3 cells using the CRISPR-Cas9 system. ΔIII₁₃ 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 ΔIII₁₃ FN was provided to Chinese hamster ovary (CHO) cells, showing that lack of III₁₃ caused the deficiency in assembly by ΔIII₁₃ cells. Addition of heparin promoted the assembly of wildtype FN by CHO cells, but it had no effect on the assembly of ΔIII₁₃ FN. Furthermore, heparin binding stabilized the folded conformation of III₁₃ and prevented it from self-associating with increasing temperature suggesting that stabilization by HS/heparin binding might regulate interactions between III₁₃ and other FN modules. This effect would be particularly important at matrix assembly sites where our data show that ΔIII₁₃ 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 III₁₃. We conclude that HS/heparin binds to III₁₃ to promote and control the nucleation and development of FN fibrils.