FIBRONECTIN MUTATIONS DECREASE SECRETION AND EXTRACELLULAR MATRIX ASSEMBLY TO ALTER CELLULAR DIFFERENTIATION IN A NOVEL SKELETAL DYSPLASIA

Abstract

The extracellular matrix (ECM) is composed of a network of fibrillar proteins that serves a critical extension of cells to control their organization and behavior. Proper assembly of the ECM is essential for normal tissue development, as mutations in ECM proteins lead to developmental disorders. The ECM glycoprotein fibronectin (FN) is one such essential protein. In the developing skeleton, FN is a first component assembled by mesenchymal progenitor cells and is necessary for their aggregation and differentiation, a process known as condensation. In part because FN is necessary for life, mutations in FN that cause disease are unprecedented. I present an individual with a unique skeletal dysplasia linked to a novel heterozygous FN mutation. This mutation disrupts a disulfide bond in the assembly domain, an essential site of intermolecular FN-FN binding. Analysis of blood plasma from the affected individual shows¬ decreased circulating FN, with wildtype FN more abundant than mutant. Dermal fibroblasts were isolated from this individual to study FN matrix assembly. Immunofluorescence staining revealed a FN assembly defect due in part to preferential retention of mutant FN in a distended endoplasmic reticulum, accompanied by increased chaperone protein levels. Treatments with protein degradation inhibitors suggest mutant FN is degraded in lysosomes. These FN decreases are sufficient to ablate assembly of fibrillar collagens that rely on a FN matrix, though collagen secretion is unaffected. The gene editing tool CRISPR was used to generate assembly domain mutations in established cell lines and showed these mutations are sufficient to induce the retention and decreased ECM phenotype. We developed a novel model of mouse embryonic stem cell condensation to study the impact of mutations on skeletal differentiation. Stem cells with homozygous FN mutations assemble almost no FN fibrils and are unable to condense unless provided exogenous FN. Analysis of additional mutants suggest condensation failure correlates with the level of secreted FN. A model is proposed in which FN assembly domain mutations decrease FN secretion and matrix assembly, alter condensation and differentiation, and lead to skeletal disease.