Supramolecular Chemistry And Self-assembly Special Feature: Fibronectin extension and unfolding within cell matrix fibrilscontrolled by cytoskeletal tension

摘要

Evidence is emerging that mechanical stretching can alter the functional states of proteins. Fibronectin (Fn) is a large, extracellular matrix protein that is assembled by cells into elastic fibrils and subjected to contractile forces. Assembly into fibrils coincides with expression of biological recognition sites that are buried in Fn's soluble state. To investigate how supramolecular assembly of Fn into fibrillar matrix enables cells to mechanically regulate its structure, we used fluorescence resonance energy transfer (FRET) as an indicator of Fn conformation in the fibrillar matrix of NIH 3T3 fibroblasts. Fn was randomly labeled on amine residues with donor fluorophores and site-specifically labeled on cysteine residues in modules FnIII7 and FnIII15 with acceptor fluorophores. Intramolecular FRET was correlated with known structural changes of Fn in denaturing solution, then applied in cell culture as an indicator of Fn conformation within the matrix fibrils of NIH 3T3 fibroblasts. Based on the level of FRET, Fn in many fibrils was stretched by cells so that its dimer arms were extended and atleast one FnIII module unfolded. When cytoskeletal tension wasdisrupted using cytochalasin D, FRET increased, indicating refolding ofFn within fibrils. These results suggest that cell-generated force isrequired to maintain Fn in partially unfolded conformations. Theresults support a model of Fn fibril elasticity based on unraveling andrefolding of FnIII modules. We also observed variation of FRET betweenand along single fibrils, indicating variation in the degree ofunfolding of Fn in fibrils. Molecular mechanisms by which mechanicalforce can alter the structure of Fn, converting tensile forces intobiochemical cues, are discussed.