Dynamics and elasticity of the fibronectin matrix in living cell culture visualized by fibronectin–green fluorescent protein

Abstract

Fibronectin (FN) forms the primitive fibrillar matrix in both embryos and healing wounds. To study the matrix in living cell cultures, we have constructed a cell line that secretes FN molecules chimeric with green f luorescent protein. These FN-green f luorescent protein molecules were assembled into a typical matrix that was easily visualized by f luorescence over periods of several hours. FN fibrils remained mostly straight, and they were seen to extend and contract to accommodate movements of the cells, indicating that they are elastic. When fibrils were broken or detached from cells, they contracted to less than one-fourth of their extended length, demonstrating that they are highly stretched in the living culture. Previous work from other laboratories has suggested that cryptic sites for FN assembly may be exposed by tension on FN. Our results show directly that FN matrix fibrils are not only under tension but are also highly stretched. This stretched state of FN is an obvious candidate for exposing the cryptic assembly sites.Assembly of the fibronectin (FN) matrix has been studied most extensively in cell cultures, in which a network of extended fibrils is demonstrated by antibody staining. The matrix consists of interconnected fibrils up to 1 m or more in diameter. Electron microscopy shows that these fibrils are bundles of thinner filaments, Ϸ5 nm in diameter, and that the fibrils can vary from Ϸ10 nm in diameter (and contain only a few filaments) to 100-1,000 nm in diameter (and contain many parallel filaments) (1, 2). The 5-nm diameter of the thin filaments is close to the Ϸ3-nm diameter of individual FN molecules (3), but the exact molecular arrangement of molecules within filaments and fibrils is not known.Visualizing the FN matrix by immunofluorescence requires fixation of the cultures and does not reveal dynamics of a living culture. Green fluorescent protein (gfp) has been used as a tag to localize many intracellular proteins in living cells. Visualization of the cytoskeleton has been particularly dramatic, and localization of proteins to the nucleus or specialized membranous compartments has had many applications. Surprisingly, we were unable to find any references using gfp to localize extracellular matrix proteins. It seemed a useful approach and feasible, and, indeed, a recent study reported localization of the protein SPARC-gfp in Caenorhabditis elegans (4). This study and our localization of FN-gfp reported below suggest that gfp should be generally useful to localize extracellular matrix molecules.To visualize the matrix in living cultures, we have made chimeras of FN and gfp. An eventual goal is to follow the assembly of the matrix, starting with freshly plated cells. In preliminary observations of more established matrices, we observed surprising movements of the FN-matrix fibrils that suggest an elasticity never before demonstrated. We report here the design of the successful FN-gfp chimera and the observations of matrix fibril elasticity.

Fibronectin extension and unfolding within cell matrix fibrils controlled by cytoskeletal tension

“…Proteins such as titan and tenascin have been unfolded by using atomic force microscopy and optical tweezers (18,19), but the question of whether cells unfold proteins or protein modules under physiological conditions remains unanswered (14,20). By measuring FRET between donor and acceptor f luorophores conjugated to Fn, we were able to investigate the effect of cell-generated tension on the structure of Fn in fibrillar matrices.…”

“…High-resolution structures of FnIII modules have shown that different modules are structurally homologous and measure 3.2 nm from N to C terminus (22)(23)(24)(25). Because FRET is limited to donors and acceptors located within 10 nm of each other (26), only donors within modules FnIII 5-9 and FnIII [13][14][15] are sufficiently close to acceptors located within FnIII 7 and FnIII 15 to permit energy transfer. From 2 M to 4 M Gdn⅐HCl, FnIII modules unfold sequentially, beginning with the least stable modules (16,27).…”

“…It has been shown that cell contractility is required for assembly of Fn into fibrils (7)(8)(9)(10)(11), and that stretching exposes cryptic sites embedded in the protein (12,13). Furthermore, Fn fibrils are highly elastic and subject to cell-generated tension (14). Thus, Fn is an excellent model protein for investigating how protein function is controlled by matrix assembly and stretching.…”

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

“…It is currently believed that Fn fibers can be stretched three-to four-fold beyond their resting length (Ohashi et al, 1999). This conclusion was derived from various cell culture measurements of the lengths of chimeric GFP or YFP-Fn fibers before and after breakage or detachment of fibers under cell-dependent strain, from fiber rupture from laser damage, or after inhibition of cell contractility with cyto-chalasin B.…”

“…The conformations Fn assumes on synthetic surfaces, however, are rather distinct from those of native Fn in soluble and fibrillar forms (reviewed in Antia et al, 2006;Baugh and Vogel, 2004;Halter et al, 2005). Fn polymerization into fibers is thought to proceed as cryptic self-assembly sites are exposed by cell contractility (Baneyx and Vogel, 1999;Mao and Schwarzbauer, 2005;Ohashi et al, 1999;Zhong et al, 1998).…”