The myokine irisin is supposed to be cleaved from a transmembrane precursor, FNDC5 (fibronectin type III domain containing 5), and to mediate beneficial effects of exercise on human metabolism. However, evidence for irisin circulating in blood is largely based on commercial ELISA kits which are based on polyclonal antibodies (pAbs) not previously tested for cross-reacting serum proteins. We have analyzed four commercial pAbs by Western blotting, which revealed prominent cross-reactivity with non-specific proteins in human and animal sera. Using recombinant glycosylated and non-glycosylated irisin as positive controls, we found no immune-reactive bands of the expected size in any biological samples. A FNDC5 signature was identified at ~20 kDa by mass spectrometry in human serum but was not detected by the commercial pAbs tested. Our results call into question all previous data obtained with commercial ELISA kits for irisin, and provide evidence against a physiological role for irisin in humans and other species.

Fibronectin (FN) is secreted as a disulfide-bonded FN dimer. Each subunit contains three types of repeating modules: FN-I, FN-II, and FN-III. The interactions of α5β1 or αv integrins with the RGD motif of FN-III repeat 10 (FN-III10) are considered an essential step in the assembly of FN fibrils. To test this hypothesis in vivo, we replaced the RGD motif with the inactive RGE in mice. FN-RGE homozygous embryos die at embryonic day 10 with shortened posterior trunk, absent tail bud–derived somites, and severe vascular defects resembling the phenotype of α5 integrin–deficient mice. Surprisingly, the absence of a functional RGD motif in FN did not compromise assembly of an FN matrix in mutant embryos or on mutant cells. Matrix assembly assays and solid-phase binding assays reveal that αvβ3 integrin assembles FN-RGE by binding an isoDGR motif in FN-I5, which is generated by the nonenzymatic rearrangement of asparagines (N) into an iso-aspartate (iso-D). Our findings demonstrate that FN contains a novel motif for integrin binding and fibril formation whose activity is controlled by amino acid modification.

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.