Quantifying the relation between adhesion ligand–receptor bond formation and cell phenotype

Abstract

One of the fundamental interactions in cell biology is the binding of cell receptors to adhesion ligands, and many aspects of cell behavior are believed to be regulated by the number of these bonds that form. Unfortunately, a lack of methods to quantify bond formation, especially for cells in 3D cultures or tissues, has precluded direct probing of this assumption. We now demonstrate that a FRET technique can be used to quantify the number of bonds formed between cellular receptors and synthetic adhesion oligopeptides coupled to an artificial extracellular matrix. Similar quantitative relations were found between bond number and the proliferation and differentiation of MC3T3-E1 preosteoblasts and C2C12 myoblasts, although the relation was distinct for each cell type. This approach to understanding 3D cell-extracellular matrix interactions will allow one to both predict cell behavior and to use bond number as a fundamental design criteria for synthetic extracellular matrices. fluorescence resonance energy transfer ͉ myoblast ͉ osteoblast ͉ proliferation ͉ RGD peptides C ell-extracellular matrix (ECM) binding interactions, via cell surface receptors such as integrins (1, 2), regulate a wide array of developmental, pathological, and regenerative processes (3-5). Both the number of these bonds that form and the specific receptor-ligand pairing may mediate this signaling (6, 7). Although identification of the pairing may be readily accomplished and clearly regulates intracellular signaling and gene expression (2,4,7,8), tools to quantify bond formation, especially for cells within a 3D environment, are lacking. Delineating this latter relation may be particularly important both in understanding the cellular behavior in different ECM and in developing a strategy to design synthetic ECM or cell-instructive materials, because biomaterials are frequently functionalized with molecules containing a receptor-binding domain to direct cell fate (5, 9). The ligand molecular structure, overall density, and spatial distribution at the micrometer and nanometer scale may all influence cell adhesion (e.g., focal adhesion formation), signaling pathways, cellular proliferation, apoptosis, migration, differentiation (9-14), and tissue formation (15, 16). It is unclear whether a subset or all of these variables alter the cellular response by simply altering bond number, but this assumption underlies many aspects of current biomaterials design. One can analyze receptor-ligand bonding with radioactive molecules (17), fluorescent molecules (18), and chemical assays (19), but these methods have been limited to analyzing cells suspended in medium or adherent to 2D substrates. Encapsulation of cells in 3D better reflects in vivo cell adhesion to ECM (20) and leads to distinct patterns of gene expression (21, 22) but is not amenable to the application of existing methods to quantify bond formation.In this study a FRET technique is demonstrated to allow one to quantify the number of receptor-ligand bonds formed between cells and adhesion peptid...

Material-based deployment enhances efficacy of endothelial progenitor cells

“…Peptides containing the arginine-glycineaspartic acid (RGD) amino acid sequence, a ubiquitous cell-binding domain found in many extracellular matrix molecules, were covalently coupled to alginate as previously described (19)(20)(21). The RGD coupling confers a specific mechanism for integrin-mediated cell adhesion to the otherwise nonadhesive polymer, and the RGD-ligand density and distribution can be manipulated to provide control over cell adhesion, proliferation, and cell fate after transplantation (22)(23)(24)(25). Vascular endothelial growth factor (VEGF) is a key regulator in new blood vessel formation, and was also investigated as a component of the material system because it regulates the survival, proliferation, and migration of endothelial cells (26).…”

Integrin-Adhesion Ligand Bond Formation of Preosteoblasts and Stem Cells in Three-Dimensional RGD Presenting Matrices

“…The number of bonds per cell was calculated using a standard curve that correlated energy transfer to peptide bond formation assessed by using iodinated RGD peptides coupled to alginate polymer chains for MC3T3 and D1 cells, as previously described. 6 Figure S1. The standard curve for MC3T3 has been previously published by Kong et al 6 …”

“…Previous control experiments for this assay were performed with MC3T3 preosteoblasts preincubated in soluble RGD peptides prior to encapsulation in RGD-TAMRA-modified alginate, and the degree of energy transfer was negligible as well. 6 Inclusion of TAMRA to the RGD peptide is not expected to affect binding to integrins as the peptide sequence used in these studies, G 4 RDASSK-TAMRA, was designed to minimize this possibility. The ASSK spacer is intended to allow the integrin to bind to the RGD sequence without interference from the TAMRA dye.…”

“…Bond formation at the cell-matrix interface was probed using an established FRET technique to quantify cell receptor-adhesion ligand bond number. 6 FRET is the process by which a donor fluorophore transfers energy to a neighboring acceptor by nonradiative dipole-dipole interaction 7 and is used to monitor a variety of biological phenomenon, including detection of receptor-ligand binding, 8 and probing the structure of single molecules. 9 In this application of FRET, cell-ligand bond formation results in a relative decrease in emission intensity from the donor due to energy transfer from the fluoresceinlabeled cells to the acceptor (RGD ligands labeled with TAMRA dye).…”

“…The relative decrease in donor emission intensity is quantitatively measured and converted to the degree of energy transfer, which is then correlated to bond number using a standard curve as previously described. 6 The integrin expression profile of MC3T3 preosteoblasts and D1 stem cells was also examined via flow cytometry (FACS) to determine whether differential expression of integrins was dependent on the stage of differentiation and ECM ligand presentation. Specific integrin-ligand interactions govern the cell response to ECM cues, as certain integrin-ligand interactions have been demonstrated to activate differing downstream signaling pathways.…”