Search

Dynamic substrates for cell culture control the spatial and temporal presentation of extracellular matrix ligands that interact with adherent cells. This paper reports a photoactive surface chemistry that can repeatedly activate regions of the substrate for cell adhesion, spreading, and migration. The approach uses self-assembled monolayers presenting the integrin ligand RGD that is caged with a nitrophenyl-based photoprotecting group. The group is also modified with a maltoheptaose oligosaccharide to prevent nonspecific protein adsorption and cell attachment. The peptide is uncaged when irradiated with a laser source at 405 nm on a microscope to reveal micron-size regions for single cell attachment. This method is applied to studies of gap junction-mediated communication between two neighboring cells and requires the patterning of an initial receiver cell population and then the patterning of a second sender population to give a culture wherein each pair of cells are separated by 30 μm. Finally, activation of the region between the cells permits cell-cell contact and gap junction assembly between the sender and receiver cells. This example demonstrates the broad relevance of this method to studying complex phenotypes in cell culture.

The extracellular matrix (ECM) is a complex, spatially inhomogeneous environment that is host to myriad cell-receptor interactions that promote changes in cell behavior. These biological systems can be probed and simulated with engineered surfaces, but doing so demands careful control over the arrangement of ligands. Here, we describe how such surfaces can be fabricated by utilizing polymer pen lithography (PPL), which is a cantilever-free scanning probe lithographic method that utilizes polymeric pen arrays to generate patterns over large areas. With the advent of PPL, fundamental questions in cell biology can be answered by recapitulating cell-ECM interactions to explore how these interactions lead to changes in cell behavior. Here, we describe an approach for the combinatorial screening of cell adhesion behavior to gain understanding of how ECM protein feature size dictates osteogenic differentiation of mesenchymal stem cells. The technique outlined here is generalizable to other biological systems and can be paired with quantitative analytical methods to probe important processes such as cell polarization, proliferation, signaling, and differentiation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

This paper describes a molecular strategy to restore adhesion of cells to surfaces that otherwise do not present ligands that can mediate adhesion. The approach is based on a carbonic anhydrase fusion protein that binds benzenesulfonamides and that also includes the RGD peptide motif that can bind to cell-surface integrin adhesion receptors. In this way, the fusion protein can bind to a monolayer that presents the benzenesulfonamide ligand, thereby positioning the RGD peptide at the surface, where it can mediate the adhesion and spreading of cells. This strategy may provide a general method for promoting the adhesion of cells to non-natural surfaces or to defective biological matrices.

Most mammalian cells must adhere to the extracellular matrix (ECM) to maintain proper growth and development. Fibronectin is a predominant ECM protein that engages integrin cell receptors through its Arg-Gly-Asp (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) peptide binding sites. To study the roles these motifs play in cell adhesion, proteins derived from the 9th (containing PHSRN) and 10th (containing RGD) type III fibronectin domains were engineered to be in frame with cutinase, a serine esterase that forms a site-specific, covalent adduct with phosphonate ligands. Self-assembled monolayers (SAMs) that present phosphonate ligands against an inert background of tri(ethylene glycol) groups were used as model substrates to immobilize the cutinase-fibronectin fusion proteins. Baby hamster kidney cells attached efficiently to all protein surfaces, but only spread efficiently on protein monolayers containing the RGD peptide. Cells on RGD-containing protein surfaces also displayed defined focal adhesions and organized cytoskeletal structures compared to cells on PHSRN-presenting surfaces. Cell attachment and spreading were shown to be unaffected by the presence of PHSRN when compared to RGD alone on SAMs presenting higher densities of protein, but PHSRN supported an increased efficiency in cell attachment when presented at low protein densities with RGD. Treatment of suspended cells with soluble RGD or PHSRN peptides revealed that both peptides were able to inhibit the attachment of FN10 surfaces. These results support a model wherein PHSRN and RGD bind competitively to integrins--rather than a two-point synergistic interaction--and the presence of PHSRN serves to increase the density of ligand on the substrate and therefore enhance the sticking probability of cells during attachment.

This work reports on the role of the synergy peptide PHSRN in mediating the adhesion of cells. The attachment of baby hamster kidney cells and 3T3 Swiss fibroblasts to model substrates presenting either GRGDS or PHSRN was evaluated using self-assembled monolayers of alkanethiolates on gold presenting the peptide ligands mixed with tri(ethylene glycol) groups. These substrates permit rigorous control over the structures and densities of peptide ligands and at the same time prevent nonspecific interactions with adherent cells. Both cell types attached efficiently to monolayers presenting either the RGD or the PHSRN peptide but not to monolayers presenting scrambled peptide GRDGS or HRPSN. Cell attachment was comparable on substrates presenting either peptide ligand but less efficient than on substrates presenting the protein fibronectin. The degree of cell spreading, however, was substantially higher on substrates presenting RGD relative to PHSRN. Staining of 3T3 fibroblasts with anti-vinculin and phalloidin revealed clear cytoskeletal filaments and focal adhesions for cells attached by way of either RGD or PHSRN. Inhibition experiments showed that the attachment of 3T3 fibroblasts to monolayers presenting RGD could be inhibited completely by a soluble RGD peptide and partially by a soluble PHSRN peptide. IMR 90 fibroblast attachment to monolayers presenting PHSRN could be inhibited with anti-integrin alpha(5) or anti-integrin beta(1) antibody. This work demonstrates unambiguously that PHSRN alone can support the attachment of cells and that the RGD and PHSRN bind competitively to the integrin receptors.

The adhesion of cells is mediated by the binding of several cell-surface receptors to ligands found in the extracellular matrix. These receptors often have overlapping specificities for the peptide ligands, making it difficult to understand the roles for discrete receptors in cell adhesion, migration, and differentiation as well as to direct the selective adhesion of cell types in tissue-engineering applications. To overcome these limitations, we developed a strategy to rewire the receptor-ligand interactions between a cell and substrate to ensure that adhesion is mediated by a single receptor with unique specificity. The strategy combines a genetic approach to engineer the cell surface with a chimeric integrin receptor having a unique ligand binding domain with a surface chemistry approach to prepare substrates that present ligands that are bound by the new binding domain. We show that Chinese hamster ovary cells that are engineered with a chimeric beta1 integrin adhere, signal, and even migrate on a synthetic matrix.

An electroactive substrate that combines dual dynamic properties is demonstrated. A monolayer is patterned to first release an immobilized ligand, and therefore adherent cells, on application of an electrical potential. Subsequently, electrical oxidation of the substrate results in immobilization of ligands, and migration of adherent cells.

Summary: Fibrinogen (Fbg) mediates platelet aggregation by binding the αIIbβ3 integrin receptor, but the interaction of the receptor with peptide motifs of Fbg remains unresolved. This paper describes the use of self-assembled monolayers (SAMs) to study the adhesion of αIIbβ3-transfected CHO cells to the GRGDS and HHLGGAKQAGDV motifs within Fbg. Cells adhered to and spread on monolayers presenting either peptide. Cell adhesion could be inhibited by either soluble peptide, demonstrating that the peptides bind competitively to the integrin. A peptide array was used to show that AGD was the minimal binding sequence in HHLGGAKQAGDV and that the receptor recognizes ligands of the form GXGDSC, where X is a hydrophobic or basic residue. This work revises our understanding of the αIIbβ3 specificity and also suggests a new class of antithrombotic agents. [Copyright &y& Elsevier]

Looks at factors which influence the efficiency of the solid-phase synthesis of peptide-substituted alkanethiols in the preparation of substrates which support cell adhesion. Role of ligands in the interactions of mammalian cells; Strategies required to protect alkanethiol; Details on the preparation of peptides.

Abstract: Significant efforts have addressed the role of vimentin intermediate filaments (VIF) in cell motility, shape, adhesion and their connections to microfilaments (MF) and microtubules (MT). The present work uses micropatterned substrates to control the shapes of mouse fibroblasts and demonstrates that the cytoskeletal elements are dependent on each other and that unlike MF, VIF are globally controlled. For example, both square and circle shaped cells have a similar VIF distribution while MF distributions in these two shapes are quite different and depend on the curvature of the shape. Furthermore, in asymmetric and polarized shaped cells VIF avoid the sharp edges where MF are highly localized. Experiments with vimentin null mouse embryonic fibroblasts (MEFs) adherent to polarized (teardrop) and un-polarized (dumbbell) patterns show that the absence of VIF alters microtubule organization and perturbs cell polarity. The results of this study also demonstrate the utility of patterned substrates for quantitative studies of cytoskeleton organization in adherent cells. [Copyright &y& Elsevier]