Sean R. Coyer

Nanopatterning Reveals an ECM Area Threshold for Focal Adhesion Assembly and Force Transmission that is regulated by Integrin Activation and Cytoskeleton Tension

Summary Integrin-based focal adhesions (FA) transmit anchorage and traction forces between the cell and the extracellular matrix (ECM). To gain further insight into the physical parameters of the ECM that control FA assembly and force transduction in non-migrating cells, we used fibronectin (FN) nanopatterning within a cell adhesion-resistant background to establish the threshold area of ECM ligand required for stable FA assembly and force transduction. Integrin–FN clustering and adhesive force were strongly modulated by the geometry of the nanoscale adhesive area. Individual nanoisland area, not the number of nanoislands or total adhesive area, controlled integrin–FN clustering and adhesion strength. Importantly, below an area threshold (0.11 µm2), very few integrin–FN clusters and negligible adhesive forces were generated. We then asked whether this adhesive area threshold could be modulated by intracellular pathways known to influence either adhesive force, cytoskeletal tension, or the structural link between the two. Expression of talin- or vinculin-head domains that increase integrin activation or clustering overcame this nanolimit for stable integrin–FN clustering and increased adhesive force. Inhibition of myosin contractility in cells expressing a vinculin mutant that enhances cytoskeleton–integrin coupling also restored integrin–FN clustering below the nanolimit. We conclude that the minimum area of integrin–FN clusters required for stable assembly of nanoscale FA and adhesive force transduction is not a constant; rather it has a dynamic threshold that results from an equilibrium between pathways controlling adhesive force, cytoskeletal tension, and the structural linkage that transmits these forces, allowing the balance to be tipped by factors that regulate these mechanical parameters.

Poly(dimethylsiloxane) elastomers with tethered peptide ligands for cell adhesion studies.

Poly(dimethylsiloxane) (PDMS) is the choice of material for a wide range of biological and non-biological applications because of its chemical inertness, non-toxicity, ease of handling and commercial availability. However, PDMS exhibits uncontrolled protein adsorption and cell adhesion and it has proved difficult to functionalize to present bioactive ligands. We present a facile strategy for functional surface modification of PDMS using commercial reagents to engineer polymer brushes of oligo(ethylene glycol) methacrylate that prevent cell adhesion and can be functionalized to display bioadhesive ligands. The polymer brushes resist biofouling and prevent cell adhesion and bioadhesive peptides can be tethered either uniformly or constrained to micropatterned domains using standard peptide chemistry approaches. This approach is relevant to various biomedical and biotechnological applications.

Large-Scale Arrays of Aligned Single Viruses

The fabrication of single virus arrays is herein demonstrated using the direct printing of unmodified anti-M13 bacteriophage antibodies on silicon with nanometer resolution, widely variable feature pitch, and flow alignment of the viruses. Organization of virus-based systems into functional, addressable arrays has many technological applications, including micro-array technology and bottom-up nano-assemblies.

Protein Tethering into Multiscale Geometries by Covalent Subtractive Printing

The engineering of surfaces functionalized with biological/bioactive components is important for medical and diagnostic applications, including protein arrays and biosensors,[1] as well as fundamental life sciences studies.[2] Spatial control at length scales of both cellular adhesive structures (e.g., focal adhesions; micrometer/sub-micrometer scale) and individual proteins (nanometer scale) has been highly sought after to produce surfaces capable of eliciting specific biological responses. A major challenge has been combining features with micrometer and nanometer dimensions onto one sample while maintaining a protein-resistant background that is stable for extended periods under cell culture conditions. Sample production must also be high-throughput and low cost in order to be broadly applicable and cost- and time-effective. Traditional microcontact printing has been successful in producing micrometer-scale patterns for biological studies quickly and inexpensively but faces limitations when approaching sub-micrometer resolution of patterns due to the diffraction limit of light, which affects the fabrication of molds prepared using photolithography, and the instability of elastomer stamp materials.[3] Scanning probe-based techniques (i.e., dippen lithography), which control protein placement by depositing or scraping off molecules using a cantilever, have enabled access to the regime with features of the size of tens of nanometers.[4] While patterns approaching single proteins produced with the mentioned techniques are desirable for a variety of applications, limitations in the writing areas achievable with standard equipment makes high-throughput sample production challenging. Colloidal lithography with diblock copolymers has provided control over the nanometer-scale spacing between ligands for studies of cell adhesion[5] and apoptosis,[6] but the pattern geometries are currently limited to spacings predetermined by micelle chemistry. In this communication, we introduce a technique with the capacity to produce multi-length-scale patterns of bioactive proteins that are covalently immobilized onto an activated surface and surrounded by a protein adsorption-resistant background. Patterns of the cell adhesive protein fibronectin were printed on a nonadhesive background to produce arrays of single cells where adhesion is constrained to the region of tethered protein. The applicability of the technique to biological studies is demonstrated by producing arrays of adherent cells on which focal adhesion size and spatial arrangement are modulated according to the geometry of the adhesive region.