Modulation of cell adhesion strengthening by nanoscale geometries at the adhesive interface

This research project focused on quantitatively analyzing the adhesive responses while systematically modulating the adhesive interface. The objective of this project was to analyze the role of nanoscale geometry of the adhesive interface in regulating integrin recruitment to adhesive contacts and modulating cell adhesion strengthening to extracellular matrices (ECM). Our central hypothesis was that the size and location of clusters of recruited integrin modulates cell adhesion strengthening in response to nanoscale organization of the adhesive interface.We first focused on developing a technique for producing high resolution patterns of proteins in biologically relevant geometries. To this end, the subtractive patterning technique was used to produce a pattern of proteins on a flat elastomer using a silicon nanotemplate which was then transferred to a final substrate by contact and release. A wide range of pattern geometries were demonstrated by printing lines, linelets, and squares with spacing between features ranging from hundreds of nanometers to 64 mum. Patterns comprising two types of antibodies with intrinsic self-alignment were produced by the successive inking, subtraction, and printing of antibodies. Our results introduce a facile, high-throughput technique for patterning proteins on surfaces that enables the production of arrays of multiple types of proteins with high resolution, high contrast, and self-alignment in geometries that are relevant to cell adhesion studies.The objective of our next study was to develop a method for producing cell adhesion arrays that constrain adhesion to nanoscale patterns of protein that are surrounded by a non-fouling background. To this end, we combined the subtractive patterning technique with mixed self-assembled monolayers. A mixed self-assembled monolayer was produced by assembling mixed carboxylic acid- and tri(ethylene glycol)-terminated alkanethiols into self-assembled monolayers on gold-coated substrates.The objective of our next study was to analyze the recruitment of integrins into adhesive clusters in response to nanoscale geometry of the adhesive interface (adhesion area, spacing, and clustering) and determine the functional implications of integrin recruitment by quantifying variations in adhesion strength. Patterns of FN consisting of features with a range of nanoscale geometries (adhesion islands with dimensions of 1000, 500, 333, and 250 nm in clusters of 1, 2, 4, or 9) were produced using the subtractive patterning technique to directly immobilize proteins by covalent tethering onto surfaces presenting mixed self-assembled monolayers of alkanethiols. Cells seeded on the arrays were limited to one cell per pattern and adhesion was constrained to the adhesion region presented by the protein pattern. Spreading in between the patterns is prevented by the non-fouling background. Patterned substrates were shown to maintain the original pattern dimensions and resist FN deposition from cells using immunostaining with FN antibodies. These results demonstrate that the patterned arrays constrain cell adhesion to defined regions and that the adhesion patterns maintain their original design throughout the experiment.This thesis project has developed a unique experimental approach to analyze recruitment of bound integrins into clusters and quantify modulation of adhesion strength in response to systematic variation of the area, spacing, and clustering of adhesion areas. We determined that integrin recruitment is directed by changes in the size, clustering, and orientation of adhesion regions. We established a threshold pattern area between 333 x 333 nm2 (0.11 mum 2) and 250 x 250 nm2 (0.06 mum2) below which integrin recruitment switches from robust integrin clusters to low frequency punctate formations. The role of area splitting in adhesion strengthening was established where adhesion strength changes despite no change to the total available adhesion area. A relationship was established between adhesion strength and area of individual adhesion islands. Patterns with adhesion areas below the threshold were unable to generate adhesion strength. Adhesion strength is seen to vary with integrin pad occupancy and not with the level of integrin clustering at adhesion regions. Furthermore, our results suggest that integrin clusters with areas between 0.25 mum2 and 1 mum 2 generate equal adhesion strengths. (Abstract shortened by UMI.)...