Separation of particular cell types from a heterogeneous cell population is highly desirable for diagnosis and therapy of many pathologies. This can be accomplished most selectively by means of differential receptor-mediated cell adhesion to ligand-coated surfaces; one example is cell affinity chromatography. The key to satisfactory performance of an affinity-based cell separation process is quantitative understanding of the parameters governing the probability of stable cell adhesion to the surface during a transient encounter in fluid shear flow. We are using quantitative experimental assays for receptor/ligand binding, cell attachment, and cell detachment kinetics to test a theoretical model for dynamic cell adhesion.

Migration of mammalian blood and tissue cells over two-dimensional and through three-dimensional substrata is a critical process in normal physiology as well as in pathological conditions. This migration appears to require dynamic interactions between the extracellular matrix, the cell membrane, and the cell cytoskeleton, mediated by transmembrane cell adhesion receptors. We are attempting to understand the ways in which biochemical and biophysical properties of these receptors influence the rate and direction of cell migration, using a combination of theoretical analysis of cell kinetic and mechanic phenomena and experimental observations on migration of cells with modified receptor characteristics.

Intracellular signals generated by receptors stimulated by growth factor binding regulate proliferation of mammalian blood and tissue cells. An important aspect of overall signal generation is the modulation of actively signalling growth factor/receptor complexes by cell surface binding and intracellular trafficking dynamics. We are developing mathematical models for the dependence of fibroblast proliferation responses to epidermal growth factor (EGF) on EGF binding and trafficking parameters. The aim is to elucidate strategies for optimal cell line and media formulations with respect to growth factor utilization. Experiments testing these models are being performed with various site-directed mutations of EGF and the EGF receptor.

Biomaterial rejection is linked to the initial protein adsorption upon contact with foreign materials with body fluids. Our objective is to minimize or control protein and biological interactions with new materials through the tailored manipulation of the molecular forces controlling the outcomes of encounters between foreign materials and biological fluids. Using a combination of direct force measurements, biochemical methods, and molecular modeling, we are quantifying the relationship between molecular surface structure and composition, surface forces, and protein adsorption. These data are intended to guide the design of new, effective antiadhesive materials.

Cell adhesion is mediated and modulated through contacts involving chemical moieties on cell surfaces. In particular, the enhanced expression of certain glycolipids on cancer cells may determine their metastatic potential. The significance of specific glycolipid interactions, however, is linked directly to the strengths of the molecular forces governing the resulting adhesion. We are using direct force measurements, fluorescence microscopy, and light scattering to quantify the magnitudes and ranges of glycolipid-mediated adhesive forces and to determine the impact of those forces on the strengths of glycolipid-mediated membrane attachments. We are testing directly the role of membrane surface components in cell adhesion and the potential utility of therapeutics designed to block their interactions.

Many biosensor designs are based on the selective binding of soluble analyte to immobilized receptors. The surface microenvironment can, however, significantly affect sensor performance. We are currently quantifying changes in protein-binding strengths in response to interfacial perturbations. Furthermore, kinetic modeling of site-selective adsorption data have demonstrated that subtle interfacial perturbations impact sensor performance. Our objective is to determine the molecular basis of altered protein function by the surface microenvironment and to optimize sensor performance through the tailored manipulation of the transducer surface properties.

Protein surface topology plays a major role in modulating the rates of protein-binding events. We are using direct force measurements to probe the impact of local protein structural motifs on the forces that control the rates of protein collisions. In particular, we are investigating the impact of surface charge distributions and protein orientation on protein electrostatic surface properties and resulting protein interactions. Use of both wild type and engineered proteins permits precise control of the surface region probed. Measurements are compared with theoretical calculations. The functional implications of these findings are being investigated by Brownian dynamics simulations and kinetic measurements.

The purpose of this project is to increase the efficiency of production of pharmaceutical proteins by yeast. Molecular chaperones are molecules that assist protein folding within the cell by stabilizing intermediates that are otherwise prone to aggregation. Foldases catalyze rate-limiting isomerizations in protein folding. We hypothesized that chaperones and foldases play a central role in determining the rate of secretion of heterologous proteins in yeast. A combination of genetic manipulation of chaperone levels, quantitative cell biological measurements, and mathematical modeling is being applied to study the process of protein secretion in yeast.

High-level production of pharmaceutical proteins in yeast requires stable maintenance of an amplified number of copies of the gene of interest. We have developed a vector that targets foreign DNA for integration into yeast chromosomal DNA at multiple dispersed sites, where it is replicated and partitioned at cell division with high fidelity. We have also mathematically modeled the amplification of an autonomously replicating circular plasmid, and are experimentally testing the predictions of that model.

Understanding the process of protein folding as it occurs within the cell is critical to the rational design of improved production organisms. We have developed a system for quantitatively studying the folding of bovine pancreatic trypsin inhibitor (BPTI) in the yeast secretory pathway. By examining the effects of genetic and environmental alterations on the rate and yield of BPTI folding, we hope to achieve significant improvements in the specific productivity of yeast cells for the production of pharmaceutical proteins.