Using mechanical force to study biological processes: from blood clotting to immune cell adhesion

Abstract

The thesis contains three distinct projects united by a loose theme of quantitative approaches of applying forces to molecules to answer biological questions. First, the behavior of the mechanically activated blood clotting protein von Willebrand Factor (VWF) was investigated in shear flow using both single-molecule imaging and Brownian dynamics simulations. A new method was developed, which uses patterns of light pulses to make multiple measurements of particles in flow to correct motion blur artifacts. With the experiments and simulations, our findings suggest that 1) free VWF in shear flow is not a responsive sensor for increases in shear stress, 2) the tension experienced by free VWF in physiological shear flow is lower than indicated by previous reports, and 3) that tethering to platelets or the vessel wall is required to mechanically activate VWF adhesive function for primary hemostasis. Secondly, the feasibility of using cystine-lysine distance measurements to identify proteins was evaluated based on a distance-measurement method developed in the Wong lab using optical tweezers. Finally, a high-throughput method for quantifying the strength of cell-cell interactions was developed using a modified centrifuge force microscope (CFM) with fluorescence imaging capabilities. Protein avidity can be measured at physiological densities by monitoring the unbinding of cells as a function of the applied centrifugal force. The preparation, instrument, and imaging pipeline are demonstrated by measuring an antibody-red blood cell interaction and an activated T-cell B-cell interaction.