Characterization of Early Membrane Adhesion Activity with a Drift-corrected Optical Trap

Open Access
Author:
Moroney, George J
Area of Honors:
Bioengineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Peter J Butler, Thesis Supervisor
  • Peter J Butler, Honors Advisor
  • William O Hancock, Faculty Reader
Keywords:
  • Focal adhesions
  • mechanotransduction
Abstract:
Vascular endothelial cells have been shown to sense the pressure and shear stress imparted by blood flow and to convert these mechanical stimuli into intracellular and extracellular chemical products such as prostacyclin and nitric oxide. Such products alter cardiovascular function by their ability to adjust vascular dilation and inhibit platelet aggregation and clotting. A leading hypothesis for mechanotransduction suggests that forces alter the interaction of integrin-based adhesions to extracellular matrix molecules. Such interactions are expected to be on the time scale of cardiac pulsatility (1-2 Hz). Thus, a greater understanding of the precise magnitude and temporal characteristics of cell adhesion would be instrumental in understanding the fundamental mechanisms of mechanotransduction. Whereas the cell interacts with its environment via the assembly of membrane proteins to form focal adhesions, studies on the early dynamics of force production by optical trapping would lead to a fundamental understanding of mechanotransduction by vascular endothelium. This thesis describes the development, testing, and optimization of protocols and instrumentation for the characterization of integrin-based adhesion to extracellular matrix proteins. Optical trapping allows for application of forces to single molecules and molecular clusters of similar magnitude (~2 pN) to the external forces that these molecules experience in vivo. More importantly for this study, the optical trap is able to detect the exact time that a force is applied to the cell. In order to monitor the activity of adhesion events, a continuous focus drift mechanism with a 100 nanometer-scale sensitivity was developed. This technique measures the changes in distance between the objective and cover slip by measuring the intensity of the light from the epi pathway of the microscope that reflects off of the cover slip. The early adhesion dynamics of the cells were studies using both a variance-based and time-to-adhesion-based assay using fibronectin-functionalized beads in contact with the membrane of human aortic endothelial cells. Preliminary results suggest that decreasing membrane fluidity with benzyl alcohol decreases time to adhesion, suggesting that a principle mechanism of force transduction depends on lateral transport of integrins to the adhesion site.