| Cells transduce forces into biochemical signals through a process termed mechanotransduction. This process involves transmembrane proteins, the activity of which is modulated by the lipid solvent surrounding them. The goal of this work was to develop the experimental, computational and theoretical infrastructure to study the direct interrelationship between force and lipid dynamics. First, fluorescence correlation spectroscopy (FCS) and fluorescence lifetime methods based on time-correlated single photon counting (TCSPC) instrumentation were developed in order to capture the forceinduced change in lipid dynamics on spatial and temporal scales relevant to mechanotransduction. In tandem, the infrastructure was developed and tested for micropipette aspiration of giant unilamellar vesicles (GUV's) stained with lipoid dyes such as DiI-C18(3). These systems will be used for direct interrogation of the relationship between membrane tension and molecular dynamics. Second, we developed atomistic computational molecular dynamics (MD) simulations of a lipoid fluorescence dye, DiI-C18(3), in a DPPC bilayer. From these simulations, we clarify, for the first time, the location of DiI-C18(3) within a bilayer and compare the computational data to experimentally available data. Third, a generalized analytical model was developed that shows an exponential relationship between membrane lateral stretch and lipid diffusion; a relationship that can be tested directly on stressed GUVs using single molecule fluorescence methods. This infrastructure provides tools to determine, from atomic to continuum scale and from nanosecond to minute time scales, the role of plasma membrane lipids in mechanotransduction. |
