Analysis of mechanical characteristics of plasma membranes using optical tweezers
Ermilov, Sergey Alexandrovich
Doctor of Philosophy
In this thesis work an optical tweezers system was designed and used to characterize viscoelastic response of plasma membranes (PMs) to an applied stress under different environmental conditions. In order to perform accurate force measurements we analyzed dynamics of an optically trapped microsphere under altering external viscous drag force using Fourier methods. Next, using optically trapped fluorescent microspheres, we recorded tethering force vs. PM displacement profiles, which revealed the tether formation process, initiated with linear deformation of the PM, followed by a nonlinear regime and terminated with the local separation of PM. Tethering force vs. displacement profiles were used to estimate tether formation force and stiffness parameter of the PM. Integration of the force-displacement profiles yielded the work of tether formation, including linear and nonlinear components. We characterized mechanical properties of the outer hair (OHC) and human embryonic kidney (HEK) cell PMs perfused with 10 mM sodium salicylate (Sal), which is known to affect electromotility of OHCs as well as PM surface charge, morphology of erythrocytes, and PM lipid diffusion. Sal reduced tether formation force, PM stiffness parameter, and equilibrium tethering force in HEK cells, explained by enhanced PM/cytoskeleton compliance. The parameters estimated for OHCs remained the same after Sal perfusion, which is consistent with the hypothesis that Sal induced reversible hearing loss appears to be more the result of its competition with essential anions and less the result of a change in PM mechanics. Finally, we found that cell perfusion with hypo- and hyperosmotic solutions did not influence equilibrium tethering force, attributed to cellular regulation of the effective PM tension. The observed reduction in magnitude of the fast tethering force relaxation process was related to the stress-minimizing redistribution of the PM lipids. Application of a non-specific transmembrane water transport blocker, mercury (II) chloride, increased steady-state and equilibrium tethering forces and suppressed slow component of the tethering force relaxation. Temporal tethering force profiles obtained from the same PM tethers pulled in several repetitive cycles exhibited different behavior and resulted in reduced forces and time constants explained by essential irreversibility of PM tether elongation involving PM-cytoskeleton dissociation and/or plastic deformations.
Cell biology; Biomedical engineering; Biophysics