Experimental Investigation of Cell Membrane Nano-mechanics and Plasma Membrane-Cytoskeletal Interactions Using Optical Tweezers

The mechanical properties of the cell components, cell plasma membrane and cytoskeleton, as well as membrane-cytoskeleton associations, determine the mechanical properties of the whole cell which is important in cellular shape change behavior and mechanical signal transduction in living cells. Examples of biologically important processes involving cellular shape changes are deformation of erythrocytes in capillaries, cell division, phagocytosis, pseudopodium and dendritic spine formation, and electromotility of the outer hair cells. This dissertation focuses on investigating the mechanical properties of the living cell plasma membrane and the local mechanical associations of the plasma membrane with the underlying cytoskeleton. Mechanical properties of the living cell plasma membrane are investigated by forming membrane nanotubes (tethers) from human embryonic kidney cells using optical tweezers technique. In order to analyze the role of membrane composition on its nano-mechanical properties, the membrane cholesterol content, the major lipid component of the plasma membrane, is manipulated and the obtained membrane mechanical properties are correlated to the membrane cholesterol content. The results reveal significant effects of membrane cholesterol in specific, and membrane composition in general, on membrane nano-mechanical properties. Specifically, decreases in membrane cholesterol content were associated with increased plasma membrane equilibrium force, plasma membrane tether stiffness and plasma membrane-cytoskeleton adhesion energy per unit area. Elevation of the membrane cholesterol content was followed by lower membrane tether equilibrium force, lower stiffness values, and lower membrane-cytoskeleton adhesion energy. The membrane bending modulus was almost unchanged upon membrane cholesterol manipulations. In order to discern the role of cell cytoskeleton on membrane mechanical properties the experiments were repeated after F-actin disruption and in control cell and cholesterol manipulated cells. The disruption of F-actin filaments showed a noticeable impact on the membrane mechanical properties and diminished the observed disparity in membrane mechanical properties upon cholesterol depletion and cholesterol enrichment. This dissertation also focuses on local cell deformations (protrusions). These deformations occur at an intermediate region between deformations at cell plasma membrane level and whole cell deformation, and are biologically important in formation of pseudopodia and filopodia, deformation of macrophages to engulf particles, and the surface protrusions on the cells preceding formation of membrane tethers. We used a combined optical tweezers-fluorescent microscopy technique to study cellular protrusions in adherent living cells. The mechanical properties of the protrusions were analyzed by obtaining the associated force-length plots and a Maxwell viscoelastic model is used to fit the force-length plots a. The experiments are performed on adherent human embryonic kidney cells, under cholesterol depleted and cholesterol enriched conditions to examine the effects of membrane cholesterol on protrusions. The experiments indicated greater maximum protrusion forces and shorter protrusion length under cholesterol depleted conditions in addition to greater values of the protrusion stiffness. Cholesterol enrichment experiments were associated with lower values of maximum protrusion force and protrusion stiffness, and formation of longer protrusions The observations suggest a significant contribution of the cytoskeletal F-actin filaments on the observed mechanical properties of protrusion regardless of membrane cholesterol content