Physical measurements of bilayer-skeletal separation forces

Bilayer membranes are intrinsically fluid in character and require stabilization by association with an underlying cytoskeleton. Instability either in the membrane-associated cytoskeleton or in the association between the bilayer and the skeleton can lead to loss of membrane bilayer and premature cell death. In this report measurements of the physical strength of the association between membrane bilayer and the membrane-associated skeleton in red blood cells are reported. These measurements involve the mechanical formation of long, thin cylinders of membrane bilayer (tethers) from the red cell surface. Ultrastructural evidence is presented indicating that these tethers do not contain membrane skeleton and, furthermore, that they are deficient in at least some integral membrane proteins. By measuring the forces on the cell as the tether is formed and the dimensions of the tether, the energy associated with its formation can be calculated. The minimum force to form a tether was found to be approximately 50 pN corresponding to an energy of dissociation of 0.2-0.3 mJ/m2. Such measurements enable critical evaluation of potential physical mechanisms for the stabilization of the membrane bilayer by the underlying cytoskeleton. It is postulated that an important contribution to the energy of association between bilayer and skeleton comes from the increase in chemical potential due to the lateral segregation of lipids and integral proteins.