Physical measurements of bilayer-skeletal separation forces

Abstract

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.

Membrane tension regulates motility by controlling lamellipodium organization

“…2D is of unknown origin, but does not seem to represent cell adjustment as it is also observed in control cells (Fig. 2D, blue curve) and has been observed in other cell types in the absence of osmotic shock (15). Under isotonic conditions, similar tether forces were obtained when tubes were pulled from the cell body or from the lamellipodium of activated sperm cells, despite the fact that the MSP cytoskeleton by fluorescence microscopy was of different densities and organizations in these different regions.…”

Hydrodynamic narrowing of tubes extruded from cells

“…Numerous works were devoted to the study of tether growth from various cells, namely red blood cells (RBCs) (5,6,8,22), neuronal growth cones (NGCs) (10,12), outer hair cells (OHCs) (23). In the vast majority of cases, experiments were aimed at measuring the static force to derive the static adhesion energy, W 0 .…”

“…Although reported force-velocity traces could generally be well fitted by a linear curve, we tested whether our model, which basically predicts a nonlinear dependence, could be applied. Beside the data relative to neutrophils, we have collected three additional sets of published data corresponding to the three different kinds of cells, namely RBCs (8,22), OHCs (23), and NGCs (10). In Fig.…”

“…Tethers can be extracted from synthetic vesicles or living cells by the application of an external point force [using a fluid drag (4,5) or pipette-tweezer system (6,7)]. In the case of living cells, where the lipidic membrane is coupled to a cytoskeleton, tethers can be used as membrane sensors to measure the membrane-cytoskeleton adhesion energy W 0 (8)(9)(10)(11)(12).…”

Membrane instability in late-stage erythropoiesis

“…An alternative to fragmentation methods for assessing membrane instability is the formation of membrane strands (tethers) from the surfaces of cells. [10][11][12] This approach provides a direct and quantitative measure of the energy required to separate the membrane bilayer from the underlying membrane-associated cytoskeleton. 13,14 In the present report we have applied this approach to demonstrate that the instability observed in immature RBCs lies in the tightness of the association between membrane bilayer and the underlying membrane skeleton, and we provide a quantitative measure of the degree of instability in terms of the work (energy) required to separate lipid bilayer from the surfaces of cells at different stages of maturation.…”