We have used an ultrasensitive force probe and optical interferometry to examine the thickness compressibility of the red cell membrane in situ. Pushed into the centers of washed-white red cell ghosts lying on a coverglass, the height of the microsphere-probe tip relative to its closest approach on the adjacent glass surface revealed the apparent material thickness, which began at ∼90 nm per membrane upon detection of contact (force ∼1-2 pN). With further impingement, the apparent thickness per membrane diminished over a soft compliant regime that spanned ∼40 nm and stiffened on approach to ∼50 nm under forces of ∼100 pN. The same force-thickness response was obtained on recompression after retraction of the probe, which demonstrated elastic recoverability. Scaled by circumferences of the microspheres, the forces yielded energies of compression per area which exhibited an inverse distance dependence resembling that expected for flexible polymers. Attributed to the spectrin component of the membrane cytoskeleton, the energy density only reached one thermal energy unit (KBT) per spectrin tetramer near maximum compression. Hence, we hypothesized that the soft compliant regime probed in the experiments represented the compressibility of the outer region of spectrin loops and that the stiff regime <50 nm was the response of a compact mesh of spectrin backed by a hardcore structure. To evaluate this hypothesis, we used a random flight theory for the entropic elasticity of polymer loops to model the spectrin network. We also examined the possibility that additional steric repulsion and apparent thickening could arise from membrane thermal-bending excitations. Fixing the energy scale to kBT/spectrin tetramer, the combined elastic response of a network of ideal polymer loops plus the membrane steric interaction correlated well with the measured dependence of energy density on distance for a statistical segment length of ∼5 nm for spectrin (i.e., free chain end-to-end length of ∼29 nm) and a hardcore limit of ∼30 nm for underlying structure.
|Original language||English (US)|
|Number of pages||12|
|State||Published - 2001|
ASJC Scopus subject areas