Recent work to understand the elasticity of large single biological polymers has indicated that molecules like DNA and titin behave as entropic springs. The mechanical character of these polymers is described by two intrinsic molecular features, contour length and inherent elasticity. In erythrocytes, spectrin is the large structural polymer which determines the mechanical properties of the membrane skeleton such as membrane rigidity. Increased interactions of spectrin with transmembrane proteins has been shown to rigidify the erythrocyte membrane but the essential question of the underlying molecular mechanism is unknown. Specifically, does erythrocyte membrane rigidification, induced by increasing network interactions, involve shortening the contour length of spectrin by restricting it's ability to extend and there by altering the topology of the network, or alternatively, by increasing spectrin's inherent stiffness and there by leaving the network topology unaltered. To answer this question, monoclonal antibody BRAC18 binding to band 3 was targeted as a model system because transmembrane protein band 3 is a major tethering point between the skeletal network and the bilayer and it regulates membrane rigidity through its interaction with the spectrin based membrane skeleton. Membrane rigidity was measured by micropipette aspiration and fluorescence imaged microdeformation (FIMD) was employed because it measures component molecular density in stretched networks and would be sensitive to topological alterations involving altered contour length. Cells were prepared by fluorescently labeling erythrocyte band 3 and actin, in situ, with fluorescein-5-maleimide (FMA) and rhodamine-phalloidin (RhPh), respectively. Actin was chosen because it is an ideal topological marker of spectrin end-to-end length and network density. Appropriateness of the model was confirmed because with increasing incubation concentrations of BRAC18 there was a dose-dependent increase in membrane rigidity. This increased rigidity was associated with increased interaction of band 3 with spectrin as observed by FIMD, following the addition of BRAC18, which showed an increased slope of entrance density minus the cap density (Pe-Pc), of FMA-labeled band 3, versus aspiration length normalized by pipette radius (L/Rp). However, and surprisingly, BRAC18 binding had no measurable effect on the density difference for RhPh-labeled actin. These results indicated that the binding of BRAC18 antibody to band 3, which produced a marked increase in membrane rigidity, did not alter the topology of the skeletal network. These data imply that the molecular mechanism for rigidification is increased inherent stiffness of spectrin rather than topological crosslinking resulting in a shortening of the contour length of spectrin.
As a potential model system for membrane rigidification, antibody binding to band 3 was targeted because this transmembrane protein is a major tethering point between the skeletal network and the bilayer and because it regulates membrane rigidity through its interaction with the spectrin based membrane skeleton. Appropriateness of the model was tested by determining whether binding the monoclonal antibody BRAC18 to band 3 increased membrane rigidity, as measured by the technique of micropipette aspiration of single red cells (Fig. 1A). Indeed, with increasing incubation concentrations of BRAC18 there was a dose-dependent increase in membrane rigidity (Fig. 1B and 1C). Within the incubation concentration range of 0 to 100ug/ml of BRAC18, the membrane rigidity increased 3.8 fold compared to nonliganded cells and plateaued around 10 µg/ml.
Finally FIMD was used to test whether BRAC18 binding altered spectrin's crosslink density by measuring the deformation maps of RhPh-labeled actin. Actin, an oligomer at the junctional complexes within the network, was chosen because it is an ideal topological marker of spectrin end-to-end length and network density. Increased crosslink density of spectrin would be realized by an altered density map of the network under deformation. Surprisingly, BRAC18 binding had no measurable effect on the density difference between RhPh-labeled actin at the entrance and the cap (Fig. 3A). Further, both the entrance and the cap densities of RhPh-labeled actin were unaltered (Fig. 3B) implying that Brac18 binding did not alter the networks ability to condense or dilate, respectively. These results indicated that the binding of BRAC18 antibody to band 3, which produced a marked increase in membrane rigidity, did not alter the topology of the network.