Surface shape change during fusion of erythrocyte membranes is sensitive to membrane skeleton agents

We previously reported that the induction of membrane fusion between pairs of erythrocyte ghosts is accompanied by the formation of a multipore fusion zone that undergoes an area expansion with condition-dependent characteristics. These characteristics allowed us to hypothesize substantial, if not major, involvement of the spectrin-based membrane skeleton in controlling this expansion. It was also found that the fusion zone, which first appears in phase optics as a flat diaphragm, has a lifetime that is also highly condition-dependent. We report here that 2,3-diphosphoglycerate, wheat germ agglutinin, diamide, and N-ethylmaleimide, all known to have binding sites primarily on skeleton components (including spectrin), have condition-dependent effects on specific components of the fusion zone diameter versus time expansion curve and the flat diaphragm lifetime. We also report a pH/ionic strength condition that causes a dramatic stabilization of flat diaphragms in a manner consistent with the known pH/ionic strength dependence of the spectrin calorimetric transition, thus further supporting the hypothesis of spectrin involvement. Our data suggest that the influence of the membrane skeleton on cell fusion is to restrain the rounding up that takes place after membrane fusion and that it may have variable, rather than fixed, mechanical properties. Data show that WGA, a known ligand for sialic acid, and DPG, a known metabolite, influences the flat diaphragm stability and late period expansion rates, raising the possibility that some of these mechanical properties are biologically regulated.