Restriction of the lateral motion of band 3 in the erythrocyte membrane by the cytoskeletal network: dependence on spectrin association state

Abstract

The effects of incubation of erythrocyte ghosts under various conditions (ionic strength or addition of ankyrin, diamines, or ATP) on the lateral motion of band 3 in the membranes were studied by using the fluorescence photobleaching recovery technique. Incubation of ghosts with exogenous ankyrin increased the immobile fraction of band 3, from 0.6 in intact ghosts to 0.8-0.9 when an average of 0.2 mol of extra ankyrin was bound per mole of band 3. Ankyrin-free band 3 proteins were mobile, but their mobility was governed by the spectrin association state in the cytoskeletal network. The diffusion constant was 5.3 X 10(-11) cm2 s-1 at a spectrin tetramer mole fraction of 0.3-0.4 in 10 mM NaCl/5 mM sodium phosphate, pH 7.8, and decreased 1 order of magnitude when the tetramer fraction increased to 0.5 in higher NaCl concentration (150 mM NaCl). A similar decrease was observed when the spectrin tetramer fraction was increased by 0.2 mM spermine in 10 mM NaCl/10 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.6. On the other hand, the rotational motion of band 3 in the membranes was not affected by the spectrin association state. Trypsin treatment of ghosts cleaved off the cytoplasmic domain of band 3 and caused a marked (8-fold) increase in the lateral mobility, D = 4.0 X 10(-10) cm2 s-1. These results indicate that the lateral mobility of ankyrin-free band 3 protein is restricted by interactions of their cytoplasmic domain with the cytoskeletal network. A model is presented that band 3 can pass the network when spectrins are in dissociated dimers and cannot pass when they are tetramers. The lateral diffusion constant is thus determined by the spectrin dimer population in the network.

Interaction of aggregated native and mutant IgE receptors with the cellular skeleton.

“…This model is related to one suggested by Tsuji and Ohnishi (32) for explaining why the altered mobility of band 3 proteins on erythrocytes depends on the state of association of spectrin (32). A physical trapping is also compatible with the observation that hyperaggregation of membrane proteins promotes insolubility (refs.…”

Influence of polyamines on membrane functions

“…In the case of Band 3, which is associated to the cytoskeleton network via ankyrin, this effect was correlated with the polyamine-induced spectrin association state, i.e. spermine and spermidine favour polymerization of spectrin (Tsuji & Ohnishi, 1986;Farmer et al, 1985). Such results could be explained if polyamines were bridging proteins and lipids in the membrane, increasing protein-protein interactions in the cytoskeletal network and bridging skeletal and membrane-bound proteins.…”

“…In erythrocytes, the lateral mobilities of fluoresceinlabelled transmembrane glycoproteins (Schindler et al, 1980) and Band 3 (Tsuji & Ohnishi, 1986) were found to be modulated by low concentrations of polyamines that interact with the cytoplasmic side of the cells. Spermine decreased dramatically their lateral diffusion independently from variations in lipid microviscosity and rotational motions.…”

Differential regulation of adherens junction dynamics during apical–basal polarization

“…Because the majority of the spot-FRAP recoveries appear to be non-diffusion based (see below), we measured the mobile fraction and half-time of recovery (t 1/2 ) using the same exponential rise equation used in whole-cell FRAP assays (see Materials and Methods) (Figs 3,5). To make our spot-FRAP data more comparable with previous studies based on the assumption of lateral membrane diffusion, we also processed the data with the diffusion-based formula (Klonis et al, 2002;Tsuji and Ohnishi, 1986) (supplementary material Fig. S5).…”