Resonance energy transfer studies of the mechanisms of microclustering of lentil lectin membrane receptors on HeLa cells

The association and dissociation mechanisms of lectin membrane receptor microclustering on HeLa cells have been studied by measuring resonance energy transfer between fluoresceinated and rhodaminated lentil lectin. Compounds known to affect membrane receptor mobility, such as Ca²⁺ ions, methylamine, cytochalasin D and nocodazole, did not modify the association kinetics nor the maximal energy transfer values at 4 and 37 °C. Dissociation of the membrane receptor microclusters was followed by measuring the temporal decrease in energy transfer values at 4 °C after preincubation for different time intervals at 37 °C. The rate of dissociation of the lectin receptors decreased in the presence of Ca²⁺ ions (10⁻³ M) and after cross-linking with anti-lectin antibodies. An increase was observed in the presence of cytochalasin D (10⁻⁶ M) and, to a lesser extent, of methylamine (10⁻² M). When cytochalasin D and methylamine were combined at subliminal concentrations, a partial synergistic effect was observed. Nocodazole (10⁻⁶ M) had no effect. The results suggest that the association of lectin membrane receptors in microclusters is mediated only by physicochemical parameters. Ca²⁺ ions, cytochalasin D (microfilaments) and methylamine (transglutaminase)-sensitive components appear, however, to play an important role in the stabilization of the receptor microclusters.