Diffusion and Patching of Macromolecules on Planar Lipid Bilayer Membranes

David E. Wolf, Joseph Schlessinger, Elliot L. Elson, Watt W. Webb, Robert Blumenthal, Pierre Henkart

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Abstract

We have developed a model system for biological membranes in which amphipathic macromolecular antigens are bound to planar lipid bilayers. The effect of antibodies on the diffusion and distribution of these antigens has been studied. The antigens used are derivatives of dextran (mol wt 82 000) to which controlled amounts of fatty acid, rhodamine, and the antigenic hapten, 2,4,6-trinitrophenyl (TNP), have been covalently bound. These fluorescent amphipathic antigens bind to artificial planar lipid bilayer membranes from the aqueous medium. The diffusion coefficients of these macromolecules were measured by fluorescence photobleaching recovery. Membrane-bound antigens at ~102 molecules/μm2 were distributed homogeneously and had a lateral diffusion coefficient D = (2.1 ± 0.9) × 10−8 cm2/s on oxidized cholesterol and (4.5 ± 2.2) × 10−8 cm2/s on egg phosphatidylcholine. On the addition of anti-TNP, small visible clumps formed similar to the “patches” observed on cell membranes. At a higher level of membrane bound antigen, ~104 molecules/μm2, the distribution was again homogeneous but lateral diffusion was slower with D = (3.1 ± 1.0) × 10−9 cm2/s on oxidized cholesterol and (6.8 ± 2.4) × 10−9 cm2/s on egg phosphatidylcholine. At this concentration, the addition of anti-TNP reduced the diffusion coefficient and immobilized a fraction of the antigens, but did not cause patching. However, further cross-linking by anti-Ig resulted in a complete cessation of lateral diffusion and at higher concentrations in some cases the formation of patches. Similar phenomena were observed using concanavalin A as the cross-linking ligand. The diffusion of a fluorescent lipid probe was unaffected by either the addition of macromolecules or their subsequent cross-linking. Crosslinking one population of dextran derivative retarded the diffusion of another non-cross-reacting population. Sodium azide, colchicine, and cytochalasin B did not affect the motion of these molecules on oxidized cholesterol bilayers.

Original languageEnglish
Pages (from-to)3476-3483
Number of pages8
JournalBiochemistry
Volume16
Issue number15
DOIs
StatePublished - Jul 1 1977
Externally publishedYes

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    Wolf, D. E., Schlessinger, J., Elson, E. L., Webb, W. W., Blumenthal, R., & Henkart, P. (1977). Diffusion and Patching of Macromolecules on Planar Lipid Bilayer Membranes. Biochemistry, 16(15), 3476-3483. https://doi.org/10.1021/bi00634a029