Lateral diffusion of specific antibodies bound to lipid monolayers on alkylated substrates.

Abstract

We have measured the lateral mobility of fluoresceinated monoclonal IgG antibodies bound specifically to a spin label lipid hapten in phospholipid monolayers supported on alkylated silicon oxide surfaces. Dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine monolayers containing 5 mol% of the lipid hapten were transferred by conventional Langmuir-Blodgett techniques onto substrates alkylated with hydrocarbon chains containing 10, 16, and 18 carbon atoms. We show that the diffusion of the bound antibodies depends on their lateral density, the composition of the lipid monolayer, and the nature of lipid coupling to hydrocarbon chains on the alkylated substrate. Antibody diffusion coefficients at low antibody densities are within a factor of 2 of those displayed by the lipid hapten in the absence of the bound antibody. High antibody densities result in reduced antibody mobility, but the lateral diffusion of unbound lipids is unaffected.There is much current interest in the design of appropriate model systems to investigate the structure and function of cellular membranes. In recent work from our laboratory, a variety of model phospholipid membranes has been used in attempts to understand the molecular mechanisms ofcell-cell communication in the immune system (1-3). The biochemical functions of some of these cells are triggered by interactions involving antibody molecules bound to suitable receptors on cell surfaces. For example, mast cells are triggered by the crosslinking of receptor-bound IgE, and macrophages can be triggered by the binding of IgG-antigen complexes to membrane-bound F, receptors (4, 5).The specific binding and subsequent interaction of antibodies bound to lipid haptens in model phospholipid membranes is representative of a general class of events involving ligands and receptors on cell surfaces. New and improved methods for reconstitution of receptors into artificial membranes have led to much progress in understanding membrane phenomena in the immune and nervous systems (6, 7). However, many fundamental questions remain unanswered due to the unavailability of suitable model systems.Epifluorescence and total internal reflection spectroscopy have been used recently to study the interaction of cells ofthe immune system with planar phospholipid membranes (3, 8, 9). Investigations using optical microscopy are much more easily carried out on substrate-supported flat membranes than on vesicles and liposomes. Planar membranes containing histocompatibility antigens have been prepared by fusion of vesicles with solid glass substrates and employed in the study of antigen recognition by T cells (10-12). Planar monolayers and bilayers, transferred onto solid substrates from the air-water interface of a Langmuir trough, have been successfully employed as target membranes in experiments involving antibody-mediated binding of macrophages and basophils. Langmuir-Blodgett techniques afford a high degree of control over the composition and packing densities of molecules in these transferred ...

Improved method of preparation of supported planar lipid bilayers as artificial membranes for antigen presentation

“…A phospholipid monolayer was then deposited using Langmuir‐Schaeffer transfer from a lipid film compressed to a surface pressure of 32 mN/m (Subramaniam et al,1986; von Tscharner and McConnell,1981). This type of monolayer has been shown to be fluid, and its fluidity is inversely correlated to the length of alkyl chain fixed on the substrate (Subramaniam et al,1986; von Tscharner and McConnell,1981). In our hands, when POPC doped with DOPE‐Rho (red fluorescence) was deposited, the monolayer was generally homogeneous under a 63x oil objective in PBS buffer (Fig.…”

Inhomogeneous translational diffusion of monoclonal antibodies on phospholipid Langmuir-Blodgett films

“…The diffusion of AN02 antibodies on supported DMPC monolayers containing a haptenated lipid similar to the one shown in Fig. 1 has previously been investigated (Subramaniam et al, 1986). The films differed from those studied here in several ways.…”

“…Antibodies specifically bind to Langmuir-Blodgett films containing hapten-conjugated phospholipids and the bound antibodies can be laterally mobile Subramaniam et al, 1985Subramaniam et al, , 1986. Antibodies bound to phospholipid Langmuir-Blodgett films for several hours can also form planar crystals (Uzgiris and Kornberg, 1983;Uzgiris, 1986).…”

Lateral mobility of tetramethylrhodamine (TMR) labelled G protein α and βγ subunits in NG 108-15 cells

“…The high Dr value could also result if the ~ subunit associates with the membrane through its N-terminal myristoyl tail. Exogenous proteins attached to lipids and attached to the cytosolic face of plasma membranes have been shown to have diffusion coefficients which approach that of the lipids to which they are attached [50].…”