Binding and calcium-induced aggregation of laminin onto lipid bilayers.

E Kalb,J Engel

doi:10.1016/s0021-9258(18)55170-x

Abstract

Direct binding of laminin in the form of its complex with nidogen to planar lipid bilayers was demonstrated with total internal reflection fluorescence microscopy. Binding occurred equally well to zwitterionic (phosphatidylcholine) and negatively charged (phosphatidylglycerol) lipids and was enhanced by sulfatides but only at nonphysiological molar ratios higher than 30 mol %. Strong interactions with lipid bilayers were also observed for bovine serum albumin. This explains a strong inhibition of laminin binding by this protein. However, binding of laminin to sulfatide-rich bilayers was not completely inhibited. Observable by the microscopic technique was the formation of laminin clusters on the surface of the bilayer which occurred concomitantly with binding. Both processes were strongly enhanced by the presence of calcium. These results show that calcium-induced laminin self-assembly is enhanced at lipid surfaces.

Highlights

Direct binding of laminin in the form of its complex with nidogento planar lipid bilayers was demonstrated with total internal reflection fluorescence microscopy
In the present work we examined nonreceptor-mediated binding of laminin and its complex with nidogen/entactin to lipid bilayers composed of different lipids including sulfatides by a physical technique
Planar supported bilayers (9) were prepared by consecutive deposition of two separate monolayers ontoaquartz slide, and bound fluorescence-labeled protein was monitored by selective illumination of the quartzbuffer interface by total internal reflection fluorescence microscopy (TIRFM (10)).This technique combines the advantages of measuring the binding at real time to a well defined lipid bilayer without disturbanceof the equilibrium with direct microscopic observation of the stateof the bilayer and protein

Summary

RESULTS

Laminin Binds to Supported Bilayers-We examined the binding of laminin in the form of its LN to lipid bilayers supportedonquartz slides. Dependence of Binding onLipid Composition-Very similar binding isotherms were observed in theabsence of BSA (Fig. 1A)for bilayers composed of zwitterionic phosphatidylcholine (POPC), negatively charged phosphatidylglycerol (POPG), and mixtures of POPC and 1 or 30 mol % sulfatide. When BSAwas added to thebuffer phase at high molar excess over lamininnidogen and lipid the binding of LN to POPC membranes was effectively abolished (Fig. 1B). Influence of Ca" ions on binding and aggregationof laminin-nidogen the protein and thebuffer by addition of an excess of EDTA at liDid bilayers led to much diminished binding (Fig. 3).

Lipid composition

DISCUSSION

Laminin Lipid Membrane Interactions