Lipid domain reorganization and receptor events Results obtained with new fluorescent lipid probes

Abstract

Binding of a ligand to the extracellular site of a membrane receptor involves conformational changes not only of the extracellular and cytoplasmic sites but also of the membrane-spanning segments of the receptor protein. The lipids forming the environment of the trans-membrane receptor segments, being flexible and mobile, must follow the shape changes of the receptor molecule to some extent. For this reason ligandreceptor binding itself can be expected to result in changes of the packing of lipid molecules surrounding the receptor. Since lipids are organized in a cooperative manner such changes may affect large areas of the membrane and hence can be registered by physical methods. It has been stated repeatedly that binding of various ligands including hormones, antibodies or lectins to membrane-associated receptors is accompanied by fluidity changes of the target membrane [1][2][3].The aim of this article is to summarize recent evidence suggesting that in highly heterogeneous biological membranes containing numerous metastable lipid domains and/or clusters conformational changes of membrane-associated proteins may result not only in global fluidity changes but also in alterations of the supramolecular lipid domain organization. Frequently such changes can be tbllowed by using, as fluorescent probe, analogs of natural membrane phospholipids or glycolipids bearing, at the end of one of the fatty acyl chains, a 9.anthrylvinyl (AV) group ( cells, and in the latter case may reside for a relatively long time in the plasma membrane without internalization [7].In comparison to most other fluorophores used in membrane research, the AV group displays important advantages. It causes little disturbance because it is nonpolar, flat, and relatively compact. When attached to the fatty acyl chain at an appropriate distance from the head group the AV-fluorophores reside uniformly and exclusively in the center of the bilayer where the host lipids are packed most loosely, and affect neither the mobility nor the orientation and conformation of the major part of the molecules of the surrounding host lipids including their head groups [8]. The AV-group is characterized also by high quantum yields and very short fluorescence lifetimes [9]. The latter property makes the AV-fluorophore suitable for fluorescence polarization measurements. Moreover, in phospholipid bilayers the restricted rotational diffusion of the AVlabeled lipid molecules is of the same time scale as the fluorescence decay. Therefore even subtle changes in the fluidity of the probes microenvironment produce considerable changes in the polarization of fluorescence. The fluorescent parameters of the AV-group (,~ 365 nm, A,~m 430 rim; high extinction coefficient) make it also a highly efficient acceptor for resonance energy transfer (RET) from nearby tryptophans. Since the distance that RET can occur over is as long as 5 nm while RET efficiency depends on the sixth power of the donoracceptor distance, RET measurements with AV-labeled lipids permits the following of even e...

Nonequilibrium Lipid Domain Growth in the Gel−Fluid Two-Phase Region of a DC16PC−DC22PC Lipid Mixture Investigated by Monte Carlo Computer Simulation, FT-IR, and Fluorescence Spectroscopy

“…22,47 The percolation threshold, which is defined as the switching point between the phases forming the connected and disconnected phase, has been proposed to possess functional significance for biological inplane bimolecular reactions and the aggregation of protein subunits to form a functioning protein complex. 20,48,49 Even though a large number of experimental studies have focused on the lipid bilayer properties within the two-phase region, a proper understanding of the thermodynamic and dynamic behavior of such lipid bilayer systems is still lacking. 5 Our findings suggest that the lateral membrane structure within the gel-fluid coexistence region can exist as a nonequilibrium long-living complex heterogeneous structure composed of coexisting gel and fluid domains characterized by a lifetime on the order of hours.…”

“…The lateral organization of two-component lipid bilayers within the phase separated regions has been investigated by a number of techniques such as ESR and FRAP, which clearly have demonstrated that the two-phase region can be characterized as a heterogeneous percolative-like structure. , The percolation threshold, which is defined as the switching point between the phases forming the connected and disconnected phase, has been proposed to possess functional significance for biological inplane bimolecular reactions and the aggregation of protein subunits to form a functioning protein complex. ,, Even though a large number of experimental studies have focused on the lipid bilayer properties within the two-phase region, a proper understanding of the thermodynamic and dynamic behavior of such lipid bilayer systems is still lacking …”