Lateral organisation of membrane lipids. The superlattice view.

Most biological membranes are extremely complex structures consisting of hundreds or even thousands of different lipid and protein molecules. The prevailing view regarding the organisation of these membranes is based on the fluid-mosaic model proposed by Singer and Nicholson in 1972. According to this model, phospholipids together with some other lipids form a fluid bilayer in which these lipids are diffusing very rapidly laterally. The idea of rapid lateral diffusion implies that, in general, the different lipid species would be randomly distributed in the plain of the membrane. However, there are recent data indicating that the components tend to adopt regular (superlattice-like) distributions in fluid, mixed bilayers. Based on this, a superlattice model of membranes has been proposed. This superlattice model is intriguing because it allows only a limited certain number of 'critical' compositions. These critical compositions could play a key role in the regulation of the lipid compositions of biological membranes. Furthermore, such putative critical compositions could explain how compositionally distinct organelles can exist despite of rapid inter-organelle membrane traffic. In this review, these intriguing predictions are discussed along with the basic principles of the model and the evidence supporting it.     

Modeling of cell adhesion and deformation mediated by receptor–ligand interactions

The current work is devoted to studying adhesion and deformation of biological cells mediated by receptors and ligands in order to enhance the existing models. Due to the sufficient in-plane continuity and fluidity of the phospholipid molecules, an isotropic continuum fluid membrane is proposed for modeling the cell membrane. The developed constitutive model accounts for the influence of the presence of receptors on the deformation and adhesion of the cell membrane through the introduction of spontaneous area dilation. Motivated by physics, a nonlinear receptor–ligand binding force is introduced based on charge-induced dipole interaction. Diffusion of the receptors on the membrane is governed by the receptor–ligand interaction via Fick’s Law and receptor-ligand interaction. The developed model is then applied to study the deformation and adhesion of a biological cell. The proposed model is used to study the role of the material, binding, spontaneous area dilation and environmental properties on the deformation and adhesion of the cell.     

On the principles of functional ordering in biological membranes

Integrating the available data on lipid-protein interactions and ordering in lipid mixtures allows to emanate a refined model for the dynamic organization of biomembranes. An important difference to the fluid mosaic model is that a high degree of spatiotemporal order should prevail also in liquid crystalline, "fluid" membranes and membrane domains. The interactions responsible for ordering the membrane lipids and proteins are hydrophobicity, coulombic forces, van der Waals dispersion, hydrogen bonding, hydration forces and steric elastic strain. Specific lipid-lipid and lipid-protein interactions result in a precisely controlled yet highly dynamic architecture of the membrane components, as well as in its selective modulation by the cell and its environment. Different modes of organization of the compositionally and functionally differentiated domains would correspond to different functional states of the membrane. Major regulators of membrane architecture are proposed to be membrane potential controlled by ion channels, intracellular Ca2+, pH, changes in lipid composition due to the action of phospholipase, cell-cell coupling, as well as coupling of the membrane with the cytoskeleton and the extracellular matrix. Membrane architecture is additionally modulated due to the membrane association of ions, lipo- and amphiphilic hormones, metabolites, drugs, lipid-binding peptide hormones and amphitropic proteins. Intermolecular associations in the membrane and in the membrane-cytoskeleton interface are further selectively controlled by specific phosphorylation and dephosphorylation cascades involving both proteins and lipids, and regulated by the extracellular matrix and the binding of growth factors and hormones to their specific receptor tyrosine kinases. A class of proteins coined architectins is proposed, as a notable example the pp60src kinase. The functional role of architectins would be in causing specific changes in the cytoskeleton-membrane interface, leading to specific configurational changes both in the membrane and cytoskeleton architecture and corresponding to (a) distinct metabolic/differentiation states of the cell, and (b) the formation and maintenance of proper three dimensional membrane structures such as neurites and pseudopods.     

On the cyclotron resonance mechanism for magnetic field effects on transmembrane ion conductivity

The cyclotron resonance model, recently proposed to account for physiological response to weak environmental magnetic fields, is shown to violate the laws of classical mechanics. Further, it is argued that the ubiquitous presence of dynamic friction in fluid media precludes significant magnetic effects on membrane ion transport.     

Calcium-Actin Waves and Oscillations of Cellular Membranes

We propose a mechanism for the formation of membrane oscillations and traveling waves, which arise due to the coupling between the actin cytoskeleton and the calcium flux through the membrane. In our model, the fluid cell membrane has a mobile but constant population of proteins with a convex spontaneous curvature, which act as nucleators of actin polymerization and adhesion. Such a continuum model couples the forces of cell-substrate adhesion, actin polymerization, membrane curvature, and the flux of calcium through the membrane. Linear stability analysis shows that sufficiently strong coupling among the calcium, membrane, and protein dynamics may induce robust traveling waves on the membrane. This result was checked for a reduced feedback scheme and is compared to the results without the effects of calcium, where permanent phase separation without waves or oscillations is obtained. The model results are compared to the published observations of calcium waves in cell membranes, and a number of testable predictions are proposed.     

The ‘fluid mosaic model’ and the ‘lattice model’ as two nonequilibrium states of the same membrane and the significance for biochemical control

Summary Biochemical control involves steep and hysteretic response. But the law of mass action does not allow for cooperativity. Therefore resort is classically made to concerted conformational change of protomers. This explanation of steepness and hysteresis by cooperativity is supported by regular surface patterns often observed by electron microscopy. But at other times the lattice appearance which gave rise to the lattice model is not observed. By contrast, a random appearance is observed and the fluid mosaic model of the membrane is assumed. So we are faced with the choice between the fluid mosaic model and the lattice model. Recently the fluid mosaic model is favoured but unlike the lattice model it does not explain the steep hyteretic response.It is suggested that the lattice model and the fluid mosaic model are in fact expression of two states of the very same membrane. The random state corresponds to a resting state. The lattice state corresponds to an active or inhibited state. Thus the transition from random distribution to hexagonal distribution provides simultaneously for triggering and hysteretic cycle with respect to both chemical production and transport across the membrane. This is a universal mechanism for rapid responsiveness and cyclic activity which is largely independent of the chemical mechanism assumed. It is based on the law of mass action supplemented by lateral diffusion. Conformational change and cooperativity are not invoked at all.     

Effect of membrane potential on the mechanical equilibrium of biological membranes

The mechanical equilibrium of a membranous sac, whose wall is sandwiched by two oppositely charged fluid layers, is investigated as a mathematical model of a living cell. In so doing, it is assumed that the space charge density in the inner and the outer charged fluid layer is constant. It is also assumed that the fluid inside and outside of the charged fluid layer is a perfect conductor. By solving Maxwell's equation, the electric field and the thickness of the inner and the outer charged fluid layer is determined as a function of the geometry of the sac. Then, the fluid pressure in the charged fluid layer is derived by considering the body force created by the electrostatic field. The condition of mechanical equilibrium of the sac membrane yields an equation which reveals the inter-relation between the geometry, the sac fluid pressure and the membrane potential. According to this equation, the change of membrane potential causes a deformation of the sac. If the wall of the membranous sac is permeable, increase (decrease) of the absolute value of the membrane potential results in swelling (shrinking) of the sac. On the other hand, the mechanical change of the sac volume results in the change of the membrane potential. This analysis provides also an explanation of how the red blood cell maintains the biconcave shape, when the red blood cell is assumed to be a fluid filled membranous sac with non-zero membrane potential.     

A continuum model of protrusion of pseudopod in leukocytes.

The morphology of human leukocytes, the biochemistry of actin polymerization, and the theory of continuum mechanics are used to model the pseudopod protrusion process of leukocytes. In the proposed model, the pseudopod is considered as a porous solid of F-actin network, the pores of which are full of aqueous solution. G-actin is considered as a "solute" transported by convection and diffusion in the fluid phase. The pseudopod grows as actin filaments elongate at their barbed ends at the tip of the pseudopod. The driving force of extension is hypothesized as being provided by the actin polymerization. It is assumed that elongation of actin filaments, powered by chemical energy liberated from the polymerization reaction, does mechanical work against opposing pressure on the membrane. This also gives rise to a pressure drop in the fluid phase at the tip of the pseudopod, which is formulated by an equation relating the work done by actin polymerization to the local state of pressure. The pressure gradient along the pseudopod drives the fluid filtration through the porous pseudopod according to Darcy's Law, which in turn brings more actin monomers to the growing tip. The main cell body serves as a reservoir of G-actin. A modified first-order equation is used to describe the kinetics of polymerization. The rate of pseudopod growth is modulated by regulatory proteins. A one-dimensional moving boundary problem based on the proposed mechanism has been constructed and approximate solutions have been obtained. Comparison of the solutions with experimental data shows that the model is compatible with available observations. The model is also applicable to growth of other cellular systems such as elongation of acrosomal process in sperm cells.     

Fluid lipid fraction in rod outer segment membrane.

Rod outer segment membrane is analyzed using the spin resonance label technique by means of two probes. The solubility of the first label,2,2,6,6-tetramethylpiperidin-1-oxyl, is correlated with the membrane fluidity which is measured using a stearic acid spin probe. The two values are compared to the solubility-fluidity relationship which characterizes a model system in which all lipids are in a fluid state. The analysis leads to the conclusion that only two thirds of the membrane lipids are fluid. This conclusion is reinforced by the observation that partial lipid removal leaves rigid lipids associated with the rhodopsin molecules.     

Fluid lipid fraction in rod outer segment membrane

Rod outer segment membrane is analyzed using the spin label technique by means of two probes. The solubility of the first label, 2,2,6,6-tetramethylpiperidin-1-oxyl, is correlated with the membrane fluidity which is measured using a stearic acid spin probe. The two values are compared to the solubility-fluidity relationship which characterizes a model system in which all lipids are in a fluid state. The analysis leads to the conclusion that only two thirds of the membrane lipids are fluid. This conclusion is reinforced by the observation that partial lipid removal leaves rigid lipids associated with the rhodopsin molecules.     

Stimulation of chloride transport by fatty acids in corneal epithelium and relation to changes in membrane fluidity.

The effect of altering cell membrane lipids on ion transport across isolated corneas was studied. Corneas mounted in Ussing-type chambers showed a rapid increase in short-circuit current following treatment with a variety of unsaturated fatty acids of varying chain length and unsaturation. Measurements of membrane fluidity which utilize immunofluorescence labelling of membrane proteins showed corneal epithelial cell membranes to be significantly more fluid following linoleic acid treatment. Uptake studies indicate rapid incorporation of [14C]linoleic acid into corneal cell membranes. Highly unsaturated fatty acids were found to have the greatest ability to stimulate chloride transport. Saturated fatty acids were tested and were found to have no effect on chloride transport at any concentration. It is proposed that unsaturated fatty acids activate chloride transport by increasing membrane lipid fluidity. The relationship of these parameters is discussed in terms of a mobile receptor model. We speculate that an increase in membrane lipid fluidity promotes lateral diffusion of membrane receptor proteins and enzymes, increasing protein-protein interactions within the membrane, ultimately resulting in the enhancement of cyclic AMP synthesis.     

FLUID TRANSPORT IN THE RABBIT GALLBLADDER : A Combined Physiological and Electron Microscopic Study

The fine structure of the rabbit gallbladder has been studied in specimens whose functional state was undetermined, which were fixed either in situ or directly after removal from the animal; in specimens whose rate of fluid absorption was determined, either in vivo or in vitro, immediately prior to fixation; and in specimens from bladders whose absorptive function was experimentally altered in vitro. Considerable variation was found in the width of the epithelial intercellular spaces in the bladders whose functional state was undefined. In bladders known to be transporting fluid, either in vivo or in vitro, the intercellular spaces were always distended, as were the subepithelial capillaries. This distension was greatest in bladders which had been functioning in vitro. When either Na⁺ or Cl^- was omitted from the bathing media, there was no fluid transport across the wall of the gallbladder studied in vitro. The epithelial intercellular spaces of biopsies taken from several bladders under these conditions were of approximately 200 A width except for minor distension at the crests of mucosal folds. The addition of the missing ion rapidly led to the reestablishment of fluid transport and the distension of the intercellular spaces throughout most of the epithelium of these bladders. Studies of sodium localization (by fixation with a pyroantimonate-OsO₄ mixture) showed high concentrations of this ion in the distended intercellular spaces. Histochemical studies of ATPase activity showed that this enzyme was localized along the lateral plasma membrane of the epithelial cells. The analogy is drawn between the structure of the gallbladder mucosa and a serial membrane model proposed by Curran to account for coupled solute-solvent transport across epithelia. It is concluded that the intercellular compartment fulfills the conditions for the middle compartment of the Curran model and that active transport of solute across the lateral plasma membrane into the intercellular space may be responsible for fluid absorption by the gall bladder.     

Effects of surfactin on membrane models displaying lipid phase separation

Surfactin, a bacterial amphiphilic lipopeptide is attracting more and more attention in view of its bioactive properties which are in relation with its ability to interact with lipids of biological membranes. In this work, we investigated the effect of surfactin on membrane structure using model of membranes, vesicles as well as supported bilayers, presenting coexistence of fluid-disordered (DOPC) and gel (DPPC) phases. A range of complementary methods was used including AFM, ellipsometry, dynamic light scattering, fluorescence measurements of Laurdan, DPH, calcein release, and octadecylrhodamine B dequenching. Our findings demonstrated that surfactin concentration is critical for its effect on the membrane. The results suggest that the presence of rigid domains can play an essential role in the first step of surfactin insertion and that surfactin interacts both with the membrane polar heads and the acyl chain region. A mechanism for the surfactin lipid membrane interaction, consisting of three sequential structural and morphological changes, is proposed. At concentrations below the CMC, surfactin inserted at the boundary between gel and fluid lipid domains, inhibited phase separation and stiffened the bilayer without global morphological change of liposomes. At concentrations close to CMC, surfactin solubilized the fluid phospholipid phase and increased order in the remainder of the lipid bilayer. At higher surfactin concentrations, both the fluid and the rigid bilayer structures were dissolved into mixed micelles and other structures presenting a wide size distribution.     

Phase transitions of the coupled membrane-cytoskeleton modify cellular shape

Formation of protrusions and protein segregation on the membrane is of a great importance for the functioning of the living cell. This is most evident in recent experiments that show the effects of the mechanical properties of the surrounding substrate on cell morphology. We propose a mechanism for the formation of membrane protrusions and protein phase separation, which may lay behind this effect. In our model, the fluid cell membrane has a mobile but constant population of proteins with a convex spontaneous curvature. Our basic assumption is that these membrane proteins represent small adhesion complexes, and also include proteins that activate actin polymerization. Such a continuum model couples the membrane and protein dynamics, including cell-substrate adhesion and protrusive actin force. Linear stability analysis shows that sufficiently strong adhesion energy and actin polymerization force can bring about phase separation of the membrane protein and the appearance of protrusions. Specifically, this occurs when the spontaneous curvature and aggregation potential alone (passive system) do not cause phase separation. Finite-size patterns may appear in the regime where the spontaneous curvature energy is a strong factor. Different instability characteristics are calculated for the various regimes, and are compared to various types of observed protrusions and phase separations, both in living cells and in artificial model systems. A number of testable predictions are proposed.     

Isolation and characterization of the tubular organelles induced by fumarate reductase overproduction in Escherichia coli

Strains of Escherichia coli amplifying the intrinsic membrane enzyme fumarate reductase accommodate the overproduced enzyme by increasing the amount of membrane material, in the form of intracellular tubular structures. These tubules have been observed in strains harbouring multicopy frd plasmids and in ampicillin hyper-resistant strains. A procedure has been developed for isolation of tubules nearly free of cytoplasmic membrane. Using protein A-gold labelling and optical diffraction of electron micrographs, a model for tubule structure is proposed. The tubules have a lower lipid/protein ratio than the cytoplasmic membrane, with the enzyme accounting for greater than 90% of the protein in the tubules. Both cytoplasmic membranes and tubules from amplified strains are enriched in cardiolipin and have a more fluid fatty acid composition than wild-type strains. Mutants defective in cardiolipin synthesis produce tubules in response to excess fumarate reductase, but these tubules have an altered appearance, indicating that lipid-protein interactions may be important for tubule assembly.     

The Suppression of the Late After-Potential in Rubidium-Containing Frog Muscle Fibers

The late after-potential that follows trains of impulses in frog muscle fibers is virtually absent when most of the intracellular potassium is replaced by rubidium and the muscle is immersed in rubidium-containing Ringer's fluid. Its amplitude is also reduced in freshly dissected, potassium-containing muscle fibers that are immersed directly in Rb-Ringer's fluid. These findings are discussed in terms of the model for muscle membrane of Adrian and Freygang (1962 a, b) and in relation to the report of Adrian (1964) that Rb-containing muscle fibers do not exhibit the variations in potassium permeability as a function of membrane potential that are found in fibers with normal intracellular potassium concentration immersed in Ringer's fluid.     

Interactions of pyrethroids with gramicidin-containing liposomal membranes

Fluorescence steady-state anisotropy and phase-modulation lifetime techniques have been utilized to study the interactions of pyrethroid compounds with fluid-phase phosphatidylcholine membranes containing the polypeptide gramicidin. This polypeptide is considered to be a model of hydrophobic regions of cellular integral membrane proteins. The pyrethroids disorder lipid packing in cellular membranes and gel-phase liposomes but do not disorder lipid packing in fluid-phase lipid (Stelzer, K.J. and Gordon, M.A. (1984) J. Immunopharmacol. 6, 381-410; (1985) Biochim. Biophys. Acta 812, 361-368) Irrespective of liposomal size, gramicidin incorporation resulted in a substantial increase in anisotropy of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), in fluid phase lipid. In the absence of gramicidin, permethrin and three other pyrethroids, allethrin, cypermethrin and fenpropathrin, increased DPH anisotropy. In these fluid phase systems, as the protein:lipid ratio was increased, the extent of the pyrethroid-mediated increase in fluorescence anisotropy diminished. Also, the pyrethroids shortened DPH fluorescence lifetimes. At high gramicidin:lipid ratios, permethrin substantially lowered anisotropy in the fluid phase lipid, relative to controls. The data suggest that pyrethroids disturb fluid-phase lipids which have been promoted to a relative state of order by proximity to an integral membrane protein. This type of order is one which is represented by DPH fluorescence anisotropy. A model based on these results is proposed to explain the effects of pyrethroids on lipid packing order in cellular membranes, as determined by DPH fluorescence anisotropy.