Scientific and practical applications of supported lipid-protein bilayers are described. Membranes can be covalently coupled to or separated from solids by ultrathin layers of water or soft polymer cushions. The latter systems maintain the structural and dynamic properties of free bilayers, forming a class of models of biomembranes that allow the application of a manifold of surface-sensitive techniques. They form versatile models of low-dimensionality complex fluids, which can be used to study interfacial forces and wetting phenomena, and enable the design of phantom cells to explore the interplay of lock-and-key forces (such as receptor-ligand binding) and universal forces for cell adhesion. Practical applications are the design of (highly selective) receptor surfaces of biosensors on electrooptical devices or the biofunctionalization of inorganic solids.
Lipid-bilayer membranes supported on solid substrates are widely used as cell-surface models that connect biological and artificial materials. They can be placed either directly on solids or on ultrathin polymer supports that mimic the generic role of the extracellular matrix. The tools of modern genetic engineering and bioorganic chemistry make it possible to couple many types of biomolecule to supported membranes. This results in sophisticated interfaces that can be used to control, organize and study the properties and function of membranes and membrane-associated proteins. Particularly exciting opportunities arise when these systems are coupled with advanced semiconductor technology.
A magnetic bead microrheometer has been designed which allows the generation of forces up to 10(4) pN on 4.5 micron paramagnetic beads. It is applied to measure local viscoelastic properties of the surface of adhering fibroblasts. Creep response and relaxation curves evoked by tangential force pulses of 500-2500 pN (and approximately 1 s duration) on the magnetic beads fixed to the integrin receptors of the cell membrane are recorded by particle tracking. Linear three-phasic creep responses consisting of an elastic deflection, a stress relaxation, and a viscous flow are established. The viscoelastic response curves are analyzed in terms of a series arrangement of a dashpot and a Voigt body, which allows characterization of the viscoelastic behavior of the adhering cell surface in terms of three parameters: an effective elastic constant, a viscosity, and a relaxation time. The displacement field generated by the local tangential forces on the cell surface is visualized by observing the induced motion of assemblies of nonmagnetic colloidal probes fixed to the membrane. It is found that the displacement field decays rapidly with the distance from the magnetic bead. A cutoff radius of Rc approximately 7 micron of the screened elastic field is established. Partial penetration of the shear field into the cytoplasm is established by observing the induced deflection of intracellular compartments. The cell membrane was modeled as a thin elastic plate of shear modulus mu * coupled to a viscoelastic layer, which is fixed to a solid support on the opposite side; the former accounts for the membrane/actin cortex, and the latter for the contribution of the cytoskeleton to the deformation of the cell envelope. It is characterized by the coupling constant chi characterizing the elasticity of the cytoskeleton. The coupling constant chi and the surface shear modulus mu * are obtained from the measured displacements of the magnetic and nonmagnetic beads. By analyzing the experimental data in terms of this model a surface shear modulus of mu * approximately 2 . 10(-3) Pa m to 4 . 10(-3) Pa m is found. By assuming an approximate plate thickness of 0.1 micron one estimates an average bulk shear modulus of mu approximately (2 / 4) . 10(-4) Pa, which is in reasonable agreement with data obtained by atomic force microscopy. The viscosity of the dashpot is related to the apparent viscosity of the cytoplasm, which is obtained by assuming that the top membrane is coupled to the bottom (fixed) membrane by a viscous medium. By application of the theory of diffusion of membrane proteins in supported membranes we find a coefficient of friction of bc approximately 2 . 10(9) Pa s/m corresponding to a cytoplasmic viscosity of 2 . 10(3) Pa s.
We measured the viscoelastic properties of the cytoplasm of J774 macrophages with a recently developed microrheometer. Ferromagnetic beads (1.3 microm in diameter) were used to determine the local viscoelastic moduli. Step-force pulses were applied to the magnetic beads and the displacement was observed by single particle tracking. By analyzing the creep response curves in terms of a triphasic mechanical equivalent circuit, we measured the shear elastic modulus, the effective viscosities, and the strain relaxation time. The values of the shear modulus vary by more than an order of magnitude within the cell population (range, 20-735 Pa; average, 343 Pa) and by a factor of 2 within single cells. The effective viscosity of the cytoplasm exhibits a relatively sharp distribution about an average of eta = 210 Pa s (+/- 143 Pa s). We measured the displacement field generated by the local forces by observing the induced motion of nonmagnetic beads. Even at distances of the order of 1 microm, no induced motion was seen, suggesting that the cytoplasm is composed of clusters of densely packed and cross-linked filaments separated by soft regions. In another series of experiments we analyzed the magnetophoretic motion of the ferromagnetic beads at a constant magnetic force. Measuring the bead velocity parallel and perpendicular to the applied force showed that local active forces on the beads varied from 50 to 900 pN.
Using specular reflection of neutrons, we investigate for the first time the structure of a single dimyristoylphosphatidylcholine bilayer adsorbed to a planar quartz surface in an aqueous environment. We demonstrate that the bilayer is strongly adsorbed to the quartz surface and is stable to phase state changes as well as exchange of the bulk aqueous phase. Our results show that the main phase transition is between the L alpha phase and the metastable L beta'* phase, with formation of the P beta' ripple phase prevented by lateral stress on the adsorbed bilayer. By performing contrast variation experiments, we are able to elucidate substantial detail in the interfacial structure. We measure a bilayer thickness of 43.0 +/- 1.5 A in the L alpha phase (T = 31 degrees C) and 46.0 +/- 1.5 A in the L beta'* phase (T = 20 degrees C). The polar head group is 8.0 +/- 1.5 A thick in the L alpha phase. The water layer between the quartz and bilayer is 30 +/- 10 A for the lipid in both the L alpha and L'* phase. Our results agree well with those previously reported from experiments using lipid vesicles and monolayers, thus establishing the feasibility of our experimental methods.
Monolayers of synthetic lecithins as well as phosphatidic acid at different stages of ionization were studied with the film balance technique at pressures above the lateral vapour pressure. Pressure (π) versus area (a) curves (isotherms) and, by application of a special technique, area (a) versus temperature (T) curves (isobars) were recorded. From these data, plots of the lateral compressibility, χ, as a function of lateral lipid density (a-1) and of the thermal expansion coefficient, α, as a function of temperature (T) were obtained. The well known transition (at T = TM) between the expanded (fluid) and the condensed (crystalline) states of the films, called the main transition, is characterized by a non-horizontal deflection of the isotherms (and isobars) in the coexistence region. A pronounced hysteresis shows that it is a first order transition. Two additional phase transitions, one at T > TM (fluid region) and one at T < TM (crystalline region), were revealed by breaks in the slopes of the isotherms and the isobars. Discontinuities in the compressibilities and in the expansion coefficients show that these transitions are of second order. A flow experiment showed : that the fluid → fluid transition is also characterized by a pronounced discontinuity in viscosity. In fatty acid monolayers the transition (at T < TM) between two crystalline states is of weak first order ( — 10 J./Mole). The signs of the discontinuities, in χ and a, were analysed using the Landau theory of phase transitions. This provided information on the symmetry of the polymorphic states of the monolayers. The symmetry is described 1) in terms of the de Gennes stretching vector J, characterizing the orientation within both the hydrophobic and the hydrophilic region of the monolayer and 2) in terms of the density wave, p, characterizing the symmetry of the lateral organization of the lipid molecules. For α-Dipalmitoyllecithin a phase diagram is established. For π > 15 dyn./cm the polymorphic states of phospholipid monolayers observed at decreasing areas are : fluid isotropic (I) (hydrocarbon chains normal to water surface) ; fluid anisotropic (II) (tilted chains) ; tilted crystalline (III) ; non tilted crystalline (IV). At π ≦ 10 dyn./cm phase II and IV are not observable. Heats of transition ( QM) of the main transition were obtained from the Clausius Clapeyron equation. QM is constant (37 kJ./Mole) at low temperatures while it converges towards zero at higher temperatures, according to QM = Q0M(T - Tc), thus defining a critical temperature. It is proposed that this is a tricritical point above which the main transition becomes of second order. This allows for a symmetry break at the fluid-to-crystalline (main) transition above this critical temperature. The behaviour is explained by the Landau theory in terms of the strength in coupling between the lateral order (density wave p) and the chain orientational order (stretching vector J). In analogy to the Rodbell-Bean effect in magnetism a decrease in coupling leads from a first to a second or...
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