Lateral redistribution of transmembrane proteins and liquid-ordered domains in lipid membranes with inhomogeneous curvature

A number of processes in living cells are accompanied by significant changes of the geometric curvature of lipid membranes. In turn, heterogeneity of the lateral curvature can lead to spatial redistribution of membrane components, most important of which are transmembrane proteins and liquid-ordered lipid-protein domains. These components have a so-called hydrophobic mismatch: the length of the transmembrane domain of the protein, or the thickness of the bilayer of the domain differ from the thickness of the surrounding membrane. In this work we consider redistribution of membrane components with hydrophobic mismatch in membranes with non-uniform geometric curvature. Dependence of the components’ energy on the curvature is calculated in terms of theory of elasticity of liquid crystals adapted to lipid membranes. According to the calculations, transmembrane proteins prefer regions of the membrane with zero curvature. Liquid-ordered domains having a size of a few nm distribute mainly into regions of the membrane with small negative curvature appearing in the cell plasma membrane in the process of endocytosis. The distribution of domains of a large radius is determined by a decrease of their perimeter upon bending; these domains distribute into membrane regions with relatively large curvature.     

Shape transformations induced by amphiphiles in erythrocytes

Shape alterations induced in human erythrocytes by cationic, anionic, zwitterionic and nonionic amphiphiles (C10-C16) at antihaemolytic concentrations (CAH50 and CAHmax) and at a slightly lytic concentration (2-10% haemolysis) were studied. Anionic (sodium alkyl sulphates) and zwitterionic amphiphiles (3-(alkyldimethylammonio)-1-propanesulfonates) proved to be potent echinocytogenic agents. Among the nonionic amphiphiles there were potent stomatocytogenicagents (octaethyleneglycol alkyl ethers, pentaethyleneglycol dodecyl ether), one potent echinocytogenic agent (dodecyl D-maltoside) and one weak echinocytogenic agent (decyl beta-D-glucopyranoside). Shape alterations induced by cationic amphiphiles (alkyltrimethylammonium bromides, cetylpyridinium chloride and dodecylamine hydrochloride) showed a strong time-dependence. These amphiphiles immediately induced strongly crenated erythrocytes which during incubation shifted to less crenated erythrocytes or to stomatocytes. All of the echinocytogenic amphiphiles induced echinocytes immediately, and there were only small alterations of the induced shape during incubation. Among the stomatocytogenic amphiphiles there were some that induced stomatocytes immediately or after a short lag time while others first passed the erythrocytes through echinocytic stages before stomatocytic shapes were attained. Erythrocytes treated with amphiphiles did not recover their normal discoid shape following repeated washing and reincubation for 1 h in amphiphile-free medium. Our study shows that shape alterations induced by amphiphiles in erythrocytes cannot be explained solely by assuming a selective intercalation of differently charged amphiphiles into the monolayers of the lipid bilayer as suggested in the bilayer couple hypothesis (Sheetz, M.P. and Singer, S.J. (1976) J. Cell Biol. 70, 247-251). We suggest that amphiphiles, when intercalated into the lipid bilayer, trigger a rapid formation of intrabilayer non-bilayer phases which protect the bilayer against a collapse and bring about a transbilayer redistribution of intercalated amphiphiles as well as of bilayer lipids.     

Bending rigidities of cell surface molecules P-selectin and PSGL-1

P-selectin is a cell adhesion molecule expressed on activated endothelial cells and platelets. P-selectin glycoprotein ligand 1 (PSGL-1) is a mucin expressed on leukocytes. The interaction of P-selectin and PSGL-1 mediates leukocyte tethering to and rolling on the vascular surface, which are initiating events in inflammatory and thrombotic processes. In the hemodynamic environment of the circulation, P-selectin and PSGL-1 are subject to a wide range of forces, which can cause deformation. For P-selectin/PSGL-1 interaction to be physically possible, these molecules may need to project above much of the glycocalyx layers of the respective cell surfaces, suggesting that they are either longer than the thickness of glycocalyx or better able to support compression than the glycocalyx. As such, the mechanical properties of these molecules and their functional implications merit investigation. Here we report determination of the bending rigidities of P-selectin and PSGL-1 by analyzing their thermally excited curvature fluctuations, whose values are of the order of magnitude of 100 pN nm².     

Lateral diffusion in inhomogeneous membranes. Model membranes containing cholesterol.

The problem of lateral diffusion in inhomogeneous membranes is illustrated by a theoretical calculation of the lateral diffusion of a fluorescent lipid probe in binary mixtures of phosphatidylcholine and cholesterol under conditions of temperature and composition such that this lipid mixture consists of alternating parallel domains of fluid and solid lipid, having separations that are small compared with the distance scale employed in photobleaching experiments. The theoretical calculations clearly illustrate how inhomogeneities in membrane composition affecting the lateral motion of membrane components on a small (10-100 nm) distance scale can give complex diffusive responses in experiments such as fluorescence photobleaching that employ comparatively macroscopic distances (10-100 micrometers) for the measurement of diffusive recovery. The theoretical calculations exhibit the unusual dependence of the apparent lateral diffusion coefficient of a fluorescent lipid probe on lipid composition in binary mixtures of cholesterol and phosphatidylcholines as reported by Rubenstein et al. (1979, Proc. Natl. Acad. Sci. U.S.A., 76:15-18).     

Hopf bifurcations in multiple-parameter space of the Hodgkin-Huxley equations II. Singularity theoretic approach and highly degenerate bifurcations

In the Hodgkin-Huxley equations (HH), we have identified the parameter regions in which either two stable periodic solutions with different amplitudes and periods and an equilibrium point or two stable periodic solutions coexist. The global structure of bifurcations in the multiple-parameter space in the HH suggested that the bistabilities of the periodic solutions are associated with the degenerate Hopf bifurcation points by which several qualitatively different behaviors are organized. In this paper, we clarify this by analyzing the details of the degenerate Hopf bifurcations using the singularity theory approach which deals with local bifurcations near a highly degenerate fixed point. Received: 23 April 1999 / Accepted in revised form: 24 September 1999     

Global bifurcations in the FitzHugh-Nagumo equations for nerve wave propagation

The FitzHugh-Nagumo equations for action potential propagation along nerve axons and the corresponding ordinary differential equations for travelling waves are solved numerically. Above a critical value, a constant bias current can drive a wave-front solution. At the critical value, a global bifurcation occurs. As a result, the wave front switches into a pulse.Based on a thesis by one of the authors (H. F.).     

Spontaneous local membrane curvature induced by transmembrane proteins

The (local) curvature of cellular membranes acts as a driving force for the targeting of membrane-associated proteins to specific membrane domains, as well as a sorting mechanism for transmembrane proteins, e.g., by accumulation in regions of matching spontaneous curvature. The latter measure was previously experimentally employed to study the curvature induced by the potassium channel KvAP and by aquaporin AQP0. However, the direction of the reported spontaneous curvature levels as well as the molecular driving forces governing the membrane curvature induced by these integral transmembrane proteins could not be addressed experimentally.Here, using both coarse-grained and atomistic molecular dynamics (MD) simulations, we report induced spontaneous curvature values for the homologous potassium channel Kv 1.2/2.1 Chimera (KvChim) and AQP0 embedded in unrestrained lipid bicelles that are in very good agreement with experiment. Importantly, the direction of curvature could be directly assessed from our simulations: KvChim induces a strong positive membrane curvature ($\approx 0.036$ nm^?1) whereas AQP0 causes a comparably small negative curvature ($\approx ?0.019$ nm^?1).Analyses of protein-lipid interactions within the bicelle revealed that the potassium channel shapes the surrounding membrane via structural determinants. Differences in shape of the protein-lipid interface of the voltage-gating domains between the extracellular and cytosolic membrane leaflets induce membrane stress and thereby promote a protein-proximal membrane curvature. In contrast, the water pore AQP0 displayed a high structural stability and an only faint effect on the surrounding membrane environment that is connected to its wedge-like shape.     

Generation of high curvature membranes mediated by direct endophilin bilayer interactions

Endophilin 1 is a presynaptically enriched protein which binds the GTPase dynamin and the polyphosphoinositide phosphatase synptojanin. Perturbation of endophilin function in cell-free systems and in a living synapse has implicated endophilin in endocytic vesicle budding (Ringstad, N., H. Gad, P. Low, G. Di Paolo, L. Brodin, O. Shupliakov, and P. De Camilli. 1999. Neuron. 24:143-154; Schmidt, A., M. Wolde, C. Thiele, W. Fest, H. Kratzin, A.V. Podtelejnikov, W. Witke, W.B. Huttner, and H.D. Soling. 1999. Nature. 401:133-141; Gad, H., N. Ringstad, P. Low, O. Kjaerulff, J. Gustafsson, M. Wenk, G. Di Paolo, Y. Nemoto, J. Crun, M.H. Ellisman, et al. 2000. Neuron. 27:301-312). Here, we show that purified endophilin can directly bind and evaginate lipid bilayers into narrow tubules similar in diameter to the neck of a clathrin-coated bud, providing new insight into the mechanisms through which endophilin may participate in membrane deformation and vesicle budding. This property of endophilin is independent of its putative lysophosphatydic acid acyl transferase activity, is mediated by its NH2-terminal region, and requires an amino acid stretch homologous to a corresponding region in amphiphysin, a protein previously shown to have similar effects on lipid bilayers (Takei, K., V.I. Slepnev, V. Haucke, and P. De Camilli. 1999. Nat. Cell Biol. 1:33-39). Endophilin cooligomerizes with dynamin rings on lipid tubules and inhibits dynamin's GTP-dependent vesiculating activity. Endophilin B, a protein with homology to endophilin 1, partially localizes to the Golgi complex and also deforms lipid bilayers into tubules, underscoring a potential role of endophilin family members in diverse tubulovesicular membrane-trafficking events in the cell.     

Biophysical perturbations induced by ethylazinphos in lipid membranes

Perturbations induced by ethylazinphos on the physical organization of dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol membranes were studied by differential scanning calorimetry (DSC) and fluorescence polarization of 2-, 6-, 12-(9-anthroyloxy) stearic acids and 16-(9-anthroyloxy) palmitic acid. Ethylazinphos (50 and 100 microM) increases the fluorescence polarization of the probes, either in the gel or in the fluid phase of DPPC bilayers, and this concentration dependent effect decreases from the surface to the bilayer core. Additionally, the insecticide displaces the phase transition to a lower temperature range and broadens the transition profile of DPPC. A shifting and broadening of the phase transition is also observed by DSC. Furthermore at insecticide/lipid molar ratios higher than 1/7, DSC thermograms, in addition to the normal transition centered at 41 degrees C, also display a new phase transition centered at 45.5 degrees C. The enthalpy of this new transition increases with insecticide concentration, with a corresponding decrease of the main transition enthalpy. Ethylazinphos in DPPC bilayers with low cholesterol (< or = 20 mol%) perturbs the membrane organization as described above for pure DPPC. However, cholesterol concentrations higher than 20 mol% prevent insecticide interaction, as revealed by fluorescence polarization and DSC data. Apparently, cholesterol significantly modulates insecticide interaction by competition for similar distribution domains in the membrane. The present results strongly support our previous hypothesis that ethylazinphos locates in the cooperativity region, i.e. the region of C1-C9 atoms of the acyl chains, and extends to the lipid-water interface, where it increases lipid packing order sensed across all the thickness of the bilayer. Additionally, and, on the basis of DSC data, a lateral regionalization of ethylazinphos is here tentatively suggested.     

Structural rearrangements in membranes induced by anesthetics]

Studies were carried out on the effects of anesthetics on the stability of membranes to sodium dodecylsulfate and trypsin. The initial rate of membrane desintegration by detergent evaluated by light scattering with stopped flow method was increased in the presence of ethanol, propanol, benzyl alcohol, procain-amide (erthrocyte membrane) and chlorpromazine (rat brain synaptosomes). Concentrations of anesthetics which correspond to half of the maximum effect were very close to that of blocking the action potential and inducing 50%--antihemolysis. Alcohols and procaine at anesthetic concentrations inhibited proteolysis of isolated erythrocyte membranes but were without effect on their ultrasonic fragments. Increase of membrane desintegtation rate is interpreted as structural rearrangements of membranes with the weakening of detergent sensitive intermolecular interactions. Proteolysis inhibition of ghosts but not the gragments indicates against the participation of the basic func of proteins in the structural membrane rearrangements.     

Shape and size changes induced by taurine depletion in neonatal cardiomyocytes

Summary Taurine is a very important organic osmolyte in most adult cells. Because of this property it has been proposed that large changes in the intracellular content of taurine can osmotically stress the cell, causing changes in its size and shape. This hypothesis was examined by measuring cell dimensions of taurine deficient cardiomyocytes using confocal microscopy. Incubation of isolated neonatal rat myocytes with medium containing 5mM-alanine led to a 55% decrease in intracellular taurine content. Associated with the loss of taurine was a reduction in cell size. Two factors contributed to the change in cell size. First, there was a shift in cell shape, favoring the smaller of the two cellular configurations commonly found in the myocyte cell culture. Second, the size of the polyhedral configuration was reduced after ßalanine treatment. These same two events also contributed to size reduction in cardiomyocytes incubated with medium containing 30mM mannitol. Nonetheless, some qualitative differences exist between cells osmotically stressed by increasing the osmolality of the incubation medium and decreasing intracellular osmolality. The results support a role for taurine in the regulation of osmotic balance in the neonatal cardiomyocyte.     

Particle displacement in epithelial cell membranes of rat prostate and pancreas induced by routine low temperature fixation

When carried out at 4 °C in contrast to 37 °C, glutaraldehyde fixation for routine freeze-fracture specimen preparation results in displacement of membrane particles of epithelial cells of rat ventral prostate. Particle-free areas are observed in endoplasmic reticulum (ER), nuclear envelope, and Golgi membranes; particle aggregation is observed in lateral and basal portions of the plasma membrane. Multilamellar arrangement of lipids can be observed in some of the large particle-free areas. Thin section observation indicates that in the ER, particle-free areas coexist with segregation of cytoplasmic ground substance from the membrane surfaces. Particle-free patches are also observed in the ER membranes of pancreatic tissues fixed at 4 °C.     

Local and nonlocal curvature elasticity in bilayer membranes by tether formation from lecithin vesicles.

Bilayer membranes exhibit an elastic resistance to changes in curvature. This resistance depends both on the intrinsic stiffness of the constituent monolayers and on the curvature-induced expansion or compression of the monolayers relative to each other. The monolayers are constrained by hydrophobic forces to remain in contact, but they are capable of independent lateral redistribution to minimize the relative expansion or compression of each leaflet. Therefore, the magnitude of the expansion and compression of the monolayers relative to each other depends on the integral of the curvature over the entire membrane capsule. The coefficient characterizing the membrane stiffness resulting from relative expansion is the nonlocal bending modulus kr. Both the intrinsic (local) bending modulus (kc) and the nonlocal bending modulus (kr) can be measured by the formation of thin cylindrical membrane strands (tethers) from giant phospholipid vesicles. Previously, we reported measurements of kc based on measurements of tether radius as a function of force (Song and Waugh, 1991, J. Biomech. Engr. 112:233). Further analysis has revealed that the contribution from the nonlocal bending stiffness can be detected by measuring the change in the aspiration pressure required to establish equilibrium with increasing tether length. Using this approach, we obtain a mean value for the nonlocal bending modulus kr of approximately 4.1 x 10(-19)J. The range of values is broad (1.1-10.1 x 10(-19)J) and could reflect contributions other than simple mechanical equilibrium. Inclusion of the nonlocal bending stiffness in the calculation of kc results in a value for that modulus of approximately 1.20 +/- 0.17 x 10(-19)J, in close agreement with values obtained by other methods.     

Intrinsic curvature in normal and inverted lipid structures and in membranes.

The intrinsic or spontaneous radius of curvature, R(o), of lipid monolayer assemblies is expressed in terms of a lipid molecular packing parameter, V/AI, for various geometries. It is shown that the equivalent lipid length, 1, in inverted hexagonal (HII) phases, defined by a cylindrical shell of equal total lipid volume, yields an expression for R o identical to that for inverted cylindrical micelles (or, equivalently, HII phases in the presence of excess hydrocarbon). This identity is used to obtain values of the effective packing parameter for various phosphatidylethanolamines. The temperature dependence of the intrinsic radius of curvature is predicted to be negative and to be considerably greater than that for the lipid length in nearly all cases. The thermal expansion coefficient is not constant but is found to vary, depending on the value of the lipid packing parameter. A possible addition rule is constructed for the intrinsic radius of curvature of lipid mixtures, based on the linear additivity of the effective molecular volumes, V, and molecular areas, A. This relation is found to hold for mixtures of dioleoyl phosphatidylcholine (DOPC) with dioleoyl phosphatidylethanolamine, and a value of R(o) of > or = 9 A (V/AI = 1.08) is obtained for DOPC. The energetics of the intrinsic curvature and lamellar-nonlamellar transitions are also discussed within the framework of the model.     

Lipid phase transitions in membranes involving intrinsic periodic curvature

A conformation of the lipid bilayer of membranes is proposed, with periodic curvature corresponding to the minimal surface structure of cubic lipid phases. Evidence is given indicating that activities of lipid synthesis/modification enzymes embedded in the membrane are controlled by the lateral "packing-pressure", so that the lipid bilayer is close to a transition from the lamellar (L alpha) type of conformation to this periodically curved conformation. Such a phase transition mechanism is assumed to be involved in numerous cooperative membrane functions.