Charge sensing and controllable tunnel coupling in a Si/SiGe double quantum dot.

We report integrated charge sensing measurements on a Si/SiGe double quantum dot. The quantum dot is shown to be tunable from a single, large dot to a well-isolated double dot. Charge sensing measurements enable the extraction of the tunnel coupling t between the quantum dots as a function of the voltage on the top gates defining the device. Control of the voltage on a single such gate tunes the barrier separating the two dots. The measured tunnel coupling is an exponential function of the gate voltage. The ability to control t is an important step toward controlling spin qubits in silicon quantum dots.

Supported membranes: scientific and practical applications.

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.

Microfluidic device for simultaneous analysis of neutrophil extracellular traps and production of reactive oxygen species.

Neutrophil extracellular traps (NETs) were first reported in 2004, and since their discovery, there has been an increasing interest in NETs, how they are formed, their role in controlling infections, and their contribution to disease pathogenesis. Despite this rapid expansion of our understanding of NETs, many details remain unclear including the role of reactive oxygen species (ROS) in the formation of NETs. Further, to study NETs, investigators typically require a large number of cells purified via a lengthy purification regimen. Here, we report a microfluidic device used to quantify both ROS and NET production over time in response to various stimulants, including live bacteria. This device enables ROS and NET analysis using a process that purifies primary human neutrophils in less than 10 minutes and requires only a few microliters of whole blood. Using this device we demonstrate the ability to identify distinct capabilities of neutrophil subsets (including ROS production and NET formation), the ability to use different stimulants/inhibitors, and the ability to effectively use samples stored for up to 8 hours. This device permits the study of ROS and NETs in a user-friendly format and has potential for widespread applications in the study of human disease.

Variation of frequency spectrum of the erythrocyte flickering caused by aging, osmolarity, temperature and pathological changes.

Frequency spectra of the surface undulations (flickering) of erythrocyte plasma membranes are measured by direct spectral analysis of the intensity fluctuations of the light passing the cells in a phase contrast microscope. Spectra are taken as a function (1) of the temperature (2) of the viscosity and osmolarity of the outer medium (3) of the aging of cells and (4) of pathological transformations. The spectra are approximately superpositions of two Lorentzian lines. At large frequencies, f, the spectra follow f-2. This behaviour can be interpreted in terms of cell thickness fluctuations caused by thermally excited membrane undulations provided the range of wavelengths is small. The undulations are determined by the membrane curvature elasticity while the lateral tension is negligibly small for cells of discoid shape. The technique presented allows accurate measurements of relative curvature (bending) elastic constants. The spectra of freshly drawn cells are remarkably reproducible. Aging of the cells in the medium leads to an increase in the curvature elastic constant. A decrease in osmolarity causes a reduction in the intensity and line width of the spectra and the flickering vanishes if the cell approaches a spherical shape. The effect of temperature between 10 and 40 degrees C is astonishingly small with the exception of a sudden increase in the amplitude with increasing temperature at 35 degrees C. The flicker spectra of a large fraction of the cells from patients suffering from cronical alcoholism exhibit a reduced line width or an increase in the curvature elastic constant.

Sensitive detection of protein adsorption to supported lipid bilayers by frequency-dependent capacitance measurements and microelectrophoresis.

In the first part, we report experiments which enable the sensitive detection of protein adsorption to lipid bilayers deposited onto chromium electrodes on glass substrates by frequency-dependent capacitance measurements. The sensitivity of the present type of sensor (better than 0.3 nm average protein layer thickness) is at least equivalent to that of ellipsometry. A high specific resistance of the supported bilayer of (1-5).10(5) omega.cm2 is achieved by deposition of a tightly packed (crystalline) cadmium arachidate monolayer in contact with the substrate, whereas the outer monolayer can be more loosely packed (fluid phase or state of fluid-solid coexistence) which is essential for the incorporation of receptors. In the present work, charged lipids are incorporated as nonspecific receptors for polylysine and cytochrome c. The capacitance measurements provide a very sensitive test of the tightness and the long-time stability of the supported bilayers and, in combination with ellipsometric thickness measurements, enable estimations of dielectric properties of protein layers (such as the permittivity). In the second part, we report first electrophoresis experiments in asymmetric bilayers on substrates which enable simultaneous measurements of lateral diffusion coefficients and frictional coefficients between monolayers. The potential application of the electrophoretic effect for the differentiation between different receptors and the amplification of signals in biosensors is discussed.

Chemically induced lipid phase separation in model membranes containing charged lipids: a spin label study.

The lipid distribution in binary mixed membranes containing charged and uncharged lipids and the effect of Ca2+ and polylysine on the lipid organization was studied by the spin label technique. Dipalmitoyl phosphatidic acid was the charged, and spin labelled dipalmitoyl lecithin was the uncharged (zwitterionic) component. The ESR spectra were analyzed in terms of the spin exchange frequency, Wex. By measuring Wex as a function of the molar percentage of labelled lecithin a distinction between a random and a heterogeneous lipid distribution could be made. It is established that mixed lecithin-phosphatidic acid membranes exhibit lipid segregation (or a miscibility gap) in the fluid state. Comparative experiments with bilayer and monolayer membranes strongly suggest a lateral lipid segregation. At low lecithin concentration, aggregates containing between 25% and 40% lecithin are formed in the fluid phosphatidic acid membrane. This phase separation in membranes containing charged lipids is understandable on the basis of the Gouy-Chapman theory of electric double layers. In dipalmitoyl lecithin and in dimyristoyl phosphatidylethanolamine membranes the labelled lecithin is randomly distributed above the phase transition and has a coefficient of lateral diffusion of D = 2.8-10(-8) cm2/s at 59 degrees C. Addition of Ca2+ dramatically increases the extent of phase separation in lecithin-phosphatidic acid membranes. This chemically (and isothermally) induced phase separation is caused by the formation of crystalline patches of the Ca2+-bound phosphatidic acid. Lecithin is squeezed out from these patches of rigid lipid. The observed dependence of Wex on the Ca2+ concentration could be interpreted quantitatively on the basis of a two-cluster model. At low lecithin and Ca2+ concentration clusters containing about 30 mol % lecithin are formed. At high lecithin or Ca2+ concentrations a second type of precipitation containing 100% lecithin starts to form in addition. A one-to-one binding of divalent ions and phosphatidic acid at pH 9 was assumed. Such a one-to-one binding at pH 9 was established for the case of Mn2+ using ESR spectroscopy. Polylysine leads to the same strong increase in the lecithin segregation as Ca2+. The transition of the phosphatidic acid bound by the polypeptide is shifted from Tt = 47.5 degrees to Tt = 62 degrees C. This finding suggests the possibility of cooperative conformational changes in the lipid matrix and in the surface proteins in biological membranes.

Local measurement of lateral motion in erythrocyte membranes by photobleaching technique.

The lateral diffusion coefficients (D) of the molecular fluorescence probe 3,3'-dioctadecylindocarbocyanine iodide (DII) in the membrane of discoiderythrocyte ghosts has been measured with the photobleaching technique between 7 degrees C and 40 degrees C. A fluorescence microscope which allows bleaching experiments within small local fields (approx. 1 micron m2) at high magnification (X1600) has been used for these measurements. The diffusion coefficient increases from D = 9 - 10-10 cm2/s to D = 7.5 - 10-9 cm2/s from 7 to 40 degrees C. An increase in membrane fluidity between 12 degrees C and 17 degrees C indicates a conformational change of the lipid bilayer moiety in this temperature region. The diffusion coefficient measured in the regions between the spicules of echinocytes is appreciably smaller than in the untransformed discoid ghosts. In the myelin tubes originating from cells, the lateral diffusion is somewhat larger (about a factor of 2) than in the non-transformed ghosts. With the fluorescence probe technique the rate of growth of myelin tubes of 0.3 micronm diameter has been estimated.

Human platelet P-235, a talin-like actin binding protein, binds selectively to mixed lipid bilayers.

The interaction of platelet talin (P-235) with mixtures of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylserine (DMPS) as well as with pure lipids was studied in reconstituted lipid bilayers. Incorporation of platelet talin into vesicles was achieved by self-assembly during cycles of freeze-thawing of co-dispersions containing vesicles and the purified protein. The yield of protein incorporation as a function of lipid composition was determined by measuring the protein/lipid ratio using protein assay, phosphate determination and gel electrophoresis in parallel. Protein-lipid interactions are monitored by high sensitive differential scanning calorimetry (DSC) measuring (i) the shifts of transition states delta Ts* and delta Tl*, where Ts represents the solidus line, the onset of lipid chain melting, and Tl the liquidus line, the endpoint of chain melting, and (ii) the heats of transition. Cytoplasmic talin differs from a membrane bound form by its ability and mode of lipid interaction. The latter partially penetrates into the hydrophobic region of the bilayer, which renders a low incorporation rate even into neutral lipids. This interaction is greatly enhanced in the presence of charged lipids: a marked shift of Tl occurs due to a selective electrostatic interaction of the protein with the membrane surface. Evidence for a selective binding is also provided by Fourier transform infrared spectroscopy (FTIR). Right-side-out oriented platelet talin can be cleaved by proteinases, which truncate the extrinsic electrostatic binding domain but not the hydrophobic. In addition, reconstituted platelet talin, like in vivo, can be cleaved by thrombin. The interaction of cytoplasmic platelet talin with lipid bilayers is purely electrostatic. Our data suggest that protein reconstitution by freeze-thawing is an equilibrium process and that the protein distribution between the membrane and water is determined by the Nernst distribution law. Consequently, the work of protein transfer from water into the bilayer can be measured as a function of charged lipids.

Three dimensional microscopic surface profiles of membranes reconstructed from freeze etching electrol micrographs.

A method of three-dimensional reconstruction of the surface profile of artificial and natural membranes from freeze quenched electron micrographs is presented. The method is based on the analysis of the variation in thickness of platinum layers, deposited under an oblique angle. In essence, it is reminiscent of the method of Eratosthenes to measure the earth's radius. The thickness of etch-like protrusions of membranes could be determined to an accuracy of about 3 A. True distances on curved surfaces rather than projections of distances are obtained. The method has been applied to both model membranes and biological membranes. The essential results are: 1. Detailed information on the symmetry and the molecular structure of the crystalline phases of dimyristoyl phosphatidylcholine was obtained. The microscopic surface profile of the ripple structure observed between the pretransition and the main transition was analysed. In accordance with a previous model we found that the ripple structure is caused by the spontaneous curvature of the monolayers. The surface profiles of the ripple structure and of the low temperature biaxial phase could be clearly distinguished. 2. The sizes and shapes of lipid domains formed by both thermically and charge-induced lateral phase separation were determined. This showed that the visual inspection of electron micrographs may lead to a considerable underestimation of the domain size. Conclusions may be drawn concerning the different phases formed upon lateral phase separation. 3. As a biological example, yeast cell membranes were studied. The method allows one to distinguish between different membrane-bound proteins by measuring the width-to-height ratio of the particles. The deformation of the lipid layer in the environment of the proteins may be determined. This deformation contains information about lipid-mediated long-range interactions between membrane proteins.

Self assembly of covalently anchored phospholipid supported membranes by use of DODA-Suc-NHS-lipids.

We present a novel preparation method for the self assembly of covalently anchored phospholipid supported membranes. The surface is gold covered by cysteamine. Vesicles containing DMPC and activated DODA-Suc-NHS-lipids assembled on this surface. The whole self-assembly process is monitored conveniently by Near Infrared Surface Plasmon Resonance (NIR-SPR). Comparing the data to those obtained by Ca(2+)-mediated vesicle fusion, confirmed this interpretation.

Solubilization of DMPC and DPPC vesicles by detergents below their critical micellization concentration: high-sensitivity differential scanning calorimetry, Fourier transform infrared spectroscopy and freeze-fracture electron microscopy reveal two interaction sites of detergents in vesicles.

The interaction of sodium deoxycholate, sodium cholate and octyl glucoside with sonicated vesicles of L alpha-dimyristoyl-phosphatidylcholine (DMPC) and L alpha-dipalmitoylphosphatidylcholine (DPPC) at concentrations below the critical micellization concentration (cmc) of the detergents was studied by high-sensitivity DSC (hs-DSC), Fourier transform infrared spectroscopy (FT-IR) and freeze-fracture electron microscopy. The two phospholipids exhibited a striking different thermotropic behaviour in the presence of these detergents. For DPPC vesicles, the detergents were found to interact exclusively in the aqueous interface region of the bilayer below the membrane saturation concentration Rsat while in DMPC vesicles two coexisting interaction sites below this concentration persist. These are detergents which interact at the aqueous interface region (site 1) and in the acyl chain region (site 2) of the DMPC vesicles. The partition coefficients K of the detergents between DPPC vesicles and the water phase were calculated from the hs-DSC results at two detergent/phospholipid molar ratios Rtot less than or equal to Rsat as 0.35, 0.049 and 0.040 mol-1 for sodium deoxycholate, sodium cholate and octyl glucoside, respectively. In contrast, for DMPC the K values for Rtot less than or equal to Rsat were found to be dependent on Rtot due to the occupation of site 2 by the detergents above a certain Rtot. The model is discussed on the basis of the detergents free energies of transfer from the water phase to site 1 and site 2 of the vesicles, respectively. The solubilization behaviour of DPPC vesicles, dependent on whether the total detergent concentration is above or below the cmc at Rsat, differed significantly as revealed by hs-DSC. This suggests that in the latter case an additional hydrophobic effect could facilitate the formation of disc shaped mixed micelles. Moreover, this different behaviour was employed to measure the cmc values of the detergents studied in the presence of the vesicles by hs-DSC.

Structural properties of a phosphatidylcholine-cholesterol system as studied by small-angle neutron scattering: ripple structure and phase diagram.

Small-angle neutron scattering has been used to study structural features of lamellar bilayer membranes of dimyristoylphosphatidylcholine (DMPC) and DMPC mixed with various amount of cholesterol. The studies were recorded at a fixed hydration level of 17% 2H2O, i.e. just below saturation. Bragg reflections gives information on the ripple structure and on the bilayer periodicity. The crystalline Lc phase, which was stabilized after long time storage at low temperature, exhibits major small angle scattering when cholesterol is mixed into the membrane. The intermediate P beta' gel-phase, which is characteristic by the rippled structure, is dramatically stabilized by the introduction of cholesterol. The ripple structure depends significantly both on the cholesterol content and on the temperature. At high temperatures, T greater than 15 degrees C, the inverse ripple periodicity varies basically linearly with cholesterol content, and approach zero (i.e. periodicity goes to infinite) at 20 mol% cholesterol, approximately. At lower temperatures the correlation is more complex. The data indicate additional phase boundaries below 2 mol% and at approx. 8 mol%. Secondary rippled structures are observed in the low temperature L beta'-phase for cholesterol content below approx. 8 mol%. The data gives detailed insight into the phosphatidylcholine cholesterol phase diagram, which is discussed on the basis of a simple model in which the cholesterol complexes are fixed to the defect stripes of the rippled structure.

Polymyxin binding to charged lipid membranes. An example of cooperative lipid-protein interaction.

The binding of polymyxin-B to lipid bilayer vesicles of synthetic phosphatidic acid was studied using fluorescence, ESR spectroscopy and electron microscopy. 1,6-Diphenylhexatriene (which exhibits polarized fluorescence) and pyrene decanoic acid (which forms excimers) were used as fluorescence probes to study the lipid phase transition. The polymyxin binds strongly to negatively charged lipid layers. As a result of lipid/polymyxin chain-chain interactions, the transition temperature of the lipid. This can be explained in terms of a slight expansion of the crystalline lipid lattice (Lindeman's rule). Upon addition of polymyxin to phosphatidic acid vesicles two rather sharp phase transitions (width deltaT = 5 degrees C) are observed. The upper transition (at Tu) is that of the pure lipid and the lower transition (at T1) concerns the lipid bound to the peptide. The sharpness of these transitions strongly indicates that the bilayer is characterized by a heterogeneous lateral distribution of free and bound lipid regions, one in the crystalline and the other in the fluid state. Such a domain structure was directly observed by electron microscopy (freeze etching technique). In (1 : 1) mixtures of dipalmitoyl phosphatidic acid and egg lecithin, polymyxin induces the formation of domains of charged lipid within the fluid regions of egg lecithin. With both fluorescence methods the fraction of lipid bound to polymyxin-B as a function of the peptide concentration was determined. S-shaped binding curves were obtained. The same type of binding curve is obtained for the interaction of Ca2+ with phosphatidic acid lamellae, while the binding of polylysine to such membranes is characterized by a linear or Langmuir type binding curve. The S-shaped binding curve can be explained in terms of a cooperative lipid-ligand (Ca2+, polymyxin) interaction. A model is proposed which explains the association of polymyxin within the membrane plane in terms of elastic forces caused by the elastic distortion of the (liquid crystalline) lipid layer by this highly asymmetric peptide.

Line defects in gel phase lipid monolayers.

We investigate various models of the hydrolysis of gel-phase phosphatidylcholine monolayers by phospholipase A2 (Grainger et al. (1989) FEBS Lett. 252, 73-82). We assume that the probability of hydrolysis of a given lipid depends only upon how many of its nearest neighbour lipids have already been hydrolysed. We find that the experimental data are consistent with a model in which line defects exist in the gel phase and that lipids on such defects are more easily hydrolysed than the other gel-phase lipids. Based on this model, we calculate the course of hydrolysis of a gel-phase region possessing line defects, and we suggest how such a structure might be made and the model tested. An experiment, similar to that proposed by us, has been carried out by Grainger et al. (1990) Biochim. Biophys. Acta 1023, 365-379). We also calculate the fractal dimension, df, of the interface created by the hydrolytic process and show that a measurement of df might identify how this process proceeds.

An optical study of the exchange kinetics of membrane bound molecules.

The kinetics of molecular exchange between lipid bilayers are studied using a special fluorescence technique. Pyrene and pyrene decanoic acid are chosen as typical examples of an apolar and amphiphilic molecule. Their property of forming dimers in the excited state (excimer) is exploited. The time dependencies of monomer and excimer intensities after rapid mixing of vesicles doped with fluorescent probe with undoped ones are studied by stopped-flow technique. The transient curves reveal the information on the exchange kinetics. A theoretical analysis shows that the molecular exchange follows a first order kinetics. Surprisingly short half life-times tex for this exchange process are obtained (for dipalmitoyl phosphatidylcholine tex=3.3 s for T=23 degrees C, tex=0.5 s for T=68 degrees C). Multilamellar systems (onion like structure) show much slower exchange rates. The exchange rates are nearly equal for polar and unpolar molecules. Addition of cholesterol has a strong reducing effect on this rate. Charging of dipalmitoyl phosphatidylcholine vesicle surfaces by the addition of (a) EuCl3 to the aqueous phase and (b) dipalmitoyl phosphatidic acid to the lipid phase reduces the exchange rate by about an order of magnitude above the phase transition. In a separate experiment it is shown that the lipid exchange or fusion for two different lipids is a much slower process compared to the label exchange. In fact vesicles kept below the phase transition temperature Ttr for both lipids, do not fuse even after 70 h. Noticeable fusion occurs after 10 h when the mixture stays above Ttr. Experiment shows that the fusion of pure lipid vesicles is not very much affected by the presence of a charged lipid. Change in concentration of the monovalent ions in the aqueous solution by two orders of magnitude does not have an appreciable effect on the exchange rate of phospholipids.

Intersecting polymers in lipid bilayers: cliques, static order parameters and lateral diffusion.

We have modelled a macrolipid polymer composed of lipid molecules (monomers) embedded in a lipid bilayer or monolayer and polymerized via their polar groups. Because of fluctuations perpendicular to the plane of the bilayer, the polar region occupied by the polymer chain possesses sufficient space so that the polymer might exhibit 'self-intersection' if its conformational state is projected onto the plane of the bilayer/monolayer. We represent the plane of the bilayer/monolayer by a triangular lattice. Each site can be occupied by a monomer or be empty (and thus occupied by one of the unpolymerizable lipids which make up the bilayer/monolayer). A macrolipid is represented by a sequence of N monomers connected by N-1 bonds. Bonds may be either short (connecting nearest neighbour monomers) or long (between second neighbour monomers), in accord with the average properties of the spacers between the polymerized lipids. We have carried out computer simulation of this system using the Carmesin-Kremer bond stretching algorithm. Although no two monomers can occupy the same site, bonds may cross each other. We analyzed the dependence of and approximately N2vc and + approximately N2 sigma c, where Nsc and Nmc are the number of bond-crossings in the same macrolipid ('self-crossing') or in two different macrolipids ('mutual-crossing'). For single macrolipids, we confirmed that vc = 3/4 and have found that sigma c approximately 0.52, which we consider supports that sigma c = 1/2. For the dense case with monomer concentration, c = 0.72, we found that vc = 1/2 and that sigma c approximately 0.52 supports that sigma c = 1/2. In the semi-dilute regime (c = 0.2) we found crossover behaviour, although sigma c = 1/2. The total number of bond crossings thus scale like N, independent of concentration. We studied the connectivity of the system by calculating the weight averaged cluster, or 'clique', size. Cliques are defined as being composed of all macrolipids which exhibit at least one crossing bond with one other member of the clique. We found that while the average clique contains about two macrolipids at low concentrations, the clique size approaches the maximum possible value at high concentrations if the macrolipids are sufficiently long. In the latter case a transition appears to occur as the macrolipid length increases. This transition occurs at length = 40 when c = 0.72. These observations should have experimental consequences for the viscoelastic properties of the system.(ABSTRACT TRUNCATED AT 400 WORDS)

Interactions of liposomes and hydrophobically-modified poly-(N-isopropylacrylamides): an attempt to model the cytoskeleton.

The interactions of small unilamellar vesicles (SUV) and water-soluble copolymers were studied by fluorescence spectroscopy, differential scanning calorimetry (DSC) and quasi-elastic light scattering (QELS). The anchoring onto liposomal bilayer membranes of copolymers of N-isopropylacrylamide, N-(2-(1-naphthyl)ethyl)-N-n-octadecylacrylamide and or N-[4-(1-pyrenyl)butyl]-N-n-octadecylacrylamide (0.5 mol% of the octadecylacrylamide comonomer) was monitored by non-radiative energy transfer between excited naphthalene and pyrene. The anchoring process occurred on zwitterionic lecithin liposomes and on negatively charged phosphatidic acid liposomes, whether the bilayer was in the crystalline or the liquid-crystalline phase. Insertion of the copolymer octadecyl groups within crystalline bilayers was attributed to the presence of packing defects. Aqueous solutions of poly-(N-isopropylacrylamide) and of its hydrophobically-modified copolymers exhibit a lower critical solution temperature (LCST). The coil to globule collapse of the polymer chains which is known to occur as the aqueous solution is heated through the LCST, also took place when the copolymers were anchored onto vesicular bilayers. The copolymers remained anchored during this collapse and the liposomes were not destroyed. The process was thermo-reversible. Detailed aspects of the reversibility of the phenomenon depended on the relative values of the phase transition temperatures of the liposomes and of the polymer LCST.

Supported membranes on soft polymer cushions: fabrication, characterization and applications.

Soft biofunctional and biocompatible interfaces on solids designed by the deposition of ultrathin soft polymer films or supported membranes have numerous scientific and practical applications. These include the immobilization of glycolipids, membrane receptors and proteins to generate models of cell and tissue surfaces. Powerful surface-sensitive techniques can be applied to study protein-protein recognition processes at membranes and the control of cell adhesion by the interplay of specific 'lock-and-key' forces and universal interfacial forces. Potential practical applications include the design of smart biosensors based on electro-optical devices and the fabrication of biofunctional surfaces for the stimulation of cell proliferation and tissue growth, or for the suppression of apoptosis.

The seventh Datta Lecture. Membrane bending energy concept of vesicle- and cell-shapes and shape-transitions.

The main objective of this lecture is to discuss the role of lipid-bilayer elasticity (1) for the self-organization of lipid/protein-bilayers (2) for the stabilization of domain structures and shapes of cell membranes and (3) for the control of shape transitions (e.g. bud- and pit-formation) and shape instabilities (vesicle fission). It is demonstrated that many complex shape transitions of cell membranes can be mimicked by single lipid bilayer vesicles by simply varying the area-to-volume ratio or by chemically induced bending moments suggesting that these processes are governed by the universal minimum bending energy concept of closed shells composed of stratified membranes. The essential role of the coupling between curvature and phase separation in mixed membranes for the formation and stabilization of local pits and buds or the fission of budded vesicles is demonstrated. Finally, we discuss the consequences of the pronounced thermally excited bending undulations of the hyperelastic membranes for the membrane tension, the material exchange at membrane surfaces and the control of the adhesion of vesicles (or cells) on solid substrates.

Talin anchors and nucleates actin filaments at lipid membranes. A direct demonstration.

Platelet talin nucleates actin assembly as we show here directly by using rhodamine-phalloidin labelling of actin filaments. Nucleation by talin still occurs after reconstitution into liposomal bilayers. This is also demonstrated directly after protein-lipid double labelling and light microscopic imaging. Talin, thus, is the first actin binding protein for which anchoring and nucleation of actin filament growth at lipid interfaces have been visualized.