Spatial organization of EphA2 at the cell-cell interface modulates trans-endocytosis of ephrinA1.

EphA2 is a receptor tyrosine kinase (RTK) that is sensitive to spatial and mechanical aspects of the cell's microenvironment. Misregulation of EphA2 occurs in many aggressive cancers. Although its juxtacrine signaling geometry (EphA2's cognate ligand ephrinA1 is expressed on the surface of an apposing cell) provides a mechanism by which the receptor may experience extracellular forces, this also renders the system challenging to decode. By depositing living cells on synthetic supported lipid membranes displaying ephrinA1, we have reconstituted key features of the juxtacrine EphA2-ephrinA1 signaling system while maintaining the ability to perturb the spatial and mechanical properties of the membrane-cell interface with precision. In addition, we developed a trans-endocytosis assay to monitor internalization of ephrinA1 from a supported membrane into the apposing cell using a quantitative three-dimensional fluorescence microscopy assay. Using this experimental platform to mimic a cell-cell junction, we found that the signaling complex is not efficiently internalized when lateral reorganization at the membrane-cell contact sites is physically hindered. This suggests that EphA2-ephrinA1 trans-endocytosis is sensitive to the mechanical properties of a cell's microenvironment and may have implications in physical aspects of tumor biology.

Dynamic domains in polymersomes: mixtures of polyanionic and neutral diblocks respond more rapidly to changes in calcium than to pH.

Chemical triggering of membrane domain dynamics is of broad relevance to cell signaling through lipid bilayers and might also be exploited in application of phase-separated vesicles. Here we describe the morphodynamics and remixing kinetics of spotted polymersomes made with mixtures of polyanionic and neutral amphiphiles plus calcium. Addition of the calcium chelator EDTA to vesicle dispersions produced a decrease in domain size within minutes, whereas increasing the pH with NaOH led to the viscous fingering of domains and decreased domain size over hours. Although the latter suggests that the charge of the polyanion contributes to domain formation, the remixing of more negative chains at high pH is surprising. Domain roughening at high pH is also accelerated by EDTA, which highlights the dominance of cross-bridging. Importantly, even though vesicles were perturbed only externally, the inner and outer leaflets remain coupled throughout, consistent with molecular dynamics simulations and suggestive of an order-disorder transition that underlies the remixing kinetics.

Dynamic sorting of lipids and proteins in membrane tubes with a moving phase boundary.

Cellular organelle membranes maintain their integrity, global shape, and composition despite vigorous exchange among compartments of lipids and proteins during trafficking and signaling. Organelle homeostasis involves dynamic molecular sorting mechanisms that are far from being understood. In contrast, equilibrium thermodynamics of membrane mixing and sorting, particularly the phase behavior of binary and ternary model membrane mixtures and its coupling to membrane mechanics, is relatively well characterized. Elucidating the continuous turnover of live cell membranes, however, calls for experimental and theoretical membrane models enabling manipulation and investigation of directional mass transport. Here we introduce the phenomenon of curvature-induced domain nucleation and growth in membrane mixtures with fluid phase coexistence. Membrane domains were consistently observed to nucleate precisely at the junction between a strongly curved cylindrical (tube) membrane and a pipette-aspirated giant unilamellar vesicle. This experimental geometry mimics intracellular sorting compartments, because they often show tubular-vesicular membrane regions. Nucleated domains at tube necks were observed to present diffusion barriers to the transport of lipids and proteins. We find that curvature-nucleated domains grow with characteristic parabolic time dependence that is strongly curvature-dependent. We derive an analytical model that reflects the observed growth dynamics. Numerically calculated membrane shapes furthermore allow us to elucidate mechanical details underlying curvature-dependent directed lipid transport. Our observations suggest a novel dynamic membrane sorting principle that may contribute to intracellular protein and lipid sorting and trafficking.

Spotted vesicles, striped micelles and Janus assemblies induced by ligand binding.

Selective binding of multivalent ligands within a mixture of polyvalent amphiphiles provides, in principle, a simple mechanism for driving domain formation in self-assemblies. Divalent cations are shown here to crossbridge polyanionic amphiphiles, which thereby demix from neutral amphiphiles and form spots or rafts within vesicles as well as stripes within cylindrical micelles. Calcium- and copper-crossbridged domains of synthetic block copolymers or natural lipid (phosphatidylinositol-4,5-bisphosphate) possess tunable sizes, shapes and/or spacings that can last for years. Lateral segregation in these 'ligand-responsive Janus assemblies' couples weakly to curvature and proves to be restricted within phase diagrams to narrow regimes of pH and cation concentration that are centred near the characteristic binding constants for polyacid interactions. Remixing at high pH is surprising, but a theory for strong lateral segregation shows that counterion entropy dominates electrostatic crossbridges, thus illustrating the insights gained into ligand-induced pattern formation within self-assemblies.

Membrane cholesterol is a biomechanical regulator of neutrophil adhesion.

OBJECTIVE: The purpose of this study was to evaluate the role of membrane cholesterol on human neutrophil and HL-60 biomechanics, capture, rolling, and arrest to P-selectin- or IL-1-activated endothelium. METHODS AND RESULTS: Methyl-beta-cyclodextrin (MbetaCD) removed up to 73% and 45% of membrane cholesterol from HL-60 cells and neutrophils, whereas MbetaCD/cholesterol complexes resulted in maximum enrichment of 65% and 40%, respectively, above control levels. Cells were perfused at a venous wall shear rate of 100 s(-1) over adherent P-selectin-coated 1-microm diameter beads, uncoated 10-mum diameter beads, P-selectin-coated surfaces, or activated endothelium. Elevated cholesterol enhanced capture efficiency to 1-microm beads and increased membrane tether growth rate by 1.5- to 2-fold, whereas cholesterol depletion greatly reduced tether formation. Elevated cholesterol levels increased tether lifetime by 17% in neutrophils and adhesion lifetime by 63% in HL-60 cells. Deformation of cholesterol-enriched neutrophils increased the contact time with 10-mum beads by 32% and the contact area by 7-fold. On both P-selectin surfaces and endothelial-cell monolayers, cholesterol-enriched neutrophils rolled more slowly, more stably, and were more likely to firmly arrest. Cholesterol depletion resulted in opposite effects. CONCLUSIONS: Increasing membrane cholesterol enhanced membrane tether formation and whole cell deformability, contributing to slower, more stable rolling on P-selectin and increased firm arrest on activated endothelium.

Bending stiffness depends on curvature of ternary lipid mixture tubular membranes.

Lipid and protein sorting and trafficking in intracellular pathways maintain cellular function and contribute to organelle homeostasis. Biophysical aspects of membrane shape coupled to sorting have recently received increasing attention. Here we determine membrane tube bending stiffness through measurements of tube radii, and demonstrate that the stiffness of ternary lipid mixtures depends on membrane curvature for a large range of lipid compositions. This observation indicates amplification by curvature of cooperative lipid demixing. We show that curvature-induced demixing increases upon approaching the critical region of a ternary lipid mixture, with qualitative differences along two roughly orthogonal compositional trajectories. Adapting a thermodynamic theory earlier developed by M. Kozlov, we derive an expression that shows the renormalized bending stiffness of an amphiphile mixture membrane tube in contact with a flat reservoir to be a quadratic function of curvature. In this analytical model, the degree of sorting is determined by the ratio of two thermodynamic derivatives. These derivatives are individually interpreted as a driving force and a resistance to curvature sorting. We experimentally show this ratio to vary with composition, and compare the model to sorting by spontaneous curvature. Our results are likely to be relevant to the molecular sorting of membrane components in vivo.

Mechanical stability of micropipet-aspirated giant vesicles with fluid phase coexistence.

Micropipet aspiration of phase-separated lipid bilayer vesicles can elucidate physicochemical aspects of membrane fluid phase coexistence. Recently, we investigated the composition dependence of line tension at the boundary between liquid-ordered and liquid-disordered phases of giant unilamellar vesicles obtained from ternary lipid mixtures using this approach. Here we examine mechanical equilibria and stability of dumbbell-shaped vesicles deformed by line tension. We present a relationship between the pipet aspiration pressure and the aspiration length in vesicles with two coexisting phases. Using a strikingly simple mechanical model for the free energy of the vesicle, we predict a relation that is in almost quantitative agreement with experiment. The model considers the vesicle free energy to be proportional to line tension and assumes that the vesicle volume, domain area fraction, and total area are conserved during aspiration. We also examine a mechanical instability encountered when releasing a vesicle from the pipet. We find that this releasing instability is observed within the framework of our model that predicts a change of the compressibility of a pipet-aspirated membrane cylinder from positive (i.e., stable) to negative (unstable) values, at the experimental instability. The model furthermore includes an aspiration instability that has also previously been experimentally described. Our method of studying micropipet-induced shape transitions in giant vesicles with fluid domains could be useful for investigating vesicle shape transitions modulated by bending stiffness and line tension.

Quantifying Membrane Curvature Generation of Drosophila Amphiphysin N-BAR Domains.

Biological membrane functions are coupled to membrane curvature, the regulation of which often involves membrane-associated proteins. The membrane-binding N-terminal amphipathic helix-containing BIN/Amphiphysin/Rvs (N-BAR) domain of amphiphysin is implicated in curvature generation and maintenance. Improving the mechanistic understanding of membrane curvature regulation by N-BAR domains requires quantitative experimental characterization. We have measured tube pulling force modulation by the N-BAR domain of Drosophila amphiphysin (DA-N-BAR) bound to tubular membranes pulled from micropipette-aspirated giant vesicles. We observed that fluorescently-labeled DA-N-BAR showed significantly higher protein density on tubules compared to the connected low-curvature vesicle membrane. Furthermore, we found the equilibrium tube pulling force to be systematically dependent on the aqueous solution concentration of DA-N-BAR, thereby providing the first quantitative assessment of spontaneous curvature generation. At sufficiently high protein concentrations, pulled tubes required no external force to maintain mechanical equilibrium, in agreement with the qualitative spontaneous tubulation previously reported for amphiphysin.

Line tension at fluid membrane domain boundaries measured by micropipette aspiration.

Line tension is a determinant of fluid phase domain formation kinetics and morphology in lipid bilayer membranes, which are models for biological membrane heterogeneity. We describe the first direct measurement of this line tension by micropipette aspiration. Our data are analyzed with a model that does not rely on independently measured (and composition dependent) secondary parameters, such as bending stiffness or membrane viscosities. Line tension is strongly composition dependent and decreases towards a critical consolute point in a quasiternary room temperature phase diagram.

Flicker spectroscopy of thermal lipid bilayer domain boundary fluctuations.

This contribution describes measurements of lipid bilayer domain line tension based on two-dimensional thermal undulations of membranes with liquid ordered/liquid disordered phase coexistence and near-critical composition at room temperature. Lateral inhomogeneity of lipid and protein composition is currently a subject of avid research aimed at determining both fundamental properties and biological relevance of membrane domains. Line tension at fluid lipid bilayer membrane domain boundaries controls the kinetics of domain growth and therefore regulates the size of compositional heterogeneities. High line tension promotes membrane domain budding and fission. Line tension could therefore be an important control parameter regulating functional aspects of biological membranes. Here the established method of fluid domain flicker spectroscopy is applied to examine thermal domain wall fluctuations of phase-separated bilayer membranes. We find a Gaussian probability distribution for the first few excited mode amplitudes, which permits an analysis by means of appropriately specialized capillary wave theory. Time autocorrelation functions are found to decay exponentially, and relaxation times are fitted by means of a hydrodynamic theory relating line tensions and excited mode relaxation kinetics. Line tensions below 1 pN are obtained, with these two approaches yielding similar results. We examine experimental artifacts that perturb the Fourier spectrum of domain traces and discuss ways to identify the number of modes that yield reliable line tension information.

Prediction of collective diffusion coefficient of bovine serum albumin in aqueous electrolyte solution with hard-core two-Yukawa potential.

A new method to predict concentration dependence of collective diffusion coefficient of bovine serum albumin (BSA) in aqueous electrolyte solution is developed based on the generalized Stokes-Einstein equation which relates the diffusion coefficient to the osmotic pressure. The concentration dependence of osmotic pressure is evaluated using the solution of the mean spherical approximation for the two-Yukawa model fluid. The two empirical correlations of sedimentation coefficient are tested in this work. One is for a disordered suspension of hard spheres, and another is for an ordered suspension of hard spheres. The concentration dependence of the collective diffusion coefficient of BSA under different solution conditions, such as pH and ionic strength is predicted. From the comparison between the predicted and experimental values we found that the sedimentation coefficient for the disordered suspension of hard spheres is more suitable for the prediction of the collective diffusion coefficients of charged BSA in aqueous electrolyte solution. The theoretical predictions from the hard-core two-Yukawa model coupled with the sedimentation coefficient for a suspension of hard spheres are in good agreement with available experimental data, while the hard sphere model is unable to describe the behavior of diffusion due to its neglect of the double-layer repulsive charge-charge interaction between BSA molecules.