Membrane Thermal Fluctuations Research Articles

Shear-induced sponge-to-lamellar transition in bicontinuous microemulsions evidenced by microfluidic-SANS

HypothesisShear flow applied to bicontinuous microemulsions is expected to induce a transition to lamellae via the suppression of surfactant monolayer fluctuations. Compared to the topologically analogous L3 (sponge) phase, composed of surfactant bilayers, this transition is likely to occur at much higher shear rates. ExperimentsWe examine the flow response of a model bicontinuous microemulsion, D2O/n-octane/C10E4 by coupling microfluidics with small-angle neutron scattering (SANS), attaining wall shear rates in excess of 105 s−1. The reduction of probed sample volumes down to ∼10 nL allows the spatial mapping of the structural and orientation changes within the microchannel, as a function of the flow field components. FindingsWith increasing flow rate, we observe a gradual increase in scattering anisotropy, accompanied by a decrease of the microemulsion domain size along the main flow orientation. A consistent description of the degree of anisotropy was obtained when considering the velocity gradient along the scattering plane perpendicular to the flow. We discuss the flow dependence of the effective bending rigidity, rationalizing a strong influence of shear on thermal membrane fluctuations. Assuming a similar shear dependence for the saddle splay modulus, the bicontinuous-to-lamellar transition can be attributed to the gradual disappearance of inter-lamellar passages.

Receptor-ligand diffusion-limited reaction rates on curved membranes

We provide an expression for the diffusion-limited reaction rate on a curved surface, or membrane, in the perturbative deformation regime. This result is applied to the specific case of calculating the receptor-ligand association rate at membrane deforming, transmembrane receptor proteins, or inclusions. The dynamics of mechanochemical coupling between chemical activity and local membrane curvature is further investigated, and the effect of thermal membrane fluctuations is also discussed. It is found for transmembrane proteins, or inclusions, that their concomitant receptor-ligand reaction rates can be typically reduced by as much as 20% due to the effect of locally induced membrane curvature or geometry.

Interleaflet Interaction in Phase Separated Asymmetric Lipid Bilayers

Ordered lipid domains (OLD) may serve as signaling platforms provided both membrane leaflets contribute. Interleaflet registration of phase separated regions in symmetric bilayers is driven by (i) the line tension around the thicker ordered domains (1) and (ii) membrane undulations (2, 3). How these forces act to generate membrane spanning domains in compositionally different leaflets is unknown. Here we use both fluorescence microscopy and fluorescence correlation spectroscopy of folded asymmetric planar lipid bilayers (4) to observe the enslavement of disordered lipids into ordered domains by ordered lipids in the opposing leaflet and, vice versa, the partial dissolution of ordered phases by disordered lipids in the opposing leaflet. These results were mirrored by the transition of lipids from the gel phase to the liquid phase and vice versa as seen by molecular dynamics simulations of compositionally symmetric, but - due to temperature differences - phase asymmetric bilayers. Our analysis of elastic lipid deformations, thermal membrane fluctuations and the structure of the fluid/gel boundary shows that the alignment of the original domain in one leaflet with the induced domain in the opposing leaflet is the energetically favored state and that the phase boundaries in the individual leaflets are shifted relative to each other by a few nanometers along the membrane surface. We conclude that similar forces govern domain registration in both symmetric and asymmetric membranes. 1. Galimzyanov, et al. Phys. Rev. Lett. 2015, 115:088101. 2. Horner, Antonenko, Y, Pohl. Biophys. J. 2009, 96:2689. 3. Galimzyanov, et al. Biophys. J. 2017, 112:339. 4. Horner; Akimov; Pohl. Phys. Rev. Lett. 2013, 110:268101.

Acetonitrile-Induced Destabilization in Liposomes

To understand the behavior of cellular interfaces, it is important to clarify the effect of chemical compounds on artificial cell membranes. In this study, an aqueous acetonitrile solution was mixed with a suspension of lipid vesicles, and the changes in vesicle behavior arising as a result of acetonitrile application were observed. The fast Fourier transformations (FFTs) of the membrane waviness/crinkliness of the vesicles were carried out, and the membrane thermal fluctuations were analyzed. The experimental results show that the addition of acetonitrile molecules enhances the fluctuation of lipid membranes. In particular, the k = 2 mode fluctuation was significantly enhanced. This finding is expected to lead us to a further understanding of the fundamental properties of living cells.

Geometric localization of thermal fluctuations in red blood cells

The thermal fluctuations of membranes and nanoscale shells affect their mechanical characteristics. Whereas these fluctuations are well understood for flat membranes, curved shells show anomalous behavior due to the geometric coupling between in-plane elasticity and out-of-plane bending. Using conventional shallow shell theory in combination with equilibrium statistical physics we theoretically demonstrate that thermalized shells containing regions of negative Gaussian curvature naturally develop anomalously large fluctuations. Moreover, the existence of special curves, "singular lines," leads to a breakdown of linear membrane theory. As a result, these geometric curves effectively partition the cell into regions whose fluctuations are only weakly coupled. We validate these predictions using high-resolution microscopy of human red blood cells (RBCs) as a case study. Our observations show geometry-dependent localization of thermal fluctuations consistent with our theoretical modeling, demonstrating the efficacy in combining shell theory with equilibrium statistical physics for describing the thermalized morphology of cellular membranes.

The modified fluorescence based vesicle fluctuation spectroscopy technique for determination of lipid bilayer bending properties

Lipid bilayer is the main constitutive element of biological membrane, which confines intracellular space. The mechanical properties of biological membranes may be characterized by various parameters including membrane stiffness or membrane bending rigidity, which can be measured using flicker noise spectroscopy. The flicker noise spectroscopy exploits the spontaneous thermal undulations of the membrane. The method is based on the quantitative analysis of a series of microscopic images captured during thermal membrane fluctuations. Thus, measured bending rigidity coefficient depends on the image quality as well as the selection of computational tools for image processing and mathematical model used. In this work scanning and spinning disc confocal microscopies were used to visualize fluctuating membranes of giant unilamellar vesicles. The bending rigidity coefficient was calculated for different acquisition modes, using different fluorescent probes and different image processing methods. It was shown that both imaging approaches gave similar bending coefficient values regardless of acquisition time. Using the developed methodology the effect of fluorescent probe type and aqueous phase composition on the value of the membrane bending rigidity coefficient was measured. Specifically it was found that the bending rigidity coefficient of DOPC bilayer in water is smaller than that determined for POPC membrane. It has been found that the POPC and DOPC bending rigidities coefficient in sucrose solution was lower than that in water. Fluorescence imaging makes possible the quantitative analysis of membrane mechanical properties of inhomogeneous membrane.

Cell membrane softening in human breast and cervical cancer cells

Biomechanical properties are key to many cellular functions such as cell division and cell motility and thus are crucial in the development and understanding of several diseases, for instance cancer. The mechanics of the cellular cytoskeleton have been extensively characterized in cells and artificial systems. The rigidity of the plasma membrane, with the exception of red blood cells, is unknown and membrane rigidity measurements only exist for vesicles composed of a few synthetic lipids. In this study, thermal fluctuations of giant plasma membrane vesicles (GPMVs) directly derived from the plasma membranes of primary breast and cervical cells, as well as breast cell lines, are analyzed. Cell blebs or GPMVs were studied via thermal membrane fluctuations and mass spectrometry. It will be shown that cancer cell membranes are significantly softer than their non-malignant counterparts. This can be attributed to a loss of fluid raft forming lipids in malignant cells. These results indicate that the reduction of membrane rigidity promotes aggressive blebbing motion in invasive cancer cells.

Time resolved membrane fluctuation spectroscopy

Probing the mechanical properties of phospholipid membranes is a fundamental characterization step for biomimetic membrane systems as well as for living cellular systems. A common method relies on the analysis of thermal membrane fluctuations, which has been implemented in video flicker spectroscopy. Here we present a new optical method that directly measures the dynamics of membrane fluctuations with nm and μs resolution, thus providing access to the bending modulus κ and the membrane tension σ for measurement times of 10 s. Our method allows the observation of bilayer membrane fluctuations of liposomes and the calculation of the power spectral density in yet unreported regimes of frequencies >50 Hz and amplitudes <10 nm. The recorded data are in agreement with the Helfrich membrane theory over 4 decades of frequencies (0.1 Hz–1000 Hz). However, we find a systematic overestimation of the buffer viscosity, which can not be simply explained by measurement errors, but unveils an effect that is not explained by the classical theory for membrane dynamics, and hence suggests that new physics must be developed in the observed frequency and amplitude range. The experimental method is easily reproducible on any optical tweezer setup equipped with a quadrant photodiode position detection sensor.

Entropic attraction of adhesion bonds toward cell boundaries

Adhesion bonds between membranes and surfaces are attracted to each other via effective interactions whose origin is the entropy loss due to the reduction in the amplitude of the membrane thermal fluctuations in the vicinity of the adhesion bonds. These fluctuation-induced interactions are also expected to drive the adhesion bonds toward the rim of the cell as well as toward the surfaces of membrane inclusions. In this paper we analyze the attraction of adhesion bonds to the cell inner and outer boundaries. Our analysis shows that the probability distribution function of a single (diffusing) adhesion bond decays algebraically with the distance from the boundaries. Upon increasing the concentration of the adhesion bonds, the attraction to the boundaries becomes strongly self-screened.

Entropy-driven aggregation of adhesion sites of supported membranes

We study, by means of mean-field calculations and Monte Carlo simulations of a lattice gas model, the distribution of adhesion sites of a bilayer membrane and a supporting flat surface. Our model accounts for the many-body character of the attractive interactions between adhesion points induced by the membrane thermal fluctuations. We show that while the fluctuation-mediated interactions alone are not sufficient to allow the formation of aggregation domains, they greatly reduce the strength of the direct interactions required to facilitate cluster formation. Specifically, for adhesion molecules interacting via a short-range attractive potential, the strength of the direct interactions required for aggregation is reduced by about a factor of two to below the thermal energy k(B)T.

A Multiscale Red Blood Cell Model with Accurate Mechanics, Rheology, and Dynamics

Red blood cells (RBCs) have highly deformable viscoelastic membranes exhibiting complex rheological response and rich hydrodynamic behavior governed by special elastic and bending properties and by the external/internal fluid and membrane viscosities. We present a multiscale RBC model that is able to predict RBC mechanics, rheology, and dynamics in agreement with experiments. Based on an analytic theory, the modeled membrane properties can be uniquely related to the experimentally established RBC macroscopic properties without any adjustment of parameters. The RBC linear and nonlinear elastic deformations match those obtained in optical-tweezers experiments. The rheological properties of the membrane are compared with those obtained in optical magnetic twisting cytometry, membrane thermal fluctuations, and creep followed by cell recovery. The dynamics of RBCs in shear and Poiseuille flows is tested against experiments and theoretical predictions, and the applicability of the latter is discussed. Our findings clearly indicate that a purely elastic model for the membrane cannot accurately represent the RBC's rheological properties and its dynamics, and therefore accurate modeling of a viscoelastic membrane is necessary.

Red blood cell thermal fluctuations: comparison between experiment and molecular dynamics simulations

We outline a new method of analysis of thermal shape fluctuations of red blood cells, based on comparison between experiments and coarse-grained molecular dynamics simulations. The fluctuations of 2D equatorial contours of red blood cells are recorded experimentally using fast phase-contrast video microscopy, from which the fluctuation spectrum is calculated. The spectrum is compared to the corresponding contour fluctuation spectrum obtained from a finite-temperature particle-dynamics simulation, modelling a cell with bending and shear elasticity and conserved volume and surface area. We demonstrate that the simulation correctly describes the mean cell shape as well as the membrane thermal fluctuations, returning physically sound values for the relevant membrane elastic moduli.

Role of molecular tilt in thermal fluctuations of lipid membranes

Long-wavelength thermal fluctuations of lipid membranes are adequately described by the Helfrich elastic model. On the other hand, fluctuations of wavelengths comparable with bilayer thickness exhibit significant deviations from the prediction of the elastic model and are typically assumed to be dominated by microscopic surface tension due to protrusion of lipid molecules into the solvent. We present evidence that the short-wavelength modes of a lipid membrane are dominated by fluctuations of the tilt of lipid molecules with respect to the membrane normal rather than the microscopic surface tension. We obtain an expression for the spectral intensity of the thermal membrane fluctuations by appealing to the Hamm-Kozlov model, which accounts for both membrane bending and lipid tilt contributions to the total membrane energy but neglects the contributions of the microscopic surface tension. The tilt and the bending fluctuations obtained from our coarse-grained molecular dynamics simulations of a dipalmitoylphosphatidylcholine lipid bilayer show good agreement with the theory. Furthermore, the obtained tilt and bending moduli are in close agreement with experimentally determined values. The magnitude of the microscopic protrusion tension estimated from our simulations is significantly smaller than that of the tilt modulus. These results indicate that the membrane fluctuations can be adequately described by a macroscopic elastic model down to scales of interlipid distance provided one accounts for the tilt fluctuations.

Hydrodynamic Damping of Membrane Thermal Fluctuations near Surfaces Imaged by Fluorescence Interference Microscopy

Hydrodynamic coupling between closely apposed lipid bilayer membranes can substantially slow the time scale of thermal fluctuations. These effects could dominate the kinetics of macromolecular interactions at membrane interfaces, and may exert important influences over the interactions between living cells. Here, we study a reconstituted membrane system consisting of two planar bilayers with average separation distances of 30-60 nm. This spacing range is comparable to that of natural intercellular junctions. Using real-time fluorescence interference contrast imaging, spatial and temporal characteristics of the thermal fluctuation spectra are monitored simultaneously. Strong hydrodynamic damping of the fluctuation time scale is observed whereas fluctuation spatial dimensions are unaltered by proximity to another surface. These observations provide experimental confirmation of recent theoretical models.

Thermal Fluctuations of Red Blood Cell Membrane via a Constant-Area Particle-Dynamics Model

We describe a model of the mechanical properties of the cell plasma membrane using a finite-temperature particle-dynamics simulation of the whole cell, in which a two-dimensional network of virtual particles embedded in a three-dimensional closed surface represents the membrane. The particles interact via harmonic potential and dihedral angle potential and are subject to a constant area constraint. The evolution of the positions of the particles yields the equilibrium state of the membrane and allows determination of the membrane thermal fluctuations and the elastic moduli. We show that time-averaging of the cell-model configurations allows quantitative comparison with experimental data on membrane fluctuations and elastic moduli of the red blood cell.

Coherent X-ray scattering and speckle pattern of solid-supported multilayers of surfactant bilayers

Charged surfactant bilayers deposited in thick multilayer films and swollen in vapor or in solution have been studied by coherent X-ray scattering. We report a pronounced static speckle pattern which persists under conditions of full hydration, despite the fact that the bilayers are fluid, relatively soft, and consisting of soluble amphiphiles (DDABr). Characteristic of the macroscopic domains size, the speckle pattern is observed in the plane of incidence (rocking curves). Contrarily, out-of-plane scans, which probe smaller lateral distances along the multilayer, exhibit a smooth diffuse scattering curve characteristic of thermal fluctuations of membranes.

Fast Lipid Disorientation at the Onset of Membrane Fusion Revealed by Molecular Dynamics Simulations

Membrane fusion is a key event in vesicular trafficking in every cell, and many fusion-related proteins have been identified. However, how the actual fusion event occurs has not been elucidated. By using molecular dynamics simulations we found that when even a small region of two membranes is closely apposed such that only a limited number of water molecules remain in the apposed area (e.g., by a fusogenic protein and thermal membrane fluctuations), dramatic lipid disorientation results within 100 ps-2 ns, which might initiate membrane fusion. Up to 12% of phospholipid molecules in the apposing layers had their alkyl chains outside the hydrophobic region, lying almost parallel to the membrane surface or protruding out of the bilayer by 2 ns after two membranes were closely apposed.

Inclusions in membranes

Recent work on membrane-inclusion and inclusion-inclusion interactions has focused on models of the membrane based on curvature elasticity. The resulting deformation profiles are non-monotonic and lead to either attractive or repulsive interactions. In addition, considerations of thermal membrane fluctuations and the membrane-inclusion contact angle have led to predictions of new long-range interactions.

How does a virus bud?

How does a virus bud from the plasma membrane of its host? Here we investigate several possible rate-limiting processes, including thermal fluctuations of the plasma membrane, hydrodynamic interactions, and diffusion of the glycoprotein spikes. We find that for bending moduli greater than 3 x 10(-13) ergs, membrane thermal fluctuations are insufficient to wrap the viral capsid, and the mechanical force driving the budding process must arise from some other process. If budding is limited by the rate at which glycoprotein spikes can diffuse to the budding site, we compute that the budding time is 10-20 min, in accord with the experimentally determined upper limit of 20 min. In light of this, we suggest some alternative mechanisms for budding and provide a rationale for the observation that budding frequently occurs in regions of high membrane curvature.