Membrane Undulations Research Articles

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.

Fluctuation spectroscopy of giant unilamellar vesicles using confocal and phase contrast microscopy.

A widely used method to measure the bending rigidity of bilayer membranes is fluctuation spectroscopy, which analyses the thermally-driven membrane undulations of giant unilamellar vesicles recorded with either phase-contrast or confocal microscopy. Here, we analyze the fluctuations of the same vesicle using both techniques and obtain consistent values for the bending modulus. We discuss the factors that may lead to discrepancies.

Detection of the mesenchymal-to-epithelial transition of invasive non-small cell lung cancer cells by their membrane undulation spectra.

A cancer cell changes its state from being epithelial- to mesenchymal-like in a dynamic manner during tumor progression. For example, it is well known that mesenchymal-to-epithelial transition (MET) is essential for cancer cells to regain the capability of seeding on and then invading secondary/tertiary regions. However, there is no fast yet reliable method for detecting this transition. Here, we showed that membrane undulation of invasive cancer cells could be used as a novel marker for MET detection, both in invasive model cell lines and repopulated circulating tumor cells (rCTCs) from non-small cell lung cancer (NSCLC) patients. Specifically, using atomic force microscopy (AFM), it was found that the surface oscillation spectra of different cancer cells, after undergoing MET, all exhibited two distinct peaks from 0.001 to 0.007 Hz that are absent in the spectra before MET. In addition, by adopting the long short-term memory (LSTM) based recurrent neural network learning algorithm, we showed that the positions of recorded membrane undulation peaks can be used to predict the occurrence of MET in invasive NSCLC cells with high accuracy (>90% for model cell lines and >80% for rCTCs when benchmarking against the conventional bio-marker vimentin). These findings demonstrate the potential of our approach in achieving rapid MET detection with a much reduced cell sample size as well as quantifying changes in the mesenchymal level of tumor cells.

Mechanics of Twisted DNA Molecule Adsorbed on a Biological Membrane

DNA is the carrier of all cellular genetic information and increasingly used in nanotechnology. The study of DNA molecule achieved in vitro while submitting the DNA to all chemicals agent capabilities to destabilize links hydrogen, such as pH, temperature. In fact, the DNA enveloped in the membrane cellular, so it is legitimate to study the influence of membrane undulations. In this work, we try to show that the fluctuations of the membrane can be considerate as a physics agent is also capable to destabilize links hydrogen. In this investigation, we assume that each pair base formed an angle an with the membrane’s surface. We have proposed a theoretical model, and we have established a relationship between the angle formed by the pair base θeq and an angle formed by the membrane and each pair base. We assume that DNA and biomembrane interact via a realistic potential of Morse type. To this end, use is made of a generalized model that extends that introduced by M. Peyrard and A. R. Bishop in the past modified by M. Zoli. This generalized model is based on the resolution of a Schrodinger-like equation. The exact resolution gives the expression of the ground state, and the associated eigenvalue (energy) that equals the free energy, in the thermodynamic limit. First, we compute the denaturation temperature of DNA strands critical temperature. Second, we deduce all critical properties that mainly depend on the parameters of the model, and we quantify the effects of the membrane undulations. These undulations renormalize all physical quantities, such as harmonic stacking, melting temperature, eigenfunctions, eigenvalues and regular part of specific heat.

Identifying Heteroprotein Complexes in the Nuclear Envelope

The nucleus is delineated by the nuclear envelope (NE), which is a double membrane barrier composed of the inner and outer nuclear membranes as well as a ∼40-nm wide lumen. In addition to its barrier function, the NE acts as a critical signaling node for a variety of cellular processes, which are mediated by protein complexes within this subcellular compartment. Although fluorescence fluctuation spectroscopy is a powerful tool for characterizing protein complexes in living cells, it was recently demonstrated that conventional fluorescence fluctuation spectroscopy methods are not suitable for applications in the NE because of the presence of slow nuclear membrane undulations. We previously addressed this challenge by developing time-shifted mean-segmented Q (tsMSQ) analysis and applied it to successfully characterize protein homo-oligomerization in the NE. However, many NE complexes, such as the linker of the nucleoskeleton and cytoskeleton complex, are formed by heterotypic interactions, which single-color tsMSQ is unable to characterize. Here, we describe the development of dual-color (DC) tsMSQ to analyze NE heteroprotein complexes built from proteins that carry two spectrally distinct fluorescent labels. Experiments performed on model systems demonstrate that DC tsMSQ properly identifies heteroprotein complexes and their stoichiometry in the NE by accounting for spectral cross talk and local volume fluctuations. Finally, we applied DC tsMSQ to study the assembly of the linker of the nucleoskeleton and cytoskeleton complex, a heteroprotein complex composed of Klarsicht/ANC-1/SYNE homology and Sad1/UNC-84 (SUN) proteins, in the NE of living cells. Using DC tsMSQ, we demonstrate the ability of the SUN protein SUN2 and the Klarsicht/ANC-1/SYNE homology protein nesprin-2 to form a heterocomplex in vivo. Our results are consistent with previously published in vitro studies and demonstrate the utility of the DC tsMSQ technique for characterizing NE heteroprotein complexes.

How a lipid bilayer membrane responds to an oscillating nanoparticle: Promoted membrane undulation and directional wave propagation

Mechanical forces acting on a plasma membrane are of essential importance to cellular functioning via inducing delicate change of the membrane shape with the underlying mechanism yet to be elucidated. Here, we introduce an oscillating nanoparticle (NP) interaction with a lipid bilayer membrane, using the coarse-grained simulation to investigate the dynamic membrane response to constrained mechanical stimulation, which is ubiquitous in biology. Our results demonstrate that, the membrane responds to an oscillating NP by generating nanoscale undulation waves, which immediately propagate through the membrane. In dynamics, propagation of the generated membrane undulation waves always starts from flattening of the region where the NP locates, thus producing a lateral force to propel the waves away from the point of stimulation. The speed of membrane undulation wave propagation is proportional to that of NP oscillation and accelerated by increasing the integral membrane surface tension, suggesting that both the membrane bending and stretching contribute to the energy driving the unique response of membrane undulation wave propagation.

Transient protein accumulation at the center of the T cell antigen-presenting cell interface drives efficient IL-2 secretion.

Supramolecular signaling assemblies are of interest for their unique signaling properties. A µm scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center of the interface of T cells activated by antigen-presenting cells. We have determined that it is composed of multiple complexes of a supramolecular volume of up to 0.5 µm3 and associated with extensive membrane undulations. To determine cSMAC function, we have systematically manipulated the localization of three adaptor proteins, LAT, SLP-76, and Grb2. cSMAC localization varied between the adaptors and was diminished upon blockade of the costimulatory receptor CD28 and deficiency of the signal amplifying kinase Itk. Reconstitution of cSMAC localization restored IL-2 secretion which is a key T cell effector function as dependent on reconstitution dynamics. Our data suggest that the cSMAC enhances early signaling by facilitating signaling interactions and attenuates signaling thereafter through sequestration of a more limited set of signaling intermediates.

Defective Pollen Wall 3 (DPW3), a novel alpha integrin-like protein, is required for pollen wall formation in rice.

In flowering plants, pollen wall is a specialized extracellular cell-wall matrix surrounding male gametophytes and acts as a natural protector of pollen grains against various environmental and biological stresses. The formation of pollen wall is a complex but well-regulated process, which involves the action of many different genes. However, the genetic and molecular mechanisms underlying this process remain largely unknown. In this study, we isolated and characterized a novel rice male sterile mutant, defective pollen wall3 (dpw3), which displays smaller and paler anthers with aborted pollen grains. DPW3 encodes a novel membrane-associated alpha integrin-like protein conserved in land plants. DPW3 is ubiquitously expressed in anther developmental stages and its protein is localized to the plasma membrane, endoplasmic reticulum (ER) and Golgi. Anthers of dpw3 plants exhibited unbalanced anther cuticular profile, abnormal Ubisch bodies, disrupted callose deposition, defective pollen wall formation such as abnormal microspore plasma membrane undulation and defective primexine formation, resulting in pollen abortion and complete male sterility. Our findings revealed a novel and vital role of alpha integrin-like proteins in plant male reproduction.

Receptor-Free Signaling at Curved Cellular Membranes.

Plasma membranes are subject to continuous deformations. Strikingly, some of these transient membrane undulations yield membrane-associated signaling hubs that differ in composition and function, depending on membrane geometry and the availability of co-factors. Here, recent advancements on this ubiquitous type of receptor-independent signaling are reviewed, with a special focus on emerging concepts and technical challenges associated with studying these elusive signaling sites.

3D Columnar Phase of Stacked Short DNA Organized by Coherent Membrane Undulations.

We report on the discovery of a new organized lipid-nucleic acid phase upon intercalation of blunt duplexes of short DNA (sDNA) within cationic multilayer fluid membranes. End-to-end interactions between sDNA leads to columnar stacks. At high membrane charge density, with the inter-sDNA column spacing (dsDNA) comparable but larger than the diameter of sDNA, a 2D columnar phase (i.e., a 2D smectic) is found similar to the phase in cationic liposome-DNA complexes with long lambda-phage DNA. Remarkably, with increasing dsDNA as the membrane charge density is lowered, a transition is observed to a 3D columnar phase of stacked sDNA. This occurs even though direct DNA-DNA electrostatic interactions across layers are screened by diffusing cationic lipids near the phosphate groups of sDNA. Softening of the membrane bending rigidity (κ), which further promotes membrane undulations, significantly enhances the 3D columnar phase. These observations are consistent with a model by Schiessel and Aranda-Espinoza where local membrane undulations, due to electrostatically induced membrane wrapping around sDNA columns, phase lock from layer-to-layer, thereby precipitating coherent "crystal-like" undulations coupled to sDNA columns with long-range position and orientation order. The finding that this new phase is stable at large dsDNA and enhanced with decreasing κ is further supportive of the model where the elastic cost of membrane deformation per unit area around sDNA columns (∝ κh2/dsDNA4, h2 = sum of square of amplitudes of the inner and outer monolayer undulations) is strongly reduced relative to the favorable electrostatic attractions of partially wrapped membrane around sDNA columns. The findings have broad implications in the design of membrane-mediated assembly of functional nanoparticles in 3D.

Nanopore Formation in the Cuticle of an Insect Olfactory Sensillum

Nanometer-level patterned surface structures form the basis of biological functions, including superhydrophobicity, structural coloration, and light absorption [1-3]. In insects, the cuticle overlying the olfactory sensilla has multiple small (50- to 200-nm diameter) pores [4-8], which are supposed to function as a filter that admits odorant molecules, while preventing the entry of larger airborne particles andlimiting water loss. However, the cellular processes underlying the patterning of extracellular matrices into functional nano-structures remain unknown. Here, we show that cuticular nanopores in Drosophila olfactory sensilla originate from a curved ultrathin film that is formed in the outermost envelope layer of the cuticle and secreted from specialized protrusions in the plasma membrane of the hair forming (trichogen) cell. The envelope curvature coincides with plasma membrane undulations associated with endocytic structures. The gore-tex/Osiris23 gene encodes an endosomal protein that is essential for envelope curvature, nanopore formation, and odor receptivity and is expressed specifically in developing olfactory trichogen cells. The 24-member Osiris gene family is expressed in cuticle-secreting cells and is found only in insect genomes. These results reveal an essential requirement for nanopores for odor reception and identify Osiris genes as a platform for investigating the evolution of surface nano-fabrication in insects.

Effects of Ethanol Addition on the Size Distribution of Liposome Suspensions in Water

Understanding the influence of ethanol on liposomes is important for insights into the physiological effects of ethanol on cell membranes and for transdermal drug delivery. In this work, different amounts of ethanol are added to liposome suspensions (1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes) of different initial sizes, and the size distributions are measured using dynamic light scattering. The average diameter reduces by 5–6% at a concentration of 20 vol % of ethanol, and the size distributions with and without ethanol are identical when scaled with the number-average diameter. The observed changes in diameter are significantly larger than the standard error of the measurements. Dilution of the liposome suspension containing 20 vol % ethanol with addition of water causes the liposomes to nearly regain their original size at that concentration; thus, the size change is reversible. The size measurements from dynamic light scattering are validated by cryo-transmission electron microscopy measurements, which show a similar reduction in the number-average diameter. ³¹P NMR studies also show a reduction in size upon addition of ethanol. The volume fraction of liposomes in the suspension, obtained from viscosity measurements, decreases by 9% with ethanol addition. A reduction of 4% in the number-average diameter is obtained upon adding 20 vol % ethanol to a suspension of giant liposomes, with the size distributions obtained by optical microscopy. Addition of cholesterol in the lipid membrane reduces the extent of shrinkage. The results indicate that the size reduction is due to shrinkage of liposomes and not due to breakage or dissolution and that the shrinkage is independent of the membrane curvature. Analysis shows that the decrease in size cannot be ascribed to membrane undulations alone when the increase in area per lipid molecule due to ethanol is taken into account. Addition of ethanol during the preparation of liposomes results in a much larger reduction in the average liposome diameter (up to 20%).

Cholesterol Depletion by MβCD Enhances Cell Membrane Tension and Its Variations-Reducing Integrity

Cholesterol depletion by methyl-β-cyclodextrin (MβCD) remodels the plasma membrane’s mechanics in cells and its interactions with the underlying cytoskeleton, whereas in red blood cells, it is also known to cause lysis. Currently it’s unclear if MβCD alters membrane tension or only enhances membrane-cytoskeleton interactions—and how this relates to cell lysis. We map membrane height fluctuations in single cells and observe that MβCD reduces temporal fluctuations robustly but flattens spatial membrane undulations only slightly. Utilizing models explicitly incorporating membrane confinement besides other viscoelastic factors, we estimate membrane mechanical parameters from the fluctuations’ frequency spectrum. This helps us conclude that MβCD enhances membrane tension and does so even on ATP-depleted cell membranes where this occurs despite reduction in confinement. Additionally, on cholesterol depletion, cell membranes display higher intracellular heterogeneity in the amplitude of spatial undulations and membrane tension. MβCD also has a strong impact on the cell membrane’s tenacity to mechanical stress, making cells strongly prone to rupture on hypo-osmotic shock with larger rupture diameters—an effect not hindered by actomyosin perturbations. Our study thus demonstrates that cholesterol depletion increases membrane tension and its variability, making cells prone to rupture independent of the cytoskeletal state of the cell.

Four-dimensional joint visualization of electrode degradation and liquid water distribution inside operating polymer electrolyte fuel cells

Understanding of degradation mechanisms present in polymer electrolyte fuel cells (PEFCs) is important to continue the integration of this clean energy technology into everyday life. Further comprehension of the interaction between various components during fuel cell operation is also critical in this context. In this work, a four-dimensional operando X-ray computed tomography method is developed for combined visualization of all PEFC components as well as transient water distribution residing in the cell, which results as a by-product of the electrochemical reaction. Time resolved, identical-location visualization through degradation stages is uniquely enabled by the non-invasive and non-destructive qualities of this method. By applying an accelerated stress test that targets cathode catalyst layer (CCL) corrosion, novel observations resulting from morphological changes of the CCL such as reduction in the water volume in the adjacent gas diffusion layer, CCL crack formation and propagation, membrane swelling, as well as quantification of local carbon loss is achieved. Additionally, insight into features that contribute to reduced fuel cell performance is enabled by the use of this specialized imaging technique, such as increased membrane undulation causing delamination and separation of the CCL from the microporous layer, which greatly affects liquid water pathways and overall device performance.

Challenges and Opportunities for Characterizing the Assembly of Nuclear Envelope Proteins by Fluorescence Fluctuation Spectroscopy

The nuclear envelope (NE), consisting of two membranes separated by a thin perinuclear space, has proven to be a challenging environment for measuring protein dynamics in living cells. First, exogenously expressed, fluorescently tagged proteins must be properly localized to the NE. Furthermore, undulations of the nuclear membranes complicate the application of fluorescence fluctuation spectroscopy (FFS) to proteins located within the perinuclear space by adding a slowly fluctuating signal. While this signal is absent for proteins associated with the nuclear membranes, distinguishing soluble from membrane-associated protein complexes by FFS presents a significant challenge since the diffusion time depends strongly on molecular size within the perinuclear space. Some proteins, such as the AAA+ ATPase TorsinA, may exist in both a membrane-associated and soluble form depending on the conditions in the cell. Determining the extent to which a protein associates with the membrane can provide valuable insight into its function within the cell. While diffusion time alone is insufficient to differentiate these populations, we explore whether the dependence of diffusion time on temperature can provide insight into a protein's localization within the NE. In addition, we explore how spatial correlations can be used to study the dynamics of proteins within the NE and the membrane undulations we previously reported. Finally, z-scan FFS provides a means to quantify the localization of proteins to the NE. These examples highlight the ways in which FFS techniques can be used to overcome the challenges of quantifying protein behavior in the NE of living cells. This work has been supported by a grant from the National Institutes of Health (R01 GM64589).

Ordered Lipid Domains Assemble via Concerted Recruitment of Constituents from Both Membrane Leaflets

Lipid rafts serve as anchoring platforms for membrane proteins. Thus far they escaped direct observation by light microscopy due to their small size. Here we used differently colored dyes as reporters for the registration of both ordered and disordered lipids from the two leaves of a freestanding bilayer. Photoswitchable lipids dissolved or reformed the domains. Measurements of domain mobility indicated the presence of 120 nm wide ordered and 40 nm wide disordered domains. These sizes are in line with the predicted roles of line tension and membrane undulation as driving forces for alignment.

The Influence of Periodic Size Effects and Membrane Undulation on Phase Separation in a DPPC/DOPC/Chol Coarse Grain Martini System

Ternary mixtures containing a high melting temperature lipid (such as di-palmitoyl-phosphatidylcholine, DPPC), a low melting temperature lipid (such as di-oleoyl-phosphatidylcholine, DOPC), and cholesterol (CHOL) form bilayers consisting of up to three different lipid phases. The potential lipid phases that can form are liquid-disordered (Ld), liquid-ordered (Lo), a gel-like phase (Lb), or any combination of the three. The phase(s) present within these membranes are dependent on the specific percentage composition of the three components within the membrane. The mixture-dependent phases formed by these specific constituents have been well mapped experimentally to construct detailed phase diagrams. In our recent work, we presented a modified version of the CG Martini DPPC and DOPC lipid parameters. These parameters not only reproduced the characteristics of individual lipid species (such as area per lipid), but also reproduced the majority of the correct experimental phase separation. Our previous studies to iteratively improve and evaluate the lipid parameters was carried out using standard system sizes of 3,000 lipids that were restrained to maintain a planar lipid bilayer.Here we further expand on this work to investigate how the size of the system, as well as membrane undulations influence the phase separating behavior of the various lipid mixtures. This work performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. Release numbers: LLNL-ABS-759357.

Thermodynamic analysis of multivalent binding of functionalized nanoparticles to membrane surface reveals the importance of membrane entropy and nanoparticle entropy in adhesion of flexible nanoparticles.

We present a quantitative model for multivalent binding of ligand-coated flexible polymeric nanoparticles (NPs) to a flexible membrane expressing receptors. The model is developed using a multiscale computational framework by coupling a continuum field model for the cell membrane with a coarse-grained model for the polymeric NPs. The NP is modeled as a self-avoiding bead-spring polymer chain, and the cell membrane is modeled as a triangulated surface using the dynamically triangulated Monte Carlo method. The nanoparticle binding affinity to a cell surface is mainly determined by the delicate balance between the enthalpic gain due to the multivalent ligand-receptor binding and the entropic penalties of various components including receptor translation, membrane undulation, and NP conformation. We have developed new methods to compute the free energy of binding, which includes these enthalpy and entropy terms. We show that the multivalent interactions between the flexible NP and the cell surface are subject to entropy-enthalpy compensation. Three different entropy contributions, namely, those due to receptor-ligand translation, NP flexibility, and membrane undulations, are all significant, although the first of these terms is the most dominant. However, both NP flexibility and membrane undulations dictate the receptor-ligand translational entropy making the entropy compensation context-specific, i.e., dependent on whether the NP is rigid or flexible, and on the state of the membrane given by the value of membrane tension or its excess area.

Simulated physiological oocyte maturation has side effects on bovine oocytes and embryos.

Oocyte maturation is a complex process involving nuclear and cytoplasmic modulations, during which oocytes acquire their ability to become fertilized and support embryonic development. The oocyte is apparently "primed" for maturation during its development in the dominant follicle. As bovine oocytes immediately resume meiosis when cultured, it was hypothesized that delaying resumption of meiosis with cyclic nucleotide modulators before in vitro maturation (IVM) would allow the oocytes to acquire improved developmental competence. We tested the Simulated Physiological Oocyte Maturation (SPOM) system that uses forskolin and 3-isobutyl-1-methylxanthine for 2h prior to IVM against two different systems of conventional IVM (Con-IVM). We evaluated the ultrastructure of matured oocytes and blastocysts and also assessed the expression of 96 genes related to embryo quality in the blastocysts. In summary, the SPOM system resulted in lower blastocyst rates than both Con-IVM systems (30 ± 9.1 vs. 35 ± 8.7; 29 ± 2.6 vs. 38 ± 2.8). Mature SPOM oocytes had significantly increased volume and number of vesicles, reduced volume and surface density of large smooth endoplasmic reticulum clusters, and lower number of mitochondria than Con-IVM oocytes. SPOM blastocysts showed only subtle differences with parallel undulations of adjacent trophectoderm plasma membranes and peripherally localized ribosomes in cells of the inner cell mass compared with Con-IVM blastocysts. SPOM blastocysts, however, displayed significant downregulation of genes related to embryonic developmental potential when compared to Con-IVM blastocysts. Our results show that the use of the current version of the SPOM system may have adverse effects on oocytes and blastocysts calling for optimized protocols for improving oocyte competence.

Nanoparticle transport across model cellular membranes: when do solubility-diffusion models break down?

The interactions of nanoparticles (NPs) with cellular membranes and subsequent transport processes have major implications for the biology, toxicology, and pharmacology of nanoscale materials. Moreover, understanding and predicting the behaviors of diverse NP designs in a physiological setting is of increasing technological and regulatory importance. Still, the current complexity of experiments and lack of a consensus in modeling and simulation preclude a clear picture of relevant NP-membrane interaction modes and mechanisms, particularly for particles on the ~1–10 nm scale. Here, we leverage detailed coarse-grained molecular dynamics simulations with advanced sampling strategies to uncover the thermodynamic driving forces and possible kinetic pathways of approximately 0.5–2.0 nm hydrophilic, hydrophobic, and ‘interfacially active’ particles with model lipid bilayer membranes. Using the simulations, we test the applicability of well-established theoretical models for the permeability of small molecule transport—Overton’s rule and the inhomogeneous solubility-diffusion model—and conclude that the former is overly-simplified for fluctuating lipid bilayers, while the latter breaks down at the larger particle sizes due to the influence of other physics like membrane undulations. We place this work in the context of recent simulation studies, and conclude with critical physical and methodological insights to guide future thermodynamic and kinetic studies of NP-membrane interactions.