Ramon Reigada

Interplay of Unsaturated Phospholipids and Cholesterol in Membranes: Effect of the Double-Bond Position

The structural and dynamical properties of lipid membranes rich in phospholipids and cholesterol are known to be strongly affected by the unsaturation of lipid acyl chains. We show that not only unsaturation but also the position of a double bond has a pronounced effect on membrane properties. We consider how cholesterol interacts with phosphatidylcholines comprising two 18-carbon long monounsaturated acyl chains, where the position of the double bond is varied systematically along the acyl chains. Atomistic molecular dynamics simulations indicate that when the double bond is not in contact with the cholesterol ring, and especially with the C18 group on its rough beta-side, the membrane properties are closest to those of the saturated bilayer. However, any interaction between the double bond and the ring promotes membrane disorder and fluidity. Maximal disorder is found when the double bond is located in the middle of a lipid acyl chain, the case most commonly found in monounsaturated acyl chains of phospholipids. The results suggest a cholesterol-mediated lipid selection mechanism in eukaryotic cell membranes. With saturated lipids, cholesterol promotes the formation of highly ordered raft-like membrane domains, whereas domains rich in unsaturated lipids with a double bond in the middle remain highly fluid despite the presence of cholesterol.

Direct mapping of nanoscale compositional connectivity on intact cell membranes

Lateral segregation of cell membranes is accepted as a primary mechanism for cells to regulate a diversity of cellular functions. In this context, lipid rafts have been conceptualized as organizing principle of biological membranes where underlying cholesterol-mediated selective connectivity must exist even at the resting state. However, such a level of nanoscale compositional connectivity has been challenging to prove. Here we used single-molecule near-field scanning optical microscopy to visualize the nanolandscape of raft ganglioside GM1 after tightening by its ligand cholera toxin (CTxB) on intact cell membranes. We show that CTxB tightening of GM1 is sufficient to initiate a minimal raft coalescence unit, resulting in the formation of cholesterol-dependent GM1 nanodomains < 120 nm in size. This particular arrangement appeared independent of cell type and GM1 expression level on the membrane. Simultaneous dual color high-resolution images revealed that GPI anchored and certain transmembrane proteins were recruited to regions proximal (< 150 nm) to CTxB-GM1 nanodomains without physical intermixing. Together with in silico experiments, our high-resolution data conclusively demonstrate the existence of raft-based interconnectivity at the nanoscale. Such a linked state on resting cell membranes constitutes thus an obligatory step toward the hierarchical evolution of large-scale raft coalescence upon cell activation.

Cholesterol induces specific spatial and orientational order in cholesterol/phospholipid membranes.

Background In lipid bilayers, cholesterol facilitates the formation of the liquid-ordered phase and enables the formation of laterally ordered structures such as lipid rafts. While these domains have an important role in a variety of cellular processes, the precise atomic-level mechanisms responsible for cholesterols specific ordering and packing capability have remained unresolved. Methodology/Principal Findings Our atomic-scale molecular dynamics simulations reveal that this ordering and the associated packing effects in membranes largely result from cholesterols molecular structure, which differentiates cholesterol from other sterols. We find that cholesterol molecules prefer to be located in the second coordination shell, avoiding direct cholesterol-cholesterol contacts, and form a three-fold symmetric arrangement with proximal cholesterol molecules. At larger distances, the lateral three-fold organization is broken by thermal fluctuations. For other sterols having less structural asymmetry, the three-fold arrangement is considerably lost. Conclusions/Significance We conclude that cholesterol molecules act collectively in lipid membranes. This is the main reason why the liquid-ordered phase only emerges for Chol concentrations well above 10 mol% where the collective self-organization of Chol molecules emerges spontaneously. The collective ordering process requires specific molecular-scale features that explain why different sterols have very different membrane ordering properties: the three-fold symmetry in the Chol-Chol organization arises from the cholesterol off-plane methyl groups allowing the identification of raft-promoting sterols from those that do not promote rafts.

Effects of Dimethyl Sulfoxide in Cholesterol-Containing Lipid Membranes: A Comparative Study of Experiments In Silico and with Cells

Dimethyl sulfoxide (DMSO) has been known to enhance cell membrane permeability of drugs or DNA. Molecular dynamics (MD) simulations with single-component lipid bilayers predicted the existence of three regimes of action of DMSO: membrane loosening, pore formation and bilayer collapse. We show here that these modes of action are also reproduced in the presence of cholesterol in the bilayer, and we provide a description at the atomic detail of the DMSO-mediated process of pore formation in cholesterol-containing lipid membranes. We also successfully explore the applicability of DMSO to promote plasma membrane permeability to water, calcium ions (Ca2+) and Yo-Pro-1 iodide (Yo-Pro-1) in living cell membranes. The experimental results on cells in culture can be easily explained according to the three expected regimes: in the presence of low doses of DMSO, the membrane of the cells exhibits undulations but no permeability increase can be detected, while at intermediate DMSO concentrations cells are permeabilized to water and calcium but not to larger molecules as Yo-Pro-1. These two behaviors can be associated to the MD-predicted consequences of the effects of the DMSO at low and intermediate DMSO concentrations. At larger DMSO concentrations, permeabilization is larger, as even Yo-Pro-1 can enter the cells as predicted by the DMSO-induced membrane-destructuring effects described in the MD simulations.

Structural and kinetic molecular dynamics study of electroporation in cholesterol-containing bilayers.

We present a numerical study of pore formation in lipid bilayers containing cholesterol (Chol) and subjected to a transverse electric field. Molecular dynamics simulations of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DOPC) membranes reveal the formation of a pore when an electric field of 325 mV/nm is applied. The minimum electric field needed for membrane permeabilization strongly increases with the addition of cholesterol above 10 mol %, reaching 750 mV/nm for 40 mol % Chol. Analysis of simulations of DOPC/Chol bilayers suggests this is caused by a substantial increment of membrane cohesion. Simulations also show that pore formation kinetics is much slower at high Chol contents.

Influence of cis double-bond parametrization on lipid membrane properties: How seemingly insignificant details in force-field change even qualitative trends

We have employed atomistic molecular dynamics simulations to investigate the effect of double-bond parametrization on lipid membrane properties. As models, we use one-component membranes composed of glycerol-based phosphatidylcholines (PCs) with monounsaturated acyl chains, and we complement these studies by additional PC/cholesterol simulations. We compare differences between double-bond parametrizations by varying the position of the double bond systematically along the lipid hydrocarbon chains. The results give rise for concern: They indicate that the double-bond description may change not only the quantitative but also the qualitative nature of membrane behavior. In particular, we find that the double-bond description which accounts for skew states in the vicinity of a double bond predicts a maximum in membrane disorder, when the double bond resides at the middle of an acyl chain, in agreement with experiments. The more commonly used description which does not accommodate skew states, however, predicts membrane disorder to decrease monotonically as the double bond is shifted from the glycerol backbone to the end of an acyl chain. The results highlight the importance of properly describing double bonds especially in many-component membranes, where the interplay of different molecule types is difficult to predict on intuitive grounds.

Size-controlled nanopores in lipid membranes with stabilizing electric fields.

Molecular dynamics (MD) has been shown to be a useful tool for unveiling many aspects of pore formation in lipid membranes under the influence of an applied electric field. However, the study of the structure and transport properties of electropores by means of MD has been hampered by difficulties in the maintenance of a stable electropore in the typically small simulated membrane patches. We describe a new simulation scheme in which an initially larger porating field is systematically reduced after pore formation to lower stabilizing values to produce stable, size-controlled electropores, which can then be characterized at the molecular level. A new method allows the three-dimensional modeling of the irregular shape of the pores obtained as well as the quantification of its volume. The size of the pore is a function of the value of the stabilizing field. At lower fields the pore disappears and the membrane recovers its normal shape, although in some cases long-lived, fragmented pores containing unusual lipid orientations in the bilayer are observed.

A Monte Carlo simulation of localized corrosion

A two‐dimensional Monte Carlo simulation algorithm is formulated following the tunneling mechanism for localized (pitting) corrosion. Depending on the values of a minimum set of control parameters, easily interpreted in terms of physicochemical variables, the two limiting regimes of stable and unstable corrosion are reproduced. Pits grown under such distinctive conditions are analyzed from the point of view of both their electrochemical response (current vs time behavior) and their morphology. In this last respect, fractal concepts are advantageously used to describe their exposed profiles as well as their distributions of local currents.

Energy relaxation in nonlinear one-dimensional lattices

We study energy relaxation in thermalized one-dimensional nonlinear arrays of the Fermi-Pasta-Ulam type. The ends of the thermalized systems are placed in contact with a zero-temperature reservoir via damping forces. Harmonic arrays relax by sequential phonon decay into the cold reservoir, the lower-frequency modes relaxing first. The relaxation pathway for purely anharmonic arrays involves the degradation of higher-energy nonlinear modes into lower-energy ones. The lowest-energy modes are absorbed by the cold reservoir, but a small amount of energy is persistently left behind in the array in the form of almost stationary low-frequency localized modes. Arrays with interactions that contain both a harmonic and an anharmonic contribution exhibit behavior that involves the interplay of phonon modes and breather modes. At long times relaxation is extremely slow due to the spontaneous appearance and persistence of energetic high-frequency stationary breathers. Breather behavior is further ascertained by explicitly injecting a localized excitation into the thermalized arrays and observing the relaxation behavior.

Robustness and Stability of the Gene Regulatory Network Involved in DV Boundary Formation in the Drosophila Wing

Gene regulatory networks have been conserved during evolution. The Drosophila wing and the vertebrate hindbrain share the gene network involved in the establishment of the boundary between dorsal and ventral compartments in the wing and adjacent rhombomeres in the hindbrain. A positive feedback-loop between boundary and non-boundary cells and mediated by the activities of Notch and Wingless/Wnt-1 leads to the establishment of a Notch dependent organizer at the boundary. By means of a Systems Biology approach that combines mathematical modeling and both in silico and in vivo experiments in the Drosophila wing primordium, we modeled and tested this regulatory network and present evidence that a novel property, namely refractoriness to the Wingless signaling molecule, is required in boundary cells for the formation of a stable dorsal-ventral boundary. This new property has been validated in vivo, promotes mutually exclusive domains of Notch and Wingless activities and confers stability to the dorsal-ventral boundary. A robustness analysis of the regulatory network complements our results and ensures its biological plausibility.