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We have imaged the formation of membrane microdomains immediately after their induction using a novel technology platform coupling high hydrostatic pressure to fluorescence microscopy. After formation, the ordered domains are small and highly dynamic. This will enhance links between model lipid assemblies and dynamic processes in cellular membranes.

Lipid bicontinuous cubic phases have attracted enormous interest as bio-compatible scaffolds for use in a wide range of applications including membrane protein crystallisation, drug delivery and biosensing. One of the major bottlenecks that has hindered exploitation of these structures is an inability to create targeted highly swollen bicontinuous cubic structures with large and tunable pore sizes. In contrast, cubic structures found in vivo have periodicities approaching the micron scale. We have been able to engineer and control highly swollen bicontinuous cubic phases of spacegroup Im3m containing only lipids by (a) increasing the bilayer stiffness by adding cholesterol and (b) inducing electrostatic repulsion across the water channels by addition of anionic lipids to monoolein. By controlling the composition of the ternary mixtures we have been able to achieve lattice parameters up to 470 Å, which is 5 times that observed in pure monoolein and nearly twice the size of any lipidic cubic phase reported previously. These lattice parameters significantly exceed the predicted maximum swelling for bicontinuous cubic lipid structures, which suggest that thermal fluctuations should destroy such phases for lattice parameters larger than 300 Å.

Understanding of cellular regulatory pathways that involve lipid membranes requires the detailed knowledge of their physical state and structure. However, mapping the viscosity and diffusion in the membranes of complex composition is currently a non-trivial technical challenge. We report fluorescence lifetime spectroscopy and imaging (FLIM) of a meso-substituted BODIPY molecular rotor localised in the leaflet of model membranes of various lipid compositions. We prepare large and giant unilamellar vesicles (LUVs and GUVs) containing phosphatidylcholine (PC) lipids and demonstrate that recording the fluorescence lifetime of the rotor allows us to directly detect the viscosity of the membrane leaflet and to monitor the influence of cholesterol on membrane viscosity in binary and ternary lipid mixtures. In phase-separated 1,2-dioleoyl-sn-glycero-3-phosphocholine-cholesterol-sphingomyelin GUVs we visualise individual liquid ordered (Lo) and liquid disordered (Ld) domains using FLIM and assign specific microscopic viscosities to each domain. Our study showcases the power of FLIM with molecular rotors to image microviscosity of heterogeneous microenvironments in complex biological systems, including membrane-localised lipid rafts.

The effect of hydrostatic pressure on lipid structure and dynamics is highly important as a tool in biophysics and bio-technology, and in the biology of deep sea organisms. Despite its importance, high hydrostatic pressure remains significantly less utilised than other thermodynamic variables such as temperature and chemical composition. Here, we give an overview of some of the theoretical aspects which determine lipid behaviour under pressure and the techniques and technology available to study these effects. We also summarise several recent experiments which highlight the information available from these approaches., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Mixtures of dsDNA and lipids, so-called lipoplexes, are widely used as less toxic alternatives to viral vectors in transfection studies. However, the transfection efficiency achieved by lipoplexes is significantly lower than that of viral vectors and is a barrier to their use in the clinic. There is now significant evidence suggesting that the molecular organization and structure (nanoarchitecture) of lipoplexes might correlate with biological activity. As a consequence, the ability to predict quantitatively the nanoarchitecture of new systems, and how these might change intracellularly, would be a major tool in the development of rational discovery strategies for more efficient lipoplex formulations. Here we report the use of a coarse-grain molecular dynamics simulation to predict the phases formed by two lipoplex systems: dsDNA-DOPE and dsDNA-DOPE-DOTAP. The predictions of the simulations show excellent agreement with experimental data from polarized light microscopy and small-angle X-ray diffraction (SAXS); the simulations predicted the formation of phases with d-spacings that were comparable to those measured by SAXS. More significantly, the simulations were able to reproduce for the first time the experimentally observed change from a fluid lamellar to an inverse hexagonal phase in the dsDNA-DOPE-DOTAP system as a function of changes in lipid composition. Our studies indicate that coarse-grain MD simulations could provide a powerful tool to understand, and hence design, new lipoplex systems.

In this paper, we report on a novel osmotic cell, developed to simultaneously subject a sample to osmotic stress and measure structural changes by small angle x-ray diffraction. The osmotic cell offers many advantages over more conventional methods of osmotically stressing soft materials to measure their structural response. In particular, a full osmotic analysis can be performed with a single small sample (25 microl). This reduces sample handling and the associated systematic errors, as well as enabling tight control and monitoring of the thermodynamic environment during osmosis, thereby increasing measurement precision. The cell design enables control of osmotic pressure to +/-0.04 bar over a pressure range of 1-100 bar, and temperature control to +/-0.05 degrees C. Under these conditions, the lattice spacing in lyotropic structures was resolved to better than +/-0.005 A. Using the osmotic cell, we demonstrate good agreement with previous conventional measurements on the energy of dehydrating the fluid lamellar phase of dioleoylphosphatidylcholine in water.

We present a cellular model of lipid biosynthesis in the plasma membrane that couples biochemical and biophysical features of the enzymatic network of the cell-wall-less Mycoplasma Acholeplasma laidlawii. In particular, we formulate how the stored elastic energy of the lipid bilayer can modify the activity of curvature-sensitive enzymes through the binding of amphipathic alpha-helices. As the binding depends on lipid composition, this results in a biophysical feedback mechanism for the regulation of the stored elastic energy. The model shows that the presence of feedback increases the robustness of the steady state of the system, in the sense that biologically inviable nonbilayer states are less likely. We also show that the biophysical and biochemical features of the network have implications as to which enzymes are most efficient at implementing the regulation. The network imposes restrictions on the steady-state balance between bilayer and nonbilayer lipids and on the concentrations of particular lipids. Finally, we consider the influence of the length of the amphipathic alpha-helix on the efficacy of the feedback and propose experimental measurements and extensions of the modeling framework.

In this paper, we give an overview of our studies by static and time-resolved X-ray diffraction of inverse cubic phases and phase transitions in lipids. In [section sign] 1, we briefly discuss the lyotropic phase behaviour of lipids, focusing attention on non-lamellar structures, and their geometric/topological relationship to fusion processes in lipid membranes. Possible pathways for transitions between different cubic phases are also outlined. In [section sign] 2, we discuss the effects of hydrostatic pressure on lipid membranes and lipid phase transitions, and describe how the parameters required to predict the pressure dependence of lipid phase transition temperatures can be conveniently measured. We review some earlier results of inverse bicontinuous cubic phases from our laboratory, showing effects such as pressure-induced formation and swelling. In [section sign] 3, we describe the technique of pressure-jump synchrotron X-ray diffraction. We present results that have been obtained from the lipid system 1:2 dilauroylphosphatidylcholine/lauric acid for cubic-inverse hexagonal, cubic-cubic and lamellar-cubic transitions. The rate of transition was found to increase with the amplitude of the pressure-jump and with increasing temperature. Evidence for intermediate structures occurring transiently during the transitions was also obtained. In [section sign] 4, we describe an IDL-based 'AXcess' software package being developed in our laboratory to permit batch processing and analysis of the large X-ray datasets produced by pressure-jump synchrotron experiments. In [section sign] 5, we present some recent results on the fluid lamellar-Pn3m cubic phase transition of the single-chain lipid 1-monoelaidin, which we have studied both by pressure-jump and temperature-jump X-ray diffraction. Finally, in [section sign] 6, we give a few indicators of future directions of this research. We anticipate that the most useful technical advance will be the development of pressure-jump apparatus on the microsecond time-scale, which will involve the use of a stack of piezoelectric pressure actuators. The pressure-jump technique is not restricted to lipid phase transitions, but can be used to study a wide range of soft matter transitions, ranging from protein unfolding and DNA unwinding and transitions, to phase transitions in thermotropic liquid crystals, surfactants and block copolymers.

Constructing higher-order vesicle assemblies has discipline-spanning potential from responsive soft-matter materials to artificial cell networks in synthetic biology. This potential is ultimately derived from the ability to compartmentalise and order chemical species in space. To unlock such applications, spatial organisation of vesicles in relation to one another must be controlled, and techniques to deliver cargo to compartments developed. Herein, we use optical tweezers to assemble, reconfigure and dismantle networks of cell-sized vesicles that, in different experimental scenarios, we engineer to exhibit several interesting properties. Vesicles are connected through double-bilayer junctions formed via electrostatically controlled adhesion. Chemically distinct vesicles are linked across length scales, from several nanometres to hundreds of micrometres, by axon-like tethers. In the former regime, patterning membranes with proteins and nanoparticles facilitates material exchange between compartments and enables laser-triggered vesicle merging. This allows us to mix and dilute content, and to initiate protein expression by delivering biomolecular reaction components. [ABSTRACT FROM AUTHOR]

Rab proteins are a large family of GTP-binding proteins that regulate cellular membrane traffic and organelle identity. Rab proteins cycle between association with membranes and binding to RabGDI. Bound on membranes, each Rab has a very specific cellular location and it is this remarkable degree of specificity with which Rab GTPases recognize distinct subsets of intracellular membranes that forms the basis of their ability to act as key cellular regulators, determining the recruitment of downstream effectors to the correct membrane at the correct time. The molecular mechanisms controlling Rab localization remain poorly understood. Here, we present a fluorescence-based assay to investigate Rab GTPase membrane extraction and delivery by RabGDI. Using EGFP-Rab fusion proteins the amount of Rab:GDI complex obtained by GDI extraction of Rab proteins from HEK293 membranes could be determined, enabling control of complex concentration. Subsequent partitioning of the Rab GTPases into vesicles made up of artificial binary lipid mixtures showed for the first time, that the composition of the target membrane plays a key role in the localization of Rab proteins by sensing the stored curvature elastic energy in the membrane. [ABSTRACT FROM AUTHOR]

Abstract: Phosphatidylglycerol (PG) is an anionic lipid commonly found in large proportions in the cell membranes of bacteria and plants and, to a lesser extent, in animal cells. PG plays an important role in the regulation and determination of the elastic properties of the membrane. Using small angle X-ray scattering experiments, we obtain that the monolayer spontaneous curvature of dioleoylphosphatidylglycerol (DOPG) is −1/150±0.021nm−1 when measured in 150mM NaCl. When the experiments are carried out in 150mM NaCl and 20mM MgCl2, the value obtained for the monolayer spontaneous curvature is −1/8.7±0.037nm−1. These values are of importance in modelling the effects of curvature elastic stress in membrane lipid homeostasis in the bacterium Acholeplasma laidlawii [Alley, S.H., Barahona, M., Ces, O., Templer, R.H., in press. Biophysical regulation of lipid biosynthesis in the plasma membrane. Biophys. J.] and indicate that divalent cations can play a significant role in altering curvature elastic stress. [Copyright &y& Elsevier]

A largevariety of data exists on lipid phase behavior; however,it is mostly in nonbuffered systems over nonbiological temperatureranges. We present biophysical data on lipid mixtures of dioleoylphosphatidylcholine(DOPC), dioleoylphosphatidylethanolamine (DOPE), and lysophosphatidylcholine(LysoPC) examining their behaviors in excess water and buffer systemsover the temperature range 4–34 °C. These mixtures arecommonly used to investigate the effects of spontaneous curvatureon integral membrane proteins. Using small-angle X-ray scattering(SAXS) and 31P NMR, we observed lamellar and vesicle phases,with the buffer causing an increase in the layer spacing. Increasingamounts of DOPE in a DOPC bilayer decreased the layer spacing of themesophase, while the opposite trend was observed for increasing amountsof LysoPC. 31P static NMR was used to analyze the DOPC:LysoPCsamples to investigate the vesicle sizes present, with evidence ofvesicle budding observed at LysoPC concentrations above 30 mol %.NMR line shapes were fitted using an adapted program accounting forthe distortion of the lipids within the magnetic field. The distortionof the vesicle, because of magnetic susceptibility, varied with LysoPCcontent, and a discontinuity was found in both the water and buffersamples. Generally, the distortion increased with LysoPC content;however, at a ratio of DOPC:LysoPC 60:40, the sample showed a levelof distortion of the vesicle similar to that of pure DOPC. This impliesan increased flexibility in the membrane at this point. Commonly,the assumption is that for increasing LysoPC concentration there isa reduction in membrane tension, implying that estimations of membranetension based on spontaneous curvature assumptions may not be accurate. [ABSTRACT FROM AUTHOR]

Mechanical properties of biological membranes are known to regulate membrane protein function. Despite this, current models of protein communication typically feature only direct protein-protein or protein-small molecule interactions. Here we show for the first time that, by harnessing nanoscale mechanical energy within biological membranes, it is possible to promote controlled communication between proteins. By coupling lipid-protein modules and matching their response to the mechanical properties of the membrane, we have shown that the action of phospholipase A2 on acyl-based phospholipids triggers the opening of the mechanosensitive channel, MscL, by generating membrane asymmetry. Our findings confirm that the global physical properties of biological membranes can act as information pathways between proteins, a novel mechanism of membrane-mediated protein-protein communication that has important implications for (i) the underlying structure of signaling pathways, (ii) our understanding of in vivo communication networks, and (iii) the generation of building blocks for artificial protein networks. [ABSTRACT FROM AUTHOR]