Key Role of Polyphosphoinositides in Dynamics of Fusogenic Nuclear Membrane Vesicles

The role of phosphoinositides has been thoroughly described in many signalling and membrane trafficking events but their function as modulators of membrane structure and dynamics in membrane fusion has not been investigated. We have reconstructed models that mimic the composition of nuclear envelope precursor membranes with naturally elevated amounts of phosphoinositides. These fusogenic membranes (membrane vesicle 1(MV1) and nuclear envelope remnants (NER) are critical for the assembly of the nuclear envelope. Phospholipids, cholesterol, and polyphosphoinositides, with polyunsaturated fatty acid chains that were identified in the natural nuclear membranes by lipid mass spectrometry, have been used to reconstruct complex model membranes mimicking nuclear envelope precursor membranes. Structural and dynamic events occurring in the membrane core and at the membrane surface were monitored by solid-state deuterium and phosphorus NMR. “MV1-like” (PC∶PI∶PIP∶PIP2, 30∶20∶18∶12, mol%) membranes that exhibited high levels of PtdIns, PtdInsP and PtdInsP2 had an unusually fluid membrane core (up to 20% increase, compared to membranes with low amounts of phosphoinositides to mimic the endoplasmic reticulum). “NER-like” (PC∶CH∶PI∶PIP∶PIP2, 28∶42∶16∶7∶7, mol%) membranes containing high amounts of both cholesterol and phosphoinositides exhibited liquid-ordered phase properties, but with markedly lower rigidity (10–15% decrease). Phosphoinositides are the first lipids reported to counterbalance the ordering effect of cholesterol. At the membrane surface, phosphoinositides control the orientation dynamics of other lipids in the model membranes, while remaining unchanged themselves. This is an important finding as it provides unprecedented mechanistic insight into the role of phosphoinositides in membrane dynamics. Biological implications of our findings and a model describing the roles of fusogenic membrane vesicles are proposed

Monitoring changes of paramagnetically-shifted 31P signals in phospholipid vesicles

Phospholipid vesicles are commonly used as biomimetics in the investigation of the interaction of various species with cell membranes. In this paper we present a 31P NMR investigation of a simple vesicle system using a paramagnetic shift reagent to probe the inner and outer layers of the lipid bilayer. Time-dependent changes in the 31P NMR signal are observed, which differ whether the paramagnetic species is inside or outside the vesicle, and on the choice of buffer solution used. An interpretation of these results is given in terms of the interaction of the paramagnetic shift reagent with the lipids

Membrainy: a ‘smart’, unified membrane analysis tool

BACKGROUND: The study of biological membranes using Molecular Dynamics has become an increasingly popular means by which to investigate the interactions of proteins, peptides and potentials with lipid bilayers. These interactions often result in changes to the properties of the lipids which can modify the behaviour of the membrane. Membrainy is a unified membrane analysis tool that contains a broad spectrum of analytical techniques to enable: measurement of acyl chain order parameters; presentation of 2D surface and thickness maps; determination of lateral and axial headgroup orientations; measurement of bilayer and leaflet thickness; analysis of the annular shell surrounding membrane-embedded objects; quantification of gel percentage; time evolution of the transmembrane voltage; area per lipid calculations; and quantification of lipid mixing/demixing entropy. RESULTS: Each analytical component within Membrainy has been tested on a variety of lipid bilayer systems and was found to be either comparable to or an improvement upon existing software. For the analytical techniques that have no direct comparable software, our results were confirmed with experimental data. CONCLUSIONS: Membrainy is a user-friendly, intelligent membrane analysis tool that automatically interprets a variety of input formats and force fields, is compatible with both single and double bilayers, and capable of handling asymmetric bilayers and lipid flip-flopping. Membrainy has been designed for ease of use, requiring no installation or configuration and minimal user-input to operate

Action of melittin on the DPPC-cholesterol liquid-ordered phase: a solid state 2H-and 31P-NMR study.

Solid-state deuterium and phosphorus-31 nuclear magnetic resonance studies of deuterium-labeled beta--[2,2',3,4,4',6-2H6]-cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine have been undertaken to monitor the action of melittin on model membranes containing 30 mol% cholesterol, both at the molecular and macroscopic level. Cholesterol totally inhibits the toxin-triggered formation of large unilamellar vesicles and strongly restricts the appearance of small discs. The latter remain stable over a wide temperature range (20-60 degrees C) because of an increase in their cholesterol content as the temperature increases. This process is related to a constant disc hydrophobic thickness of approximately 29 A. The system, when not in the form of discs, appears to be composed of very large vesicles on which melittin promotes magnetically induced ellipsoidal deformation. This deformation is the greatest when the maximum of discs is observed. A model to describe both the disc formation and stability is proposed

Interfacial hydration of ceramide in stratum corneum model membrane measured by

2H NMR of D2O was used to determine the interfacial hydration of bilayers modelizing the stratum corneum lamellar sheets. Our model membranes are composed of equimolar amounts of ceramide and cholesterol sulfate and the study was realized at 70 °C, temperature for which the system is in a lamellar phase. The two-site exchange theory applied to the spin lattice relaxation rate, 1/TlZ, led us to conclude that 1 molecule of ceramide and 1 molecule of cholesterol sulfate bind ca. 11 molecules of D2O. The comparison of this value to previous works reporting that cholesterol sulfate binds ca. 12 molecules of water (Faure et al., 1996, Biophysical J., 70, 1380–1390), indicates that ceramide is little hydrated. This result provides a basis to account for the non-existence of a pure lamellar phase with ceramide-water or ceramide-cholesterol-water systems

Effects of pH and cholesterol on DMPA membranes: a solid state 2H- and 31P-NMR study.

The effect of pH and cholesterol on the dimyristoylphosphatidic acid (DMPA) model membrane system has been investigated by solid state 2H- and 31P-NMR. It has been shown that each of the three protonation states of the DMPA molecule corresponds to a 31P-NMR powder pattern with characteristic delta sigma values; this implies additionally that the proton exchange on the membrane surface is slow on the NMR time scale (millisecond range). Under these conditions, the 2H-labeled lipid chains sense only one magnetic environment, indicating that the three spectra detected by 31P-NMR are related to charge-dependent local dynamics or orientations of the phosphate headgroup or both. Chain ordering in the fluid phase is also found to depend weakly on the charge at the interface. In addition, it has also been found that the first pK of the DMPA membrane is modified by changes in the lipid lateral packing (gel or fluid phases or in the presence of cholesterol) in contrast to the second pK. The incorporation of 30 mol% cholesterol affects the phosphatidic acid bilayer in a way similar to what has been reported for phosphatidylcholine/cholesterol membranes, but to an extent comparable to 10-20 mol % sterol in phosphatidylcholines. However, the orientation and molecular order parameter of cholesterol in DMPA are similar to those found in dimyristoylphosphatidylcholine

Restatement of order parameters in biomembranes: calculation of C-C bond order parameters from C-D quadrupolar splittings.

An expression for the C-C bond order parameter, SCC, of membrane hydrocarbon chains has been derived from the observed C-D bond order parameters. It allows calculation of the probability of each of the C-C bond rotamers and, consequently, the number of gauche defects per chain as well as their projected average length onto the bilayer normal, thus affording the calculation of accurate hydrophobic bilayer thicknesses. The effect of temperature has been studied on dilauroyl-, dimyristoyl-, and dipalmitoylphosphatidylcholine (DLPC, DMPC, DPPC) membranes, as has the effect of cholesterol on DMPC. The salient results are as follows: 1) an odd-even effect is observed for the SCC versus carbon position, k, whose amplitude increases with temperature; 2) calculation of SCC, from nonequivalent deuterons on the sn-2 chain of lipids, SCC2, leads to negative values, indicating the tendency for the C1-C2 bond to be oriented parallel to the bilayer surface; this bond becomes more parallel to the surface as the temperature increases or when cholesterol is added; 3) calculation on the sn-2 chain length can be performed from C1 to Cn, where n is the number of carbon atoms in the chain, and leads to 10.4, 12.2, and 13.8 A for DLPC, DMPC, and DPPC close to the transition temperature, TC, of each of the systems and to 9.4, 10.9, and 12.6 for T-TC = 30-40 degrees C, respectively; 4) separation of intra- and intermolecular motions allows quantitation of the number of gauche defects per chain, which is equal to 1.9, 2.7, and 3.5 for DLPC, DMPC, and DPPC near TC and to 2.7, 3.5, and 4.4 at T-TC = 30-40 degrees C, respectively. Finally, the validity of our model is discussed and compared with previously published models

Comparative effects of cholesterol and cholesterol sulfate on hydration and ordering of dimyristoylphosphatidylcholine membranes.

The comparative effect of cholesterol (CH) versus cholesterol sulfate (CS) on dimyristoylphosphatidylcholine (DMPC) membranes has been investigated by optical microscopy, freeze-fracture electron microscopy, x-ray diffraction, and solid state 2H and 31P nuclear magnetic resonance (NMR). The sulfate analogue extends the lamellar phase domain toward high water contents, and substitution of 30 mol % CH by CS in DMPC lamellae induces the trapping of 30 wt % additional water. The greater swelling of the CS-containing systems is evidenced by determination of lamellar repeat distances at maximal hydration: 147 +/- 4 A and 64 +/- 2 A in the presence of CS and CH, respectively. 2H-NMR of heavy water demonstrates that CS binds approximately 12 more water molecules at the interface than CH whereas NMR of deuterium-labeled DMPC chains reveals that 30 mol % CS orders the membrane as 15 mol % CH at high temperature and disorders much more than CH at low temperatures. The various effects of CS versus CH are discussed by taking into account attractive Van der Waals forces and repulsive steric/electrostatic interactions of the negatively charged sulfate group