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

Hypercholesterolemia Impaired Sperm Functionality in Rabbits

Hypercholesterolemia represents a high risk factor for frequent diseases and it has also been associated with poor semen quality that may lead to male infertility. The aim of this study was to analyze semen and sperm function in diet-induced hypercholesterolemic rabbits. Twelve adult White New Zealand male rabbits were fed ad libitum a control diet or a diet supplemented with 0.05% cholesterol. Rabbits under cholesterol-enriched diet significantly increased total cholesterol level in the serum. Semen examination revealed a significant reduction in semen volume and sperm motility in hypercholesterolemic rabbits (HCR). Sperm cell morphology was seriously affected, displaying primarily a “folded head”-head fold along the major axe-, and the presence of cytoplasmic droplet on sperm flagellum. Cholesterol was particularly increased in acrosomal region when detected by filipin probe. The rise in cholesterol concentration in sperm cells was determined quantitatively by Gas chromatographic-mass spectrometric analyses. We also found a reduction of protein tyrosine phosphorylation in sperm incubated under capacitating conditions from HCR. Interestingly, the addition of Protein Kinase A pathway activators -dibutyryl-cyclic AMP and iso-butylmethylxanthine- to the medium restored sperm capacitation. Finally, it was also reported a significant decrease in the percentage of reacted sperm in the presence of progesterone. In conclusion, our data showed that diet-induced hypercholesterolemia adversely affects semen quality and sperm motility, capacitation and acrosomal reaction in rabbits; probably due to an increase in cellular cholesterol content that alters membrane related events

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

Cholesterol stabilizes the hexagonal type II phase of 1-palmitoyl-2-oleoyl sn glycero-3-phosphoethanolamine. A solid state

The effect of 30 mol % cholesterol on the thermotropism and hydration of 1- palmitoyl-2-oleoyl sn glycero-3-phosphoethanolamine (POPE) was monitored by solid state 31P-NMR of the phospholipid and 2H-NMR of D2O. For a water content of 15 molecules per total lipid, it is observed that cholesterol lowers by ~ 30°C the temperature of the lamellar-to-hexagonal phase transition and promotes dehydration of both the lamellar and hexagonal phases

Comparison of methods to determine aliphatic chain length in biomembranes from selectively or perdeuterated systems. A

Comparison of the different methods to determine aliphatic chain length in biomembranes from the C-D quadrupolar splittings obtained with solid state 2H-NMR is reported. It is shown that perdeuteration of the saturated lipid acyl chains is the alternative to selectively labelled chains without loss of accuracy. Accuracy in chain length determination may be then estimated to be of the order of 0.2 Å

An efficient method for detection of energetically low lipid phase transitions : deuterium NMR of D

2H NMR of sn-2 pcrdeuteriated DMPC was used to compare the respective effect of 30 mol% of cholesterol (CH) and cholesterol sulfate (CS) on DMPC membranes. Both steroids act as membrane ordering regulators but CS is less potent than CH. However, at variance to CH, CS does not totally abolish the DMPC gel-to-fluid phase transition. NMR of deuteriated chains fails to evidence this residual transition whereas 2H NMR of heavy water confirms DSC results by exhibiting a minimum in the temperature dependence of the D2O quadrupolar splitting

On the relationship between C-C and C-D order parameters and its use for studying the conformation of lipid acyl chains in biomembranes

We have used the order tensor method to establish relationships between carbon-carbon and carbon-deuterium order parameters (SCC and SCD, respectively) in saturated and unsaturated lipid acyl chains in biomembranes. We thus confirm the existence of a recursion relation between these order parameters ( - 2S(k)(CD) = S-k(CC) + S-k+1(CC)) as was; previously shown using Wigner matrices formalism [Douliez, Leonard, and Dufourc, Biophys. J. 68, 1727 (1995)]. This leads to the determination of S-k(CC) as a function of the position k along the chain having experimentally measured the corresponding S-k(CD). Making use of a mean field approach for computing the energy of chain conformers in the frame of the rotational isomeric model (RIS) we have also calculated S-CC and S-CD order profile for the sn-l chain of dipalmitoylphosphatidylcholine. A very good agreement is obtained between computational and predicted S-k(CC) values, reinforcing the validity of the recursion relation. A marked odd-even effect is evidenced on the S-CC order profile for positions near the polar head, as was previously experimentally reported for dimyristoylphosphatidylcholine. We propose that this effect reveals an average bent orientation of the beginning of the chain with respect to the normal of the bilayer. Finally, it is shown that our formalism can be applied to some extent to cis or trans unsaturated lipid