SummaryCell and organelle membranes consist of a complex mixture of phospholipids (PLs) that determine their size, shape, and function. Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic membranes, yet how cells sense and regulate its levels in vivo remains unclear. Here we show that PCYT1A, the rate-limiting enzyme of PC synthesis, is intranuclear and re-locates to the nuclear membrane in response to the need for membrane PL synthesis in yeast, fly, and mammalian cells. By aligning imaging with lipidomic analysis and data-driven modeling, we demonstrate that yeast PCYT1A membrane association correlates with membrane stored curvature elastic stress estimates. Furthermore, this process occurs inside the nucleus, although nuclear localization signal mutants can compensate for the loss of endogenous PCYT1A in yeast and in fly photoreceptors. These data suggest an ancient mechanism by which nucleoplasmic PCYT1A senses surface PL packing defects on the inner nuclear membrane to control PC homeostasis.

The recent suggestion of phosphine in Venus' atmosphere has regenerated interest in the idea of life in clouds. However, such analyses usually neglect the role of water activity, which is a measure of the relative availability of water, in habitability. Here, we compute the water activity within the clouds of Venus and other Solar-System planets from observations of temperature and water-vapour abundance. We find water-activity values of sulphuric acid droplets, which constitute the bulk of Venus' clouds, of ≤0.004, two orders of magnitude below the 0.585 limit for known extremophiles. Considering other planets, ice formation on Mars imposes a water activity ≤0.537, slightly below the habitable range, whereas conditions are biologically permissive (>0.585) at Jupiter's clouds (although other factors such as their composition may play a role in limiting their habitability). By way of comparison, the Earth's troposphere conditions are, in general, biologically permissive, whereas the atmosphere becomes too dry for active life above the middle stratosphere. The approach used in the current study can also be applied to extrasolar planets.

While it is widely accepted that the lipid composition of eukaryotic membranes is under homeostatic control, the mechanisms through which cells sense lipid composition are still the subject of debate. It has been postulated that membrane curvature elastic energy is the membrane property that is regulated by cells, and that lipid composition is maintained by a ratio control function derived from the concentrations of type II and type 0 lipids, weighted appropriately. We assess this proposal by seeking a signature of ratio control in quantified lipid composition data obtained by electrospray ionization mass spectrometry from over 40 independent asynchronous cell populations. Our approach revealed the existence of a universal 'pivot' lipid, which marks the boundary between type 0 lipids and type II lipids, and which is invariant between different cell types or cells grown under different conditions. The presence of such a pivot species is a distinctive signature of the operation in vivo, in human cell lines, of a control function that is consistent with the hypothesis that membrane elastic energy is homeostatically controlled.

One of the mostly widely cited theories of phospholipid homeostasis is the theory of homeoviscous adaptation (HVA). HVA states that cells maintain membrane order (frequently discussed in terms of membrane fluidity or viscosity) within tight conditions in response to environmental induced changes in membrane lipid composition. In this article we use data driven modelling to investigate membrane order, using methodology we previously developed to investigate another theory of phospholipid homeostasis, the intrinsic curvature hypothesis. A set of coarse-grain parameters emerge from our model which can be used to deconstruct the relative contribution of each component membrane phospholipid to net membrane order. Our results suggest, for the membranes in the mammalian cells we have studied, that a ratio control function can be used to model membrane order. Using asynchronous cell lines we quantify the relative contribution of around 130 lipid species to net membrane order, finding that around 16 of these phospholipid species have the greatest effect in vivo. Then using lipidomic data obtained from partially synchronised cultures of HeLa cells we are able to demonstrate that these same 16 lipid species drive the changes in membrane order observed around the cell cycle.Our findings in this study suggest, when compared with our previous work, that cells maintain both membrane order and membrane intrinsic curvature within tight conditions.

The addition of saturated fatty acids (FA) to phosphatidylcholine lipids (PC) that have saturated acyl chains has been shown to promote the formation of lyotropic liquid-crystalline phases with negative mean curvature. PC/FA mixtures may exhibit inverse bicontinuous cubic phases (Im3m, Pn3m) or inverse topology hexagonal phases (HII), depending on the length of the acyl chains/fatty acid. Here we report a detailed study of the phase behavior of binary mixtures of dioleoylphosphatidylcholine (DOPC)/oleic acid (OA) and dioleoylphosphatidylethanolamine (DOPE)/oleic acid at limiting hydration, constructed using small-angle X-ray diffraction (SAXD) data. The phase diagrams of both systems show a succession of phases with increasing negative mean curvature with increasing OA content. At high OA concentrations, we have observed the occurrence of an inverse micellar Fd3m phase in both systems. Hitherto, this phase had not been reported for phosphatidylethanolamine/fatty acid mixtures, and as such it highlights an additional route through which fatty acids may increase the propensity of bilayer lipid membranes to curve. We also propose a method that uses the temperature dependence of the lattice parameters of the HII phases to estimate the spontaneous radii of curvature (R0) of the binary mixtures and of the component lipids. Using this method, we calculated the R0 values of the complexes comprising one phospholipid molecule and two fatty acid molecules, which have been postulated to drive the formation of inverse phases in PL/FA mixtures. These are -1.8 nm (±0.4 nm) for DOPC(OA)2 and -1.1 nm (±0.1 nm) for DOPE(OA)2. R0 values estimated in this way allow the quantification of the contribution that different lipid species make to membrane curvature elastic properties and hence of their effect on the function of membrane-bound proteins.

One of the most developed theories of phospholipid homeostasis is the intrinsic curvature hypothesis, which, in broad terms, postulates that cells regulate their lipid composition so as to keep constant the membrane stored curvature elastic energy. The implication of this hypothesis is that lipid composition is determined by a ratio control function consisting of the weighted sum of concentrations of type II lipids in the numerator and the weighted sum of concentrations of Type 0 lipids in the denominator. In previous work we used a data-driven approach, based on lipidomic data from asynchronous cell cultures, to determine a criterion that allows the different lipid species to be assigned to the set of type 0 or of type II lipids, and hence construct a ratio control function that serves as a proxy for the lipid contribution to total membrane stored curvature elastic energy in vivo.Here we apply the curvature elastic energy proxy to the analysis of lipid composition data from synchronous HeLa cells as they traverse the cell cycle. Our analysis suggests HeLa cells modify their membrane stored elastic energy through the cell cycle. In S-phase type 0 lipids are the most abundant, whilst in G2 type II lipids are most abundant. Changes in our proxy for membrane stored elastic energy correlate with membrane curvature dependent processes in the HeLa cell around division, providing some insights into the interplay between the individual lipid and protein contributions to membrane free energy.

Recently we reported a method for estimating the spontaneous curvatures of lipids from temperature dependent changes in the lattice parameter of inverse hexagonal liquid crystal phases of binary lipid mixtures. This method makes use of 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine, (DOPE) as a host lipid, which preferentially forms an inverse hexagonal phase, to which a guest lipid of unknown spontaneous curvature is added. The lattice parameters of these binary lipid mixtures are determined by small angle X-ray diffraction at a range of temperatures and the spontaneous curvature of the guest lipid is determined from these data. Here we report the use of this method on a wide range of lipids under different ionic conditions. We demonstrate that our method provides spontaneous curvature values for DOPE, cholesterol and monoolein that are within the range of values reported in the literature. Anionic lipids 1,2-dioleoyl-sn-glycerol-3-phosphatidic acid (DOPA) and 1,2-dioleoyl-sn-glycerol-3-phosphoserine (DOPS) were found to exhibit spontaneous curvatures that depend on the concentration of divalent cations present in the mixtures. We show that the range of curvatures estimated experimentally for DOPA and DOPS can be explained by a series of equilibria arising from lipid-cation exchange reactions. Our data indicate a universal relationship between the spontaneous curvature of a lipid and the extent to which it affects the lattice parameter of the hexagonal phase of DOPE when it is part of a binary mixture. This universal relationship affords a rapid way of estimating the spontaneous curvatures of lipids that are expensive, only available in small amounts or are of limited chemical stability.

The alkyllysophospholipid (ALP) analogues Mitelfosine and Edelfosine are anticancer drugs whose mode of action is still the subject of debate. It is agreed that the primary interaction of these compounds is with cellular membranes. Furthermore, the membrane-associated protein CTP: phosphocholine cytidylyltransferase (CCT) has been proposed as the critical target. We present the evaluation of our hypothesis that ALP analogues disrupt membrane curvature elastic stress and inhibit membrane-associated protein activity (e.g. CCT), ultimately resulting in apoptosis. This hypothesis was tested by evaluating structure -activity relationships of ALPs from the literature. In addition we characterized the lipid typology, cytotoxicity and critical micelle concentration of novel ALP analogues that we synthesized. Overall we find the literature data and our experimental data provide excellent support for the hypothesis, which predicts that the most potent ALP analogues will be type I lipids.