Structural basis of membrane bending by the N-BAR protein endophilin.

Functioning as key players in cellular regulation of membrane curvature, BAR domain proteins bend bilayers and recruit interaction partners through poorly understood mechanisms. Using electron cryomicroscopy, we present reconstructions of full-length endophilin and its N-terminal N-BAR domain in their membrane-bound state. Endophilin lattices expose large areas of membrane surface and are held together by promiscuous interactions between endophilin's amphipathic N-terminal helices. Coarse-grained molecular dynamics simulations reveal that endophilin lattices are highly dynamic and that the N-terminal helices are required for formation of a stable and regular scaffold. Furthermore, endophilin accommodates different curvatures through a quantized addition or removal of endophilin dimers, which in some cases causes dimerization of endophilin's SH3 domains, suggesting that the spatial presentation of SH3 domains, rather than affinity, governs the recruitment of downstream interaction partners.

Varying the position of phospholipid acyl chain unsaturation modulates hopanoid and sterol ordering.

The cell membrane must balance mechanical stability with fluidity to function as both a barrier and an organizational platform. Key to this balance is the ordering of hydrocarbon chains and the packing of lipids. Many eukaryotes synthesize sterols, which are uniquely capable of modulating the lipid order to decouple membrane stability from fluidity. Ancient sterol analogs known as hopanoids are found in many bacteria and proposed as ancestral ordering lipids. The juxtaposition of sterols and hopanoids in extant organisms prompts us to ask why both pathways persist, especially in light of their convergent ability to order lipids. In this work, simulations, monolayer experiments, and cellular assays show that hopanoids and sterols order unsaturated phospholipids differently based on the position of double bonds in the phospholipid acyl chain. We find that cholesterol and diplopterol's methyl group distributions lead to distinct effects on unsaturated lipids. In Mesoplasma florum, diplopterol's constrained ordering capacity reduces membrane resistance to osmotic stress, unlike cholesterol. These findings suggest that cholesterol's broader lipid-ordering ability may have facilitated the exploration of a more diverse lipidomic landscape in eukaryotic membranes.

Surface viscosities of lipid bilayers determined from equilibrium molecular dynamics simulations.

Lipid membrane viscosity is critical to biological function. Bacterial cells grown in different environments alter their lipid composition in order to maintain a specific viscosity, and membrane viscosity has been linked to the rate of cellular respiration. To understand the factors that determine the viscosity of a membrane, we ran equilibrium all-atom simulations of single component lipid bilayers and calculated their viscosities. The viscosity was calculated via a Green-Kubo relation, with the stress-tensor autocorrelation function modeled by a stretched exponential function. By simulating a series of lipids at different temperatures, we establish the dependence of viscosity on several aspects of lipid chemistry, including hydrocarbon chain length, unsaturation, and backbone structure. Sphingomyelin is found to have a remarkably high viscosity, roughly 20 times that of DPPC. Furthermore, we find that inclusion of the entire range of the dispersion interaction increases viscosity by up to 140%. The simulated viscosities are similar to experimental values obtained from the rotational dynamics of small chromophores and from the diffusion of integral membrane proteins but significantly lower than recent measurements based on the deformation of giant vesicles.

Full scale structural, mechanical and dynamical properties of HIV-1 liposomes.

Enveloped viruses are enclosed by a lipid membrane inside of which are all of the components necessary for the virus life cycle; viral proteins, the viral genome and metabolites. Viral envelopes are lipid bilayers that adopt morphologies ranging from spheres to tubes. The envelope is derived from the host cell during viral replication. Thus, the composition of the bilayer depends on the complex constitution of lipids from the host-cell's organelle(s) where assembly and/or budding of the viral particle occurs. Here, molecular dynamics (MD) simulations of authentic, asymmetric HIV-1 liposomes are used to derive a unique level of resolution of its full-scale structure, mechanics and dynamics. Analysis of the structural properties reveal the distribution of thicknesses of the bilayers over the entire liposome as well as its global fluctuations. Moreover, full-scale mechanical analyses are employed to derive the global bending rigidity of HIV-1 liposomes. Finally, dynamical properties of the lipid molecules reveal important relationships between their 3D diffusion, the location of lipid-rafts and the asymmetrical composition of the envelope. Overall, our simulations reveal complex relationships between the rich lipid composition of the HIV-1 liposome and its structural, mechanical and dynamical properties with critical consequences to different stages of HIV-1's life cycle.

Humpback whales (Megaptera novaeangliae) attend both Mexico and Hawaii breeding grounds in the same winter: mixing in the northeast Pacific.

Humpback whales that assemble on winter breeding grounds in Mexico and Hawaii have been presumed to be, at least, seasonally isolated. Recently, these assemblies were declared Distinct Population Segments under the US Endangered Species Act. We report two humpback whales attending both breeding grounds in the same season-one moving from Hawaii to Mexico and the other from Mexico to Hawaii. The first was photo-identified in Maui, Hawaii on 23 February 2006 and again, after 53 days and 4545 km, on 17 April 2006 in the Revillagigedo Archipelago, Mexico. The second was photo-identified off Guerrero, Mexico on 16 February 2018 and again, 49 days and 5944 km later, on 6 April 2018 off Maui. The 2006 whale was identified in summer off Kodiak Island, Alaska; the 2018 whale off British Columbia. These Mexico-Hawaii identifications provide definitive evidence that whales in these two winter assemblies may mix during one winter season. This, combined with other lines of evidence on Mexico-Hawaii mixing, including interchange of individuals year to year, long-term similarity of everchanging songs, one earlier same-season travel record, and detection of humpback whales mid-ocean between these locations in winter, suggests reassessment of the 'distinctiveness' of these populations may be warranted.

Cholesterol Dependent Activity of the Adenosine A2A Receptor Is Modulated via the Cholesterol Consensus Motif.

BACKGROUND: Membrane cholesterol dysregulation has been shown to alter the activity of the adenosine A2A receptor (A2AR), a G protein-coupled receptor, thereby implicating cholesterol levels in diseases such as Alzheimer's and Parkinson's. A limited number of A2AR crystal structures show the receptor interacting with cholesterol, as such molecular simulations are often used to predict cholesterol interaction sites. METHODS: Here, we use experimental methods to determine whether a specific interaction between amino acid side chains in the cholesterol consensus motif (CCM) of full length, wild-type human A2AR, and cholesterol modulates activity of the receptor by testing the effects of mutational changes on functional consequences, including ligand binding, G protein coupling, and downstream activation of cyclic AMP. RESULTS AND CONCLUSIONS: Our data, taken with previously published studies, support a model of receptor state-dependent binding between cholesterol and the CCM, whereby cholesterol facilitates both G protein coupling and downstream signaling of A2AR.

Activation of G-protein-coupled receptors is thermodynamically linked to lipid solvation.

Preferential lipid solvation of the G-protein-coupled A2A adenosine receptor (A2AR) is evaluated from 35 µs of all-atom molecular dynamics simulation. A coarse-grained transition matrix algorithm is developed to overcome slow equilibration of the first solvation shell, obtaining estimates of the free energy of solvation by different lipids for the receptor in different activation states. Results indicate preference for solvation by unsaturated chains, which favors the active receptor. A model for lipid-dependent G-protein-coupled receptor activity is proposed in which the chemical potential of lipids in the bulk membrane modulates receptor activity. The entropies associated with moving saturated and unsaturated lipids from bulk to A2AR's first solvation shell are evaluated. Overall, the acyl chains are more disordered (i.e., obtain a favorable entropic contribution) when partitioning to the receptor surface, and this effect is augmented for the saturated chains, which are relatively more ordered in bulk.

Changes in pairwise correlations during running reshape global network state in the main olfactory bulb.

Neural codes for sensory inputs have been hypothesized to reside in a broader space defined by ongoing patterns of spontaneous activity. To understand the structure of this spontaneous activity in the olfactory system, we performed high-density recordings of neural populations in the main olfactory bulb of awake mice. We observed changes in pairwise correlations of spontaneous activity between mitral and tufted (M/T) cells when animals were running, which resulted in an increase in the entropy of the population. Surprisingly, pairwise maximum entropy models that described the population activity using only assumptions about the firing rates and correlations of neurons were better at predicting the global structure of activity when animals were stationary as compared to when they were running, implying that higher order (3rd, 4th order) interactions governed population activity during locomotion. Taken together, we found that locomotion alters the functional interactions that shape spontaneous population activity at the earliest stages of olfactory processing, one synapse away from the sensory receptors in the nasal epithelium. These data suggest that the coding space available for sensory representations responds adaptively to the animal's behavioral state.NEW & NOTEWORTHY The organization and structure of spontaneous population activity in the olfactory system places constraints of how odor information is represented. Using high-density electrophysiological recordings of mitral and tufted cells, we found that running increases the dimensionality of spontaneous activity, implicating higher order interactions among neurons during locomotion. Behavior, thus, flexibly alters neuronal activity at the earliest stages of sensory processing.

Nanoscale dynamics of cholesterol in the cell membrane.

Cholesterol constitutes ∼30-40% of the mammalian plasma membrane, a larger fraction than of any other single component. It is a major player in numerous signaling processes as well as in shaping molecular membrane architecture. However, our knowledge of the dynamics of cholesterol in the plasma membrane is limited, restricting our understanding of the mechanisms regulating its involvement in cell signaling. Here, we applied advanced fluorescence imaging and spectroscopy approaches on in vitro (model membranes) and in vivo (live cells and embryos) membranes as well as in silico analysis to systematically study the nanoscale dynamics of cholesterol in biological membranes. Our results indicate that cholesterol diffuses faster than phospholipids in live membranes, but not in model membranes. Interestingly, a detailed statistical diffusion analysis suggested two-component diffusion for cholesterol in the plasma membrane of live cells. One of these components was similar to a freely diffusing phospholipid analogue, whereas the other one was significantly faster. When a cholesterol analogue was localized to the outer leaflet only, the fast diffusion of cholesterol disappeared, and it diffused similarly to phospholipids. Overall, our results suggest that cholesterol diffusion in the cell membrane is heterogeneous and that this diffusional heterogeneity is due to cholesterol's nanoscale interactions and localization in the membrane.

Local Enrichment of Unsaturated Chains around the A2A Adenosine Receptor.

Two 15 µs all-atom simulations of the A2A adenosine receptor were obtained in a ternary mixture of cholesterol, saturated phosphatidylcholine lipids, and unsaturated phosphatidylcholine lipids. An analysis of local lipid solvation is reported on the basis of a Voronoi tessellation of the upper and lower leaflets, identifying first and second solvation shells. The local environments of both the inactive state and the partially active state of the receptor are significantly enriched with unsaturated chains but depleted of cholesterol and saturated chains, relative to the bulk membrane composition. In spite of the local depletion of cholesterol, the partially active receptor binds cholesterol at three locations during the entire simulation trajectory. These long-lived interactions represent the extreme of a very broad distribution of first-solvation shell lipid lifetimes, confounding sharp distinctions between lipid interactions. The broad distributions of lifetimes also make equilibrating the local lipid environment difficult, necessitating long simulation times.

From Dynamics to Membrane Organization: Experimental Breakthroughs Occasion a "Modeling Manifesto".

New experimental techniques, especially in the context of observing molecular dynamics, reveal the plasma membrane to be heterogeneous and "scale rich," from nanometers to microns and from microseconds to seconds. This is critical information, which shows that scale-dependent transport governs the molecular encounters that underlie cellular signaling. The data are rich and reaffirm the importance of the cortical cytoskeleton, protein aggregates, and lipidomic complexity on the statistics of molecular encounters. Moreover, the data demand simulation approaches with a particular set of features, hence the "manifesto." Together with the experimental data, simulations that satisfy these requirements hold the promise of a deeper understanding of membrane spatiotemporal organization. Several experimental breakthroughs in measuring molecular membrane dynamics are reviewed, the constraints that they place on simulations are discussed, and the status of simulation approaches that aim to meet them are detailed.

Identification of Two New Cholesterol Interaction Sites on the A2A Adenosine Receptor.

By mole, cholesterol is the most abundant component of animal cell plasma membranes. Many membrane proteins have been shown to be functionally dependent on cholesterol, several of which have also been shown to bind cholesterol at well-defined locations on their membrane-facing surface. In this work, a combination of coarse-grained "Martini" and all-atom simulations are used to identify two, to our knowledge, new cholesterol-binding sites on the A2A adenosine receptor, a G-protein-coupled receptor that is a target for the treatment of Parkinson's disease. One of the sites is also observed to bind cholesterol in several recent, high-resolution crystal structures of the protein, and in the simulations, interacts with cholesterol only when bound to the inverse agonist ZM241385. Cataloguing cholesterol-binding sites is a vital step in the effort to understand cholesterol-dependent function of membrane proteins. Given that cholesterol content in plasma membranes varies with cell type and on administration of widely prescribed pharmaceuticals, such as statins, understanding cholesterol-dependent function is an important step toward exploiting membrane compositional variation for therapeutic purposes.

Toward Hydrodynamics with Solvent Free Lipid Models: STRD Martini.

Solvent hydrodynamics are incorporated into simulations of the solvent-free Dry Martini model. The solvent hydrodynamics are modeled with the stochastic rotation dynamics (SRD) algorithm, a particle-based method for resolving fluid hydrodynamics. SRD does not require calculation of particle-particle distances in the solvent, and so is scalable to arbitrary volumes of solvent with minimal additional computational overhead. The viscosity of the solvent is easily tuned via parameters of the algorithm to span an order of magnitude in viscosity around the viscosity of water at room temperature. The combination "Stochastic Thermostatted Rotation Dynamics (STRD) with Martini" was implemented in Gromacs v.5.01. Simulations of an SRD/palmitoyloleoylphosphatidylcholine membrane demonstrate that the solvent may be included without reparametrizing the lipid model, with minimal perturbation to the thermodynamics. A recent generalization of Saffman-Delbruck theory to periodic geometries by Camley and Brown indicates that lipid dynamics are contaminated by a finite-size effect in typical molecular dynamics (MD) simulations, and that very large systems are required for quantitative simulation of dynamics. Analysis of lipid translational diffusion in this work shows good agreement with the theory, and with explicitly solvated simulations. This indicates that STRD Martini is a viable approach for quantitative simulation of membrane dynamics and does not require massive computational overhead to model the solvent.

Hexagonal Substructure and Hydrogen Bonding in Liquid-Ordered Phases Containing Palmitoyl Sphingomyelin.

All-atom simulation data are presented for ternary mixtures of palmitoyl sphingomyelin (PSM), cholesterol, and either palmitoyl oleoyl phosphatidyl choline or dioleoyl phosphatidyl choline (DOPC). For comparison, data for a mixture of dipalmitoyl phosphatidyl choline (DPPC), cholesterol, and DOPC are also presented. Compositions corresponding to the liquid-ordered phase, the liquid-disordered phase, and coexistence of the two phases are simulated for each mixture. Within the liquid-ordered phase, cholesterol is preferentially solvated by DOPC if it is available, but if DOPC is replaced by POPC, cholesterol is preferentially solvated by PSM. In the DPPC mixtures, cholesterol interacts preferentially with the saturated chains via its smooth face, whereas in the PSM mixtures, cholesterol interacts preferentially with PSM via its rough face. Interactions between cholesterol and PSM have a very particular character: hydrogen bonding between cholesterol and the amide of PSM rotates the tilt of the amide plane, which primes it for more robust hydrogen bonding with other PSM. Cholesterol-PSM hydrogen bonding also locally modifies the hexagonal packing of hydrocarbon chains in the liquid-ordered phase of PSM mixtures.

The molecular structure of the liquid-ordered phase of lipid bilayers.

Molecular dynamics simulations reveal substructures within the liquid-ordered phase of lipid bilayers. These substructures, identified in a 10 µs all-atom trajectory of liquid-ordered/liquid-disordered coexistence (L(o)/L(d)) are composed of saturated hydrocarbon chains packed with local hexagonal order and separated by interstitial regions enriched in cholesterol and unsaturated chains. Lipid hydrocarbon chain order parameters calculated from the L(o) phase are in excellent agreement with (2)H NMR measurements; the local hexagonal packing is also consistent with (1)H-MAS NMR spectra of the L(o) phase, NMR diffusion experiments, and small-angle X-ray and neutron scattering. The balance of cholesterol-rich to local hexagonal order is proposed to control the partitioning of membrane components into the L(o) regions. The latter have been frequently associated with formation of so-called rafts, platforms in the plasma membranes of cells that facilitate interaction between components of signaling pathways.

Varying the position of phospholipid acyl chain unsaturation modulates hopanoid and sterol ordering.

The cell membrane must balance mechanical stability with fluidity to function as both a barrier and an organizational platform. Key to this balance is the thermodynamic ordering of lipids. Most Eukaryotes employ sterols, which are uniquely capable of modulating lipid order to decouple membrane stability from fluidity. Ancient sterol analogues known as hopanoids are found in many bacteria and are proposed as ancestral ordering lipids. The juxtaposition of sterols and hopanoids in extant organisms prompts us to ask why both pathways persist, especially in light of their convergent ability to order lipids. We reveal that both hopanoids and sterols order unsaturated phospholipids differently based on the position of double bonds in the phospholipid's acyl chain. We find that cholesterol and diplopterol's methyl group distributions lead to distinct effects on unsaturated lipids. In Mesoplasma florum, diplopterol's constrained ordering capacity reduces membrane resistance to osmotic stress, unlike cholesterol. These findings suggest cholesterol's broader lipid ordering ability may have facilitated the exploration of a more diverse lipidomic landscape in eukaryotic membranes.

Membrane mimetic-dependence of GPCR energy landscapes.

We leveraged variable-temperature 19F-NMR spectroscopy to compare the conformational equilibria of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor (GPCR), across a range of temperatures ranging from lower temperatures typically employed in 19F-NMR experiments to physiological temperature. A2AAR complexes with partial agonists and full agonists showed large increases in the population of a fully active conformation with increasing temperature. NMR data measured at physiological temperature were more in line with functional data. This was pronounced for complexes with partial agonists, where the population of active A2AAR was nearly undetectable at lower temperature but became evident at physiological temperature. Temperature-dependent behavior of complexes with either full or partial agonists exhibited a pronounced sensitivity to the specific membrane mimetic employed. Cellular signaling experiments correlated with the temperature-dependent conformational equilibria of A2AAR in lipid nanodiscs but not in some detergents, underscoring the importance of the membrane environment in studies of GPCR function.

Homeocurvature adaptation of phospholipids to pressure in deep-sea invertebrates.

Hydrostatic pressure increases with depth in the ocean, but little is known about the molecular bases of biological pressure tolerance. We describe a mode of pressure adaptation in comb jellies (ctenophores) that also constrains these animals' depth range. Structural analysis of deep-sea ctenophore lipids shows that they form a nonbilayer phase at pressures under which the phase is not typically stable. Lipidomics and all-atom simulations identified phospholipids with strong negative spontaneous curvature, including plasmalogens, as a hallmark of deep-adapted membranes that causes this phase behavior. Synthesis of plasmalogens enhanced pressure tolerance in Escherichia coli, whereas low-curvature lipids had the opposite effect. Imaging of ctenophore tissues indicated that the disintegration of deep-sea animals when decompressed could be driven by a phase transition in their phospholipid membranes.

Bellwethers of change: population modelling of North Pacific humpback whales from 2002 through 2021 reveals shift from recovery to climate response.

For the 40 years after the end of commercial whaling in 1976, humpback whale populations in the North Pacific Ocean exhibited a prolonged period of recovery. Using mark-recapture methods on the largest individual photo-identification dataset ever assembled for a cetacean, we estimated annual ocean-basin-wide abundance for the species from 2002 through 2021. Trends in annual estimates describe strong post-whaling era population recovery from 16 875 (± 5955) in 2002 to a peak abundance estimate of 33 488 (± 4455) in 2012. An apparent 20% decline from 2012 to 2021, 33 488 (± 4455) to 26 662 (± 4192), suggests the population abruptly reached carrying capacity due to loss of prey resources. This was particularly evident for humpback whales wintering in Hawai'i, where, by 2021, estimated abundance had declined by 34% from a peak in 2013, down to abundance levels previously seen in 2006, and contrasted to an absence of decline in Mainland Mexico breeding humpbacks. The strongest marine heatwave recorded globally to date during the 2014-2016 period appeared to have altered the course of species recovery, with enduring effects. Extending this time series will allow humpback whales to serve as an indicator species for the ecosystem in the face of a changing climate.