Cryo-EM images of phase-separated lipid bilayer vesicles analyzed with a machine-learning approach.

Lateral lipid heterogeneity (i.e., raft formation) in biomembranes plays a functional role in living cells. Three-component mixtures of low- and high-melting lipids plus cholesterol offer a simplified experimental model for raft domains in which a liquid-disordered (Ld) phase coexists with a liquid-ordered (Lo) phase. Using such models, we recently showed that cryogenic electron microscopy (cryo-EM) can detect phase separation in lipid vesicles based on differences in bilayer thickness. However, the considerable noise within cryo-EM data poses a significant challenge for accurately determining the membrane phase state at high spatial resolution. To this end, we have developed an image-processing pipeline that utilizes machine learning (ML) to predict the bilayer phase in projection images of lipid vesicles. Importantly, the ML method exploits differences in both the thickness and molecular density of Lo compared to Ld, which leads to improved phase identification. To assess accuracy, we used artificial images of phase-separated lipid vesicles generated from all-atom molecular dynamics simulations of Lo and Ld phases. Synthetic ground-truth data sets mimicking a series of compositions along a tieline of Ld + Lo coexistence were created and then analyzed with various ML models. For all tieline compositions, we find that the ML approach can correctly identify the bilayer phase with >90% accuracy, thus providing a means to isolate the intensity profiles of coexisting Ld and Lo phases, as well as accurately determine domain-size distributions, number of domains, and phase-area fractions. The method described here provides a framework for characterizing nanoscopic lateral heterogeneities in membranes and paves the way for a more detailed understanding of raft properties in biological contexts.

Seeing the Membrane from Both Sides Now: Lipid Asymmetry and Its Strange Consequences.

Almost all biomembranes are constructed as lipid bilayers and, in almost all of these, the two opposing monolayers (leaflets) have distinct lipid compositions. This lipid asymmetry arises through the concerted action of a suite of energy-dependent enzymes that maintain living bilayers in a far-from-equilibrium steady-state. Recent discoveries reveal that lipid compositional asymmetry imparts biophysical asymmetries and that this dualistic organization may have major consequences for cellular physiology. Importantly, while transbilayer asymmetry appears to be an essential, near-ubiquitous characteristic of biological membranes, it has been challenging to reproduce in reconstituted or synthetic systems. Although recent methodological developments have overcome some critical challenges, it remains difficult to extrapolate results from available models to biological systems. Concurrently, there are few experimental approaches for targeted, controlled manipulation of lipid asymmetry in living cells. Thus, the biophysical and functional consequences of membrane asymmetry remain almost wholly unexplored. This perspective summarizes the current state of knowledge and highlights emerging themes that are beginning to make inroads into the fundamental question of why life tends toward asymmetry in its bilayers.

Building Asymmetric Lipid Bilayers for Molecular Dynamics Simulations: What Methods Exist and How to Choose One?

The compositional asymmetry of biological membranes has attracted significant attention over the last decade. Harboring more differences from symmetric membranes than previously appreciated, asymmetric bilayers have proven quite challenging to study with familiar concepts and techniques, leaving many unanswered questions about the reach of the asymmetry effects. One particular area of active research is the computational investigation of composition- and number-asymmetric lipid bilayers with molecular dynamics (MD) simulations. Offering a high level of detail into the organization and properties of the simulated systems, MD has emerged as an indispensable tool in the study of membrane asymmetry. However, the realization that results depend heavily on the protocol used for constructing the asymmetric bilayer models has sparked an ongoing debate about how to choose the most appropriate approach. Here we discuss the underlying source of the discrepant results and review the existing methods for creating asymmetric bilayers for MD simulations. Considering the available data, we argue that each method is well suited for specific applications and hence there is no single best approach. Instead, the choice of a construction protocol-and consequently, its perceived accuracy-must be based primarily on the scientific question that the simulations are designed to address.

Sampling Bottleneck in Validating Membrane Dynamics.

Molecular dynamics (MD) simulations have become increasingly impactful in membrane biophysics because they offer atomistic resolution into the atomistic fluctuations of lipid assemblies. Validation of the simulation trajectories with experimental data is crucial for interpretation and application of MD results. As an ideal benchmarking technique, NMR spectroscopy delivers order parameters of the carbon-deuterium bond fluctuations along the lipid chains. Additionally, NMR relaxation can access lipid dynamics providing yet another point for validation of simulation force fields. Here we performed short resampling simulations of membrane trajectories to investigate the lipid CH bond fluctuations on sub-40-ps timescales to explore the local fast dynamics. We recently established a robust framework for analysis of NMR relaxation rates from MD simulations, which improves upon current approaches and shows excellent agreement of experimental and theoretical results. The calculation of relaxation rates from simulations presents a universal challenge that we addressed by hypothesizing the existence of fast CH bond dynamics that evade the analysis of simulation data with temporal resolution of 40 ps (or lower). Indeed, our results support this hypothesis confirming the validity of our solution to the sampling problem. Furthermore, we show that the fast CH bond dynamics occur on timescales at which carbon-carbon bond conformations appear nearly stationary and unaffected by cholesterol. Lastly, we discuss the correspondence to the CH bond dynamics of liquid hydrocarbons and relate their existence to the apparent microviscosity of the bilayer hydrocarbon core.

Molecular simulations and NMR reveal how lipid fluctuations affect membrane mechanics.

Lipid bilayers form the main matrix of functional cell membranes, and their dynamics underlie a host of physical and biological processes. Here we show that elastic membrane properties and collective molecular dynamics (MD) are related by the mean-square amplitudes (order parameters) and relaxation rates (correlation times) of lipid acyl chain motions. We performed all-atom MD simulations of liquid-crystalline bilayers that allow direct comparison with carbon-hydrogen (CH) bond relaxations measured with NMR spectroscopy. Previous computational and theoretical approaches have assumed isotropic relaxation, which yields inaccurate description of lipid chain dynamics and incorrect data interpretation. Instead, the new framework includes a fixed bilayer normal (director axis) and restricted anisotropic motion of the CH bonds in accord with their segmental order parameters, enabling robust validation of lipid force fields. Simulated spectral densities of thermally excited CH bond fluctuations exhibited well-defined spin-lattice (Zeeman) relaxations analogous to those in NMR measurements. Their frequency signature could be fit to a simple power-law function, indicative of nematic-like collective dynamics. Moreover, calculated relaxation rates scaled as the squared order parameters yielding an apparent κC modulus for bilayer bending. Our results show a strong correlation with κC values obtained from solid-state NMR studies of bilayers without and with cholesterol as validated by neutron spin-echo measurements of membrane elasticity. The simulations uncover a critical role of interleaflet coupling in membrane mechanics and thus provide important insights into molecular sites of emerging elastic properties within lipid bilayers.

Probing the Link between Pancratistatin and Mitochondrial Apoptosis through Changes in the Membrane Dynamics on the Nanoscale.

Pancratistatin (PST) is a natural antiviral alkaloid that has demonstrated specificity toward cancerous cells and explicitly targets the mitochondria. PST initiates apoptosis while leaving healthy, noncancerous cells unscathed. However, the manner by which PST induces apoptosis remains elusive and impedes the advancement of PST as a natural anticancer therapeutic agent. Herein, we use neutron spin-echo (NSE) spectroscopy, molecular dynamics (MD) simulations, and supporting small angle scattering techniques to study PST's effect on membrane dynamics using biologically representative model membranes. Our data suggests that PST stiffens the inner mitochondrial membrane (IMM) by being preferentially associated with cardiolipin, which would lead to the relocation and release of cytochrome c. Second, PST has an ordering effect on the lipids and disrupts their distribution within the IMM, which would interfere with the maintenance and functionality of the active forms of proteins in the electron transport chain. These previously unreported findings implicate PST's effect on mitochondrial apoptosis.

Vesicle Viewer: Online visualization and analysis of small-angle scattering from lipid vesicles.

Small-angle X-ray and neutron scattering are among the most powerful experimental techniques for investigating the structure of biological membranes. Much of the critical information contained in small-angle scattering (SAS) data is not easily accessible to researchers who have limited time to analyze results by hand or to nonexperts who may lack the necessary scientific background to process such data. Easy-to-use data visualization software can allow them to take full advantage of their SAS data and maximize the use of limited resources. To this end, we developed an internet-based application called Vesicle Viewer to visualize and analyze SAS data from unilamellar lipid bilayer vesicles. Vesicle Viewer utilizes a modified scattering density profile (SDP) analysis called EZ-SDP in which key bilayer structural parameters, such as area per lipid and bilayer thickness, are easily and robustly determined. Notably, we introduce a bilayer model that is able to describe an asymmetric bilayer, whether it be chemically or isotopically asymmetric. The application primarily uses Django, a Python package specialized for the development of robust web applications. In addition, several other libraries are used to support the more technical aspects of the project; notable examples are Matplotlib (for graphs) and NumPy (for calculations). By eliminating the barrier of downloading and installing software, this web-based application will allow scientists to analyze their own vesicle scattering data using their preferred operating system. The web-based application can be found at https://vesicleviewer.dmarquardt.ca/.

Ca2+-dependent mechanism of membrane insertion and destabilization by the SARS-CoV-2 fusion peptide.

Cell penetration after recognition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus by the ACE2 receptor and the fusion of its viral envelope membrane with cellular membranes are the early steps of infectivity. A region of the Spike protein of the virus, identified as the "fusion peptide" (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor-binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca2+ ions to the FPs of coronaviruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca2+ binding to the SARS-CoV-2-FP, the role of Ca2+ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca2+-bound SARS-CoV-2-FP, and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca2+-bound peptide models with lipid membranes, SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif, which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca2+ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca2+ ions. These findings provide novel mechanistic insights regarding the role of Ca2+ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry.

Model Membrane Systems Used to Study Plasma Membrane Lipid Asymmetry.

It is well known that the lipid distribution in the bilayer leaflets of mammalian plasma membranes (PMs) is not symmetric. Despite this, model membrane studies have largely relied on chemically symmetric model membranes for the study of lipid-lipid and lipid-protein interactions. This is primarily due to the difficulty in preparing stable, asymmetric model membranes that are amenable to biophysical studies. However, in the last 20 years, efforts have been made in producing more biologically faithful model membranes. Here, we review several recently developed experimental and computational techniques for the robust generation of asymmetric model membranes and highlight a new and particularly promising technique to study membrane asymmetry.

Structure and Interdigitation of Chain-Asymmetric Phosphatidylcholines and Milk Sphingomyelin in the Fluid Phase.

We addressed the frequent occurrence of mixed-chain lipids in biological membranes and their impact on membrane structure by studying several chain-asymmetric phosphatidylcholines and the highly asymmetric milk sphingomyelin. Specifically, we report trans-membrane structures of the corresponding fluid lamellar phases using small-angle X-ray and neutron scattering, which were jointly analyzed in terms of a membrane composition-specific model, including a headgroup hydration shell. Focusing on terminal methyl groups at the bilayer center, we found a linear relation between hydrocarbon chain length mismatch and the methyl-overlap for phosphatidylcholines, and a non-negligible impact of the glycerol backbone-tilting, letting the sn1-chain penetrate deeper into the opposing leaflet by half a CH2 group. That is, penetration-depth differences due to the ester-linked hydrocarbons at the glycerol backbone, previously reported for gel phase structures, also extend to the more relevant physiological fluid phase, but are significantly reduced. Moreover, milk sphingomyelin was found to follow the same linear relationship suggesting a similar tilt of the sphingosine backbone. Complementarily performed molecular dynamics simulations revealed that there is always a part of the lipid tails bending back, even if there is a high interdigitation with the opposing chains. The extent of this back-bending was similar to that in chain symmetric bilayers. For both cases of adaptation to chain length mismatch, chain-asymmetry has a large impact on hydrocarbon chain ordering, inducing disorder in the longer of the two hydrocarbons.

Ca 2+ -dependent mechanism of membrane insertion and destabilization by the SARS-CoV-2 fusion peptide.

Cell penetration after recognition of the SARS-CoV-2 virus by the ACE2 receptor, and the fusion of its viral envelope membrane with cellular membranes, are the early steps of infectivity. A region of the Spike protein (S) of the virus, identified as the "fusion peptide" (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca 2+ ions to the FPs of corona viruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca 2+ binding to the SARS-CoV-2-FP, the role of Ca 2+ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca 2+ -bound SARS-CoV-2-FP and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics (MD) simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca 2+ -bound peptide models with lipid membranes SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca 2+ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca 2+ ions. These findings provide novel mechanistic insights regarding the role of Ca 2+ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry. STATEMENT OF SIGNIFICANCE: SARS-CoV-2, the cause of the COVID-19 pandemic, penetrates host cell membranes and uses viral-to-cellular membrane fusion to release its genetic material for replication. Experiments had identified a region termed "fusion peptide" (FP) in the Spike proteins of coronaviruses, as the spearhead in these initial processes, and suggested that Ca 2+ is needed to support both functions. Absent structure and dynamics-based mechanistic information these FP functions could not be targeted for therapeutic interventions. We describe the development and determination of the missing information from analysis of extensive MD simulation trajectories, and propose specific Ca 2+ -dependent mechanisms of SARS-CoV-2-FP membrane insertion and destabilization. These results offer a structure-specific platform to aid the ongoing efforts to use this target for the discovery and/or of inhibitors.

Structural and functional consequences of reversible lipid asymmetry in living membranes.

Maintenance of lipid asymmetry across the two leaflets of the plasma membrane (PM) bilayer is a ubiquitous feature of eukaryotic cells. Loss of this asymmetry has been widely associated with cell death. However, increasing evidence points to the physiological importance of non-apoptotic, transient changes in PM asymmetry. Such transient scrambling events are associated with a range of biological functions, including intercellular communication and intracellular signaling. Thus, regulation of interleaflet lipid distribution in the PM is a broadly important but underappreciated cellular process with key physiological and structural consequences. Here, we compile the mounting evidence revealing multifaceted, functional roles of PM asymmetry and transient loss thereof. We discuss the consequences of reversible asymmetry on PM structure, biophysical properties and interleaflet coupling. We argue that despite widespread recognition of broad aspects of membrane asymmetry, its importance in cell biology demands more in-depth investigation of its features, regulation, and physiological and pathological implications.

How cholesterol stiffens unsaturated lipid membranes.

Cholesterol is an integral component of eukaryotic cell membranes and a key molecule in controlling membrane fluidity, organization, and other physicochemical parameters. It also plays a regulatory function in antibiotic drug resistance and the immune response of cells against viruses, by stabilizing the membrane against structural damage. While it is well understood that, structurally, cholesterol exhibits a densification effect on fluid lipid membranes, its effects on membrane bending rigidity are assumed to be nonuniversal; i.e., cholesterol stiffens saturated lipid membranes, but has no stiffening effect on membranes populated by unsaturated lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). This observation presents a clear challenge to structure-property relationships and to our understanding of cholesterol-mediated biological functions. Here, using a comprehensive approach-combining neutron spin-echo (NSE) spectroscopy, solid-state deuterium NMR (2H NMR) spectroscopy, and molecular dynamics (MD) simulations-we report that cholesterol locally increases the bending rigidity of DOPC membranes, similar to saturated membranes, by increasing the bilayer's packing density. All three techniques, inherently sensitive to mesoscale bending fluctuations, show up to a threefold increase in effective bending rigidity with increasing cholesterol content approaching a mole fraction of 50%. Our observations are in good agreement with the known effects of cholesterol on the area-compressibility modulus and membrane structure, reaffirming membrane structure-property relationships. The current findings point to a scale-dependent manifestation of membrane properties, highlighting the need to reassess cholesterol's role in controlling membrane bending rigidity over mesoscopic length and time scales of important biological functions, such as viral budding and lipid-protein interactions.

Direct label-free imaging of nanodomains in biomimetic and biological membranes by cryogenic electron microscopy.

The nanoscale organization of biological membranes into structurally and compositionally distinct lateral domains is believed to be central to membrane function. The nature of this organization has remained elusive due to a lack of methods to directly probe nanoscopic membrane features. We show here that cryogenic electron microscopy (cryo-EM) can be used to directly image coexisting nanoscopic domains in synthetic and bioderived membranes without extrinsic probes. Analyzing a series of single-component liposomes composed of synthetic lipids of varying chain lengths, we demonstrate that cryo-EM can distinguish bilayer thickness differences as small as 0.5 Å, comparable to the resolution of small-angle scattering methods. Simulated images from computational models reveal that features in cryo-EM images result from a complex interplay between the atomic distribution normal to the plane of the bilayer and imaging parameters. Simulations of phase-separated bilayers were used to predict two sources of contrast between coexisting ordered and disordered phases within a single liposome, namely differences in membrane thickness and molecular density. We observe both sources of contrast in biomimetic membranes composed of saturated lipids, unsaturated lipids, and cholesterol. When extended to isolated mammalian plasma membranes, cryo-EM reveals similar nanoscale lateral heterogeneities. The methods reported here for direct, probe-free imaging of nanodomains in unperturbed membranes open new avenues for investigation of nanoscopic membrane organization.

Molecular Structure of Sphingomyelin in Fluid Phase Bilayers Determined by the Joint Analysis of Small-Angle Neutron and X-ray Scattering Data.

We have determined the fluid bilayer structure of palmitoyl sphingomyelin (PSM) and stearoyl sphingomyelin (SSM) by simultaneously analyzing small-angle neutron and X-ray scattering data. Using a newly developed scattering density profile (SDP) model for sphingomyelin lipids, we report structural parameters including the area per lipid, total bilayer thickness, and hydrocarbon thickness, in addition to lipid volumes determined by densitometry. Unconstrained all-atom simulations of PSM bilayers at 55 °C using the C36 CHARMM force field produced a lipid area of 56 Å2, a value that is 10% lower than the one determined experimentally by SDP analysis (61.9 Å2). Furthermore, scattering form factors calculated from the unconstrained simulations were in poor agreement with experimental form factors, even though segmental order parameter (SCD) profiles calculated from the simulations were in relatively good agreement with SCD profiles obtained from NMR experiments. Conversely, constrained area simulations at 61.9 Å2 resulted in good agreement between the simulation and experimental scattering form factors, but not with SCD profiles from NMR. We discuss possible reasons for the discrepancies between these two types of data that are frequently used as validation metrics for molecular dynamics force fields.

Regulation of lipid saturation without sensing membrane fluidity.

Cells maintain membrane fluidity by regulating lipid saturation, but the molecular mechanisms of this homeoviscous adaptation remain poorly understood. We have reconstituted the core machinery for regulating lipid saturation in baker's yeast to study its molecular mechanism. By combining molecular dynamics simulations with experiments, we uncover a remarkable sensitivity of the transcriptional regulator Mga2 to the abundance, position, and configuration of double bonds in lipid acyl chains, and provide insights into the molecular rules of membrane adaptation. Our data challenge the prevailing hypothesis that membrane fluidity serves as the measured variable for regulating lipid saturation. Rather, we show that Mga2 senses the molecular lipid-packing density in a defined region of the membrane. Our findings suggest that membrane property sensors have evolved remarkable sensitivities to highly specific aspects of membrane structure and dynamics, thus paving the way toward the development of genetically encoded reporters for such properties in the future.