Minimal model of plasma membrane heterogeneity requires coupling cortical actin to criticality

We present a minimal model of plasma membrane heterogeneity that combines criticality with connectivity to cortical cytoskeleton. The development of this model was motivated by recent observations of micron-sized critical fluctuations in plasma membrane vesicles that are detached from their cortical cytoskeleton. We incorporate criticality using a conserved order parameter Ising model coupled to a simple actin cytoskeleton interacting through point-like pinning sites. Using this minimal model, we recapitulate several experimental observations of plasma membrane raft heterogeneity. Small (r ∼ 20 nm) and dynamic fluctuations at physiological temperatures arise from criticality. Including connectivity to the cortical cytoskeleton disrupts large fluctuations, prevents macroscopic phase separation at low temperatures (T ≤ 22°C), and provides a template for long-lived fluctuations at physiological temperature (T = 37°C). Cytoskeleton-stabilized fluctuations produce significant barriers to the diffusion of some membrane components in a manner that is weakly dependent on the number of pinning sites and strongly dependent on criticality. More generally, we demonstrate that critical fluctuations provide a physical mechanism for organizing and spatially segregating membrane components by providing channels for interaction over large distances.     

Criticality in cell differentiation

Cell differentiation is an important process in living organisms. Differentiation is mostly based on binary decisions with the progenitor cells choosing between two specific lineages. The differentiation dynamics have both deterministic and stochastic components. Several theoretical studies suggest that cell differentiation is a bifurcation phenomenon, well-known in dynamical systems theory. The bifurcation point has the character of a critical point with the system dynamics exhibiting specific features in its vicinity. These include the critical slowing down, rising variance and lag-1 autocorrelation function, strong correlations between the fluctuations of key variables and non-Gaussianity in the distribution of fluctuations. Recent experimental studies provide considerable support to the idea of criticality in cell differentiation and in other biological processes like the development of the fruit fly embryo. In this review, an elementary introduction is given to the concept of criticality in cell differentiation. The correspondence between the signatures of criticality and experimental observations on blood cell differentiation in mice is further highlighted.     

Computer simulations of lipid regulation by molecular semigrand canonical ensembles

The plasma membrane is the interface between cells and exterior media. Although its existence has been known for a long time, organization of its constituent lipids remain a challenge. Recently, we have proposed that lipid populations may be controlled by chemical potentials of different lipid species, resulting in semigrand canonical thermodynamic ensembles. However, the currently available molecular dynamics software packages do not facilitate the control of chemical potentials at the molecular level. Here, we propose a variation of existing algorithms that efficiently characterizes and controls the chemical nature of each lipid. Additionally, we allow coupling with collective variables and show that it can be used to dynamically create asymmetric membranes. This algorithm is openly available as a plugin for the HOOMD-Blue molecular dynamics engine.     

Destabilization of a lipid non-bilayer phase by high pressure

Pressure is found to destabilize the non-bilayer phase with respect to the bilayer in a model lipid system. The lamellar to inverted hexagonal (H₁₁) phase transition of aqueous egg phosphatidylethanolamine is shifted to higher temperatures by hydrostatic pressure. The slope of the increase in transition temperature is constant to beyond 300 bar, and is greater than that seen for other lipid phase transitions. This behavior is consistent with the hypothesis that increasing chain disorder drives the conversion from the bilayer into the hexagonal phase. If this non-bilayer lipid phase is an intermediate in membrane fusion, then pressure should inhibit the process. This may explain the inhibition of chemical transmission at neural synapses by pressure.     

Phase behavior of lipid bilayers under tension

Given the proposed importance of membrane tension in regulating cellular functions, we explore the effects of a finite surface tension on phase equilibrium using a molecular theory that captures the quantitative structure of the phase diagram of the tensionless DPPC/DOPC/Cholesterol lipid bilayer. We find that an increase in the surface tension decreases the temperature of the transition from liquid to gel in a pure DPPC system by ～1.0 K/(mN/m), and decreases the liquid-disordered to liquid-ordered transition at constant chemical potentials by approximately the same amount. Our results quantitatively isolate the role of tension in comparison to other thermodynamic factors, such as pressure, in determining the phase behavior of lipid bilayers.     

Direct Visualization of the Action of Triton X-100 on Giant Vesicles of Erythrocyte Membrane Lipids

The raft hypothesis proposes that microdomains enriched in sphingolipids, cholesterol, and specific proteins are transiently formed to accomplish important cellular tasks. Equivocally, detergent-resistant membranes were initially assumed to be identical to membrane rafts, because of similarities between their compositions. In fact, the impact of detergents in membrane organization is still controversial. Here, we use phase contrast and fluorescence microscopy to observe giant unilamellar vesicles (GUVs) made of erythrocyte membrane lipids (erythro-GUVs) when exposed to the detergent Triton X-100 (TX-100). We clearly show that TX-100 has a restructuring action on biomembranes. Contact with TX-100 readily induces domain formation on the previously homogeneous membrane of erythro-GUVs at physiological and room temperatures. The shape and dynamics of the formed domains point to liquid-ordered/liquid-disordered (Lo/Ld) phase separation, typically found in raft-like ternary lipid mixtures. The Ld domains are then separated from the original vesicle and completely solubilized by TX-100. The insoluble vesicle left, in the Lo phase, represents around 2/3 of the original vesicle surface at room temperature and decreases to almost 1/2 at physiological temperature. This chain of events could be entirely reproduced with biomimetic GUVs of a simple ternary lipid mixture, 2:1:2 POPC/SM/chol (phosphatidylcholine/sphyngomyelin/cholesterol), showing that this behavior will arise because of fundamental physicochemical properties of simple lipid mixtures. This work provides direct visualization of TX-100-induced domain formation followed by selective (Ld phase) solubilization in a model system with a complex biological lipid composition.     

Direct Visualization of the Action of Triton X-100 on Giant Vesicles of Erythrocyte Membrane Lipids

The raft hypothesis proposes that microdomains enriched in sphingolipids, cholesterol, and specific proteins are transiently formed to accomplish important cellular tasks. Equivocally, detergent-resistant membranes were initially assumed to be identical to membrane rafts, because of similarities between their compositions. In fact, the impact of detergents in membrane organization is still controversial. Here, we use phase contrast and fluorescence microscopy to observe giant unilamellar vesicles (GUVs) made of erythrocyte membrane lipids (erythro-GUVs) when exposed to the detergent Triton X-100 (TX-100). We clearly show that TX-100 has a restructuring action on biomembranes. Contact with TX-100 readily induces domain formation on the previously homogeneous membrane of erythro-GUVs at physiological and room temperatures. The shape and dynamics of the formed domains point to liquid-ordered/liquid-disordered (Lo/Ld) phase separation, typically found in raft-like ternary lipid mixtures. The Ld domains are then separated from the original vesicle and completely solubilized by TX-100. The insoluble vesicle left, in the Lo phase, represents around 2/3 of the original vesicle surface at room temperature and decreases to almost 1/2 at physiological temperature. This chain of events could be entirely reproduced with biomimetic GUVs of a simple ternary lipid mixture, 2:1:2 POPC/SM/chol (phosphatidylcholine/sphyngomyelin/cholesterol), showing that this behavior will arise because of fundamental physicochemical properties of simple lipid mixtures. This work provides direct visualization of TX-100-induced domain formation followed by selective (Ld phase) solubilization in a model system with a complex biological lipid composition.     

Probing membrane permeabilization by the antibiotic lipopeptaibol trichogin GA IV in a tethered bilayer lipid membrane

The lipopeptaibol trichogin GA IV (TCG) can be incorporated in the lipid bilayer moiety of a mercury-supported tethered bilayer lipid membrane (tBLM) at a non-physiological transmembrane potential of about -240mV, negative on the trans side of the bilayer. Once incorporated in the tBLM, TCG is stable over the range of physiological transmembrane potentials and permeabilizes the membrane at transmembrane potentials negative of -80÷-90mV. The chronocoulometric behavior is consistent with a kinetics of nucleation and growth of bundles of TCG building blocks with ion-channel properties. The TCG building blocks also permeabilize the lipid bilayer, albeit at more negative transmembrane potentials, and can be tentatively regarded as dimers of aligned TCG helical monomers. The cyclic voltammograms of tBLMs incorporating TCG point to a voltage-gated behavior of the TCG channel, similar to that exhibited by the peptaibol alamethicin.     

Molecular simulation of dioleoylphosphatidylcholine lipid bilayers at differing levels of hydration.

The structure and dynamics of the lipid and water components of dioleoylphosphatidylcholine bilayers at various levels of hydration were studied using molecular dynamics (MD) simulations. Equilibration of these systems proceeded by use of a hybrid MD and configurational-bias Monte Carlo technique using one atmosphere of pressure normal to the membrane and a set point for the lateral area derived from experimental Bragg spacings, combined with experimentally derived specific volumes for each of the system components. Membrane surface tensions were observed to be of the order of tens of dyn/cm. The transbilayer molecular fragment peak positions at low hydration were found to agree with experimental neutron and x-ray scattering profiles and previously published simulations. For hydration levels of 5.4, 11.4, and 16 waters/lipid, molecular fragment distributions and order parameters for the headgroup, lipid chains, and water were quantified. Spin-lattice relaxation rates and lateral self-diffusion coefficients of water agreed well with results from experimental nuclear magnetic resonance studies. Relaxation rates of the choline segments and chemical shift anisotropies for the phosphate and carbonyls were computed. Headgroup orientation, as measured by the P-N vector, showed enhanced alignment with the membrane surface at low hydration. The sign of the membrane dipole potential reversed at low hydration, with the membrane interior negative relative to the interlamellar region. Calculation of the number of water molecules in the headgroup hydration shell, as a function of hydration level, supports the hypothesis that the break point in the curve of Bragg spacing versus hydration level near 12 waters/lipid, observed experimentally by Hristova and White (1988. Biophys. J. 74:2419-2433), marks the completion of the first hydration shell.     

Membrane effects of ethanol: bulk lipid versus lipid domains

It has been well-established that ethanol fluidizes the bulk lipid of membranes and that this effect may alter cell function and be involved in ethanol sensitivity and tolerance. This hypothesis has been supported in several studies, however, there is also a considerable amount of data that do not support such an explanation, e.g., direct effect of ethanol on proteins, other membrane acting drugs, temperature effects, effects of ethanol on aged membranes and inconsistent effects of chronic ethanol consumption on lipid content. This review examined the bulk membrane fluidization hypothesis in light of those data and proposed a modification of the bulk membrane hypothesis that is based on recent data that show that ethanol and other alcohols have a specific effect on the structural properties of different membrane domains. This specific effect of ethanol is discussed within the context of how changes in fluidity of domains may alter membrane function.     

Small structural alterations greatly influence the membrane affinity of lipophilic ligands: Membrane interactions of bafilomycin A1 and its desmethyl derivative bearing 19F-labeling

Molecular behavior under bilayer membrane environments is one of the important research topics concerning how organic molecules exert their biological activities when interacting with cellular membranes. However, chemistry-based approaches to this property have not been successful when compared with the structural biological strategy on ligand-receptor interactions. Here, we investigated the molecular behavior of the lipophilic ATPase inhibitor bafilomycin A₁ and its derivatives under a lipid environment from a chemical point of view. Our results revealed significant differences in membrane affinity and dynamics among ligands having different inhibitory potencies, suggesting the specific contribution of ligand-membrane interactions to their biological activity.     

Membrane hydrophobicity determines the activation free energy of passive lipid transport

The collective behavior of lipids with diverse chemical and physical features determines a membrane’s thermodynamic properties. Yet, the influence of lipid physicochemical properties on lipid dynamics, in particular interbilayer transport, remains underexplored. Here, we systematically investigate how the activation free energy of passive lipid transport depends on lipid chemistry and membrane phase. Through all-atom molecular dynamics simulations of 11 chemically distinct glycerophospholipids, we determine how lipid acyl chain length, unsaturation, and headgroup influence the free energy barriers for two elementary steps of lipid transport: lipid desorption, which is rate limiting, and lipid insertion into a membrane. Consistent with previous experimental measurements, we find that lipids with longer, saturated acyl chains have increased activation free energies compared to lipids with shorter, unsaturated chains. Lipids with different headgroups exhibit a range of activation free energies; however, no clear trend based solely on chemical structure can be identified, mirroring difficulties in the interpretation of previous experimental results. Compared to liquid-crystalline phase membranes, gel phase membranes exhibit substantially increased free energy barriers. Overall, we find that the activation free energy depends on a lipid’s local hydrophobic environment in a membrane and that the free energy barrier for lipid insertion depends on a membrane’s interfacial hydrophobicity. Both of these properties can be altered through changes in lipid acyl chain length, lipid headgroup, and membrane phase. Thus, the rate of lipid transport can be tuned through subtle changes in local membrane composition and order, suggesting an unappreciated role for nanoscale membrane domains in regulating cellular lipid dynamics.     

Do annexins participate in lipid messenger mediated intracellular signaling? A question revisited

Abstract

Annexins are physiologically important proteins that play a role in calcium buffering but also influence membrane structure, participate in Ca²⁺-dependent membrane repair events and in remodelling of the cytoskeleton. Thirty years ago several peptides isolated from lung perfusates, peritoneal leukocytes, neutrophiles and renal cells were proven inhibitory to the activity of phospholipase A₂. Those peptides were found to derive from structurally related proteins: annexins AnxA1 and AnxA2. These findings raised the question whether annexins may participate in regulation of the production of lipid second messengers and, therefore, modulate numerous lipid mediated signaling pathways in the cell. Recent advances in the field of annexins made also with the use of knock-out animal models revealed that these proteins are indeed important constituents of specific signaling pathways. In this review we provide evidence supporting the hypothesis that annexins, as membrane-binding proteins and organizers of the membrane lateral heterogeneity, may participate in lipid mediated signaling pathways by affecting the distribution and activity of lipid metabolizing enzymes (most of the reports point to phospholipase A₂) and of protein kinases regulating activity of these enzymes. Moreover, some experimental data suggest that annexins may directly interact with lipid metabolizing enzymes and, in a calcium-dependent or independent manner, with some of their substrates and products. On the basis of these observations, many investigators suggest that annexins are capable of linking Ca²⁺, redox and lipid signaling to coordinate vital cellular responses to the environmental stimuli.     

Collective predator evasion: Putting the criticality hypothesis to the test

According to the criticality hypothesis, collective biological systems should operate in a special parameter region, close to so-called critical points, where the collective behavior undergoes a qualitative change between different dynamical regimes. Critical systems exhibit unique properties, which may benefit collective information processing such as maximal responsiveness to external stimuli. Besides neuronal and gene-regulatory networks, recent empirical data suggests that also animal collectives may be examples of self-organized critical systems. However, open questions about self-organization mechanisms in animal groups remain: Evolutionary adaptation towards a group-level optimum (group-level selection), implicitly assumed in the “criticality hypothesis”, appears in general not reasonable for fission-fusion groups composed of non-related individuals. Furthermore, previous theoretical work relies on non-spatial models, which ignore potentially important self-organization and spatial sorting effects. Using a generic, spatially-explicit model of schooling prey being attacked by a predator, we show first that schools operating at criticality perform best. However, this is not due to optimal response of the prey to the predator, as suggested by the “criticality hypothesis”, but rather due to the spatial structure of the prey school at criticality. Secondly, by investigating individual-level evolution, we show that strong spatial self-sorting effects at the critical point lead to strong selection gradients, and make it an evolutionary unstable state. Our results demonstrate the decisive role of spatio-temporal phenomena in collective behavior, and that individual-level selection is in general not a viable mechanism for self-tuning of unrelated animal groups towards criticality.     

Lipid modulation of protein-induced membrane domains as a mechanism for controlling signal transduction

The reason for the enormous lipid variety present in eukaryotic membranes remains largely an enigma. We suggest that its role is to provide an on-off switch for a signaling event at the membrane level. This is achieved through lipid-lipid interactions that convert membrane protein binding and association events into very cooperative processes while maintaining reversibility. We have previously shown [Hinderliter, A., at al. (2001) Biochemistry 40, 4181-4191] that thermodynamic linkage between an intrinsic tendency for lipid demixing and a preferential interaction of a protein with a specific lipid within the mixture leads to dramatic changes in lipid and protein domain formation. Here, we tested the hypothesis that small alterations in lipid chemical structure alter the magnitude of the net interaction free energy (omega(AB)) between unlike lipids in a predictable manner, and that even very small changes in omega(AB) lead to dramatic changes in bilayer organization when coupled with protein binding. We systematically varied the chemical structure of phosphatidylcholine (PC), in mixtures with a fixed phosphatidylserine (PS), by changing the PC acyl chain length and the degree of unsaturation, and examined domain formation upon addition of a peripheral protein, the synaptotagmin I C2A motif. Experimental excimer/monomer ratios (E/M) of pyrene-substituted lipids mimicking the PS were interpreted using Monte Carlo computer simulations. E/M is larger if the PC melting temperature is lower, suggesting that domain formation is a thermodynamic consequence of weak interactions between PC and PS. Consistent with our hypothesis, only very small changes in omega(AB) were required for prediction of large changes in lipid and protein domain formation.     

Structural and functional changes in erythrocyte membranes in experimental atherosclerosis

Structural and functional characteristics of erythrocyte membranes were studied in rabbits with experimental atherosclerosis. In animals with single lipid spots in the aorta, a significant rise of the plasma cholesterol level was associated with the increased cholesterol/phospholipid (CS/PL) ratio and diminished activity of erythrocyte membrane Na+, K+-ATPase. EPMR spin probe data point to changes in structural membrane characteristics--an increase in order parameter for fatty acid chains of lipids and expansion of the temperature interval of the transition phase in the membranes. In rabbits with total aorta injury, a further increase both in the plasma cholesterol concentration and in the CS/PL ratio as well as in structural changes in erythrocyte membranes does not lead to another decrease in the enzymatic activity. In aorta homogenates of the experimental animals, the activity of Na+, K+-ATPase correlated with that in the erythrocyte membrane. This suggests the existence of similar chemical and structural changes in aorta cell membranes. The data may provide an indirect evidence in favour of the hypothesis of the involvement of smooth muscle cells and membrane enzymatic activity alterations in atherosclerosis.     

Differential effects of propofol and ketamine on critical brain dynamics

Whether the brain operates at a critical “tipping” point is a long standing scientific question, with evidence from both cellular and systems-scale studies suggesting that the brain does sit in, or near, a critical regime. Neuroimaging studies of humans in altered states of consciousness have prompted the suggestion that maintenance of critical dynamics is necessary for the emergence of consciousness and complex cognition, and that reduced or disorganized consciousness may be associated with deviations from criticality. Unfortunately, many of the cellular-level studies reporting signs of criticality were performed in non-conscious systems (in vitro neuronal cultures) or unconscious animals (e.g. anaesthetized rats). Here we attempted to address this knowledge gap by exploring critical brain dynamics in invasive ECoG recordings from multiple sessions with a single macaque as the animal transitioned from consciousness to unconsciousness under different anaesthetics (ketamine and propofol). We use a previously-validated test of criticality: avalanche dynamics to assess the differences in brain dynamics between normal consciousness and both drug-states. Propofol and ketamine were selected due to their differential effects on consciousness (ketamine, but not propofol, is known to induce an unusual state known as “dissociative anaesthesia”). Our analyses indicate that propofol dramatically restricted the size and duration of avalanches, while ketamine allowed for more awake-like dynamics to persist. In addition, propofol, but not ketamine, triggered a large reduction in the complexity of brain dynamics. All states, however, showed some signs of persistent criticality when testing for exponent relations and universal shape-collapse. Further, maintenance of critical brain dynamics may be important for regulation and control of conscious awareness.     

Failure of adaptive self-organized criticality during epileptic seizure attacks

Critical dynamics are assumed to be an attractive mode for normal brain functioning as information processing and computational capabilities are found to be optimal in the critical state. Recent experimental observations of neuronal activity patterns following power-law distributions, a hallmark of systems at a critical state, have led to the hypothesis that human brain dynamics could be poised at a phase transition between ordered and disordered activity. A so far unresolved question concerns the medical significance of critical brain activity and how it relates to pathological conditions. Using data from invasive electroencephalogram recordings from humans we show that during epileptic seizure attacks neuronal activity patterns deviate from the normally observed power-law distribution characterizing critical dynamics. The comparison of these observations to results from a computational model exhibiting self-organized criticality (SOC) based on adaptive networks allows further insights into the underlying dynamics. Together these results suggest that brain dynamics deviates from criticality during seizures caused by the failure of adaptive SOC.