Article Cite This: J. Phys. Chem. B 2018, 122, 8193−8200
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Presence and Role of Midplane Cholesterol in Lipid Bilayers Containing Registered or Antiregistered Phase Domains Michael D. Weiner† and Gerald W. Feigenson*,‡ †
Department of Physics and ‡Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, United States
J. Phys. Chem. B 2018.122:8193-8200. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 09/01/18. For personal use only.
S Supporting Information *
ABSTRACT: Three-component lipid mixtures can produce coexisting liquid ordered and liquid disordered phases, a model for eukaryotic plasma membrane rafts. In compositionally symmetric bilayers with two phase-separated leaflets, phase domains of the two leaflets may align through registration, where domains are found across from domains of the same phase, or else antiregistration, where domains are found across from domains of the opposite phase. This alignment could serve as a method of information communication across the plasma membrane. We used coarse-grained molecular dynamics simulations to study ternary mixtures of a high-melting-temperature phospholipid, a low-melting-temperature phospholipid, and cholesterol. We found a significant presence of cholesterol molecules at the bilayer midplane rather than in a leaflet in some systems, corresponding to a lack of registration. Increasing the length of the acyl chains from 16 to 24 carbons in high-melting-temperature phospholipids or increasing the concentration of cholesterol from 20 to 35 mol % in the bilayer produced a transition from registration to antiregistration and gave rise to significant populations of midplane cholesterol.
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INTRODUCTION Membrane rafts can provide an environment, distinct from the surroundings, that is part of recognition and signaling processes and can transmit information across the membrane. Rafts are thought to be regions of the eukaryotic plasma membrane (PM) with physical properties distinct from their surroundings. Typically, they are a separate phase with a large fraction of high-melting-temperature lipid. Model membranes are widely used to study rafts in a controlled setting, both in vitro and in silico. Simple lipid mixtures in bilayers can reproduce the key situation of coexisting domains of different physical properties that defines raft-containing membranes.1−3 In these models, rafts are represented by liquid−liquid phase coexistence with lipids in both phases diffusing rapidly. However, the high order of the liquid ordered (Lo) phase is closer to that of typical solids, whereas the liquid disordered (Ld) phase has the low order characteristic of most liquids. A system with as few as three lipid components, in the proper ratios, has coexisting Lo and Ld domains. These components are a phospholipid with a melting temperature above the experimental conditions (high-Tm lipid), a phospholipid with a melting temperature below the experimental conditions (lowTm lipid), and cholesterol. Experimentally solved phase diagrams (Figure 1) for such mixtures reveal a rich variety of behaviors. The most simple model membranes are compositionally symmetric, i.e., they contain the same lipids in the same ratios in each leaflet of the bilayer. If these lipids phase separate, then that separation occurs in each leaflet. Registration means that phases align across the bilayer, with an ordered domain in one leaflet apposing an ordered domain in the other leaflet. © 2018 American Chemical Society
Antiregistration, in contrast, occurs when phases avoid alignment across the bilayer, with an ordered domain in one leaflet apposing a disordered domain in the other leaflet (Figure 2). Complete antiregistration is possible only if the two phases have exactly equal area fractions of the bilayer as in this figure, which could be the case for symmetric models. In an uncorrelated system, domains in one leaflet are randomly arranged with respect to those in the other leaflet. Registration and antiregistration demonstrate the ability of the bilayer lipids to transmit information across the membrane. This provides a model for a type of signal transfer between the cell’s interior and its surroundings because a signal could induce the formation or the loss of a raft, which could reorganize the other leaflet, prompting a signal on the other side of the PM and thereby transferring information across it. Coupling of the leaflets in a lipid bilayer has been the subject of extensive research.5−7 Prior investigators have employed experimental,8−11 theoretical,12−15 and computational2,16−18 methods to observe conditions under which lipid physical chemistry in one leaflet affects properties in the other. The experimental studies have examined both symmetric8 and asymmetric9−11 compositions and found numerous cases of an ordered or cholesterol-enriched region inducing a distinct region of an apposed leaflet. In the asymmetric case, where studies use either supported bilayers or vesicles subject to cyclodextrin-mediated exchange, this can even include inducing regions of differentiated physical properties in Received: April 26, 2018 Revised: August 4, 2018 Published: August 10, 2018 8193
DOI: 10.1021/acs.jpcb.8b03949 J. Phys. Chem. B 2018, 122, 8193−8200
Article
The Journal of Physical Chemistry B
term is included, so such findings depend on implicit inclusion through a well-determined force field. Cholesterol is an essential component of the animal cell plasma membrane. Its interaction with phospholipids creates the Lo phase, offering a combination of the stiffness and order of the solid phase and the fluidity of the Ld phase. Many computational studies have been performed with relatively low concentrations of cholesterol, such as 20 mol %,2,17,18 which lies along the tieline at the edge of the two-phase coexistence region, farthest from the critical point. This long tieline reflects the maximum compositional difference between coexisting domains, which become more similar as shrinking tielines at higher cholesterol concentration move toward the critical point. A mammalian plasma membrane contains 30−45 mol % cholesterol, making 20 mol % a less-than-representative model. Additionally, the distribution of cholesterol between the cytoplasmic and exoplasmic leaflets of the PM is not known for any cell. For these reasons, it is necessary to understand the different behaviors that arise when a bilayer contains a higher cholesterol concentration. Importantly, there is a third position for cholesterol: within the bilayer but in neither leaflet, at the bilayer midplane. We find that this “midplane cholesterol” represents a significant and influential part of the bilayer at certain compositions. The presence of such a population, especially in bilayers with a large fraction of unsaturated phospholipids, has been measured experimentally using neutron scattering.20−23 A theory developed by Olmsted and colleagues explains registration as the result of two competing coupling interactions:15,17 direct coupling is the interaction between domains at the bilayer midplane, where contacts between domains of the same phase are favored, promoting registration; indirect coupling is quite different, being the result of hydrophobic mismatch at the boundary between phases in a leaflet. Domain interaction at the bilayer midplane could include, but is not limited to, chain interdigitation.16 Since the Lo phase is taller than the Ld phase, antiregistration is favored by indirect coupling to produce a bilayer of constant thickness, which avoids exposing the bilayer’s hydrophobic interior to the hydrophilic region at the phase interface. Our work considers the extension of this framework to lipids and compositions beyond those previously studied. These researchers measured the effects of 12- to 20-carbon saturated acyl chains using compositions containing 20 mol % cholesterol. We extend this work to longer acyl chains and most significantly to higher concentrations of cholesterol. An earlier study, by Perlmutter and Sachs,18 observed the rise of antiregistration in the presence of longer acyl chains, also using the Martini model (see Methods). The authors explored the difference in bilayer thickness as an explanation for the behavior of their systems, which contained saturated acyl chains of four, five, or six beads, corresponding to acyl chain lengths of 16, 20, or 24 carbons, respectively, as do ours. Those systems mostly contained at total of 20 mol % cholesterol, though a few other systems were examined along the same tieline, thus with compositions where coexisting Lo + Ld domains matched those of a system of 20 mol % cholesterol at equal phase fractions. We simulated bilayers on highercholesterol tielines and used a much larger system size. Making both of these modifications enabled study of the behavior of midplane cholesterol, which was not observed in the previous study,18 although it has been observed in single-phospholipid systems.22−26
Figure 1. Phase diagram and molecules in these simulations. This experimentally derived phase diagram, modified from ref 4 for distearoylphosphatidylcholine (DSPC)/dioleoylphosphatidylcholine (DOPC)/cholesterol, displays the behavior of a three-component lipid system similar to those used in this study, with the Ld + Lo region marked. Small gray symbols represent the compositions chosen for this study, containing high-Tm lipid/low-Tm lipid/cholesterol: circle (40:40:20), triangle (31:40:29), and diamond (30:35:35). They were chosen to represent approximately equal fractions of Ld and Lo, so they do not fall along a straight line. Surrounding the phase diagram are Martini bead representations of all of the lipids employed in these simulations. Their configurations are taken from actual snapshots of the simulation, rather than idealized models. The three on the bottom (dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DBPC), dilignoceroylphosphatidylcholine (DXPC)) are high-Tm lipids, the lower axis of the triangle, which was DSPC in the experimental phase diagram. At left, low-Tm dilinoleoylphosphatidylcholine (DUPC) takes the place of the experimental DOPC; at right, cholesterol is shown. Shapes and colors employed in this figure are used consistently throughout the article.
Figure 2. Registered domains in the bilayer. This cartoon shows how domains in each leaflet can align through registration, apposing each other, or through antiregistration, avoiding each other. If domain location in one leaflet is uncorrelated with the other leaflet’s domains, then their degree of contact will be random.
compositions where none existed in the symmetric equivalent. The role of intrinsic curvature in coupling has also been considered.19 The theoretical studies have considered the effect of interacting compositions (discussed further in the following paragraphs), where the composition and phase behavior of each leaflet can restrict or bolster domain formation depending on interleaflet coupling. Computational studies often make use of coarse-grained molecular dynamics (MD) (see Methods) to reproduce phase separation, as we do in this study, and examine the resultant alignment of domains. Using common force fields means that no explicit coupling 8194
DOI: 10.1021/acs.jpcb.8b03949 J. Phys. Chem. B 2018, 122, 8193−8200
Article
The Journal of Physical Chemistry B
distribution of the three lipid components within each leaflet of a lamellar system, allowing for demixing to occur spontaneously during equilibration and production simulations. Each bilayer contained approximately 4600 lipids solvated with Martini water equivalent to approximately 60 water molecules per lipid. These systems were energyminimized and then equilibrated for 4.75 ns with position restraints along the bilayer normal to maintain the lamellar phase during equilibration, using the equilibration template provided by CHARMM-GUI, which begins with an NVT ensemble and then transitions to an NPT ensemble. Following equilibration, each system engaged in a production run of 9.6 μs with a Berendsen thermostat and barostat in an NPT ensemble.40 Each system was simulated at both 295 K, for comparison to room-temperature experiments and simulations, and 310 K, for modeling behavior under human physiological conditions. Lipid and solvent were coupled to separate temperature baths. The semi-isotropic barostat was set at 1 bar in either case. All simulations were conducted with a time step of 20 or 30 fs. The smaller time step was used for systems that were unstable under the longer one. Both van der Waals and electrostatic interactions used a potential shift with a 1.1 nm cutoff under the Verlet scheme. The results described are taken from the last microsecond of a 9.6 μs simulation, to ensure fully equilibrated measurements. A few systems were run to 15 μs to ensure that the 9.6 μs simulations presented the full picture of the systems’ behavior. In each case, no change in physical properties was observed, suggesting that the measured state is the preferred state and not a picture of a kinetically trapped system. The Supporting Information contains graphs of some key bilayer properties over the full 9.6 μs, confirming that the results presented represent an equilibrium condition. Registration fraction equilibrated slowly in a few systems, but much more quickly in others (Figure S7). Nine different systems were studied, containing three different high-Tm lipids and three different relative quantities of the three lipid components. The high-Tm lipids used were all fully saturated, nonhybrid phosphatidylcholines (PCs). The Martini models used represent dipalmitoyl (di-16:0, DPPC), diarachidoyl (di-20:0, DBPC), and dilignoceroyl (di-24:0, DXPC) PCs. All systems used the same low-Tm lipid, dilinoleoyl PC (di-18:2, DUPC), and cholesterol. Figure 1 shows the Martini representation of each lipid in the study as well as a phase diagram. This phase diagram is generic, as it reflects experiments on a similar, but not identical, set of lipids. We have found the Lo + Ld critical point from Martini to be higher in cholesterol fraction than that of experiments, and we have one more double bond in each acyl chain of the low-Tm lipid compared to the experiment, as is standard,3 since the monounsaturated acyl chain will not form coexisting domains in the Martini model. Finally, the experimental phase diagram reflects a single high-Tm lipid selection, and the phase diagram will change with the length of the high-Tm acyl chains, with boundaries shifting in a nonlinear fashion. We also find the Martini tielines with di-18:2 PC to be longer than those of the experimental system with di-18:1 PC, with phase domains exhibiting less high-Tm lipid in Ld and less low-Tm lipid in Lo. For the tielines we consider, the changing concentration of cholesterol is the most significant variation. Unfortunately, we are not aware of any experimental three-component phase diagrams for mixtures containing DUPC or DXPC.
Previous work has also revealed that domain registration occurs only when a minimum domain size of about 15 nm diameter has been surpassed, requiring a large simulation.2,17 Our finding of a correlation between registration and midplane cholesterol reveals the necessity of the large simulation box we employ. The consideration of domain size naturally turns our focus to the line tension between the coexisting domains in a leaflet. Compositions with high line tension produce fewer, larger domains to minimize the amount of interface between phases, whereas those with low line tension produce numerous nanodomains.27 We have extensively characterized this behavior in both simulations and experiments.2,4,28 In this study, all of the compositions employed produce macrodomains, so only two domains, one of each phase, are found in each leaflet, just as in systems where registration has been found. The animal cell PM is compositionally asymmetric, with different lipid components on the exoplasmic and cytoplasmic leaflets.29 We aim to study models of such systems, but that first requires a thorough understanding of the symmetric case, whose simplicity serves as a control to inform interpretation of asymmetric studies. Additionally, current methods for forming asymmetric membranes in vitro are limited both in complexity, often having one leaflet with only a single lipid component, and in degree of asymmetry, with only 50−70% asymmetry achieved.19,30 Due to these challenges, symmetric computational results offer extensive opportunities for comparison to in vitro measurements. We use coarse-grained molecular dynamics simulations. This method successfully replicates experimentally seen phase separation,3,4,31,32 with coexisting Lo and Ld phases spontaneously demixing from an initially homogeneous mixture. The efficiency that coarse graining offers over all-atom simulations enables phase coexistence to be studied for large systems over long times with available resources, yet with key physical properties remaining. However, it is important to note that the coarse-grained model may not accurately reproduce the prevalence or behavior of midplane cholesterol, as its design was not based on this particular behavior.33 Additionally, different rates of cholesterol flip flop have been reported for experiments, all-atom simulations, and coarse-grained simulations.34 We have found that increasing the length of the acyl chains in the more ordered phospholipid component or increasing the fraction of cholesterol in the system produces a transition from registration to antiregistration and the growth of the midplane as a location of cholesterol molecules.
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METHODS Our molecular dynamics (MD) simulations used GROMACS 5.1.3.35 All simulations used the Martini force field with updated cholesterol parameters.33,36,37 This coarse-grained force field represents groups of approximately four nonhydrogen atoms as a single bead, where each bead is parameterized to account for polarity and charge, as in phospholipid headgroups, and for the presence or absence of double bonds, as in the acyl chains. The simplification provided by Martini allows for long-distance diffusion of lipids in a bilayer, which makes studies of phase separation possible using conventional supercomputer resources and MD methods. Each bilayer to be studied was created using CHARMMGUI.38,39 CHARMM-GUI produced an initially random 8195
DOI: 10.1021/acs.jpcb.8b03949 J. Phys. Chem. B 2018, 122, 8193−8200
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The Journal of Physical Chemistry B
Figure 3. Example of the method for determining domains and registration. Left, a leaflet of lipids is shown, containing DPPC (magenta), DUPC (blue), and cholesterol (yellow). Center, a Voronoi tesselation is performed, and each lipid is labeled Lo (yellow) or Ld (green) based on local composition. Considering periodic boundary conditions, this system contains two large domains. Right, by comparison with the apposed leaflet, each lipid is labeled registered (black) or antiregistered (white). This example contains DPPC and 29 mol % cholesterol, shown as the magenta triangle symbol in other figures. The boxes shown are 34.0 nm on each side.
phosphate bead (PO4). This proved to be a reliable method for classification, as cholesterols in a leaflet are found oriented with their hydroxyl beads farthest from the midplane, close to the water. As described in Figure 8 and the following section, the wide distribution of cholesterol orientations precludes using orientation to identify midplane cholesterol. Figure 8 also details the definition of cholesterol’s orientation vector, which is compared to the local value of the bilayer normal. For phospholipids, the orientation vector points from the sn2 chain (C4A bead) to the phosphate bead. This bilayer normal value is determined locally for each phospholipid and cholesterol in the system based on the orientation of lipids with phosphate groups within 1 nm in the xy-plane of the lipid of interest (where the z-axis is the initial bilayer normal). Using this method, rather than assuming that the bilayer normal lies along the z-axis, allows for accurate identification of each leaflet and the midplane regardless of curvature.
Three different relative quantities of the three components were simulated. Each composition was on a different tieline within the two-phase region and was selected to produce approximately equal mole fractions of each phase, where interpretation of registered and antiregistered is most straightforward. The three tielines explored the full twophase region, beginning with a tieline just above the threephase region, with maximally distinct phase compositions. This composition contained 40 mol % high-Tm lipid, 40 mol % lowTm lipid, and 20 mol % cholesterol. The second composition examined the center of the two-phase region at 31 mol % highTm lipid, 40 mol % low-Tm lipid, and 29 mol % cholesterol. The final composition neared the experimental critical point and approached physiological cholesterol levels at 30 mol % high-Tm lipid, 35 mol % low-Tm lipid, and 35 mol % cholesterol. The resulting demixed phase compositions are presented in the Supporting Information. It is notable that the demixing in Martini exceeds the experimental expectation, giving longer tielines than those seen in Figure 1. Simulated bilayers were analyzed using custom Python scripts employing the MDTraj library41 and available at https://github.com/mdweiner/midplanechol. The plane of the outer leaflet was divided into Voronoi cells centered on each lipid, the phosphate bead for phospholipids, and the hydroxyl bead for cholesterol. Each lipid was identified as Lo or Ld by local composition (Figure 3). Specifically, for each lipid the local composition was taken from the lipid of interest together with all of the other lipids sharing a Voronoi edge. If that set of lipids had a prevalence of high-Tm lipid and cholesterol exceeding that of the bilayer, it was identified as Lo. This process was iterated twice to remove small clusters below the size of a domain. At the conclusion of the process, the domain boundary was determined to consist of any Voronoi cell sharing an edge with a cell of the opposite phase. By comparing lipids to their neighbors in the other leaflet, Voronoi cells were identified as registered or antiregistered. Autocorrelation measurements were made to determine the appropriate sampling frequency of the results to ensure accurate uncertainty calculations. All graphs were produced using eight frames from the final microsecond, 108 ns apart, which was determined to offer independent (uncorrelated) values.42 Each plot marks the standard error of the value measured. Further detail is available in the Supporting Information. Cholesterols were classified as being in one leaflet, the other leaflet, or the midplane. Midplane cholesterol was defined as those cholesterols for which the hydroxyl bead (ROH) was located more than 1.4 nm from the nearest phospholipid
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RESULTS AND DISCUSSION The composition of the simulated bilayer determines the prevalence of registered domains observed (Figure 4). When simulations are performed using DPPC at 20 mol % cholesterol, near-perfect registration is observed. Interface regions at phase boundaries, which are excluded from the bulk
Figure 4. Registration fraction decreases with longer chains or more cholesterol. For each system studied, the fraction of the bilayer exhibiting registration is shown. A value of 1 represents a perfectly registered system; a value of 0 represents a perfectly antiregistered system; and a value of 0.5 represents an uncorrelated system. These fractions represent the bulk phase regions, excluding lipids within 1 nm of a phase interface. Shapes on the x-axis refer to the phase diagram in Figure 1. Longer acyl chains (left to right, by color) or increased cholesterol (left to right within a color, by shape) decrease registration. The shapes, as in Figure 1, represent mixtures containing high-Tm lipid/low-Tm lipid/cholesterol: circle (40:40:20), triangle (31:40:29), and diamond (30:35:35). 8196
DOI: 10.1021/acs.jpcb.8b03949 J. Phys. Chem. B 2018, 122, 8193−8200
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The Journal of Physical Chemistry B
For highly registered systems with few midplane cholesterols, the population is composed almost entirely of highly transitory molecules, which we define as those with a persistence time in the midplane less than the 13.5 ns between measured frames (see Supporting Information). For highly antiregistered systems with plentiful midplane cholesterol, dwell times at the midplane may be longer (Figure S3). However, these antiregistered systems still exhibit a larger number of cholesterols that flip all of the way through the midplane, from one leaflet to another (Figure S6). As shown in Figure 5, the fraction of cholesterols found at the midplane increases with the length of the high-Tm phospholipid or the increased concentration of cholesterol. This behavior holds for both room temperature (295 K) and human physiological temperature (310 K). For the mixture most commonly used in simulations, DPPC at 20 mol % cholesterol, less than 2% of the cholesterol is found at the midplane, so the effect is not large. For the eight-carbon-longer DXPC and 35 mol % cholesterol, 16% of the cholesterol is found at the midplane. This partitioning of the cholesterol molecules between the in-leaflet and the midplane positions can be evaluated as a difference in the energy of cholesterol in these environments. In every case, in-leaflet cholesterol is more prevalent. Since the system is believed to be at equilibrium, this partition coefficient can be converted into a free energy, presented in Figure 6. This
registration fraction, ease the hydrophobic mismatch caused by the thickness difference between phases with small antiregistered regions. As the high-Tm phospholipid is lengthened or cholesterol concentration is increased, this registration lessens, and combining these factors leads to nearly complete antiregistration of phase domains. This effect occurs at both 295 K, which represents the room temperature at which many experiments are conducted, and 310 K, which represents the human physiological temperature at which the system being modeled exists (graphs of data collected at 310 K are in the Supporting Information). There was a slight increase in registration at the higher temperature. This may be due to an avoidance of kinetic trapping in an antiregistered state. However, strongly antiregistered systems maintained their configurations even beyond 15 μs, suggesting that antiregistration may truly be the favored state. We performed simulations with three different choices of high-Tm phospholipid: DPPC, DBPC, and DXPC and three different three-component relative compositions. Each composition was selected to be the midpoint of a tieline in the liquid−liquid coexistence region. We refer to them by the cholesterol concentrations at these tieline midpoints, ranging from 20 to 35 mol % cholesterol. The range of cholesterol compositions is important, as our results reveal the shortcomings of conducting experiments and simulations along the common 20 mol % cholesterol tieline. This tieline, the lowest and longest in the two-phase liquid−liquid coexistence region, provides for maximally distinct Lo and Ld phase compositions and the clearest domains. However, physiological cholesterol concentrations are significantly higher.32 As our results show, increasing the cholesterol concentration to more physiological levels can result in significantly different biophysical outcomes, including a transition from registration to antiregistration. The location of cholesterol in the bilayer depends on the phospholipid composition and the concentration of cholesterol in the system. Most cholesterol is found clearly within one leaflet or the other, in any of numerous orientations, although most are in the “canonical” orientation aligned roughly with the bilayer normal, with the hydroxyl group closest to the surface. A fraction of the cholesterol molecules, however, is located at the bilayer midplane (Figure 5). The midplane population includes molecules rapidly flipping from one leaflet to the other, but it also contains molecules that remain positioned in the local energy minimum with an orientation perpendicular to the bilayer normal for tens of nanoseconds.
Figure 6. Free-energy difference for cholesterol molecules at the midplane compared to in a leaflet. Symbol shapes on the x-axis refer to the phase diagram in Figure 1. Longer acyl chains (left to right, by color) or increased cholesterol (left to right within a color, by shape) decrease the energy penalty of entering the midplane. Lower values on the y-axis indicate more favorable midplane location.
ΔG value represents the free energy required in each simulation to transfer a single cholesterol molecule into the midplane, given the position of all other molecules, and is always unfavorable. The increase in midplane cholesterol mirrors the decrease in registration, which is presented in Figure 7. Although the right, mostly registered portion of the graph appears roughly linear, the antiregistered systems cluster around a single registration fraction, regardless of midplane cholesterol fraction. Apparently, this low registration fraction is close to the minimum feasible registration fraction, since perfect antiregistration would require exactly equal area fractions of Lo and Ld. If one domain occupies a bit more than half of the bilayer, there would need to be at least a small region where it aligned with the same phase in the other leaflet. We can clearly demonstrate the correlation between midplane cholesterol and registration. Both factors depend on the interaction energies of the leaflets and of the cholesterol within them. We hypothesize that the midplane cholesterol
Figure 5. Fraction of the cholesterols at the bilayer midplane increases for longer chains and higher cholesterol. Symbol shapes on the x-axis refer to the phase diagram in Figure 1. Longer acyl chains (left to right, by color) or increased cholesterol (left to right within a color, by shape) increase the presence of cholesterol at the midplane. 8197
DOI: 10.1021/acs.jpcb.8b03949 J. Phys. Chem. B 2018, 122, 8193−8200
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The Journal of Physical Chemistry B
Figure 7. Data from Figures 4 and 5 are plotted against each other. Registration and midplane cholesterol fraction are negatively correlated. The clustering at the left of the plot may indicate reaching a minimum registration fraction for the phase areas included. Error bars are smaller than the symbol width. Figure 8. Orientation angle of cholesterols in each portion of bilayer. The angle between the cholesterol orientation vector (inset) and the local bilayer normal (directed outward from the upper leaflet) is plotted. Molecules in the upper (black) or lower (red) leaflet are mostly aligned with the bilayer normal and their surrounding phospholipids, whereas those in the midplane (blue) exhibit a wide range of orientations, although orientations perpendicular to the normal are most prevalent. Data shown are from the DBPCcontaining, 29 mol % cholesterol simulation, though all mixtures exhibit nearly the same behavior (see Supporting Information).
disrupts the favorable interleaflet interactions that promote registration, leaving the system to favor an antiregistered state. With many cholesterols at the midplane, the acyl chain tails of one leaflet no longer interact with those of the other leaflet, precluding the direct coupling of the model in ref 17. The remaining indirect coupling will produce the antiregistered state observed. However, midplane cholesterol is not necessary to produce antiregistration. A preceding study18 identified antiregistered behavior without noticing the presence of cholesterol at the midplane. We studied an antiregistered system of comparable size, much smaller than those presented here, and also noted the absence of midplane cholesterol. This suggests that at least for the acyl chain portion of the effect, antiregistration may arise solely due to other domain behaviors, especially an increased hydrophobic mismatch. With increasing hydrophobic mismatch that causes antiregistration, the environment between the leaflets becomes less and less energetically unfavorable for cholesterol; i.e., the free energy required to insert a cholesterol molecule into the midplane decreases. This contrasts with the case of registered domains, where favorable interleaflet interactions may exclude cholesterol from fitting into the midplane. We measured the orientation of cholesterol relative to the local bilayer normal for each population (Figure 8). Orientation angle is defined as the angle between the cholesterol’s orientation vector and the bilayer normal vector at that point. Those cholesterols identified as in a leaflet exhibit a small range of angles around the bilayer normal, reflecting the canonical orientation of cholesterol with the hydroxyl group farthest from the midplane and the molecule aligned with its neighboring phospholipids. Midplane cholesterol displays a wider range of angles, though values are clustered around an orientation laying along the bilayer midplane, perpendicular to the normal vector. This variability accounts for cholesterol captured midtransition, as it moves to or from a leaflet and has not settled into the flat orientation, and gives a clearer picture of the situation at the bilayer midplane. At the same time, the fact that all three sets of molecules exhibit a significant variability in orientation reveals the necessity of defining midplane cholesterol by distance from phosphates, rather than by orientation angle. Midplane cholesterol is unevenly distributed. Table 1 displays the preference of midplane cholesterol for the different locations: near a phase interface, between registered Lo domains, between registered Ld domains, and between antiregistered domains. Each of these values presents whether
Table 1. Midplane Cholesterol by Locationa location interface registered Lo registered Ld (DPPC) registered Ld (DBPC) registered Ld (DXPC) antiregistered
preference of midplane cholesterol 1.23 0.48 1.42 0.67 0.28 1.07
± ± ± ± ± ±
0.09 0.06 0.10 0.08 0.04 0.09
a
Higher numbers in the right column indicate preference of midplane cholesterol for each type of location found at the bilayer midplane. A value of 1 represents no preference, i.e., a fraction of cholesterol in that location that matches the fraction of the bilayer it occupies. The interfacial location contains regions within 1 nm of a phase interface in either leaflet. The preference for the registered Ld location is dependent on the length of the high-Tm lipid, so results are presented separately. Other locations do not exhibit this dependence, so a single average over all systems is used. For all cases, only locations representing at least 5% of the bilayer are included. Values include standard error.
midplane cholesterol prefers or avoids the environment in question. We find that midplane cholesterol prefers interfacial locations, defined as being within 1 nm of a domain boundary in either leaflet. It disfavors fitting between registered Lo domains, whereas there is no significant preference for antiregistered regions. In the case of registered Ld domains, the midplane cholesterol preference for this location is dependent on the high-Tm lipid. Lengthening the high-Tm lipid produces a change from favoring to disfavoring this location. This dependence is not seen for other locations. In the discussion of the interfacial location, we point out the significant finite-size effect from the simulation box size. Although physical chemical studies of coexisting phases frequently assume the interface to represent an insignificant fraction of the system, that is not the case here. Even the case of minimum interface, where the interface is a straight line fully aligned between the leaflets, would leave the interfacial 8198
DOI: 10.1021/acs.jpcb.8b03949 J. Phys. Chem. B 2018, 122, 8193−8200
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between leaflets and the role cholesterol plays in that behavior. Understanding the forces driving registration and antiregistration reveals the connection between the two leaflets of the plasma membrane, that is, between the leaflet that interacts with the cell’s external environment and the one which faces inward, binding molecules and vesicles from the cytoplasm. Although the plasma membrane is not thought to form rafts on the cytoplasmic leaflet, our findings provide insight into how a raft could induce a signal inside the cell, or how an event interior to the cell could create a raft on the exterior. We plan to conduct future studies of more physiological asymmetric compositions to better understand the nature of this influence. In sum, we show that a change in the coupling behavior of leaflets, from registration to antiregistration, may be driven by a change from shorter to longer acyl chains in a high-Tm phospholipid or by increasing the presence of cholesterol in the bilayer. This transition to antiregistration is accompanied by the increasing localization of cholesterol to the bilayer midplane. At the midplane, this cholesterol prefers locales near domain interfaces in the leaflets and avoids squeezing between registered Lo domains. Its association with Ld domains decreases as the high-Tm lipid is lengthened. The behaviors of registered domains and midplane cholesterol occur within the context of coexisting liquid phases modeling PM rafts.
location occupying approximately 15% of the bilayer for these simulations. The fraction of each system that represents each location can be found in Figure S5 of the Supporting Information. It is also possible to analyze the data into only three locations, where no distinct interfacial region is delineated. Such a choice would change the apparent midplane cholesterol preference, obscuring the fact that much of it is driven by domain boundaries. Finally, we mention curvature in the bilayer, which may be noted in the Table of Contents figure. Although the highly registered bilayers are approximately flat, we observe significant curvature developing in the highly antiregistered systems. In each case, we find the Lo domain to have negative curvature and the Ld domain positive curvature. This may reflect the intrinsic curvature of the prevalent lipids in the Martini model. Although a registered system would not allow the domains to bend in this way, having a domain with complementary intrinsic curvature apposed allows for curvature to develop and possibly to promote antiregistration in the first place. Registration is frequently presumed to be the default or near-universal behavior of compositionally symmetric phaseseparated bilayers. Our work and that of others17,18 show that both registration and antiregistration can depend upon the composition of lipids in the bilayer. In a three-component system, both lengthening the acyl chains of the high-Tm lipid and increasing the presence of cholesterol can turn antiregistration into the favored state. This underscores the importance of carefully selected compositions for computational and experimental research that attempts to model natural systems. Even within the two-phase liquid−liquid coexistence region of the phase diagram, other physical properties of the system can undergo a transition, even as phase of matter remains constant. A future extension of this work should consider the impact of compositional asymmetry on interleaflet interactions. In a more representative mixture, where the cytoplasmic leaflet consists primarily of phosphatidylethanolamines and phosphatidylserines along with cholesterol, registration as we define it here cannot exist, as such mixtures do not exhibit the same liquid−liquid phase coexistence that requires high-T m phosphatidylcholines or sphingomyelins. However, the idea that an exoplasmic leaflet domain could induce either like or unlike behavior in the other leaflet deserves further study. At the same time, the mechanism for this interaction may require reconsideration, as an asymmetric bilayer will have a lesser, or at least different, penalty for hydrophobic mismatch. More broadly, our study sheds light on the topic of interleaflet interactions and communication across the plasma membrane. Registration and antiregistration demonstrate that rearrangement of lipids in one leaflet can change the other leaflet, thereby transmitting a signal between the inside and outside of the cell. This signaling between a cell and its surroundings may be transmitted directly by the membrane itself, and cholesterol may play a key role in mediating that signal. The Supporting Information presents additional data on the 310 K simulations, revealing highly similar results to the 295 K simulations, with some variations for 35 mol % cholesterol systems.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.8b03949. Experiments at 310 K and presentation of autocorrelation data, plus additional analysis of experiments at 295 K (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: +1 (607) 255-4744. ORCID
Michael D. Weiner: 0000-0002-7601-0702 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by funding from the U.S. National Science Foundation (MCB-1410926) and the U.S. National Institutes of Health (GM105684) to G.W.F. This work used the Extreme Science and Engineering Discovery Environment (XSEDE), 43 which is supported by National Science Foundation grant number ACI-1548562, on Bridges at the Pittsburgh Supercomputing Center (TG-MCB130010 to G.W.F.). M.D.W. was supported by NIH Training Grant 1T32-GM08267. The authors thank Peter Olmsted, John Williamson, Edward Lyman, and David Ackerman for useful conversations.
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REFERENCES
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CONCLUSIONS Our studies make use of registration in symmetric bilayers as a tool to reveal the less-understood coupling interactions 8199
DOI: 10.1021/acs.jpcb.8b03949 J. Phys. Chem. B 2018, 122, 8193−8200
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DOI: 10.1021/acs.jpcb.8b03949 J. Phys. Chem. B 2018, 122, 8193−8200
