Comparison of Cholesterol and 25-Hydroxycholesterol in Phase

Sep 4, 2014 - Langmuir monolayer studies combined with fluorescence microscopy provide powerful insights into the phase behavior of cholesterol and ...
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Comparison of Cholesterol and 25-Hydroxycholesterol in PhaseSeparated Langmuir Monolayers at the Air−Water Interface Benjamin L. Stottrup,*,† Luis H. Hernandez-Balderrama,† Joan C. Kunz,‡ Andrew H. Nguyen,†,‡ and Benjamin J. Sonquist§ †

Department of Physics, ‡Department of Chemistry, and §Department of Biology, Augsburg College, Minneapolis, Minnesota 55454, United States ABSTRACT: Langmuir monolayer studies combined with fluorescence microscopy provide powerful insights into the phase behavior of cholesterol and cholesterol analogue/phospholipid monolayer systems at the air−water interface. These studies have established the ability of cholesterol and similar molecules to condense the average molecular area of the monolayer as well as to laterally organize the monolayer into sterol-rich and sterolpoor regions. Oxysterols are one class of molecules that deserve particular attention due to their metabolic and physical effects on the membrane and the functioning of mammalian cells. We systematically explore the miscibility of 25-hydroxycholesterol (25OH) with 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC) in lipid monolayers. Like cholesterol, the 25OH/DMPC system exhibits phase separation; however, due to the difunctional nature of 25OH, there are significant differences. Using fluorescence microscopy and traditional Langmuir techniques, we investigate the average molecular packing and micron scale phase separation within a 25OH/phospholipid monolayer. We present evidence of the nucleation and growth of domain formation, the expansion of the monolayer induced by 25OH, and a model to describe our results. We conclude that 25OH and other similar hydroxysterols provide a useful and independent test of cholesterol’s behavior within monolayer leaflets.

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introduction to this field, see the reviews of Möhwald and McConnell.1−3 In addition to traditional pressure−area studies, experimental approaches using microscopy (Brewster’s angle and fluorescence) as well as theoretical investigations proved important in understanding the competition of forces that stabilizes the resulting nanometer and micron scale domains with distinct chemical composition from the background phase.4 In the past decade, monolayer studies have been complemented by investigations of model lipid bilayer systems,5−7 creating intense interest in the use of these model systems to mimic and engineer mechanisms of lateral organization. An important area of study is the role that cholesterol analogues and precursors (broadly termed sterols) play in the biophysical behavior of the cell membrane. Cholesterol resulted from an evolutionary process to refine the physical properties of biological membranes.8−11 Cholesterol increases the order of acyl chains packed against it and decreases the overall order of phospholipids within a membrane. This results in a modulation of membrane permeability, elasticity, and compressibility.12 With interest in the “raft” hypothesis, increased attention has been focused on cholesterol’s ability to laterally organize

INTRODUCTION The machinery of biological membranes includes a selfassembled lipid bilayer formed through the amphiphilic interactions of lipids with water. In addition to providing a barrier between a cell/organelle and its surroundings, the membrane is responsible to regulate the flow of material and signaling across the two-dimensional interface. An understanding of the interactions between this system’s components is an important goal of membrane biophysics and drives model membrane studies. Within the membrane, the diversity of phospholipids that carry out these various functions is apparent. However, cholesterol stands out singularly in its presence and abundance. Every mammalian membrane contains cholesterol, and this cholesterol within membranes is subject to oxidation (both enzymatic and nonenzymatic). Model systems (in vitro) have been employed to gain an understanding of the roles these sterols play within the membrane by elucidating their biochemical and biophysical properties. Our work utilizes the well-defined compositions of self-assembled Langmuir monolayers at the air−water interface to investigate the mixing of phospholipids and other sterol molecules as a model for membrane behavior. Investigations of (chole)sterol/phospholipid mixtures at the air−water interface have deepened our understanding of fundamental physics in two-dimensional systems as well as provided insights into the biophysical role of cholesterol. For an © 2014 American Chemical Society

Received: July 2, 2014 Revised: September 3, 2014 Published: September 4, 2014 11231

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data of the phase fraction and morphology from fluorescence microscopy. Finally, we propose a mechanism for the behavior of 25OH/DMPC monolayers.

regions of distinct chemical composition within the membrane.3,13 Cholesterol is not unique in its membrane altering properties. By contrasting the phase behavior of cholesterol with molecules of slightly different molecular structure, we may tease out features of cholesterol’s own structure which allow it to function optimally within the cell membrane.11 Different scientific approaches are taken to study the importance of one sterol over another or the relative importance of sterols to that of cholesterol. Some approaches focus on structural similarity to cholesterol.14 Others focus on sterols like lanosterol and ergosterol which are naturally occurring.15,16 Langmuir monolayer studies reflect a diversity of perspectives.17−20 Side-chain oxysterols are particularly important and have attracted special attention; see reviews by Brown and Olsen.21,22 Oxysterols, the oxygenated derivatives of cholesterol, are produced naturally by both enzymatic and nonenzymatic (air oxidation, heat, ionizing radiation, oxidizing agents) reactions.23 They are known to play an important role in the regulation of cholesterol homeostasis through several mechanisms (e.g., gene expression levels and the regulation of exchange between the endoplasmic reticulum (ER) and plasma membrane).22 Here we focus on the behavior of 25-hydroxycholesterol (25OH). The structure of 25OH is identical to cholesterol with the addition of a hydroxyl group at the 25-position, making 25OH a dihydroxy sterol. In vivo, like other oxysterols, 25OH is crucial to the transfer of cholesterol between the ER and the plasma membrane.24 Additionally, 25OH enhances monocyte adherence to human aortic endothelial cells and has been linked to cardiovascular diseases.23,25 In vitro, experimental studies and molecular dynamics simulations have focused on the range of orientations 25OH takes within the lipid membrane and the potential perturbation in membrane physical properties. Experimental studies have shown that low concentrations of 25OH increase the permeability of the membrane to glucose26 and also promoted the formation of immiscible phases within lipid bilayers.27 Recent molecular dynamic simulations report 25OH c an expand POPC (1-palmitoyl-2-ole oylphosphatidylcholine) lipid bilayers at low concentrations.28 25-Hydroxycholesterol has been used as a model oxysterol in several previous monolayer studies.17−20 Independent experiments have reported that 25OH is responsible for both a condensing effect similar to that of cholesterol26 and an expansion of the monolayer at low surface pressures.17 It has also been reported that the modulus for monolayers of 25OH and POPC is generally less than monolayers of cholesterol and POPC (except at 25OH mole fraction 0.7), suggesting that 25OH forms more elastic monolayers.20,29,30 25-Hydroxycholesterol spontaneously transfers from monolayer to subphase vesicles at rates substantially faster than cholesterol, in the absence or the presence of nonspecific lipid transfer protein.25 The present work seeks to explore in more detail the phase behavior of 25OH/DMPC monolayers and contrast that with the phase behavior of cholesterol/DMPC monolayers. At the air−water interface, 25OH both mirrors and contrasts the behavior of cholesterol.17−19 It has been previously shown that 25OH exhibits unique phase behavior when combined with phospholipids in the lipid membrane.19 However, no formal study focusing on the phase behavior and morphology at the air−water interface has been done. Here we attempt to provide a more complete understanding of 25OH/DMPC monolayer systems using the experimental measurements of pressure- and potential-area isotherms as well as qualitative and quantitative

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METHODS AND MATERIALS Isotherms. Experiments were carried out at the air−water interface using either a Nima 612D or a LL Langmuir film balance (Coventry, England). No appreciable or systematic differences were observed between the two instruments. Both systems were housed on vibration isolation tables and enclosed in home-built Faraday cages. Temperature control was provided by a VWR circulating water bath, and temperatures were held at 22.5 ± 0.5 °C. Surface potential measurements were made using a Trek 320C electrostatic voltmeter (Medina, NY) interfaced directly to the Nima IU4 control box. Unless otherwise noted, the 320C response rate was set to slow to decrease the noise in the signal. All isotherms were logged using the Nima software and, unless otherwise noted, were carried out during compression at a speed of 50 cm2/min. Microscopy. An Olympus fluorescence microscope was coupled to the Langmuir trough system as previously described.31,32 A small fraction of Texas Red DHPE, 1 mol %, was incorporated into the monolayer. Previous studies have indicated that fluorescent probe concentrations between 0.2 and 2.0 mol % do not significantly alter the phase behavior.33 However, the incorporation of a fluorescent probe has been observed to shift miscibility transition temperatures in ternary bilayer systems as observed with NMR.34 Images. Images were recorded with a cooled CCD Retiga EXi 1394 camera from QImaging (Surrey, BC, Canada) coupled to an upright Olympus BX-FLA fluorescence microscope positioned above the Langmuir trough using a custom mount. Images were collected using 2 × 2 binning resulting in an image size of 696 × 520. Unless otherwise noted, all imaging data were based on a representative sample of at least five images. Photoshop was used to ensure there was no overlap between images analyzed and hence no multiple counting of domains for size distribution or area fraction measurements. Images were processed using ImageJ (NIH Image) to convert to binary, measure size distributions, and assess the area fraction of dark and bright phase. Size distribution histograms were plotted and analyzed using Matlab. Materials. All lipids were purchased and used without further purification. Phospholipids were obtained from Avanti Polar Lipids (Alabaster, AL), 25-hydroxycholesterol was purchased from Steraloids (New Port, RI), and Texas Red DHPE was purchased from Invitrogen (Carlsbad, CA). Lipids were suspended in chloroform and deposited at the air−water interface using a Hamilton Syringe (Reno, NV). An evaporation time of 10 min was waited before experiments began. All experiments were completed within an hour after deposition to limit the effects of photo-oxidation.35 All experiments were performed on phosphate-buffered saline (PBS) subphases made through a dilution of a Mediatech stock solution (Manassas, VA) with purified water at 18 MΩ·cm using a Direct Q3 filtration system (EMD Millipore Corporation, Billerica, MA).

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RESULTS In lipid monolayers the behavior of cholesterol is easily observed using fluorescence microscopy to see phase separation and through the condensing effect recorded in isobaric cuts 11232

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pressure from one phase to two phases at low pressures and the pressure at which the system transitions from two phases to one at high pressures. The transition pressures are generally higher than what has previously been reported for the 25OH/ DPPC system and extend to higher sterol concentrations. However, DPPC has a phase transition of its own at room temperature, and the novel phase behavior might have originated in part due to the LE/LC transition of the DPPC system itself.43 In the case of DMPC, the phospholipid is entirely in a liquid expanded phase, making it a more suitable system for study. The data from the lower transition of Figure 1C (SQUARES) comes directly from the pressure−area isotherms shown in Figure 2. Here, pressure−area isotherms were

taken from pressure−area isotherms. Using these tools, a model for the packing/mixing behavior of cholesterol/PC (or SM) has been developed and phase diagrams mapped by McConnell and colleagues.3 One variation on the phase diagram proposed by McConnell is Figure 1A.36 In the well-investigated

Figure 1. Unique phase behavior of 25OH: The phase behavior of a 25OH/phospholipid system is significantly different from the phase behavior of the more commonly investigated cholesterol/phospholipid system. (A) Cartoon sketch of a miscibility phase diagram for cholesterol/phospholipid mixtures as observed by fluorescence microscopy36 from 0 to 100 mol %. At low cholesterol concentrations and low pressures the monolayer exhibits two phases in coexistence. (B) Cartoon sketch of the miscibility phase diagram for a 25OH/ phospholipid monolayer (25OH concentrations from 0 to ∼40 mol %). At low pressures the monolayer appears homogeneous; upon compression a second phase appears and grows with continued compression.19 (C) Real data for transition pressures in the 25OH/ DMPC monolayer system. Lower transition pressures are identified using fluorescence microscopy or from pressure−area isotherms equivalently.

cholesterol/phospholipid (and SM lipid) monolayer systems,30,35,37−39 the two-phase region is observed to extend from a monolayer surface pressure of 0 mN/m through a transition to a single miscibility phase at higher pressures. Similar miscibility behavior has been observed in several sterol/ phospholipid systems,19,40 and several studies have extended this work to bilayer systems.27,41,42 Mixed monolayers containing 25OH and phospholipids exhibit similar phase separation measured by fluorescence microscopy. Figure 1B illustrates a phase diagram that has previously been reported for related 25OH/DPPC monolayers.19 We have systematically studied the phase diagram for 25OH/DMPC systems, and the results are shown in Figure 1C. In contrast to a cholesterol/phospholipid system mixed monolayers of 25OH/DMPC are homogeneous at low pressures. Upon compression, a second dark phase forms in the monolayer; the dark fraction of this phase grows with increasing compression until transitioning to a single phase at high pressures. The data in Figure 1C show the transition

Figure 2. (A) Pressure−area isotherms for binary mixtures of 25OH/ DMPC. 25OH concentration increases from right to left in increments of 5 mol % (10, 15, 20, 25, 30, 35, and 40). Inset highlights the kink (arrows) in the pressure−area isotherm for compositions of 15 and 20 mol % 25OH otherwise obscured in the main figure. (B) Inverse compressibility as a function of molecular area for compositions of 15 and 25 mol % 25OH. (C) Plot of measured surface potential and surface pressure vs average molecular area for 30 mol % 25OH. Note the correlation of the change in potential with the pressure−area isotherm kink. 11233

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collected for binary mixtures of 25OH/DMPC for compositions from 10 to 40 mol % 25OH. Isotherms in Figure 2 are a best collection taken from multiple isotherms run for each lipid composition (at least three isotherms per concentration and from two different samples). As the mole fraction of 25OH increases, the molecular area decreases at higher pressures. A “kink” was observed in each isotherm, which is highlighted in the inset of Figure 2A. The onset of the two phase region coincides with the “kink” pressure, which is observed simultaneously by fluorescence microscopy. The surface pressure at which the kink occurs decreases with increasing 25OH mole fraction, until it is no longer observable in the 40% or higher percent 25OH monolayers. A striking difference between the cholesterol and 25OH system is the observation of this kink. In the 25OH/DMPC system the kink observed in the pressure−area isotherm corresponds directly to the first appearance of a phaseseparated system. Figure 2B shows the inverse compressibility for compositions of 15 and 25 mol % 25OH. Inverse compressibility is calculated from pressure area isotherms by taking the first derivative, C−1 = −1/A(dP/dA). The presence of the kink is confirmed by the striking shift in slope on the inverse compressibility plot. We hypothesized that the observed kink in the pressure−area isotherm should similarly be observed in the surface potential− area isotherm. We coupled a surface potential sensor to the Langmuir trough to enable simultaneous measurements of surface potential and pressure−area isotherms. The result is displayed in Figure 2C, where a change in surface potential is seen to accompany the kink in the surface pressure. Further investigations of the pressure−area isotherm indicate other important differences between 25OH and cholesterol in phospholipid monolayers. Cholesterol and other sterols have been known to create a significant condensing effect within the monolayer.3 These sterols include mono-25OH, a molecule with a single hydroxyl group located on the sterol’s tail20 (note that this molecule does not have a hydroxyl group at the third carbon position, as observed in cholesterol). With only one hydroxyl group per sterol, the location of the hydroxyl group influences the condensing effect, with cholesterol showing a greater condensing effect than mono-25OH; but both sterols show a condensing effect.20 In general, greater condensation is observed with increasing cholesterol concentration20,29,30 and greater condensation with saturated lipid chains in the PC component of the mixture.29,30,44 In contrast, the difunctional 25OH shows expansion in the monolayer at pressures below the kink, as shown in Figure 3. Isobaric cuts at pressures of 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 mN/m were taken from the pressure−area isotherms in Figure 2A and are displayed in Figure 3 as area vs composition plots. The isobaric cuts are increasing in pressure from top to bottom in Figure 3. At pressures above the “kink” pressure observed in Figure 2, the system shows an expected decrease in average molecular area, as observed in cholesterol−phospholipid systems (the shaded area in Figure 3). At pressures below the “kink” pressure, the monolayer clearly exhibits area expansion not observed in cholesterol−phospholipid systems. Data from the pressure−area isotherms clearly indicate two significant features that are present in the 25OH system and not the cholesterol system: area expansion at low pressures in a one-phase region and a kink in the pressure−area isotherm marking the transition to a two-phase region observed with fluorescence microscopy. These features suggest a significantly

Figure 3. Area expansion at low pressures: Isobaric cuts were taken from pressure−area isotherms in Figure 2 at pressures of 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 mN/m (●, □, ×, +, ∗, ◇, ▽, ☆, and +, respectively). Below the kink the monolayer exhibits an area expansion in contrast to a cholesterol phospholipid system; see inset of Figure 2. Shaded area highlights pressures and compositions when the monolayer is in a twophase region.

different behavior of 25OH within the model membrane. This data suggest the diol 25OH lays flat to the monolayer, standing up at the kink pressure and compressing into DMPC’s hydrophobic tails at increasing pressures until the system transitions to a single phase.17,20,22 Results from fluorescence microscopy studies likewise show differences when comparing the behavior of 25OH to cholesterol. Qualitatively, it appears that the morphology of the systems is different (Figure 4). The left panel confirms the

Figure 4. Domain morphology: the contrasting nature of the phase transitions in the 25OH and cholesterol system is also illustrated using fluorescence microscopy images. The left panel is a monolayer image of cholesterol/DMPC (30:70) at a surface pressure of ∼5 mN/m. The right panel is a monolayer of 25OH/DMPC (30:70) at pressures of ∼4 mN/m. Scale bar, lower right, is 20 μm.

often reported morphology of a two-phase cholesterol/lipid system. Domains appear within domains, and the monolayer resembles a phase-separated system that has gone through spinodal decomposition. However, the 25OH/DMPC monolayer exhibits a somewhat different morphology, as captured in the right panel of Figure 4. Notably, there is considerably less variation in the domain sizes within the 25OH/DMPC monolayer. A second difference qualitatively observable in Figure 4 and quantified in Figure 5 is the pressure dependence of the phase fraction of the 25OH/lipid monolayers. While a cholesterol/ lipid system maintains phase fraction that is constant with changing pressure, the 25OH system increases in dark fraction as the monolayer is compressed. This effect is present in all 25OH compositions we investigated. The fraction of dark phase was determined from the images recorded while collecting the isotherms using ImageJ. The fraction of dark phase is plotted against surface pressure in Figure 5, showing an increase in the dark fraction with increasing pressure. As 11234

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DISCUSSION

Despite the similarity in appearances when imaged using fluorescence microscopy, the results above indicate a clear difference between phospholipid monolayers containing cholesterol and 25OH. In typical cholesterol/phospholipid systems a single-phase monolayer separates into two phases as the area per molecule expands. This process occurs rapidly through spinodal decomposition. The contrast between the two phases is highlighted through the uneven partitioning of a fluorescent probe. Varying the composition of the monolayer reveals that the dark phase is cholesterol-rich.33 On expansion from a tightly packed state, the 25OH system behaves similarly and exhibits a similar rapid transition from single homogeneous state to a two-phase state (Figure 6). Likewise, Figure 5 indicates that the dark phase is rich in 25OH, like the cholesterol system. The similarities between the two systems end here. In the cholesterol system, as the monolayer continues to expand and the pressure decreases, the monolayer morphology remains largely unchanged. However, the 25OH/phospholipid monolayer shows a steady decrease in the amount of dark phase with decreasing pressure. Particularly clarifying is the data presented in Figure 5. Here the area fraction of the 25OH/DMPC monolayer steadily shifts phase between dark and bright regions. The dark phase entirely vanishes at pressures below the kink transition pressure, as can be seen from 10 mol % 25OH (diamonds, Figure 5). To explain the differences between the two systems, we propose the following model: at the air−water interface the 25OH sterol is positioned flat against the water surface at low pressures. As the sterol/lipid monolayer is compressed, the average molecular area available decreases and the monolayer becomes harder to compress. This can be seen from a plot of inverse compressibility, which increases as molecular area is reduced (Figure 2B). At the kink, we hypothesize that 25OH molecules begin to stand upright. This conversion continues until all of the 25OH molecules are standing upright. While 25OH is likely to have a preferred vertical orientation at the air−water interface both orientations have been demonstrated possible in previous experiments.20 Hence, the reorientation process is likely to be statistical in nature. Unfortunately, our experimental tools are not sensitive to the statistical distribution of 25OH vertical orientations at the present time. We have highlighted this ambiguity in our table of contents figure. Work by Janout and colleagues on a related system strongly supports our hypothesis.20 In that study, the hydroxyl group has been removed from the 3-position of the steroid ring and been placed at the 25-position of the steroid tail, mono-25OH. The sterol remains monofunctional. The researchers found that the mono-25OH system was more compressive than the cholesterol system alone. The increased flexibility/compressibility of the tail over the steroid ring was offered as a plausible explanation. Both the cholesterol and the mono-25OH system exhibit an area condensation or deviation below the line of ideal mixing. The structural contrast between 25OH and mono25OH provides a test of consistency for our model. At very low surface pressures, 25OH is anchored to the monolayer at two points, and the flat orientation expands the monolayer. This is consistent with the results presented in Figure 3 and consistent with Gale et al.18 We further show that at increased surface pressure the expansion effect on the monolayer is reduced and ultimately mitigated. At the molecular area of the kink the 25OH molecule transitions to an orientation that is vertical

Figure 5. As the monolayer is compressed, the domains grow until the upper transition pressure is reached. The fraction of dark phase is plotted against surface pressure. Compositions of 10, 25, and 40 mol % of 25OH are included. Direct comparison at any surface pressure demonstrates that an increase in sterol concentration increases the observed dark fraction.

expected, the dark fraction also increases with increasing 25OH mole fraction indicating that, like cholesterol, the dark phase is rich in sterols. This can be seen by comparing 25OH concentrations at constant pressure (vertical lines on plot). The circular domain shapes of Figure 4 lead to the assumption that both dark and bright phases are in a fluid state. We test this hypothesis by observing the fluctuations in phase boundaries as the system passes through the upper phase transition. Figure 6 illustrates that the boundaries rapidly fluctuate and the monolayer contains coexisting liquids.

Figure 6. Liquid−liquid coexistence: the dark and bright phases are both fluid. This is observed most clearly near the upper transition where both phases can be observed to fluctuate as the monolayer passes through the transition. The highlighted region is the same in panels A−D; however, the overall shape changes. Images are taken from a monolayer held at 22 mN/m and are separated in time by 0.5 s. Translational motion is the result of the monolayer’s lateral motion below the objective. 11235

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within the monolayer. In this orientation some of the flexibility of the isooctyl chain lying flat against the air−water interface is lost, and the monolayer is less flexible (Figure 2B). This increase in compressibility is consistent with molecular dynamic simulations in lipid bilayers where 25OH is predicted to aggregate.45 The model we propose is also consistent with our results from fluorescence microscopy. At low pressures, prior to the reorientation of 25OH, a homogeneous lipid environment is presented to the Texas Red DHPE molecule and the fluorescent probe is uniformly distributed. It is only after the 25OH begins to stand up and align itself within the monolayer that we observe a two-phase system consistent with a cholesterol-like molecule. The compression of the monolayer dictates the fraction of laying flat against the air−water interface vs those molecules that have stood up. This continuous reorientation of the monolayer explains the steady increase of dark phase as the monolayer is compressed. The fluorescence microscopy studies presented here also provide new insight into work on similar systems by Kauffman and colleagues.17 In those studies, experiments were carried out with mixed monolayers of 25OH and POPC (1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine). Without the benefit of a morphological view of the monolayer (which our microscopy studies provide) the significance of the kink was barely mentioned, and POPC was reported to be miscible with 25OH.17 To test our model, we coupled a surface potential sensor to the Langmuir trough so simultaneous measurements of surface potential and pressure−area isotherms could be obtained. We hypothesized that the kink observed in the pressure−area isotherm would similarly be observed in the potential−area isotherm. This would indicate a reorientation in the dipole moment at the air−water interface resulting from 25OH standing vertical. At the air−water interface, measurements of surface potential are a proxy for measurements of the dipole moment. The dipole moment at the air−water interface can be considered to have origins in three dipole moments of the monolayer. The first is the reorientation of water molecules (and hence their dipole moment) by the monolayer itself. The second is the contribution of the dipole moment of the head itself. Third, the dipole moment of the hydrophobic tails can contribute to the overall dipole moment of the monolayer. Testing our hypothesis though surface potential measurements revealed somewhat mixed results. This is likely due to the sensitivity of surface potential measurements as well as the strength of the signal.17 As can be seen from Figure 2C, a discontinuity in the potential−area isotherm often accompanies the kink in the pressure−area isotherm. However, noise in the system often overwhelms the signal.

25OH upon compression. The 25OH system then provides an independent test of our understanding of phase separation in cholesterol/phospholipid monolayer systems. The second hydroxyl group becomes a mechanism by which, when the monolayer is compressed, we can turn on the cholesterol-like behavior of the molecule. Tuning this behavior through movement of the 25OH molecule may be a useful way to independently test the role of the steroidal ring structure within the monolayer and suggests an avenue for future study. The results above suggest two important areas for future membrane studies. First, how is the phase behavior of 25OH distinct from that of cholesterol, and what does this mean? Second, might we look to the structure of the 25OH sterol itself to build insight into the behavior of side-chain oxysterols?

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; phone (612) 330-1035 (B.L.S.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Ravi Tavakley and undergraduates Jessica McKay, Eleni Beyene, and Emil Eldo for supporting experiments and work in the lab. This project was supported by the National Science Foundation awards DMR 1207544 and MRI 1040126 as well as private support from the Eppley Foundation for Research, Dixie Shafer and Augsburg College’s office of Undergraduate Research and Graduate Opportunities, and Dean and Amy Sundquist.

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REFERENCES

(1) McConnell, H. Structures and Transitions in Lipid Monolayers at the Air-Water-Interface. Annu. Rev. Phys. Chem. 1991, 42, 171−195. (2) Möhwald, H. Phospholipid and Phospholipid-Protein Monolayers at the Air/Water Interface. Annu. Rev. Phys. Chem. 1990, 41, 441−476. (3) McConnell, H. M.; Radhakrishnan, A. Condensed Complexes of Cholesterol and Phospholipids. Biochim. Biophys. Acta, Biomembr. 2003, 1610, 159−173. (4) McConnell, H. M.; Moy, V. T. Shapes of Finite TwoDimensional Lipid Domains. J. Phys. Chem. 1988, 92, 4520−4525. (5) Dietrich, C.; Bagatolli, L. A.; Volovyk, Z. N.; Thompson, N. L.; Levi, M.; Jacobson, K.; Gratton, E. Lipid Rafts Reconstituted in Model Membranes. Biophys. J. 2001, 80, 1417−1428. (6) Baumgart, T.; Hess, S. T.; Webb, W. W. Imaging Coexisting Fluid Domains in Biomembrane Models Coupling Curvature and Line Tension. Nature 2003, 425, 821−824. (7) Veatch, S. L.; Cicuta, P.; Sengupta, P.; Honerkamp-Smith, A.; Holowka, D.; Baird, B. Critical Fluctuations in Plasma Membrane Vesicles. ACS Chem. Biol. 2008, 3, 287−293. (8) Bloch, K. The Biological Synthesis of Cholesterol. Science 1965, 150, 19−28. (9) Bloch, K. Sterol Molecule: Structure, Biosynthesis, and Function. Steroids 1992, 57, 378−383. (10) Miao, L.; Nielsen, M.; Thewalt, J.; Ipsen, J. H.; Bloom, M.; Zuckermann, M. J.; Mouritsen, O. G. From Lanosterol to Cholesterol: Structural Evolution and Differential Effects on Lipid Bilayers. Biophys. J. 2002, 82, 1429−1444. (11) Mouritsen, O. G.; Zuckermann, M. J. What’s So Special about Cholesterol? Lipids 2004, 39, 1101−1113. (12) Maxfield, F. R.; van Meer, G. Cholesterol, the Central Lipid of Mammalian Cells. Curr. Opin. Cell Biol. 2010, 22, 422−429.

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CONCLUSION Here we examined and compared the miscibility phase behavior of 25OH and cholesterol within lipid monolayers. Like cholesterol, 25OH monolayers undergo liquid−liquid phase separation observable with fluorescence microscopy. However, the degree to which the 25OH molecule drives this phase separation is directly dependent on the average molecular area. Further as the monolayer is compressed from large average molecular areas, the domain size grows, indicating a nucleation and growth. At smaller average molecular areas, the 25OH system behaves as expected in cholesterol. The evidence presented here supports the hypothesis that differences in phase behavior result from a reorientation of the difunctional 11236

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