Macroscopic and Nanoscopic Heterogeneous Structures in a Three

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Macroscopic and Nanoscopic Heterogeneous Structures in a Threecomponent Lipid Bilayer Mixtures Determined by Atomic Force Microscopy Nawal K Khadka, Chian Sing Ho, and Jianjun Pan Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b02863 • Publication Date (Web): 27 Oct 2015 Downloaded from http://pubs.acs.org on October 30, 2015

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Macroscopic and Nanoscopic Heterogeneous Structures in a Three-component Lipid Bilayer Mixtures Determined by Atomic Force Microscopy Nawal K. Khadkaa, Chian Sing Hoa, and Jianjun Pan*

Department of Physics, University of South Florida, Tampa, FL 33620, USA a

These authors contributed equally to this work

*To whom correspondence should be addressed: Jianjun Pan, Ph.D. Email: [email protected]

Keywords: membrane rafts, phase coexistence, tie-line, critical point, lipid bilayer

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Abstract Much of lipid raft properties can be inferred from phase behavior of multicomponent lipid membranes. We use liquid compatible atomic force microscopy (AFM) to study a threecomponent system composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), egg sphingomyelin (eSM), and cholesterol. Specifically, we obtain macroscopic and nanoscopic heterogeneous structures in a broad compositional space of DOPC/eSM/cholesterol (23oC). In the macroscopic liquid coexisting region, we quantify area fraction of the coexisting phases and determine a set of thermodynamic tie-lines. When lipid compositions are near the critical point, we obtain fluctuation-like nanoscopic structures. We also use AFM height images to explore the hypothetical three phase coexisting region. Finally, we use fluorescence microscopy to compare the phase behavior from our AFM measurements to that in free-floating giant unilamellar vesicles (GUVs). Our results highlight the role of lipid composition in mediating lipid domain formation and stability.


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Introduction Lipid rafts were conceptualized as an organization principle to compartmentalize membrane constituents, thus providing a localized environment for optimal functions involving specific lipids and proteins. In particular, lipid rafts have been proposed to play important roles in membrane trafficking, cell signaling, pathogen entry and egress, and amyloid deposition, to name a few. It is the contemporary consensus that the formation and stability of lipid rafts are governed by specific lipid-lipid and lipid-protein interactions 1. The latter often involves proteins with specific motifs that have large binding affinity to lipid components. Structural compatibility between proteins’ transmembrane segment(s) and membrane hydrophobic layer serves as an additional mechanism to mediate lipid-protein interactions 2, 3. The close interplay of proteins and lipids, therefore, is expected to alter membrane heterogeneity 4, and consequently protein function. In contrast to chemical and structural specificity that controls lipid-protein interactions, thermodynamics are the fundamental principles that dictate lipid-lipid interactions. Indeed, studies of model lipid membranes employing a few lipid species have contributed greatly to enhancing our understanding of lipid raft properties in living cells 5, 6, 7. One intriguing finding in lipid membrane research is that micron-sized raft-mimicking domains are readily formed by mixing a high-melting (high-Tm) lipid, a low-melting (low-Tm) lipid, and cholesterol (Chol). By exploring lipid mixtures in a broad compositional space, phase diagrams of many three-component systems have been reported 8, 9. Although phase boundaries are often susceptible to experimental techniques used for phase diagram construction, the reported phase diagrams are informative to illustrate qualitative lipid-lipid interactions. On the other hand, phase boundaries only indicate the emergence or disappearance of certain lipid phase when the boundaries are crossed over. They do not provide quantitative information of lipid composition in each phase, which is crucial for understanding lipid-lipid interactions. Tie-lines in phase coexisting region are special lines with their endpoints located at phase boundary. For any point along a tie-line, lipid compositions of coexisting phases are determined by tie-line endpoints. Moreover, mole fractions of the coexisting phases at any point along a tieline are determined by the distances of the tie-line endpoints to that point (i.e., Lever rule). Collectively, tie-lines provide critical information of lipid composition and phase fraction in phase coexisting region. Several experimental approaches have been developed to estimate tieline orientation and/or location: (1) fluorescence microscopy examines area fraction of coexisting phases in giant unilamellar vesicles (GUVs). Assuming lipid areas are known in each phase, mole fractions of coexisting phases can be inferred. Tie-lines can then be determined by model fitting to mole fractions of coexisting phases 10; (2) Förster resonance energy transfer (FRET) signal is dependent on partition coefficients of donor and acceptor probes, as well as mole fractions of coexisting phases. Model fitting to FRET signal has been used to obtain mole fractions of coexisting phases, which can be used to determine tie-lines 11, 12; (3) NMR 13, 14, 15 and ESR 16 spectroscopies measure lipid composition dependent spectra. Assuming that the spectrum along a tie-line is a linear superimposition of the two spectra corresponding to tie-line endpoints, tie-lines can be determined by model fitting to composition dependent spectra; and (4) repeat spacing of multilamellar vesicles (MLVs) with different water contents can be measured by X-ray diffraction. Hydration curves of lamellar repeat spacing can be used to estimate tie-line orientation 17. It is noteworthy that each method has its own disadvantages. For example, fluorescence microscopy suffers from compositional uncertainty of GUVs and artifacts


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associated with fluorescent probes. FRET, NMR, and ESR spectroscopies rely on indirect signals caused by membrane heterogeneity. Different treatments of contributions that are dependent or independent on membrane heterogeneity can result in large uncertainties of phase contents. For X-ray diffraction, only tie-line orientation can be inferred. In addition, X-ray method can fail for some compositions even when phase coexistence occurs. In view of this, alternative methods are constantly sought after for accurate tie-line determination, as well as atomic-level domain structures. Liquid compatible atomic force microscopy (AFM) has long been used to study lipid membrane structural and mechanical properties. In particular, AFM based force spectroscopy has been broadly used to assess membrane mechanical stability 18 and membrane bending modulus 19. In addition, surface sensitivity renders AFM very useful in studying lipid membrane remodeling after being exposed to foreign molecules 20. In the field of lipid membrane lateral heterogeneity, AFM provides a superb platform to visually detect membrane heterogeneous organization down to nanometer-scale. Another unique advantage of AFM is that it senses membrane heterogeneity by detecting height contrast across the membrane surface; therefore, AFM is not susceptible to artifacts associated with external probes. Similar to fluorescence microscopy, AFM directly visualizes membrane fractions of different phases, albeit with better resolution. This makes AFM a suitable tool to determine tie-lines 21. In this paper we use AFM to study composition dependent heterogeneous structures of lipid bilayers composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), egg sphingomyelin (eSM), and cholesterol. Note that eSM is a sphingomyelin (SM) mixture containing 86% 16:0, 6% 18:0, 3% 22:0, 3% 24:1, and 2% unknown species. Therefore, the system we report here is a quasi-ternary mixture. We also use fluorescence microscopy to compare phase behavior in solid supported bilayers and in free-floating GUVs. All lipid compositions are given as mole ratio, fraction, or percentage.

Materials and Methods DOPC, eSM, cholesterol, and rhodamine-DPPE (1,2-dipalmitoyl-sn-glycero-3phosphoethanolamine) were purchased as lyophilized powders from Avanti Polar Lipids (Alabaster, AL). Stock solutions were prepared by dissolving lipids in organic solvents (chloroform or chloroform/methanol). AFM experiment. AFM measurements were performed at 23oC. Lipid mixtures were prepared by mixing appropriate ratios of stock solutions in glass test tube, followed by brief vortexing. Organic solvents were removed by a gentle stream of argon gas using a 12-position N-EVAP evaprator, and then placed under vacuum for > 2 h. Lipid dry films were hydrated and ultrasonicated using a Sonic Dismembrator operated at 40 W with total duration of 12 min. The obtained small unilamellar vesicles (SUVs) were centrifuged, and characterized by dynamic light scattering using a Zetasizer Nano S (Malvern Instruments, Worcestershire, UK). The ultrasonication prepared SUVs have an average diameter of 30 nm. AFM images were acquired using a Multimode 8 AFM (Bruker, Santa Barbara, CA) operated at the PeakForce quantitative nanomechanics (QNM) mode. In this mode, the probe oscillates at a frequency (e.g., 2 kHz, much lower than the probe’s resonant frequency) while it scans across


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the sample. By adjusting the maximum normal force the tip experiences (the peak force, typically a few hundreds of pN), sample deformation depths are tightly controlled. During the scan, the tip only contacts the sample for a small fraction of the time, thus keeping the lateral force negligible. A bungee cord supported platform sitting on a vibration-isolation optical table was used to reduce environmental noise. SUVs were injected into the AFM liquid cell using a syringe pump. The liquid cell is formed by a freshly cleaved mica substrate, a liquid compatible probe holder, and a silicone O-ring. After incubation for > 30 min, either at room temperature or at 50oC using a heating accessary (Bruker model: MMHC-A60), a solid supported planar bilayer is formed by vesicle fusion22, 23, 24. Bilayers were scanned using a special Si3N4 cantilever designed to work with the PeakFroce QNM mode (Bruker model: ScanAsyst-Fluid +). Square images ranging from 500 nm to 10 µm were acquired at a scan rate of 0.5-1.0 Hz. Multiple scans were performed at different regions on the bilayer surface to gain good statistics. AFM images were exported using a Matlab utility provided by the Nanoscope Analysis software. Polynomial background was subtracted to level the image. The height difference between coexisting phases was obtained from the histogram of the height distribution. The area fraction of the Lo phase frac was determined from leveled images (10 µm×10 µm in size) by setting a threshold that is half of the height difference. frac equals to the fraction of the pixels that have height larger than the threshold. Fluorescence microscopy. GUVs were prepared using electroformation method 25, 26. Briefly, lipid films with 0.1% fluorophore rhodamine-DPPE were deposited on glass slides (ITO coated at one side), and swelled at 60oC for 2 h in 100 mM sucrose with an AC field of 1.5 V at 10 Hz. After cooling to room temperature, GUVs were suspended in 100 mM glucose and allowed to settle for 1 h. An aliquot of GUVs was transferred into a silicone-gel well, which was sandwiched by a coverslip and a glass slide. Fluorescence micrographs were acquired using an inverted microscope (Nikon Eclipse Ti-U), a CFI Super Fluor ELWD 60× objective, and an EMCCD camera (Andor iXon Ultra 897).

Results Overall phase behavior. We study composition dependent phase behavior in the quasi-ternary mixture DOPC/eSM/Chol (Fig. S1, SI). The overall phase behavior resembles that of many three-component mixtures containing DOPC 10, 12, 26, 27, 28, 29, 30, 31. Solid domains are observed in bilayers containing low fractions of cholesterol when the ratio of DOPC/eSM is ≤ 3:2. The solidcontaining phase persists to higher cholesterol fraction with increasing eSM. Interestingly, solid containing bilayers with no cholesterol can exhibit more than two types of structural features as shown in Fig. 1A. Since the mixture is expected to show gel+Ld phase coexistence. This observation highlights that the phase behavior of binary mixtures of DOPC/eSM might be influenced by the mica substrate 32. Addition of cholesterol to solid-containing bilayers yields Lo+Ld phase coexistence. The transition is identified by examining domain morphology. Compared to Lo domains, the solid domains often exhibit irregular shapes (Fig. 1). We note that the method described here is not rigorous in distinguishing the solid and Lo domains. This gives rise to uncertainty in determining the lower boundary of the Lo+Ld coexisting region. When eSM is dominant over DOPC, Lo+Ld coexistence is observed at cholesterol fractions as high as 0.45. Near the DOPC vertex, Lo+Ld


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coexistence is observed at cholesterol fractions ≥ 0.05, and a uniform phase is observed at cholesterol fractions > 0.3. A third region showing nanoscopic structures is identified near the consolute critical point. We will refer to this region as the “critical region” in the rest of the paper.

Figure 1 Domain morphology for DOPC/eSM 1:1 with different cholesterol contents (23oC). Panels A, B, and C contain islands with irregular shapes (solid+Ld coexistence), which are in contrast to those in panels D and E (Lo+Ld coexistence). Height scale indicated by the color bar at the right is 3.0 nm for A-B and 2.2 nm for C-E. Scale bars are 200 nm. Macroscopic domains. Domain morphologies in the macroscopic Lo+Ld coexisting region are illustrated in Fig. 2. Each column corresponds to a compositional trajectory with DOPC/eSM ratio fixed. At 3:1 DOPC/eSM, round shaped Lo domains are observed at small cholesterol content. The domain size first increases and then decreases with increasing cholesterol content. The bilayer eventually becomes a uniform Ld phase after crossing the phase boundary.


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Figure 2 Height images of DOPC/eSM/Chol exhibiting macroscopic Lo+Ld phase coexistence (23oC). Lipid composition is indicated by the ratio of DOPC/eSM + cholesterol mole percentage. Height scale varies from 0.6 to 1.2 nm. Scale bars are 1 µm. At intermediate ratios of DOPC/eSM (i.e., 2:1, 3:2, and 1:1), Lo domains also exhibit round shapes at low cholesterol content. (Round shapes are a result of minimizing domain interfacial energy.) Increase of cholesterol content leads to (1) larger area fraction of the Lo phase and (2) elongated shape of Lo domains. It seems that there is a threshold of frac, above which Lo domains prefer noncircular shapes. For DOPC/eSM 1:1, the elongated Lo domains merge together at frac > 0.6, and form a mesh-like network that segregates Ld domains (Fig. 2, D3 and D4). Further increase of cholesterol content along these three trajectories disrupts Lo phase, resulting in nanoscopic structures that will be discussed in the next section. At DOPC/eSM 1:2 and 1:3, a different type of morphological transition is observed. After crossing the lower miscibility boundary, large frac is immediately observed. Increase of cholesterol content causes the Lo domains to merge together. When frac reaches ~0.8, the Ld domains evolve into stripes and are segregated by the continuous Lo phase. Round shaped Ld domains are observed when frac becomes even larger. Nanoscopic heterogeneity. Nanoscopic heterogeneous structures are observed along compositional trajectories that pass through the critical region. Along each trajectory, increase of cholesterol content disrupts Lo domains by roughening domain boundary and perforating domain interior. For trajectories that pass through the critical point, there is an equal probability of forming Lo and Ld phases. Therefore, the mole fraction of the Lo phase should remain constant near the critical point. One good candidate is DOPC/eSM 3:2 (Fig. 3). It seems that the frac remains similar when the Lo domains are disintegrated into nanoscopic structures (Fig. 3D and 3E). Another evidence supporting the proximity of the trajectory to the critical point is that frac is ~0.45 when domain disintegration occurs (Fig. 3D). Considering that (1) Lo and Ld phases have equal mole fraction at the critical point and (2) Lo phase has smaller average lipid area compared to that of Ld phase due to unequal cholesterol distribution, a frac of 0.45 is consistent with the trajectory intersecting the liquid coexisting boundary at or very close to the critical point.


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Figure 3 AFM Height images for three trajectories of DOPC/eSM/Chol passing through the critical region (23oC). Lipid composition is indicated by the ratio of DOPC/eSM + cholesterol mole percentage. Scale bars are 200 nm.

Height difference. AFM probes bilayer structure in the third dimension (i.e., normal direction of the bilayer surface) that is not accessible to fluorescence microscopy. Height difference ∆h between coexisting phases can be directly determined from AFM height images without any model intervening. Height difference between coexisting phases along seven trajectories is shown in Fig. 4. For trajectories that go through the critical region (i.e., DOPC/eSM at 2:1, 12:7, 3:2, 6:5, and 1:1), when cholesterol content is low, the height difference is 1.0-1.2 nm. The height difference gradually decreases to ~0.2 nm in the critical region. We did not try to determine ∆h for bilayers with height variation less than 0.2 nm, since these bilayers no long exhibit phase coexistence, but rather compositional fluctuations (Fig. 3C). For trajectories that do not pass through the critical region, the height difference remains large even at large cholesterol concentrations (Fig. 4, DOPC/eSM at 1:3 and 1:6). Overall, the height difference between Lo and Ld phases are dependent on lipid composition. We should not attempt to assign a single height difference for a given three-component system 33.


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Figure 4 (A) Height differences between Lo and Ld phases for trajectories that intersect the critical region (i.e., DOPC/eSM at 2:1, 12:7, 3:2, 6:5 and 1:1), and for those that are far away from the critical region (i.e., DOPC/eSM at 1:3 and 1:6). (B) Corresponding lipid compositions shown in (A). Experimental temperature is 23oC. Area fraction of the Lo phase and tie-lines. Area fraction of the Lo phase frac in macroscopic Lo+Ld coexisting region is calculated from AFM height images (see Materials and Methods). The result is shown in Fig. 5. Two-dimensional linear interpolation is used to obtain the surface plot. It is clear that frac becomes smaller when the lipid composition moves toward the DOPC vertex, and larger when the lipid composition moves toward the middle of the eSM/Chol binary axis. This behavior is further illustrated by contour lines with constant frac (Fig. S2, SI). Interestingly, the contour lines with frac < 0.4 are curved toward the lower left corner, and the contour lines with frac > 0.6 are curved toward the upper right side. The contour lines are more or less linear when frac is near 0.5. Here we use a procedure reported by Dimova and coworkers to locate tie-line endpoints based on experimental frac (see SI). The result is shown in Fig. 5. Based on estimation of uncertainties associated with total lipid composition, measured phase area fraction, and area per lipid, the upper boundary of our tie-line uncertainties (i.e., location of endpoints) is ~5%. We note that larger uncertainties are associated with tie-lines near the lower boundary. Detailed tie-line parameters are listed in Table S1 (SI). Tie-line inclination angle varies between 17 and 25o. Lipid composition at tie-line endpoints indicates that Ld phase is rich in the low-Tm lipid DOPC ௅ௗ ௅ௗ ௅ௗ (ߕ஽ை௉஼ ~ 0.61-0.76) and poor in the high-Tm lipid eSM (ߕ௘ௌெ ~ 0.12-0.27). (Here ߕ஽ை௉஼ and


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௅ௗ ߕ௘ௌெ are mole fractions of the respective lipids in Ld phase. Similar notions are used for Lo ௅௢ ௅௢ phase.) Conversely, Lo phase is rich in eSM (ߕ௘ௌெ ~ 0.39-0.69) and poor in DOPC (ߕ஽ை௉஼ ~ 0.03-0.16). Along each tie-line, DOPC exhibits stronger partitioning into the Ld phase, compared to the partitioning of eSM into the Lo phase. The difference can be explained by the location of the Lo+Ld phase boundary, which is biased toward the right. ௅௢ The positive tie-line slopes suggest stronger partitioning of cholesterol into the Lo phase (ߕ஼௛௢௟ ~ ௅ௗ 0.25-0.44) than into the Ld phase (ߕ஼௛௢௟ ~ 0.06-0.26), thus highlighting preferential interactions between cholesterol and eSM 34. We also notice that the mole fraction difference of cholesterol ௅௢ ௅ௗ െ ߕ஼௛௢௟ ) remains similar (e.g., ~ 0.2) as the tile-line moves from the in the two phases (i.e., ߕ஼௛௢௟ lower to the upper boundary.

Figure 5 Surface plot of Lo phase area fraction and the determined tie-lines (23oC). The endpoints and centers of the tie-lines are indicated by green and dark circles, respectively. Compositions exhibiting nanoscopic structures are shown as square symbols. Fluorescence microscopy. To compare the phase behavior of the three-component system in GUVs and in solid supported bilayers, we use fluorescence microscopy to examine phase behavior of DOPC/eSM 3:2 with different cholesterol concentrations. At each lipid composition, variation of phase properties is observed. This includes the area fraction of the Lo phase, number of domains, and the composition at which critical fluctuations emerge or vesicles become uniform. These uncertainties are most likely due to compositional heterogeneity between different vesicles. In general, the phase behavior in GUVs is in qualitative agreement with the results from our AFM measurements (Fig. 6). For example, the area fraction of the Lo phase increases with cholesterol content (Fig. 6A-D); critical fluctuations are observed when the composition is near the critical point (Fig. 6E) 35; GUVs become homogeneous once structural features are below the diffraction limit (Fig. 6F).


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Figure 6 Fluorescence micrographs of GUVs of DOPC/eSM 3:2 with different cholesterol concentrations (23oC). Bright regions correspond to Ld phase, into which rhodamine-DPPE preferentially partition. Scale bars are 10 µm. Three-phase coexistence? Binary mixtures of low-Tm and high-Tm lipids can exhibit solid+Ld coexistence 36. Addition of cholesterol leads to Lo+Ld coexistence (Fig. S1, SI). How does the transition take place? A triangle-shaped three-phase coexisting region has been postulated between the solid+Ld and Lo+Ld coexisting regions. Previous studies mainly used spectroscopic techniques (e.g., NMR, EPR, and FRET) to explore the boundary of the hypothetical three-phase region 15, 31, 37, 38.

Figure 7 AFM height images of DOPC/eSM 1:4 as a function of cholesterol content (23oC). Solid+Ld coexistence is gradually replaced by Lo+Ld coexistence. Scale bars are 200 nm. If the three-phase coexisting region does exist, several trajectories we study should go through it. One plausible trajectory is DOPC/eSM 1:4 (Fig. 7). At 0% Chol, the solid domains are segregated by wire-shaped Ld crevices. Addition of 8% Chol widens the Ld crevices. The perimeters of the solid domains become corrugated at 12% Chol. In addition, the area fraction of the Ld phase becomes noticeably larger. It seems that the transition from 8 to 12% Chol is achieved by converting disrupted solid domains into Ld phase. The solid domains are further disrupted at 14% Chol. Within the protruded islands, regions with slightly different heights are observed (green arrow). They could correspond to the solid and Lo phases. Further increase of cholesterol to 16% results in smaller area fraction of the Ld phase. The same trend is observed at 18% Chol. It seems that Lo phase prevails once the cholesterol content reaches 16%. Together, our AFM topographic images show that the area fraction of the higher regions first decreases, then increases as cholesterol content increases. This transition is consistent with the solid phase being gradually replaced by the Lo phase. On the other hand, our data indicate that the postulated three-phase coexisting structure does not correspond to three isolated entities, such that the Lo and solid domains are surrounded by the Ld phase. The well-established phase rule states that the number of phases can only change by ±1 when a phase boundary is crossed 39. Based on this rule, there must exist an intervening region (or more than one) between the binary axis of DOPC/eSM (gel+Ld coexistence) and the Lo+Ld coexisting region. The intervening region can either be in gel+Ld+Lo phase coexistence or Ld phase only. Since we do not observe a uniform Ld phase in the intervening region along all the trajectories studied, the intervening region is expected to exhibit three-phase coexistence. Yet, our AFM height images do not clearly show three types of structural features in this intervening region. Since AFM distinguishes different phases based on height contrast, one plausible explanation is the height similarity between gel and Lo phases in the three-phase region.


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Discussion Influence of the solid substrate Solid supported planar bilayer is a valuable platform to study various membrane associated phenomena. By tailoring lipid composition in the three-component system, we observe bilayer heterogeneous structures ranging from micron to nanometer scale. One question pertaining to our study is the influence of the solid substrate40. AFM based force spectroscopy measurements revealed that there is a water layer of ~ 2-3 nm thickness residing between the substrate and the bilayer 19, 41. The absence of direct contact helps to reduce the substrate influence, and is consistent with translational diffusion measurements, which showed that lipids in liquid coexisting bilayers deposited on solid substrate are mobile, and can exchange between different phases with little energy cost 42, 43. Lipid diffusion in solid supported planar bilayers can also be observed using AFM. This is illustrated in Fig. 8, which shows two images acquired at 30 min apart. It is clear that Lo islands gradually change their shapes. In addition, islands can merge as highlighted by the green circle.

Figure 8 AFM height images of DOPC/eSM 3:2 + 20% Chol collected at two time points (30 min apart). Experimental temperature is 23oC. Scale bars are 200 nm. Two experimental observations support the validity of using solid supported bilayers to examine membrane phase behavior. Bhatia et al. used a procedure to rapidly capture GUVs onto a mica substrate using divalent cation 44. It was found that domain structures and area fractions present in free-floating GUVs are preserved in solid supported bilayers after settling. The second evidence comes from the measurement of frac, the area fraction of the Lo phase. Using confocal fluorescence microscopy, Dimova and coworkers measured frac for GUVs prepared from DOPC/eSM 1:1 + 20% Chol at 23oC. The most probable frac in their experiment is 0.3-0.4 10. The uncertainty is due to vesicle compositional heterogeneity. We obtained a frac of 0.38 at the same lipid composition, which is in good agreement with the GUV result. It seems that the solid substrate only slows the dynamics of domain coarsening, but does not change phase fractions. The impaired domain dynamics is consistent with hydrodynamic analysis, which predicts that there is a dragging force acting on membrane inclusions due to the interplay of the viscous membrane, the intermediate water layer, and the solid substrate 45. It is known that planar bilayers are not totally uncoupled from the substrate 42, 46. This is evidenced by domain shapes and sizes in our study. After loading into the liquid cell, SUVs are attracted onto the solid surface. Based on the observation that domain sizes in the planar bilayer are larger than the size of SUVs, it is conceivable that domain patches from different SUVs rapidly merge together through pathways mimicking nucleation and growth 47. Once the planar


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bilayer is formed, segregated domains diffuse and coalesce with slower rate compared to substrate-free systems 21. Substrate effect is not the only source that can antagonize domain kinetics. Domains trapped at a certain size for an extended period of time is commonly observed in free-floating GUVs. Theoretical explanations are mainly based on membrane mediated interdomain interactions 48. Substrate effect is not always undesirable. In fact, bilayer-substrate interaction may have some similarity to the coupling between plasma membrane and cytoskeleton matrix. It was postulated that lipid raft size and dynamics could be regulated by actin cytoskeleton 49, which can rapidly polymerize or depolymerize in response to stimuli. In addition, actin cytoskeleton can control raft size by forming fences with anchoring points 50. The cytoskeleton effect on bilayer phase separation has been explored by Monte Carlo simulation 51, 52. Enhanced diffusion barrier was observed for some membrane components, in addition to the disruption of long-range fluctuations and the prevention of macroscopic phase separation. The simulation also shows that cytoskeleton-bilayer coupling does not induce phases 51. This result lends additional support to the use of solid supported bilayers in our study. In accordance with the simulation, actin matrix was found to prevent macroscopic phase separation and hinder lipid diffusion in an actin supported lipid bilayer 53. Tie-lines and critical point Tie-line orientation is indicative of molecular interactions in the ternary mixture. Our tie-line inclination angles for DOPC/eSM/Chol are different from a previous study using electrofusion of GUVs 10. The authors obtained a tie-line of 24o near the hypothetical three-phase region. The angles of subsequent tie-lines increase to 50o, and then decrease to 41.5o near the critical point. One source of discrepancy is the location of the critical point. Their estimated critical composition is DOPC/eSM/Chol 52/15/33, which resides at the lower left side of the critical region obtained in our study. This displacement yields a larger tangent angle along the phase boundary at their estimated critical point 10. Tie-line angles have also been reported for SM mixed with DOPC/Chol. FRET measurements of MLVs estimated a tie-line angle of 33o for DOPC/brain SM/Chol 31. A tie-line angle of 37o was reported for DOPC/stearoyl SM/Chol using GUV imaging 30. All these values are larger than the angles obtained in our study. It is interesting to note that our tie-line angles are more similar to those obtained from DOPC/Chol mixed with a glycerol based high-Tm phospholipid 12, 15, 17. Tie-lies terminate at the critical point where tie-line endpoins merge together. Since the critical region is associated with the critical point, it is interesting to compare our results with published data. Several estimated critical points for DOPC/Chol mixed with natural or synthetic SM are shown in Fig. 9. As mentioned before, the electrofusion experiment predicted a critical point located at the lower left side of our critical region 10. A cluster of three critical points reported by three groups using three types of SM are identified at the right side of, but close to our critical region 30, 31, 54. Interestingly, an AFM study using temperature ramping predicted a critical point residing at the middle of our critical edge 21. One source contributing to the disparate critical points is the SM species used in each study. It was found that chemical structure of SM imparts a noticeable effect on membrane spatial organization 31. The second source contributing to the difficulty in locating critical point is the spatial sensitivity of different techniques. This is particularly relevant when probing nanoscopic structures taking place near the critical point. In


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addition, our data indicate that nanoscopic structures can be observed in a large region near the critical point. This gives rise to uncertainty in determining critical point when only a few samples are examined near this region.

Figure 9 Superimposing reported critical points and the liquid coexisting region determined in this study. Red inverted triangle is from Bezlyepkina et al. 10, purple star is from Connell et al. 21, magenta circle is from Petruzielo et al. 31, yellow triangle is from Farkas et al. 30, and green square is from Tian et al. 54. Different SM species used in these studies are indicated. Nanoscopic structures near the critical point. We observe nanoscopic heterogeneous structures in a broad compositional space of DOPC/eSM/Chol. Similar nanoscopic structures have been reported 21. The main difference is that these authors examined temperature effect by fixing lipid composition. Morphological transition from macroscopic to nanoscopic structures was obtained by choosing a composition in the vicinity of the critical region. We find that nanoscopic heterogeneity can be readily observed by altering lipid composition at a given temperature. This is important when considering the source contributing to the formation of nanoscopic assemblies in cell plasma membranes. If temperature is the parameter that cells use to induce lipid rafts, membrane temperature needs to be constantly altered in order to be compatible with rafts’ dynamic characteristic. Such a mechanism is not desirable, and adverse effects are expected for membrane proteins and their associated complexes. On the other hand, alteration of membrane composition can be achieved in a short time scale through mechanisms such as lipid recycling 55. Our finding contributes to the recognition of lipid role in mediating membrane raft formation and stability. Generation of membrane nanoscopic structures is essential to lipid raft hypothesis. Our results show that nanoscopic heterogeneity can be obtained by altering lipid composition near the critical point. Cholesterol plays an important role in this process by modulating ∆h 56. Other lipids can play a similar role. It was found that POPC partitions well into both the Lo and Ld phases. This reduces the interfacial line tension and sets the stage for nanodomain formation 57. The same effect can be used to explain domain size transition induced by the short chain lipid 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) 58. Biological relevance. Lipid rafts are proposed as the organization principle of membrane constituents, especially transmembrane (TM) proteins. One mechanism of raft dependent protein sorting is the thickness difference between raft and non-raft domains; it is energetically favorable


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to sequester proteins with long TM motifs into raft domains and proteins with short TM motifs into non-raft domains 2, 3. Based on the large number of membrane proteins, it is conceivable that there are always some proteins that have an intermediate thickness compared to those of raft and non-raft domains. The question is how cells decide where these proteins should go? Our result of lipid composition dependent thickness difference between coexisting phases provides the first hint, i.e., the thickness difference between raft and non-raft domains is not a constant, but rather can be actively tuned by adjusting raft composition and size. Correct protein sorting can then be initiated after the thicknesses of raft and non-raft domains, as well as other associated physicochemical parameters, are altered. Finally, we note that the height information of coexisting phases obtained from AFM measurements only corresponds to the surface of the distal leaflet with respect to the substrate. This raises the question of whether the obtained height difference reflects the bilayer as a whole or just the distal leaflet. Scattering techniques (e.g., Xray and neutron) have been broadly used to determine bilayer thickness. The total thickness difference between macroscopic Lo and Ld phases is ~1 nm 59, 60. This value is comparable to the height differences obtained in our study when lipid compositions are not near the upper miscibility boundary. This observation implies that the distal and proximal leaflets in solid supported bilayers are well coupled.

Supporting Information Three figures and one table are provided in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Acknowledgments We thank Drs. Gerald Feigenson and Shih Lin Goh for showing us how to make GUVs. J. Pan acknowledges the startup fund from the University of South Florida.


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Table of Contents Graphic


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Macroscopic and Nanoscopic Heterogeneous Structures in a Three

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