Measuring Raft Size as a Function of Membrane Composition in PC

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Measuring Raft Size as a Function of Membrane Composition in PC-Based Systems: Part II s Ternary Systems Angela C. Brown, Kevin B. Towles, and Steven P. Wrenn* Department of Chemical and Biological Engineering, Drexel UniVersity, 3141 Chestnut Street, Philadelphia, PennsylVania 19104 ReceiVed March 5, 2007. In Final Form: July 23, 2007 The heterogeneity of cell membranes, specifically the presence of lipid rafts, has been hypothesized to play a role in a large number of cellular processes. Although extensive work has been carried out to show the function of lipid rafts in these processes, the characterization of lipid rafts has proven to be extremely difficult. It is known that raft size is relevant to the function of cellular processes and that raft coalescence may be a driving factor for these processes; however, it remains unclear what factors influence raft size and coalescence in natural cell membranes. In this work, we study two ternary model phospholipid and cholesterol systems using two steady-state fluorescent techniques to detect and characterize membrane domains. Domain size is determined through the use of a model to relate experimental Fo¨rster resonance energy transfer (FRET) measurements to domain size. Domains in the range of 3-15 nm were detected in a dioleoylphosphatidylcholine-dipalmitoylphosphatidylcholine-cholesterol (DOPC-DPPC-Chol) system, while only a very small region containing domains was detected in a 1-palmitoyl-2-oleoyl-phosphatidylcholinedipamitoylphosphatidylcholine-cholesterol (POPC-DPPC-Chol) system. In addition, the polarity-dependent emission maximum shift of the acceptor 1-myristoyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-snglycero-3-phosphocholine (DAN-PC) was used to detect the type of liquid phase(s) present in the membrane. It was found that, even in the case in which no two-phase coexistence was observed (POPC-DPPC-Chol), two liquid phases are present, although not necessarily in coexistence. These steady-state fluorescent techniques provide a method for detecting the presence of very small domains in model membranes and provide previously inaccessible detail about the phase behavior of these two systems.

Introduction Since the introduction of the lipid raft hypothesis, which states that many eukaryotic cell membranes are heterogeneous,1,2 there has been an extensive amount of research into biological processes that are believed to be controlled by rafts, as well as factors that affect the formation of rafts. However, studies in natural membranes are limited to indirect methods, such as detergent extraction or intrusive methods, such as protein cross-linking, as lipid rafts are believed to be very small (nanometer-scale) and transient.3,4 To combat the limitations of studying rafts in natural membranes, many researchers have turned to model membranes, composed of two or three components, to study domain formation more systematically. Many techniques are suitable to the study of domain formation in model membranes, such as electron spin resonance (ESR),5 differential scanning calorimetry (DSC),6 nuclear magnetic resonance (NMR),7 atomic force microscopy (AFM),8 fluorescence recovery after photobleaching (FRAP),9 fluorescence microscopy,10 fluorescence anisotropy,11 and Fo¨rster resonance energy transfer (FRET);12 however, each of these * To whom correspondence should be addressed. Telephone: 215-8956694. Fax: 215-895-5837. E-mail: [email protected]. (1) Brown, D. A.; London, E. Biochem. Biophys. Res. Commun. 1997, 240. (2) Simons, K.; Ikonen, E. Nature 1997, 387. (3) London, E. Biochim. Biophys. Acta 2005, 1746. (4) Varma, R.; Mayor, S. Nature 1998, 294. (5) Chiang, Y. W.; Zhao, J.; Wu, J.; Shimoyama, Y.; Freed, J. H.; Feigenson, G. W. Biochim. Biophys. Acta 2005, 1668. (6) McMullen, T. P.; McElhaney, R. N. Biochim. Biophys. Acta 1995, 1234. (7) Oradd, G.; Westerman, P. W.; Lindblom, G. Biophys. J. 2005, 89. (8) Tokumasu, F.; Jin, A. J.; Feigenson, G. W.; Dvorak, J. A. Biophys. J. 2003, 84. (9) Chen, Y.; Lagerholm, B. C.; Yang, B.; Jacobson, K. Methods 2006, 39. (10) Veatch, S. L.; Keller, S. L. Biochim. Biophys. Acta 2005, 1746. (11) Gidwani, A.; Holowka, D.; Baird, B. Biochemistry 2001, 40. (12) Silvius, J. R. Biophys. J. 2003, 85.

techniques has led to differing conclusions regarding the nature of membrane domains. Particularly troubling is the inability of these techniques to reach a consensus regarding domain size. In liquid-liquid model ternary systems, size measurements of domains have varied widely, ranging from tens of nanometers to micrometers in size.7,13-15 Even more disconcerting is the fact that similar techniques have resulted in strikingly different domain size measurements. Recent NMR studies have found liquid domain sizes of 80-160 nm15 and greater than 1 µm.7 Similarly, different AFM studies have found liquid domains of 33-48 nm8 or several µm.16 These discrepancies indicate that the issue of domain size has not yet been resolved. While no technique is perfect, FRET appears to be one of the most promising methods to study domain size due to its sensitivity to distances in the nanometer size range.17 Recent work in measuring domain sizes in binary and ternary systems using a time-resolved FRET model has shown these domains to be on the order of tens of nanometers.13,14 Unfortunately, this particular model requires the use of a laser with a very narrow pulse width that is beyond the means of many laboratories. Another limitation of FRET to measure domain size is the difficulty in selecting appropriate probes.12,18 In this paper, we present a steady-state FRET model which is able to measure nanoscopic domain sizes in both binary and ternary systems. The model is applicable to any FRET pair that (13) de Almeida, R. F. M.; Federov, A.; Prieto, M. Biophys. J. 2003, 85. (14) Loura, L. M. S.; Federov, A.; Prieto, M. Biophys. J. 2001, 80. (15) Veatch, S. L.; Polozov, I. V.; Gawrisch, K.; Keller, S. L. Biophys. J. 2004, 86. (16) Blanchette, C. D.; Lin, W. C.; Ratto, T. V.; Longo, M. L. Biophys. J. 2006, 90. (17) Fung, B. K.; Stryer, L. Biochemistry 1978, 17. (18) Heberle, F. A.; Buboltz, J. T.; Stringer, D.; Feigenson, G. W. Biochim. Biophys. Acta 2005, 1746.

10.1021/la7006342 CCC: $37.00 © 2007 American Chemical Society Published on Web 09/22/2007

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partitions differentially between the two liquid phases. We have previously shown that the FRET pair 1-myristoyl-2-[12-[(5dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-snglycero-3-phosphocholine (DAN-PC)/dehydroergosterol (DHE) is a favorable FRET pair for this purpose.19 Here, we expand our technique to investigate two similar but slightly different ternary systems, a 1,2-dioleoyl-sn-glycero-3-phosphocholine-1,2-dipalmitoyl-sn-glycero-3-phosphocholine-cholesterol (DOPCDPPC-Chol) system and a 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine-DPPC-cholesterol (POPC-DPPC-Chol) system. Our main goal is to determine the effect of chain saturation in the third component (that is, DOPC or POPC) on domain formation and size, using our steady-state FRET model. Materials and Methods Materials. DPPC, DOPC, POPC, and DAN-PC were purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol, DHE, sodium chloride (NaCl), calcium chloride (CaCl2), sodium azide (NaN3), and 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) were purchased from Sigma Chemical Co. (St. Louis, MO). All chemicals were used without further purification. Sample Descriptions. Spectral shift samples were created along lines of constant DPPC composition at 5% cholesterol increments. The DOPC-DPPC-Chol samples contained 0%, 20%, 30%, 40%, 60%, or 80% DPPC (Figure 1A), and the POPC-DPPC-Chol samples contained 0%, 20%, 40%, 60%, or 80% DPPC (Figure 1B). In all of these spectral shift samples, DAN-PC was used at an acceptorto-lipid ratio (ALR) of 0.03, as a fraction of DOPC or POPC. FRET samples were created along lines of constant cholesterol composition with varying DPPC compositions. In the DOPC-DPPCChol system, samples contained 20%, 30%, 40%, or 60% cholesterol and 0%, 20%, 30%, 40%, 50%, or 60% DPPC (Figure 1C). In the POPC-DPPC-Chol system, samples contained 20%, 40%, or 60% cholesterol and 0%, 20%, 40%, or 60% DPPC (Figure 1D). Each FRET sample contained DAN-PC (as a fraction of DOPC or POPC) at an ALR of 0.00-0.12 and DHE (as a fraction of cholesterol) at 5%. All vesicle solutions were prepared using the rapid solvent exchange (RSE) technique.20 Stock solutions of phospholipids, cholesterol, and fluorescent probe(s) were dissolved in chloroform and added to 20 mL flat bottom vials in the necessary amounts. A volume of 3 mL of aqueous buffer (0.15 M NaCl, 5 mM CaCl2, 5 mM HEPES, and 3 mM NaN3; pH of 7.4) at 60 °C was added, and the solution was vortex mixed while exposed to a vacuum of 4.92 in. of mercury absolute pressure for 1 min. The solutions were then diluted with buffer to make a final lipid concentration of 1 mM. The DAN-PC/DHE FRET pair was chosen for this work because of some unique properties of the pair. First, we have shown that DAN-PC partitions almost exclusively in the liquid disordered (ld) phase as long as some ld phase is present in the membrane. Only when the membrane becomes entirely liquid ordered (lo) does DANPC enter the lo phase.19 In addition, it is likely that DHE partitions among both the lo and ld phases similarly to cholesterol. This differential partitioning of DAN-PC and DHE between the two phases allows for detection of membrane heterogeneities with FRET as the two phases form and the average distance between probes increases. Fluorescence Measurements. Fluorescence measurements were obtained using a steady-state fluorescence spectrometer (Photon Technology International, Ontario, Canada, model Q-5/W-601) with a circulating water bath to maintain the sample temperature to ( 0.5 °C. The temperature was read on a cuvette thermometer (Fisher Corp., Philadelphia, PA, model 15-078J). Spectral shift experiments required DAN-PC-labeled multilamellar vesicles (MLVs) to be excited at 350 nm with the emission spectrum recorded from 400 to 600 nm. FRET experiments were carried out by exciting DAN-PC- and DHE-labeled MLVs at 300 nm and (19) Brown, A. C.; Towles, K. B.; Wrenn, S. P. Langmuir 2007, 23, 11180. (20) Buboltz, J. T.; Feigenson, G. W. Biochim. Biophys. Acta 1999, 1417.

Figure 1. Sample compositions used in this study. The samples used in the spectral shift study of the DOPC-DPPC-Chol system are shown in (A), and the samples used in the spectral shift study of the POPC-DPPC-Chol system are shown in (B). The samples used in the FRET study of the DOPC-DPPC-Chol system are shown in (C), and the samples used in the FRET study of the POPC-DPPC-Chol system are shown in (D). recording the emission spectrum in the wavelength range 325-550 nm. All emission scans were conducted at 30 °C. The emission profiles were smoothed using a Savitsky and Golay protocol.21

Theory We have previously developed a model to relate steady-state FRET measurements to cholesterol-rich domain sizes in mem(21) Savitsky, A.; Golay, J. E. Anal. Chem. 1964, 36.

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branes, which correlates an experimentally determined FRET efficiency to a predicted FRET efficiency where domain size is the fitted parameter.19 This model is valid for the determination of discrete lo domain sizes for any system in which the donor partitions into the lo phase and the acceptor is excluded from the lo phase. Here, we include a summary of the relevant equations; a complete explanation of the theory and fitting methodology is included in part I of this article.19 The experimental FRET efficiency (Eexp) is calculated from the measured fluorescence intensity of the donor at its maximal emission wavelength (373 nm for DHE) in the presence and absence of acceptor (FDA and FD, respectively):22

Eexp(%) ) 1 -

FDA FD

(1)

Eexp can then be fit to a model which describes energy transfer from one donor to an array of acceptors in a membrane, accounting for the fact that domains are present within the membrane:

E)1-

1 τo

()

∫0∞ exp -t τo

[ [∫

exp -σ

∞

LL1

×

1 - exp

(( )( ) ) ∫ (( )( ) ) ]] -t Ro τo r exp

6

2πrdr +

-t Ro τo r

∞

LL2

1-

6

2πrdr dt (2)

where τo is the lifetime of the donor in the absence of acceptor, σ is the acceptor surface density, Ro is the Fo¨rster distance, r is the distance between the probes, τ is the decay time, LL1 is the distance of closest approach for probes in the same bilayer, and LL2 is the distance of closest approach for probes in opposite bilayers. The values of LL1 and LL2 depend on the partitioning of the donor because of the differing acceptor arrays in each of the two phases. It is assumed that all acceptors reside in the ld phase and the donors reside either in the ld phase or within the lo domain (at an average radial diameter of 0.67n). Because of this specific partitioning, a donor in the ld phase will see a different array of acceptors than a donor in the lo phase. When the donor is in the ld phase, it is in the same phase as the acceptors. In this scenario, giving rise to Esame, LL1 is given by the sum of the molecular radii, a, and LL2 is given by the bilayer thickness, h. When the donor is in the lo phase, it is in the opposite phase of that of the acceptors. In this scenario, giving rise to Ediff, LL1 is given by 0.33n, where n is the domain radius, and LL2 is given by [(0.33n)2 + h2]1/2. The energy transfer of the system is due to energy transfer between probes in the two donor partitioning scenarios in the following manner:

Epred ) ddEsame + doEdiff

(3)

where do is the fraction of donors in the ordered phase and dd is the fraction of donors in the disordered phase. This model assumes lo domains and a ld continuous phase, an assumption which is only valid for values of Xo that are less than 0.5. At higher Xo fractions, the system is more likely composed of ld domains in a lo continuous phase, but because this scenario is not biologically relevant, it is not considered explicitly here. The Fo¨rster distance, the distance at which energy transfer is 50% efficient, in Å, is given by the following: (22) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Plenum: New York, 1999.

Ro ) 9.79 × 103Jκ2Qonr-4

(4)

where J is the spectral overlap of the donor emission and acceptor absorption, κ2 is the orientation factor, Qo is the quantum yield of the donor, and nr is the refractive index of the medium.22 Effect of Domain Size Polydispersity on Model Predictions. The model above (and in part I) implicitly assumes monodispersity in domain size. Given the sixth-power dependence of FRET efficiency on distance, the model therefore serves as a lower limit on the (FRET-weighted) average domain radius. That is, smaller domains contribute disproportionately more to the measured FRET profile than do larger domains, so that any degree of polydispersity necessarily increases the FRET profile relative to that expected for a monodisperse system. This is analogous to (but the opposite of) light scattering, wherein larger scatterers in a polydisperse system contribute more heavily to the measured correlation function (due to the sixth-power dependence of scattering intensity on length) than do smaller scatterers, giving rise to a larger measured (intensity-weighted) average particle size than would be observed in a monodisperse system. We are interested in measuring raft sizes precisely because the sizes, and hence size distributions, are unknown. Not knowing the distributions a priori, we have no reason to assume any particular distribution. We do recognize, however, that if a distribution were known, then our model for domain sizes could be easily extended to account explicitly for any polydispersity present in the system. In particular, the predicted FRET profile would be obtained by simple integration over the domain size distribution

Epred,PD )

∫0n

max

Ni(ni)E(ni)dni

(5)

where Ni(ni) is the number fraction of domains of radius ni, nmax is the largest possible domain that could be obtained if all the cholesterol and lo lipids were packed into a single domain, and Ei(ni) is the energy transfer efficiency expected for domains of radius ni (as calculated by eqs 2 and 3 in the monodisperse limit). As a simple example, consider a normal size distribution. Equation 5 can be approximated by a simple weighted summation, where the weightings are given by a Gaussian probability density function: nmax

Epred,PD )

∑

[

]

-(ni - µ)2

1 σx2π

exp

ni)0

2σ2

E(ni)∆ni

(6)

where µ is the mean domain radius and σ is the standard deviation. Figure 2 shows the predicted FRET profiles for polydispersities of 0.0-0.5 for mean domain sizes of 3, 6, 9, and 12 nm (Figure 2A-D, respectively), where the polydispersity (PD) is defined as the standard deviation divided by twice the mean domain size

PD )

σ 2µ

(7)

For each mean domain size, the predicted FRET profiles increase with increasing degrees of polydispersity. As the mean domain size increases, the variation due to polydispersity decreases because FRET is a short-range interaction, and the model is therefore most accurate at predicting smaller domain sizes. When the mean domain size is small, the model predicts different FRET profiles for each domain size in the distribution, but when the mean domain size is large, resolution is lost and the FRET profiles for each domain size are very similar.

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The monodisperse case always predicts the lowest FRET profile, as seen in Figure 2; when polydispersity is included in the model, the predicted FRET profiles increase. To fit the experimental FRET profile, a larger domain size must be used at higher degrees of polydispersity than at lower degrees of polydispersity, resulting in a larger predicted domain size. As a result, the monodisperse model predicts the lower limit domain size, and any variation due to polydispersity will result in a larger domain size.

Results

Figure 2. Predicted FRET profiles for domain sizes of 3 nm (A), 6 nm (B), 9 nm (C), and 12 nm (D) with polydispersities ranging from 0.0 to 0.5 for samples with 20% sterol, do ) 0.8, and Ro ) 9 Å.

Demarcation of the ld-lo Boundary Using the DAN-PC Spectral Shift. We have seen in a binary DMPC-Chol system that the emission maximum of DAN-PC is constant until a particular cholesterol composition, where it undergoes a substantial blue shift. This cholesterol composition at which the blue shift begins appears to line up along the two-phase-to-lo transition or the ld-to-lo transition on the binary phase diagram. It appears that DAN-PC is not affected by the change in polarity in the lo phase as cholesterol is added to the membrane until this particular cholesterol composition, indicating that it is shielded in some way from the polarity effects. We interpret this to indicate that DAN-PC resides in the ld phase as long as some ld phase exists. As soon as the membrane becomes entirely lo, DAN-PC must enter the lo phase, and as a result a blue shift is observed. In other words, the cholesterol composition at which the blue shift begins represents the composition at which the membrane becomes entirely lo.19 Here, we apply this technique to the study of two ternary systems, DOPC-DPPC-Chol and POPC-DPPCChol. DOPC-DPPC-Chol System. The DOPC-DPPC-Chol system, which contains a di-unsaturated phospholipid (DOPC) and a disaturated phospholipid (DPPC), has been shown to exhibit liquid-liquid coexistence at a range of temperatures and compositions, with the published phase diagram for this system, which was determined through fluorescence microscopy, showing the presence of a substantial two-phase region.15 The DAN-PC emission maximum as a function of cholesterol composition for the DOPC-DPPC-Chol system is shown in Figure 3A for several DPPC compositions. In the sample containing 0% DPPC, the maximum wavelength remains constant through all cholesterol compositions, indicating that the binary DOPC-Chol edge of this system does not form a pure lo phase at any composition. The remaining samples, which contain increasing amounts of DPPC, exhibit blue shifts at particular cholesterol compositions, which, as shown in prior studies, signifies entry of the membrane into the lo phase. The point of discontinuity, or the cholesterol composition at which the blue shift occurs (and the membrane enters the lo phase), was determined using a semicontinuous piecewise fit, where each data set was fit to two lines using the least-squares method, varying the point of discontinuity. The point of discontinuity leading to the smallest error was taken as the intersection between the two linear data sets. These points of discontinuity were found to exist at cholesterol compositions of 50%, 41%, 36%, and 30% for samples containing 20%, 30%, 40%, and 60% DPPC, respectively, and are graphed on a ternary phase diagram for this system in the inset of Figure 3A. No point of discontinuity was observed in the 0% DPPC sample. A point of discontinuity was not calculated for the 80% DPPC sample because there were too few data points to reliably calculate a discontinuity. For most of the compositions studied, the spectral shift data appear to mark the edges of the two-phase region on the published

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Brown et al. Table 1. Model Parameters in the DOPC-DPPC-Chol System

Figure 3. (A) Emission maximum wavelength of DAN-PC-labeled DOPC-DPPC-Chol MLVs as a function of cholesterol composition for samples containing 0% ([), 20% (0), 30% (4), 40% (×), 60% (O), or 80% (-) DPPC. The lines indicate the semicontinuous piecewise fit for each composition. The inset shows the points of discontinuity in the emission maximum wavelength on a phase diagram for the DOPC-DPPC-Chol system.10 (B) Emission maximum wavelength of DAN-PC-labeled POPC-DPPC-Chol MLVs as a function of cholesterol composition for samples containing 0% ([), 20% (0), 40% (×), 60% (O), or 80% (-) DPPC. The lines show the semicontinuous piecewise fits. The points of discontinuity are shown on a phase diagram for the POPC-DPPC-Chol system10 in the inset.

phase diagram. However, these data extend beyond the reported two-phase region slightly, indicating that a direct ld-to-lo phase transition may be present at high cholesterol and low DPPC compositions or that the two-phase region extends beyond the reported boundary. POPC-DPPC-Chol System. The DAN-PC spectral shift assay was also applied to another ternary system containing the disaturated phospholipid, DPPC, but with an asymmetric phospholipid, POPC, instead of a di-unsaturated phospholipid. This ternary POPC-DPPC-Chol system has also been studied previously by fluorescence microscopy, and it was shown to exhibit no evidence of liquid-liquid coexistence.10 The emission maximum of DAN-PC-labeled POPC-DPPCChol membranes is shown in Figure 3B. Each sample exhibits a fairly constant emission maximum until a particular cholesterol composition, at which point a steep blue shift in emission maximum occurs. This point of discontinuity was found using a semicontinuous piecewise fit to occur at cholesterol compositions of 41%, 31%, 25%, and 20% for samples containing 0%, 20%, 40%, and 60% DPPC, respectively. A point of discontinuity was not determined for the 80% DPPC samples.

sample

% chol

% DPPC

Xo

do

Ro (Å)

n (nm)

A B

20 20

30 50

0.30 0.59

0.80 0.82

9.0 9.0

3.5 3.5

C D E F G

30 30 30 30 30

0 20 30 40 50

0.00 0.12 0.39 0.56 0.72

0.00 0.64 0.71 0.80 0.77

11.0 11.0 11.0 11.0 11.0

3.5 7.5 15 15

H I J K

40 40 40 40

0 20 30 40

0.00 0.74 0.63 0.72

0.00 0.57 0.62 0.71

12.0 12.0 12.0 12.0

4.5 5.5 3.5

L M

60 60

0 20

0.00 1.00

0.00 1.00

12.5 12.5

These points of discontinuity are shown on a phase diagram for this system10 in the inset of Figure 3B. It appears that a ld phase exists at low cholesterol compositions, below the points indicated, while a lo phase exists at higher cholesterol compositions, above the points marked on the diagram. The previously published phase diagram for this system does not show evidence of two coexisting phases; rather, it shows the presence of just one unidentified (solid, lo, or ld) phase in all of the composition space.23 The DAN-PC spectral shift results reported here indicate that while no region of two-phase coexistence may exist, two different phases exist at different compositions in this system, and the phase boundary between these two phases can be seen clearly. This phenomena of a direct phase transition between two liquid phases is similar to behavior observed in binary systems,23 but it has not previously been reported in this ternary system. Detection and Size Estimation of Membrane Domains Using FRET. The FRET pair DHE/DAN-PC has been shown by our group to be able detect nanoscopic membrane heterogeneities in both binary systems and ternary systems due to their differential partitioning between the two phases. We have seen that because DAN-PC partitions strongly in the ld phase and DHE partitions similarly to cholesterol, primarily in the lo phase, a decrease in FRET is observed within the two-phase region in relation to the FRET observed when the membrane contains only a single ld or lo phase.19 Here, we apply the DHE/DAN-PC FRET pair to membranes comprised of the DOPC-DPPC-Chol and POPCDPPC-Chol ternary systems with varying amounts of cholesterol and fit the data with our model to detect domains and predict domain size within the two-phase region. Detailed fitting procedures are provided in part I.19 In summary, an experimental FRET efficiency (eq 1) was determined and compared to a predicted FRET efficiency (eq 3) with the domain radius, n, being the fitted parameter. In both ternary systems studied, the Fo¨rster distance, Ro, was found to vary slightly as the amount of cholesterol in the membrane was increased. For each cholesterol composition, a value of Ro was determined for a sample lying outside the two-phase region (the sample containing 0% DPPC) and therefore containing no domains. This value of Ro was assumed to be independent of phospholipid composition (that is, one value of Ro was used for all samples with the same cholesterol composition). The Ro values that were used in the DOPC-DPPC-Chol and POPC-DPPC-Chol systems are included in Tables 1 and 2, respectively. The data were fit to both the monodisperse model (where domain size is the only fitted parameter) and the polydisperse model (where domain size and polydispersity are the fitted parameters), and both results are presented here. (23) Veatch, S. L.; Keller, S. L. Phys. ReV. Lett. 2005, 94.

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Table 2. Model Parameters in the POPC-DPPC-Chol System sample

% chol

% DPPC

Ro (Å)

A B C D E F G H I

20 20 20 20 40 40 40 60 60

0 20 4 60 0 20 40 0 20

10.0 10.0 10.0 10.0 10.5 10.5 10.5 10.5 10.5

DOPC-DPPC-Chol System. The DOPC-DPPC-Chol system has shown evidence of two-phase coexistence at 30 °C.24 Previously, we have detected domains of 3-6 nm in samples comprised of 20% sterol, as well as DOPC and DPPC.19 Here, we extend our study to additional compositions within the DOPCDPPC-Chol system. The model fits of the DOPC-DPPC-Chol experimental data and the parameters used in the model are shown in Table 1. In general, it was observed that the highest FRET profiles occurred in samples that were located outside of the two-phase region. Within the two-phase region, samples showed a relative restriction in energy transfer, with the largest restrictions (and largest domain sizes) occurring toward the center of the twophase region and the smallest restrictions (and smallest domain sizes) occurring toward the edges of the two-phase region. Samples containing 20% sterol and 0%, 20%, 40%, or 60% DPPC have already been reported, and they were found to contain no domains, 3 nm domains, 6 nm domains, and 3 nm domains, respectively, using the monodisperse model.19 The FRET profiles of additional samples containing 20% sterol (30% DPPC and 50% DPPC) are shown in Figure 4A, along with the theoretical FRET profile for each sample. Theoretical monodisperse fits of the data predict domain sizes of 3.5 nm for both 20% cholesterol samples. Polydisperse fits of the data predict an average domain size of 4.5 nm with a polydispersity of 0.4 for the 30% DPPC sample and an average domain size of 3.5 nm with a polydispersity of 0.1 for the 50% DPPC sample. The FRET profiles of the 30% cholesterol samples, shown in Figure 4B, show a similar trend in FRET profiles as those of the 20% sterol samples. The sample containing 0% DPPC contains no domains. The relative restriction of energy transfer that is observed at higher DPPC compositions indicates domain presence. Theoretical monodisperse fits of the data are shown, and domain sizes of 3.5, 7.5, 15, and 15 nm were calculated for samples containing 20% DPPC, 30% DPPC, 40% DPPC, and 50% DPPC, respectively. Polydisperse fits predict average domain sizes of 3.5 nm (PD ) 0.2) and 7.5 nm (PD ) 0.0) for the 20% DPPC samples and the 30% DPPC samples, respectively. A polydisperse fit of the 40% DPPC and 50% DPPC samples was not performed because the predicted monodisperse sizes are so large that the variation due to polydispersity is undetectable. The FRET profiles of the 40% cholesterol samples and their fitted monodisperse domain sizes are shown in Figure 4C. The 0% DPPC sample again is assumed to contain no domains, and a restriction of energy transfer is observed as the amount of DPPC is increased. Theoretical monodisperse fits of the data predict domain sizes of 4.5, 5.5, and 3.5 nm for samples containing 20% DPPC, 30% DPPC, and 40% DPPC, respectively. The polydisperse fit did not change the predicted domain sizes for any of the 40% cholesterol samples. Polydisperse fits predict average domain sizes of 4.5 nm (PD ) 0.5), 5.5 nm (PD ) 0.5), (24) Troup, G. M.; Wrenn, S. P. Chem. Phys. Lipids 2004, 131.

Figure 4. FRET profiles and fitted domain sizes of DOPC-DPPCChol MLVs. The 20% cholesterol samples (A) contained 30% ([) or 50% (filled gray squares) DPPC. The 30% cholesterol samples (B) contained 0% ([), 20% (filled gray squares), 30% (4), 40% (×), or 50% (O) DPPC. The 40% cholesterol samples (C) contained 0% ([), 20% (filled gray squares), 30% (4), or 40% (×) DPPC. The 60% cholesterol samples (D) contained 0% ([) or 20% (filled gray squares) DPPC.

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Brown et al. Table 3. Predicted Monodisperse Domain Sizes and Polydisperse Domain Sizes PD (nm)

Figure 5. Fitted domain sizes of DOPC-DPPC-Chol MLVs on a phase diagram for the system.10 The shaded region indicates the two-phase region as determined by fluorescence microscopy, and the dashed line indicates the border of the assumed two-phase region in this study.

and 3.5 nm (PD ) 0.0) for the 20% DPPC, 30% DPPC, and 40% DPPC samples, respectively. In the 60% cholesterol samples, no restriction in energy transfer is observed, as shown in Figure 4D. At this cholesterol composition, it appears that no domains are formed, because the system is outside of the two-phase region and is entirely lo or ld for all DPPC compositions. An unexpected phenomenon was observed in this study. The FRET profiles of samples containing no domains (the 0% DPPC samples for all cholesterol compositions) were seen to increase as the amount of cholesterol in the samples was increased. For example, the sample containing 30% cholesterol and 0% DPPC shows an efficiency of 44.9% at an ALR of 0.12 (Figure 4B), while the sample containing 60% cholesterol and 0% DPPC shows an efficiency of 75.8% at the same ALR (Figure 4D). It is assumed that all of these 0% DPPC samples do not contain any domains, due to the fact that no evidence of a lo phase was observed in the spectral shift results for any composition along the 0% DPPC line (Figure 3A) and the fact that the 0% DPPC line is well outside of the reported two-phase region.10 The observed increase in FRET with increasing cholesterol composition is therefore most likely due to changes in Ro (and more specifically, the order parameter κ2) as the membrane becomes more ordered with cholesterol. Figure 5 shows the previously described fitted domain sizes as a function of composition in the DOPC-DPPC-Chol system. The published two-phase region10 is shaded, and a dashed line is used to show a possible extension of the two-phase region that is suggested by the FRET and spectral shift results reported here. It is apparent that, toward the edges of the two-phase region, very small domains exist, while in the center of the two-phase region larger domains exist. The model is limited when domain sizes reach approximately 10 times the Fo¨rster distance. At these large distances, the model loses resolution and can no longer distinguish between domain sizes. The two samples shown in Figure 5 with predicted domain sizes of 15 nm may, in fact, have different sizes that are indistinguishable using this model When polydispersity is included in the model as a second fitted parameter, predicted domain sizes are not significantly larger than those for the monodisperse case. Table 3 shows a

% chol

% DPPC

0.0

0.1

0.2

0.3

0.4

0.5

20 20 20 20 20 30 30 30 30 40 40 40

20 40 60 30 50 20 30 40 50 20 30 40

3 6 3 3.5 3.5 3.5 7.5 15 15 4.5 5.5 3.5

3 7 3 3.5 3.5 3.5 7.5

3.5 8 3.5 4.5 4 3.5 7.5

3.5 8.5 3.5 4.5 4.5 3.5 7.5

3.5 9 3.5 4.5 4.5 3.5 8

3.5 10 4 4.5 4.5 3.5 8.5

4.5 5.5 3.5

4.5 5.5 3.5

4.5 5.5 3.5

4.5 5.5 3.5

4.5 5.5 3.5

summary of predicted monodisperse domain sizes (where domain size is the only fitted parameter) as well as predicted polydisperse domain sizes and degrees of polydispersity (where both domain size and polydispersity are fitted parameters) for all of the DOPCDPPC-Chol samples described in this paper and in part I.19 POPC-DPPC-Chol System. The POPC-DPPC-Chol system is unique in that it has not been shown to contain liquid-liquid coexistence at any temperature.23 The DHE-DAN-PC FRET assay was applied to this system to determine whether this might be due to domain sizes being too small to detect with other techniques. The results for this system vary dramatically from the results of the DOPC-DPPC-Chol system. Here, little variation in FRET profiles is observed, indicating that a large two-phase region does not exist for this system. The FRET profiles of the 20% sterol samples are shown in Figure 6A. The 0% DPPC, 20% DPPC, and 40% DPPC samples display very similar FRET profiles indicating no domain formation at any of these compositions. The 60% DPPC sample shows slightly restricted energy transfer, which may be due to a small region of liquid-liquid coexistence that was not detectable by fluorescence microscopy and is therefore not seen in the phase diagram for the system, or it may be due to solid-liquid coexistence. The 40% cholesterol samples display similar energy transfer profiles as seen in Figure 6B. The fact that there is no restriction in energy transfer as the amount of DPPC is increased suggests that there are no domains at 30 °C at any of these compositions. Likewise, both 60% cholesterol samples display unrestricted energy transfer, as seen in Figure 6C, suggesting that there are no domains at this composition. As in the DOPC-DPPC-Chol system, the FRET profiles were observed to increase with increasing cholesterol composition, a fact which could be explained by Ro values varying depending on the order of the membrane. These Ro values are shown in Table 2. The FRET results suggest that a very small region of liquidliquid coexistence may exist in this ternary POPC-DPPC-Chol system in the area near the 20% cholesterol/60% DPPC sample. However, the data and technique do not allow for differentiation of this possibility or for the possibility that the observed restriction in energy transfer at this composition may be due to solidliquid coexistence. Also, it is impossible to predict a domain size for the sample containing restricted energy transfer due to lack of information regarding the two-phase region and tie-lines for this system. Model SensitiVity to Input Parameters. The major input parameters for the model are do and Ro, neither of which is explicitly known. Assumptions made about both value were found to impact the results in very different ways.

Measuring Raft Size in Ternary Systems

Langmuir, Vol. 23, No. 22, 2007 11195 Table 4. Effect of Donor Partitioning on Predicted Domain Size for 20% Cholesterol/30% DPPC Samples DHE partitioning (do)

predicted domain size (nm)

0.8 0.7 0.9 0.6

3.5 3.5 3.5 4.5

Table 5. Effect of Ro on Predicted Domain Size for 20% Cholesterol/30% DPPC Samples Ro

predicted domain size (nm)

9.0 10.0 8.0

3.5 4.5 2.5

effect of the variation of R on the predicted domain size of the 20% cholesterol/30% DPPC sample. An increase or decrease in Ro of 1 Å causes the predicted domain radius to increase or decrease by 1 nm. While it is clear that Ro could cause significant error in the predicted domain sizes, the domain sizes would be the same order of magnitude as those presented here. Also, any possible variation in Ro is not expected to change the trend in predicted domain sizes.

Discussion

Figure 6. FRET profiles of POPC-DPPC-cholesterol MLVs. The 20% cholesterol samples (A) contained 0% ([), 20% (filled gray squares), 40% (4), or 50% (×) DPPC. The 40% cholesterol samples (B) contained 0% ([), 20% (filled gray squares), or 40% (4) DPPC. The 60% cholesterol samples (C) contained 0% ([) or 20% (filled gray squares) DPPC.

The value of do was determined by assuming that DHE partitions similarly to cholesterol and that the tie-lines and phase boundaries in the DOPC-DPPC-Chol system are the same as those in the DOPC-SM-Chol system.10 This assumption does not have significant impact on the model results, with variation in do of (0.1 causing only negligible effects on the predicted domain radius. An illustration of this is shown in Table 4, where the 20% cholesterol/30% DPPC sample experimental data are fit as described previously with varying values of do. The value of R was determined for the 0% DPPC sample (which was assumed to have no domains) at each cholesterol composition. The variation of R has a much greater impact on predicted domain radii than does the variation of do; however, the predicted domain sizes vary only slightly. Table 5 shows the

The DAN-PC spectral shift results highlight one extremely useful aspect of the DAN-PC probe. Because of its extremely strong partitioning in the ld phase, it is possible to determine information about the state of the membrane which may be impossible to determine with other methods. For example, the phase diagrams for DOPC-DPPC-Chol and POPC-DPPC-Chol that we have referenced in this work show only the existence or absence of a two-phase region.10 The fluorescence microscopy technique is unable to detect direct phase transitions however, and for that reason the published phase diagrams are able to provide little information about the lipid behavior outside of the two-phase region. The spectral shift results presented here provide a significant amount of additional detail in this regard. Of course, the techniques presented in this paper are not able to tell the full story by themselves, either. Our model cannot predict domain size without information about the tie-lines in the system, and it is unable to distinguish between liquid-liquid domains and solid-liquid domains. However, the results presented in this paper, in combination with the results of others, provide a very detailed view of the phase behavior of these systems. With the DAN-PC spectral shift, we are able to determine the point in composition space at which the membrane becomes 100% lo (either a direct ld-to-lo transition or the exit from a twophase region to a pure lo phase). This information provides valuable insight into the behavior of the lipids. For instance, the behavior of the two lipid systems studied in this work varies dramatically along the binary DOPC-Chol or POPC-Chol edge of the phase diagrams. In the binary DOPC-Chol system, the DAN-PC emission maximum was constant for all cholesterol compositions, while, in the binary POPC-Chol system, a blue shift was observed at a cholesterol composition of 41%. These results show that DOPC and cholesterol do not form a pure lo phase at any composition, a fact which is to be expected due to the unsaturated nature of the DOPC acyl chains. However, just a slight change in the saturation of the acyl chain, in this case, changing one unsaturated chain to a saturated chain, has major

11196 Langmuir, Vol. 23, No. 22, 2007

implications in the ability of the phospholipid to order. The blue shift that is observed at 41% cholesterol in the POPC-Chol system shows that the membrane becomes entirely lo at this composition. These results show that while DOPC and cholesterol are unable to form a lo phase at any cholesterol composition, POPC and cholesterol do form a lo phase at cholesterol compositions above 41%. The partitioning of DAN-PC in the ld phase has another important use, along with DHE, which partitions similarly to cholesterol into the lo and ld phases; DAN-PC has the ability to relate changes in FRET efficiency to domain size. The DOPCDPPC-Chol system displays restricted energy transfer profiles when domains are present, while the POPC-DPPC-Chol system shows evidence that it contains either a very small or no region of liquid-liquid coexistence. (Unfortunately, FRET is unable to discriminate between solid-liquid domains and liquid-liquid domains.) While our results are consistent with previously established phase diagrams for these two systems which show phase coexistence in the DOPC-DPPC-Chol system, but not in the POPC-DPPC-Chol system,10 we have also shown that the steady-state FRET profiles can also be used to measure the sizes of domains. This technique provides a straightforward method for determining domain sizes, and it will be useful in future studies of domain growth and stability. During this study, it was observed that Ro changed with cholesterol composition, probably because of the change in phospholipid order induced by cholesterol. While the quantum yield, spectral overlap, and refractive index are most likely the same in all of these cases because all of the measurements were made using the same two probes in the same type of system, it is very possible that the orientation factor, κ2, could vary with cholesterol composition. It is not surprising that κ2, which describes the relative positions of the donor and acceptors, could change with cholesterol composition. We have seen that DANPC resides at different depths within the membrane depending

Brown et al.

on cholesterol composition,24 and this effect could lead to variations in κ2 as the DAN-PC molecule shifts its location. It is generally assumed that the Fo¨rster distance is a constant for each particular FRET pair (a value that can be obtained from the literature), but this study indicates that this is not necessarily a valid assumption. The study highlights the importance of verifying that literature values of Ro were obtained under similar conditions or verifying that any composition-dependent changes in Ro are negligible. In this work, we have found two-phase coexistence at the same compositions as those in the fluorescence microscopy study,10 but we detected nanometer-scale domains in smaller MLVs. Veatch et al. detected larger nanometer-scale domains in MLVs in the DOPC-DPPC-Chol system (80-160 nm) using NMR,15 and Silvius also found evidence of domains on the order of tens of nanometers in MLVs using a different FRET assay than the one used here.12 It is likely that domains of a wide variety of sizes coexist in these membrane systems and that each technique is useful in the observation of domains of a particular size range. Studies in natural membranes have also found evidence of different domain sizes depending on the technique used.25 These systems are notoriously difficult to study, and it is not surprising that different techniques produce different results. Each technique has its own advantages and disadvantages, and to characterize these systems fully several techniques should be applied. The two steady-state fluorescent techniques that we applied to these systems provide significant additional detail regarding phase coexistence and domain size in the two ternary systems studied. Acknowledgment. This work was supported by National Institutes of Health (NIH) Grant 1 R01 GM071355. LA7006342 (25) Anderson, R. G. W.; Jacobson, K. Science 2002, 296.