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 1 - Binary 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 2, 2007 This work applied two steady-state fluorescence techniques to detect nanoscopic membrane domains in a binary dimyristoylphosphocholine (DMPC)-cholesterol system and a ternary dioleoylphosphocholine (DOPC)-dipalmitoylphosphocholine (DPPC)-cholesterol system. A polarity-induced spectral shift in the emission spectra of 1-myristoyl2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (DAN-PC) in combination with a Fo¨rster resonance energy transfer (FRET) assay agreed with the phase diagrams that have been published for these systems and were observed to be useful tools in the detection of membrane heterogeneities. The DAN-PC/dehydroergosterol (DHE) FRET pair was found to be best suited for use with these steady-state techniques because of their differential partitioning between phases, although a high acceptor concentration was needed to obtain accurate measurements. In the binary system, this high probe concentration was found to be perturbing, but in more representative ternary systems, the high probe concentration no longer disrupted the phase behavior of the system. This FRET pair allowed for the calculation of nanometer-scale domain sizes in model ternary systems, using the two steady-state fluorescence techniques along with a clear and straightforward model.

Introduction Cholesterol crystals play an important role in human disease.1-4 For example, cholesterol crystals, which are known to nucleate from phospholipid vesicles in the gallbladder, are precursors to gallstones. Although the mechanism by which cholesterol nucleates from vesicles is not known, it seems clear that the nuclei must form either in the aqueous space or within the phospholipid bilayer. It is the latter possibility that has interested us, and we previously developed a Fo¨rster resonance energy transfer (FRET) assay to describe putative cholesterol nanodomain formation in phospholipid bilayers.5 Given that the existence of such pure cholesterol domains (which we believe could arise as metastable structures in supersaturated or otherwise nonequilibrium vesicle systems) is not firmly established and that no reports of cholesterol critical nucleus size are available, we tested the capabilities of our FRET model against simulations in which we created cholesterol nanodomains of known size.6 In the present study, we build on these ideas and adapt the FRET model to examine a different class of cholesterol domain that is known to exist both in real (that is, cell) and model (that is, made-in-the-laboratory) membranes. In particular, we now focus our attention on cholesterol-rich “lipid rafts,” which occur in cell membranes and resemble the well-known liquid-ordered domains known to exist in model membranes. Although it is possible that rafts could play a role in cholesterol nucleation, that is not our primary interest here. Rather, we are interested in developing a reliable measure of cholesterol domain (raft) size * Corresponding author. E-mail: [email protected]. Tel: 215-895-6694. Fax: 215-895-5837. (1) Tabas, I. J. Clin. InVest. 2002, 110. (2) Admirand, W. H.; Small, D. M. J. Clin. InVest. 1968, 47. (3) Fuster, V.; Badimon, L.; Badimon J. J.; Chesebro, J. H. N. Engl. J. Med. 1992, 326. (4) Wrenn, S. P.; Gudheti, M.; Veleva, A. N.; Kaler, E. W.; Lee, S. P. J. Lipid Res. 2001, 42. (5) Troup, G. M.; Tulenko, T. N.; Lee, S. P.; Wrenn, S. P. Colloids Surf., B 2003, 29, 217. (6) Troup, G. M.; Tulenko, T. N.; Lee, S. P.; Wrenn, S. P. Colloids Surf., B 2003, 33.

so that we can proceed to understand what factors (e.g., lipid composition, line tension, and vesicle curvature) govern domain size and how. The question of what influences domain size is not a trivial one, as evidenced by the fact that an unsettlingly large range of raft sizes has been measured in both natural and model membranes, and a consensus on raft size has not yet been reached. Reported raft sizes vary widely with rafts of 26 to 700 nm being measured in natural cell membranes using a variety of analytical techniques.7-12 It is possible that this range in reported size could be due to the presence of different raft states such as clusters or aggregates.13 In addition, large distributions in raft size have been observed in a single cell, indicating that in natural cells there may exist a wide size distribution and that raft size may be constantly changing.8 Complicating the question further is the significant, and currently unexplained, variation in domain sizes between natural and model membranes that has been observed. Micrometer-scale domains have been observed and measured in giant model membranes consisting of a ternary lipid system (that is, a lipid with a high phase-transition temperature (Tm), a low-Tm lipid, and cholesterol), although not at physiological conditions,14 and evidence of nanometer-scale separations in these ternary systems has been detected at physiological conditions but not measured.15 Binary systems containing a phospholipid and cholesterol have shown evidence of heterogeneities that have been estimated to be on the nanometer scale;16 however, some researchers believe that these heterogeneities are simply con(7) Dietrich, C.; Yang, B.; Fujiwara, T.; Kusumi, A.; Jacobson, K. Biophys. J. 2002, 82. (8) Pralle, A.; Keller, P.; Florin, E. L.; Simons, K.; Horber, J. K. H. J. Cell Biol. 2000, 148. (9) Prior, I.; Muncke, A. C.; Parton, R. G.; Hancock, J. F. J. Cell Biol. 2003, 160. (10) Sharma, P.; Sabharanjak, S.; Satyajit, M. Sem. Cell DeV. Biol. 2002, 13. (11) Tokumasu, F.; Jin, A. J.; Feigenson, G. W.; Dvorak, J. A. Biophys. J. 2003, 84. (12) Yuan, C.; Furlong, J.; Burgos, P. Biophys. J. 2002, 82. (13) Simons, K.; Vaz, W. L. C. Ann. ReV. Biophys. Biomol. Struct. 2004, 2004. (14) Veatch, S. L.; Keller, S. L. Biophys. J. 2003, 85. (15) London, E. Curr. Opin. Struct. Biol. 2002, 12. (16) Loura, L. M. S.; Federov, A.; Prieto, M. Biophys. J. 2001, 80.

10.1021/la700633b CCC: $37.00 © 2007 American Chemical Society Published on Web 09/22/2007

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centration fluctuations rather than actual domains.17 Despite the recent spate of work in measuring raft size, the ambiguity that remains necessitates more systematic studies of the factors that govern nanoscopic domain size. The determination of nanoscopic domain size is not a simple matter. Although direct size measurement is preferential, indirect measurement methods are generally required in the study of raft size for two reasons: (1) many direct techniques, such as fluorescence microscopy, do not have the resolution to detect nanoscopic domains and (2) the two phases of interest in raft studies are both liquid phases and differentiation between them is very difficult.18 One of the most promising techniques for detecting and measuring nanoscopic raft size is FRET because of its relative simplicity19 and its sensitivity to nanometer-sized distances.20 Unfortunately, FRET has proven difficult to use in studying liquid-liquid coexistence (or rafts) because of difficulties in probe selection.19,21 In this work, the applicability of three donor and acceptor pairs to detect membrane heterogeneity using two steady-state fluorescence techniques was investigated in a 1,2dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-cholesterol system for which the phase diagram has already been established. The techniques were then extended to more realistic model systems by investigating their applicability in a ternary 1,2dioleoyl-sn-glycero-3-phosphocholine (DOPC)-1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC)-cholesterol system. Both techniques, in combination, were found to be capable of detecting membrane heterogeneities on the nanometer scale in both binary and ternary systems containing cholesterol. In addition, a clearcut mathematical model was developed to relate steady-state FRET measurements in a model ternary system to domain size. Materials and Methods Materials. Cholesterol, dehydroergosterol (DHE), sodium chloride (NaCl), calcium chloride (CaCl2), sodium azide (NaN3), and 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) were purchased from Sigma Chemical Co. (St. Louis, MO). DMPC, DOPC, DPPC, and 1-myristoyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (DANPC) were purchased from Avanti Polar Lipids (Alabaster, AL). 2-(3-Diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero3-phosphocholine (β-DPH-HPC) and N-(5-dimethylaminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DAN-DHPE) were purchased from Molecular Probes/ Invitrogen (Carlsbad, CA). All chemicals were used without further purification. The fluorescent probes used as acceptors in this study varied in both the fluorescent moiety itself and the location of the moiety. Both β-DPH-HPC and DAN-PC are tail-labeled probes, and DANDHPE is a head-labeled probe. In all of the FRET studies, DHE, which is a fluorescent analog of cholesterol, was used as the donor. Sample Preparation. Vesicle solutions were prepared using the rapid solvent exchange technique.22 Stock solutions of DMPC, cholesterol, and fluorescent probe(s) dissolved in chloroform were added to 20 mL flat-bottomed vials in the required proportions. Aqueous buffer (3 mL) at 60 °C was added, and the solution was vortex mixed while being exposed to vacuum (4.92 in. mercury absolute pressure) for 1 min. The solutions were then diluted with buffer to a final lipid concentration of 1 mM.39 The buffer, with a (17) Karmakar, S.; Sarangi, B. R.; Raghunathan, V. A. Solid State Commun. 2006, 139. (18) de Almeida, R. F. M.; Federov, A.; Prieto, M. Biophys. J. 2003, 85. (19) Heberle, F. A.; Buboltz, J. T.; Stringer, D.; Feigenson, G. W. Biochim. Biophys. Acta 2005, 1746. (20) Fung, B. K.; Stryer, L. Biochemistry 1978, 17. (21) Silvius, J. R. Biophys. J. 2003, 85. (22) Buboltz, J. T.; Feigenson, G. W. Biochim. Biophys. Acta 1999, 1417. (23) Savitsky, A.; Golay, J. E. Anal. Chem. 1964, 36.

Langmuir, Vol. 23, No. 22, 2007 11181 pH of 7.4, contained 0.15 M NaCl, 5 mM CaCl2, 5 mM HEPES, and 3 mM NaN3. Sample Descriptions. All binary samples contained DMPC and cholesterol. Binary spectral shift samples additionally contained acceptor (DAN-PC or DAN-DHPE) at an acceptor to lipid ratio (ALR) of 0.03 or 0.18, and binary FRET samples contained 5% donor (DHE) and varying amounts and types of acceptor (DAN-PC, DAN-DHPE, or β-DPH-HPC). All ternary samples contained DOPC, DPPC, and cholesterol. The ternary spectral shift samples also contained DAN-PC at an ALR of 0.03, and the ternary FRET samples contained DHE (5%) and DAN-PC (at ALRs of 0.00 to 0.12).40 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- or DAN-DHPElabeled 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-labeled MLVs at 300 nm and recording the emission spectrum in the wavelength range of 325 to 550 nm or exciting β-DPH-HPC-labeled MLVs at 310 nm and recording the emission spectrum in the wavelength range of 340 to 500 nm. All emission scans were conducted at 30 °C unless otherwise specified. The emission profiles were smoothed using a Savitsky and Golay protocol.23 Fluorescence Techniques. Two steady-state fluorescence spectroscopic techniques were used in this work: the polarity-dependent emission spectral shift and FRET. Emission Spectral Shift. Some fluorophores exhibit a polarityinduced spectral shift when the polarity of their local environment changes. As the polarity of the local environment decreases, the emission wavelength at which the fluorophore gives maximal fluorescence decreases. Similarly, an increase in local polarity produces an increase in the maximal emission wavelength.24 This phenomena is particularly useful in determining the phase in which the probe resides as a result of the polarity difference between the two phases caused by the cholesterol condensation effect.5,25-27 FRET. FRET provides a measure of the average distance between an array of donor and acceptor molecules and thus can be used to detect membrane heterogeneities if two probes with varying partition (24) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Plenum: New York, 1999. (25) Parasassi, T.; DiStefano, M.; Loiero, M.; Ravagnan, G.; Gratton, E. Biophys. J. 1994, 66. (26) Philips, M. C. The Physical State of Phospholipids and Cholesterol in Monolayers, Bilayers, and Membranes. In Progress in Surface and Membrane Science; Academic Press: New York, 1972. (27) Stryer, L. Ann. ReV. Biochem. 1978, 47. (28) Vogel, S. S.; Thaler, C.; Koushik, S. V. Science STKE 2006, 331. (29) Veatch, S. L.; Keller, S. L. Biochim. Biophys. Acta 2005, 1746. (30) Lee-Gau Chong, P.; Liu F.; Wang, M. M.; Truong, K.; Sugar, I. P.; Brown, R. E. J. Fluoresc. 1996, 6, 221-230. (31) Tampe, R.; von Luka, A.; Galla, H. J. Biochemistry 1991, 30. (32) Troup, G. M.; Wrenn, S. P. Chem. Phys. Lipids 2004, 131. (33) Brown, A. C.; Towles, K. B.; Wrenn, S. P. Langmuir 2007, 23, 11188. (34) Radhakrishnan, A.; McConnell, H. M. Biophys. J. 1999, 77. (35) London, E.; Brown, D. A. Biochim. Biophys. Acta 2000, 1508. (36) Almeida, P. F. F.; Vaz, W. L. C.; Thompson, T. E. Biochemistry 1992, 31. (37) Baumgart, T.; Hunt, G.; Farkas, E. R.; Webb, W. W.; Feigenson, G. W. Biochim. Biophys. Acta 2007, In Press. (38) Mukherjee, S.; Zha, X.; Tabas, I.; Maxfield, F. R. Biophys. J. 1998, 1915. (39) The lipid concentration of 1 mM would normally necessitate correction for inner filter effects. However, this work uses the efficiency of energy transfer, a relative measure, rather than raw intensity values. We have shown that the efficiency of energy transfer is identical for samples with a total lipid concentration of 1, 0.5, 0.25, or 0.125 mM. For this reason, we have not corrected this data for inner filter effects. (40) We recognize that a DHE composition of 5% is high. However, DHE is not an intrinsic probe but is instead a fluorescent analog of cholesterol. Because of the low quantum yield of DHE, we use 5%, but we have found that DHE does not self-quench at these compositions. The large amount of DHE necessitates a large amount of acceptor (DAN-PC in most of these studies), but we have also found that DAN-PC does not self-quench at compositions of up to 12%.

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Figure 1. Energy transfer geometries in membranes without domains (a) and with domains (b). The lateral and transverse distances of closest approach between donors and acceptors increase as domains are formed. (c) Top view of the domain, where donors are assumed to reside on a ring with a radius that is 0.67 times the domain radius, representing the average radial location of the randomly distributed donors. coefficients between the two phases are used. In the case where the partition coefficients for the two probes differ significantly, membrane heterogeneities can be detected by a change in the efficiency of energy transfer as the second phase forms and the average distance between the two probes thereby increases. The largest source of error in fluorescence spectroscopy measurements is the lack of standardization of intensity measurements. The intensity output for a sample may vary from one machine to another or from measurement to measurement on the same machine.28 To avoid this, the efficiency of energy transfer, which is a relative value, was used in this work. FRET efficiency is calculated from the measured steady-state fluorescence intensity of the donor at its maximal emission wavelength (373 nm for DHE) in the presence and absence of acceptor

E)1-

FDA E(%) ) 1 FD

(1)

where FDA and FD are the donor emission intensities in the presence and absence of acceptor, respectively.24

Theory The relation of FRET to distance in systems containing a single donor and acceptor pair at a fixed distance is straightforward and well established.27 FRET studies in membrane systems are more complex because they contain multiple donors and acceptors with a different distribution of acceptors surrounding each donor. These complexities have been accounted for in a steady-state FRET model that predicts the efficiency of energy transfer from a donor to a random distribution of acceptor molecules in an infinite monolayer:20

E)1-

1 τo

∫0∞ e-t/τ e-σS(t) dt 0

(2)

τo is the excited-state lifetime of the donor in the absence of acceptor, and S(t) is the energy transfer term, which is given by

S(t) )

∫a∞[1 - e-(t/τ )(R /r) ]2πr dr 0

o

In eq 4, J is the spectral overlap, κ2 is the dipole orientation factor, QD is the quantum yield of the donor in the absence of acceptor, and n is the refractive index of the medium.24 This theory is now applied to membrane systems containing cholesterol-rich domains, and three simplifying assumptions are made: (1) donors are distributed randomly within the domain, (2) the domains are disklike, and (3) the domains are symmetric about the plane of the bilayer, as illustrated in Figure 1b. Energy transfer within a bilayer is due to energy transfer from donors and acceptors in the same monolayer and energy transfer from donors and acceptors in opposite monolayers. Equation 2 is then modified to describe these two types of energy transfer

6

(3)

In this equation, the lower limit of integration, a, represents the sum of the molecular radii, and σ is the surface density of acceptors. The Fo¨rster distance, Ro (the distance at which energy transfer is 50% efficient), in angstroms, is given by

Ro ) 9.79 × 103(Jκ2QDn-4)1/6

(4)

∫0∞ e-t/τ

1 τo

o

e-σ[S1(t)+S2(t)] dt

(5)

S1(t) )

∞ [1 - e-(t/τ )(R /r) ]2πr dr ∫LL1

(6)

S2(t) )

∞ [1 - e-(t/τ )(R /r) ]2πr dr ∫LL2

(7)

0

0

o

o

6

6

where S1 represents energy transfer within the same monolayer and S2 represents energy transfer across the bilayer. The lateral lower limit of integration (LL1) represents the smallest possible lateral distance between a donor and acceptor in the same leaflet, and the transverse lower limit of integration (LL2) represents the smallest possible transverse distance between a donor and acceptor in opposite leaflets. In both integrals, the upper limit of integration is given by infinity to represent the maximal distance between donor and acceptor. In a membrane system containing domains, two cases are possible: both probes are located in the same phase, or the probes are located in opposite phases. When both donor and acceptor are located in the same phase (giving rise to an energy transfer Esame), LL1 is given by the sum of the molecular radii (i.e., molecular contact), a, and LL2 is given by the bilayer thickness, h (3.6 nm), as seen in Figure 1a. If donor and acceptor are in different phases (giving rise to an energy transfer Ediff), then the lower limits of integration must change to account for probe separation; both LL1 and LL2 become functions of domain radius, n, as seen in Figure 1b. If the donor is assumed to be located at the center of the domain, then LL1 is given by the domain radius, n, and LL2 is given by (n2 + h2)1/2. Because the donors are not necessarily located at the center of the domain and are instead randomly distributed within the domain, the donors were

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assumed to reside on a circle that is concentric with respect to the domain with a radius of 0.67n to represent the average location of a donor within the domain as shown in Figure 1c. Using this assumption, LL1 is given by 0.33n, and LL2 is given by [h2 + (0.33n)2]1/2. This assumption that the donors reside on a ring with a radius of 0.67n is valid when the number of domains is large (as is the case in this work). Because LL1 is always smaller than LL2, lateral energy transfer term S1 contributes more heavily to the overall predicted efficiency of energy transfer than does lateral-energy-transfer term S2. When both probes are in the same phase, S1 is integrated from a (approximately 8 A) to infinity and S2 is integrated from only h (36 Å) to infinity. This difference leads to a 100-fold difference in contribution between S1 and S2. The difference between the energy transfer terms in the case where the probes are located in different phases is significant, although not as large as in the previous case. In this case, LL2 (the hypotenuse of the triangle, as shown in Figure 1) is greater than LL1 (a leg of the triangle), and S1 is approximately 10 times greater than S2. The overall predicted energy transfer is a weighted average between the two cases (probes located in the same phase and in different phases), which depends on the partitioning of the donor between the ordered (domain) and disordered phases.

Epred ) ddEsame + doEdiff

(8)

Esame, the energy transfer between donor and acceptor in the same phase, is calculated using eqs 5-7 with LL1 equal to a and LL2 equal to h. The energy transfer between donor and acceptor in different phases, Ediff, is calculated with eqs 5-7 using LL1 ) 0.33n and LL2 ) [h2 + (0.33n)2]1/2. The fractions of donor in the ordered and disordered phases, do and dd, respectively, are determined from a phase diagram. In particular, they are found from the known tie lines within the two-phase region, which allows the determination of fractions Xo and Xd using the reverse lever rule, along with the actual mole fractions of donor in each of those phases. Simple material balance then gives the values of do and dd. It is important to note that this model assumes that the domains are lo and the continuous phase is ld. At large values of Xo, this assumption is likely no longer valid. At these high lo fractions, the system is more likely composed of ld domains in an lo continuous phase. However, such a scenario is not common biologically and is therefore not considered explicitly here. Data Fitting. Before the experimental data were fit with the model, several input parameters were determined experimentally or by assumption. Overall lipid partitioning (Xo, Xd) values were obtained from the published phase diagram and tie lines for the ternary sphingomyelin-DOPC-cholesterol system.29 DHE, being structurally similar to cholesterol, was assumed to partition similarly to cholesterol, and its partitioning values, do and dd, were obtained from the published phase diagram.29 An approximate value of the unquenched donor lifetime, τo, was taken to be 1 ns.30 The parameter Ro was determined for a sample lying outside the two-phase region. This value of Ro was assumed to be independent of phospholipid composition within the two-phase region so that this Ro value was used for each sample with the same cholesterol composition. This assumption enables a singleparameter fit, where the fitted parameter is the domain radius. The experimental efficiencies of samples within the two-phase region were compared to efficiencies predicted by the model (eq 9), and the domain radius, n, was varied to fit the data.

Figure 2. Influence of domain size on predicted FRET profiles. For domain sizes larger than about 10Ro, the model loses sensitivity and can no longer accurately predict domain size.

Model Sensitivity to Domain Size. Because FRET depends on the distance between the donor and acceptor to the sixth power, the FRET signal is most strongly sensitive to distances on smaller length scales. As the domains grow, model resolution is lost, and the predicted FRET profiles at large values of domain radius do not vary with domain size, as seen in Figure 2. For example, domains of 100 and 1000 Å give the same energytransfer profile and cannot be resolved with this model. To minimize the error in the model fits, it is desirable to obtain efficiency values that are greater than 30%. Unfortunately, this is not always possible because one must also take care to minimize the probe composition as much as possible. In this work, it was not always possible to maintain high efficiency measurements because of the already high probe compositions needed to give a suitable signal-to-noise ratio; to increase the reliability in this measurement, samples were run a minimum of five times. The median value was used in the calculations and is plotted in the graphs with error bars representing the standard deviation.

Results Detection of Nanoscale Heterogeneities in Binary DMPCCholesterol Membranes. The use of FRET to characterize membrane domain size requires knowledge of probe partitioning and the phase behavior of the system. For that reason, the two previously described fluorescence techniques were employed first in a binary DMPC-cholesterol system in order to characterize the probes’ behavior fully in a simple system containing liquidliquid coexistence. Partitioning of Dansylated Lipids into Liquid Domains. Figure 3a shows the DAN-PC emission maximum as a function of cholesterol loading at four temperatures between 30 and 60 °C. In the absence of cholesterol and at low cholesterol loadings, the DAN-PC emission maximum occurs at 513 nm at all temperatures studied. At higher cholesterol compositions, the DAN-PC emission maximum undergoes a dose-dependent blue shift to reach a value of 460 nm. The cholesterol composition at which this blue shift occurs depends on the temperature of the sample, and this point of discontinuity was calculated using a semicontinuous piecewise fit. Each data set was assumed to be composed of two sets of linear data with an unknown point of discontinuity between the two sets. The data was fit to a line 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. The points of discontinuity occur at cholesterol loadings of 21, 30, 36, and 42% for samples at 30, 40, 50, and 60 °C,

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Figure 3. (a) Emission maximum wavelength for DAN-PC at an ALR of 0.03 in DMPC-cholesterol vesicles at 30 ([), 40 (0), 50 (4), and 60 °C (×). The inset shows the points of discontinuity superimposed on a DMPC-cholesterol phase diagram.31 The points of discontinuity were determined using a semicontinuous piecewise fit. (b) Emission maximum wavelengths of DAN-PC ([) and DANDHPE (0) at an ALR of 0.03 in DMPC-cholesterol vesicles at 30 °C.

respectively. The inset of Figure 3a shows the points of discontinuity superimposed on one of the established phase diagrams for this binary system.31 At each temperature, the DANPC emission maximum is constant until this point of discontinuity, indicating that the local environment of DAN-PC does not change polarity until this point. This phenomenon suggests that throughout the two-phase region DAN-PC resides almost exclusively in the ld phase where it is shielded from the polarity change that occurs in the lo phase and that it enters the lo phase only when there is no longer an ld phase present. It is interesting that the DAN-PC blue shift occurs gradually rather than in a step-change fashion. If, as we state, DAN-PC is truly excluded from the lo phase, then as long as there is some ld in which it is able to reside, the blue shift would be expected to occur suddenly, resulting in a step change in the DAN-PC emission maximum wavelength rather than a gradual decrease as was seen. However, it has been well established by our group that the DANSYL moiety, when it is attached to the chain of a phospholipid molecule, resides in different positions within the membrane depending on the local cholesterol composition.32 As the membrane becomes more ordered with more cholesterol, the DANSYL moiety is forced further down into the membrane. The spectral shift results validate this finding, with the gradual decrease in polarity due to the movement of the DANSYL moiety into the interior of the membrane. The observed DAN-PC blue shift occurs only when the DANSYL moiety is located on the tail of a phospholipid. Head-

Brown et al.

Figure 4. (a) DAN-PC/DHE cholesterol-dependent FRET behavior (ALR ) 0.03) in a DMPC-cholesterol membrane at 30 ([) and 60 °C (0). The lines are guides for the eyes. (b) β-DPH-HPC/DHE cholesterol-dependent FRET behavior in a DMPC-cholesterol membrane at 30 °C. The line is a guide for the eye.

labeled DANSYL (DAN-DHPE) displays a behavior at 30 °C that is strikingly different from that of tail-labeled DANSYL (DAN-PC), as seen in Figure 3b. The emission maximum of DAN-DHPE decreases slightly before undergoing a red shift. The point of discontinuity, where the red shift begins, occurs at a cholesterol composition of 21%, consistent with the previously described DAN-PC blue shift. These changes in the emission spectrum of DAN-DHPE through the two-phase region indicate that the probe partitions into both phases, unlike DAN-PC. Detection of Liquid-Liquid Coexistence by FRET. The presumed exclusion of DAN-PC from the lo phase led to its study as a potential FRET donor with DHE to detect and measure membrane heterogeneities. DHE, a fluorescent analog of cholesterol, likely partitions predominately in the lo phase, making this FRET pair potentially ideal for the detection of nanoscopic domains because of their differential partitioning. The DAN-PC/DHE FRET pair was used to probe the same DMPC-cholesterol system at 30 and 60 °C to investigate the applicability of this FRET pair in detecting membrane heterogeneities. The results of this DAN-PC/DHE FRET assay are shown in Figure 4a. At 30 °C, the minimum efficiency of the energy transfer value is observed, indicating donor and acceptor separation. This minimum occurs within the two-phase region predicted by the phase diagram for this system and indicates that DAN-PC and DHE partition differentially between the two phases. At 60 °C, the efficiency of energy transfer remains fairly constant throughout all cholesterol compositions. This result indicates that there is no probe separation at any cholesterol composition at this high temperature, which agrees with the phase diagram and indicates that the probe separation observed at 30 °C is primarily due to phase behavior and not some other factor.

Measuring Raft Size

Figure 5. FRET profiles of three acceptors with DHE in DMPCcholesterol membranes containing 40% cholesterol and 5% DHE (donor) at 30 °C. β-DPH-HPC ([) shows the highest energy transfer, followed by DAN-DHPE (0). DAN-PC (4) displays a restricted FRET profile in comparison with the other two probes. Solid lines represent the best-fit Ro values, which were determined for a cholesterol composition of 45% for a system in which both the donor and acceptor are in the same phase (i.e., outside of the twophase region).

A second FRET pair, β-DPH-HPC and DHE, was used to study the same DMPC-cholesterol system. Because of its larger spectral overlap with DHE, a smaller amount of β-DPH-HPC can be used in comparison to the amount of DAN-PC while retaining a sufficient signal-to-noise ratio; in this case, a β-DPHHPC ALR of 0.005 was used compared to an ALR of 0.03 for DAN-PC. The results, presented in Figure 4b, show an almost constant FRET efficiency for all cholesterol compositions, indicating that no probe separation occurs, even as two phases form, making this FRET pair unsuitable for use in determining domain size. The results of both FRET pairs agree with the previously described DAN-PC data. DAN-PC and DHE separate through the two-phase region (as evidenced by the minimum in energy transfer), but β-DPH-HPC and DHE do not. A likely explanation for this is that DAN-PC resides preferentially in the ld phase and DHE resides in the lo phase. Effect of High Probe Concentration on Binary DMPCCholesterol Membranes. Although DAN-PC and DHE appear to be an ideal FRET pair to use in studying nanoscopic domain formation, the pair is not without weakness, the most glaring of which is the high probe concentration that is necessary to minimize the signal-to-noise ratio. The use of 5% DHE and up to 12% DAN-PC is required because of the low quantum yield of DHE. Because of the high DHE composition that is required, a high DAN-PC composition (up to 12%) is required as well. Although these compositions are large, we have found that DHE does not self-quench at compositions up to 5% and that DAN-PC does not self-quench at compositions up to 12%. Figure 5 shows the efficiency of energy transfer as a function of ALR for β-DPH-HPC/DHE, DAN-DHPE/DHE, and DANPC/DHE in DMPC membranes containing 40 mol % cholesterol. Each of these three FRET pairs displays a markedly different FRET profile as a result of variations in Ro (eq 4). The Ro values for each FRET pair are displayed in Figure 5 The variation of Ro is due to several factors. The β-DPHHPC/DHE FRET pair displays a higher FRET profile than does either DAN-DHPE or DAN-PC/DHE, which is likely due to its significant spectral overlap (J) with DHE. The location of the DANSYL moiety plays a large role in the strength of the FRET profile of the two dansylated probes, which must be due to the difference in the dipole orientation factor (κ2) between the two

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Figure 6. Emission maximum wavelength for DAN-PC at an ALR of 0.18 in DMPC-cholesterol vesicles at 30 ([), 40 (0), 50 (4), and 60 °C (×). The lines are guides for the eye. The inset shows the points of discontinuity (determined using a semicontinuous piecewise fit) superimposed on a DMPC-cholesterol phase diagram.31

probes because all other variables in eq 5 are the same for both probes. Although the higher FRET profile of DAN-DHPE may make it appear to be an ideal choice in future FRET studies, its previously described spectral shift behavior indicates that it is not excluded from the lo phase. Because DAN-PC is excluded from the lo phase, it is a better candidate than DAN-DHPE to use in FRET studies. However, the low FRET profile of the DAN-PC/DHE FRET pair requires that a fairly large ALR be used when this FRET pair is chosen, which leads to concerns about probe effects. To investigate these possible probe effects with DAN-PC, the spectral shift and FRET experiments were repeated using a high ALR of 0.18. The results of both the spectral shift and FRET experiments show similar qualitative behavior as described previously for lower DAN-PC ALRs, but the data is shifted and is no longer accurate for determining two-phase coexistence or phase transitions. These results indicate that at this high composition DANPC acts as a third component and therefore perturbs the binary system. As seen in Figure 6, DAN-PC at an ALR of 0.18 displays the same qualitative spectral shift behavior as at an ALR of 0.03, as seen in Figure 6. The emission maximum is constant until a certain cholesterol composition is reached, at which point it undergoes a blue shift. As in the case of an ALR of 0.03, this point of discontinuity depends on the temperature of the sample. However, at an ALR of 0.18, the point of discontinuity is extremely shifted to lower cholesterol compositions and no longer maps entry of the membrane into the lo phase as seen in the inset of Figure 6. A semicontinuous piecewise linear fit was used to calculate the points of discontinuity, which were found to occur at cholesterol compositions of 10, 16, 19, and 21% for temperatures of 30, 40, 50, and 60 °C, respectively. Likewise, the DAN-PC/DHE FRET assay was repeated with an ALR of 0.18 (Figure 7) and shows similar cholesteroldependent FRET behavior as observed for an ALR of 0.03. A broad dip is observed, with a minimum energy transfer value that occurs at a cholesterol composition just outside the expected two-phase region (as marked by the solid vertical lines in Figure 7). At this high ALR, the FRET pair is still able to detect the presence of membrane heterogeneities, although the cholesterol compositions at which heterogeneities appear to exist are different from the low-ALR case. This is because DAN-PC is no longer acting as a probe of a binary system; rather, it is acting as the third component in a ternary system.

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Brown et al. Table 1. Theoretical Fits of 20% Sterol Samples in a Ternary DOPC-DPPC-Cholesterol Systema

a

Figure 7. DAN-PC/DHE cholesterol-dependent FRET behavior (ALR ) 0.18) in a DMPC-cholesterol membrane at 30 °C. The line is a guide for the eye. The solid vertical lines represent the edges of the two-phase region in the binary DMPC-cholesterol system,31 and the dashed vertical lines represent the edges of the two-phase region in the ternary DOPC-DPPC-cholesterol system.29

Figure 8. FRET profiles of four samples containing 20% sterol, DOPC, and DPPC [0 ([), 20 (0), 40 (4), or 60% (×)]. The fits were obtained using Xo values of 0.00, 0.05, 0.43, and 0.78, respectively; do values of 0, 0.71, 0.77, and 0.70, respectively; and Ro of 11 Å.

This point is validated by comparing Figure 7 with the published DPPC-DOPC-cholesterol phase diagram.29 If DAN-PC is considered to be the unsaturated lipid (DOPC) at a composition of 18 mol % and DMPC is considered to be the saturated lipid (DPPC) in this phase diagram, then the points of discontinuity align exactly with the two-phase region in the phase diagram. From Figure 7, discontinuities are observed at cholesterol compositions of 15 and 45% (marked by dashed lines), which are exactly the same compositions that mark the edges of the two-phase region in the published DOPC-DPPC-cholesterol phase diagram at a DOPC composition of 18%. Detection of Nanoscale Heterogeneities in Ternary DOPCDPPC-Cholesterol Membranes. Because DAN-PC appears to be perturbing at a high ALR in binary systems, it seems to be better suited to probing ternary systems that contain an unsaturated component. DAN-PC acts similarly to the unsaturated component and is therefore less perturbing of the phase behavior. Figure 8 shows the FRET profiles of four samples in the ternary DOPC-DPPC-cholesterol system. Each of these samples contains 20% cholesterol with varying amounts of DPPC. The sample that contains 0% DPPC displays the highest efficiency of energy transfer; this sample is located outside of the two-phase region of the published phase diagram29 and therefore contains no domains. On average, the probes are as close to one another as

% DPPC

Xo

do

n (nm)

20 40 60

0.05 0.43 0.78

0.71 0.77 0.70

3 6 3

Ro was determined to be 9 Å for all three compositions.

possible. As the amount of DPPC is increased to 20% DPPC, the system enters the two-phase region. The efficiency of energy transfer for this composition is lower than for the 0% DPPC sample, indicating that two phases have formed and the average distance between the probes has increased. At a DPPC composition of 40%, the sample is located in the center of the two-phase region where the maximum phase separation would be expected to occur. This sample displays very low energy transfer, consistent with the presence of large domains. Finally, at a DPPC composition of 60%, the sample again resides on the edge of the two-phase region and a higher, but still restricted (relative to the 0% DPPC sample), efficiency of energy transfer is observed This ternary FRET data was fit with the model (eqs 6-9) to relate the experimental FRET profiles using domain size as the fitted parameter. For these calculations, it was assumed that DHE partitions similarly to cholesterol and that the proposed tie lines for the sphingomyelin-DOPC-cholesterol phase diagram29 hold for this ternary system as well. These assumptions allowed for the determination of DHE partitioning into the lo phase (do) and the overall lipid partitioning into the lo phase (Xo) using the reverse lever rule. The Fo¨rster distance was found to vary depending on the state of the membrane; specifically, it increases as the cholesterol composition increases (and the membrane becomes more ordered). We assume that the Fo¨rster distance is constant for each cholesterol composition. The model fits of the experimental data and the model parameters are shown in Table 1. This work in the ternary system is a proof of concept to demonstrate the fit of the model to initial ternary data. Exhaustive studies to characterize domain size throughout this ternary system are described in part II of this article.33

Discussion The results of this work, specifically the DAN-PC spectral shift experiment, highlight the ambiguities that continue to plague the binary phase diagram. Even slight differences in data interpretation have been shown to result in entirely different phase diagrams being presented for similar systems, a fact that is made evident by two published phase diagrams of the DMPCcholesterol system.29,31 These two phase diagrams are similar in that they show a region of two-phase coexistence but differ in the exact boundaries of this region and in the presence or absence of a direct ld-to-lo phase transition at higher temperatures.29,31 Adding to the ambiguity is the belief held by some researchers that there is no equilibrium two-phase region in binary systems at all and that what has previously been interpreted as phase coexistence is instead due to concentration fluctuations17 or nanometer-scale “condensed complexes”.34 This confusion relating to the binary phase diagrams likely stems from the lack of differences in the physical properties of the two liquid phases, making it difficult to distinguish between the two.35 The fluorescence techniques described in this article eliminate that ambiguity. It appears from the DAN-PC spectral shift results that DANPC partitions preferentially in the ld phase. At higher cholesterol compositions, where the membrane is predominately lo, DAN-

Measuring Raft Size

PC is forced into the lo phase, as evidenced by the blue shift. Because of this preferential partitioning into the ld phase, the DAN-PC blue shift can be used to observe the entry of the membrane into the lo phase. At 50 and 60 °C, the DAN-PC spectral shift data is consistent with the Tampe´ phase diagram, which shows a direct ld-to-lo phase transition,31 which was not observed in the Almeida phase diagram.36 Here, the utility of the DAN-PC spectral shift becomes evident for it is not only consistent with classical techniques but also can detect phase transitions that are inaccessible by some methods. In addition to validating the exact phase behavior of the system, the DAN-PC spectral shift elucidates the partitioning of the probe between the two phases, two pieces of information that are vital for domain size determination. The partitioning of DAN-PC predominately in the ld phase was found to be particularly useful in analyzing FRET results using DAN-PC/DHE. The minimum efficiency value that was found to occur in the binary system at 30 °C occurs within the two-phase region for this system and is a result of probe partitioning between the two phases, which is additional evidence that DAN-PC partitions preferentially in the ld phase. This minimum efficiency value allows for the determination of phase formation as well as the calculation of domain size. It is not surprising that DAN-PC partitions preferentially in the ld phase because it contains a large, bulky group on one chain. This bulky group should be expected to act similarly to a kink in the chain of an unsaturated phospholipid. Just as unsaturated phospholipids partition preferentially in the ld phase because of packing restrictions, a chain-labeled phospholipid would be expected to partition predominately in the ld phase. A recent systematic study of fluorescent probe partitioning has found that probes containing either an unsaturated chain or a labeled chain always partition predominately in the ld phase.37 Not all FRET pairs are such good markers of membrane heterogeneities, however. The FRET pair β-DPH-HPC and DHE showed little variation in energy transfer as the membrane changed from one phase to two, indicating that the partition coefficient of β-DPH-HPC into the ld phase is not as strong as that of DANPC. DPH, when used as an extrinsic chromophore, has been found to partition equally between the lo and ld phases,37 and it appears from our results that β-DPH-HPC shows similar behavior. This FRET pair is therefore unable to detect any heterogeneity in the system. It is clear from these experiments that DAN-PC has several qualities that make it an ideal probe in studies of domain size. The unique partitioning of DAN-PC allows for the determination of the lo phase transition, even in cases in which there is a direct

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phase transition rather than a two-phase region. The DAN-PC/ DHE FRET pair is likewise notable in its ability to show membrane heterogeneities with its cholesterol-dependent FRET behavior. Although DAN-PC has many desirable qualities that make its use in these studies ideal, the fact that such a large probe concentration is required is troubling. However, it was observed that at high probe concentrations DAN-PC displays the same qualitative behavior as it does at low probe concentrations. The fact that the behavior is similar, but shifted, indicates that DANPC acts as a third component in a “binary” system. This was, in fact, verified by comparing the DAN-PC-DHE FRET results to a ternary phase diagram. The presence of a third component to measure a binary system is obviously undesirable; by changing a thermodynamic variable in the binary system, size measurements become extremely difficult if not impossible. However, in a ternary system, DAN-PC acts as a constituent of the third component and is a more accurate probe of the phase behavior than in a binary system. Additionally, ternary systems provide better models of natural systems than do binary systems, and phase diagrams for several ternary systems have been established in recent years. The application of this technique to natural cell membranes is possible and could provide enlightening results for a comparison of domain size between model and natural cell membranes. It has been shown that cells can be labeled with DHE38 and labeling with DAN-PC could be accomplished in a similar manner. An analysis of the data would be more complicated than in a model system because of the lack of phase behavior information, and a new model would be required to evaluate the data. However, this could provide a very interesting comparison between previous studies in natural membranes, which generally use fluorescent proteins rather than the fluorescent lipids used in this technique. Steady-state fluorescence spectroscopy is a simple technique that holds promise for detecting and measuring liquid domains. The nature of the techniques involves some membrane perturbation, and care must therefore be used in interpreting results. In addition, not all fluorescent probes are ideally suited for use in domain size determination. DAN-PC and DHE were found to be valuable for this purpose, and their use in the two steady-state fluorescent techniques described in this article will be vital in studying domain size in ternary systems. Acknowledgment. This work was supported in part by National Science Foundation (NSF) grant CTS-0346638 and National Institutes of Health (NIH) grant 1 R01 GM071355. LA700633B