Langmuir 2007, 23, 8135-8141
8135
Domain Shapes, Coarsening, and Random Patterns in Ternary Membranes Mikkel Herholdt Jensen,† Eliza J. Morris,‡ and Adam Cohen Simonsen*,§ Department of Physics, Boston UniVersity, Boston, Massachusetts 02215, SEAS, HarVard UniVersity, Cambridge, Massachusetts 02138, and MEMPHYSsCenter for Biomembrane Physics, Institute of Physics and Chemistry, UniVersity of Southern Denmark, CampusVej 55, DK-5230 Odense M, Denmark ReceiVed March 6, 2007. In Final Form: April 24, 2007 A number of morphological and statistical aspects of domain formation in singly and doubly supported ternary membranes have been investigated. Such ternary membranes produce macroscopic phase separation in two fluid phases and are widely used as raft models. We find that membrane interactions with the support surface can have a critical influence on the domain shapes if measures are not taken to screen these interactions. Combined AFM and fluorescence microscopy demonstrate small (500 nm) irregular domains and incomplete formation of much larger (5 µm) round domains. These kinetically trapped structures are the result of interactions between the membrane and the support surface, and they can be effectively removed by employing doubly supported membranes under physiological salt concentrations. These decoupled supported membranes display macroscopic round domains that are easily perturbed by fluid shear flow. The system allows a quantitative characterization of domain coarsening upon being cooled into the coexistence region. We determine the domain growth exponent R ) 0.31, which is in close agrement with the theoretical value of 1/3. Analysis of the spatial domain pattern in terms of Voronoi polygons demonstrates a close similarity to equilibrated cellular structures with a maximized configurational entropy.
1. Introduction The significance of lateral lipid organization in membranes has been intensely studied in the past decade since the proposition of the raft hypothesis.1-4 Lipid rafts were originally defined as the insoluble membrane fragments resulting from treatment with cold detergent.5 This operational definition has recently been challenged6,7 partly because the influence of the detergent on the membrane domains is ambiguous. Lipid organization in membranes will undoubtedly turn out to be significantly more complex than the initial raft concept and may encompass several domain types, domain sizes, and characteristic time scales and integrate the role of proteins in the understanding of these phenomena.8,9 Despite these considerations, there is growing evidence that membrane domains are playing a crucial role in native biological membranes such as the pulmonary surfactant system.10 This study exemplifies that certain biologically functional membranes display the coexistence of two fluid phases and that this behavior is linked to the monolayer spreading capability, which in turn is important for the biological function. Key membrane components such as cholesterol were found to control this morphology rather than the proteins. However, we are still far from a complete understanding of the mechanisms that control the size, shape, and dynamics of membrane domains. Binary and ternary model †
Boston University. Harvard University. § University of Southern Denmark. ‡
(1) Simons, K.; Ikonen, E. Nature 1997, 387, 569-572. (2) Edidin, M. Annu. ReV. Biophys. Biomol. Struct. 2003, 32, 257-283. (3) Mayor, S.; Rao, M. Traffic 2004, 5, 231-240. (4) Jacobson, K.; Mouritsen, O. G.; Anderson, R. G. W. Nat. Cell Biol. 2007, 9, 7-14. (5) Brown, D.; Rose, J. Cell 1992, 68, 533-544. (6) Lichtenberg, D.; Goni, F.; Heerklotz, H. Trends Biochem. Sci. 2005, 30, 430-436. (7) Heerklotz, H. Biophys. J. 2002, 83, 2693-2701. (8) Hancock, J. F. Nat. ReV. Mol. Cell. Bio. 2006, 7, 456-462. (9) Munro, S. Cell 2003, 115, 377-388. (10) de la Serna, J.; Perez-Gil, J.; Simonsen, A. C.; Bagatolli, L. A. J. Biol. Chem. 2004, 279, 40715-40722.
membranes have helped to develop a fundamental understanding of how domain formation and phase separation can be dependent upon the lipid components.11 The two fluid phases observed in native and ternary model membranes are often denoted the liquid-ordered (lo) and the liquid-disordered (ld) phases, and we follow this convention. The lo phase is often associated with rafts, although no conclusive evidence exists that rafts are in this phase state. The lo phase requires the presence of cholesterol and was originally defined to be a phase in binary phospholipid/cholesterol membranes.12 Ternary model membranes are the simplest class of membrane systems that have been observed to form two macrosopic fluid domains. Such ternary model membranes are typically composed of an unsaturated or short-chain phospholipid, a sterol, and a saturated phospholipid or sphingolipid that colocalizes with the sterol. The formation of two fluid phases in giant unilamellar vesicles (GUVs)13 has been intensely studied by means of fluorescence microscopy.14 Studies on ternary GUVs have revealed the fluid phases to be highly dynamic and demonstrated that the domains relax to circular shapes in an effort to reduce line tension. Unfortunately, a quantitative study of domain patterns in these membranes is complicated by their curvature and limited by the techniques that can be applied. Atomic force microscopy (AFM) is not possible, and since GUVs are 3D objects, a significant amount of image processing is required to obtain quantitative data when they are observed with techniques such as confocal microscopy. The planar geometry of supported membranes facilitates image analysis. Phase separation in singly supported ternary model membranes has been studied with AFM, but often these studies15-17 do not reproduce the domain sizes and perfectly (11) Veatch, S. L.; Keller, S. L. Biochim. Biophys. Acta 2005, 1746, 172-185. (12) Ipsen, J.; Karlstrom, G.; Mouritsen, O.; Wennerstrom, H.; Zuckermann, M. Biochim. Biophys. Acta 1987, 905, 162-172. (13) Angelova, M.; Soleau, S.; Meleard, P.; Faucon, J.; Bothorel, P. Prog. Colloid Polym. Sci. 1992, 89, 127-31. (14) Bagatolli, L. Biochim. Biophys. Acta 2006, 1758, 1541-1556.
10.1021/la700647v CCC: $37.00 © 2007 American Chemical Society Published on Web 06/23/2007
8136 Langmuir, Vol. 23, No. 15, 2007
round shapes found in ternary GUVs.18 For time scales on the order of many hours, such domains do not evolve with time, and little or no domain coarsening takes place. Furthermore, complete or partial decoupling between the membrane leaflets can be observed in supported membranes.19,20 This is directly related to interactions between the membrane and the solid support. Such effects can limit the validity of singly supported membranes as model systems. However, the significant advantages of supported model membranes related to image analysis and imaging techniques call for a better understanding of the role of the support and improved methods for decoupling of the planar membrane from the support. Domain coarsening or Ostwald ripening21,22 is one of the fundamental physical mechanisms which can alter the distibution of domain sizes. It describes the observation that, after nucleation of domains, large crystals grow at the expense of smaller ones because the system seeks to minimize the overall interface free energy. In fluid 2D systems the driving force for domain coarsening is line tension, which seeks to minimize the total perimeter length around the domains. Under conditions of conserved order parameter, the increase with time of the mean domain area 〈A〉 is described by a power law, 〈A〉 ∝ t2R. The exponent R is 1/3 when the coarsening dynamics is diffusionlimited23 and 1/2 in the case of interface-limited dynamics.24 Domain coarsening has been observed in coexisting liquid phases of phospholipid/cholesterol monolayer films,25,26 which is a fluid 2D system similar to the present system. This system was shown to exhibit a coarsening dynamics with an exponent of R ) 0.28 ( 0.0125 close to the diffusion-limited theoretical prediction. Domain coarsening has been suggested in binary supported membranes with solid-ordered (so)/ld phase coexistence.27 There are some indications that the spherical geometry of vesicles may alter the domain coarsening dynamics as compared to that of planar membranes.28-30 The spatial pattern of the domains can be analyzed statistically in terms of the Voronoi construction.31,32 Such an analysis has been performed on monolayer systems,25 but to our knowledge not on bilayers. Several methods exist for the preparation of supported membranes. The two most common are (1) Langmuir-Blodgett transfer33 of a lipid monolayer at the air-water interface to the solid surface and (2) the fusion and explosion of 30-100 nm vesicles34 on the substrate. Recently, we have developed a procedure to prepare supported bilayers based on the hydration of spin-coated lipid films.35 This procedure is somewhat different (15) van Duyl, B. Y.; Ganchev, D.; Chupin, V.; de Kruijff, B.; Killian, J. A. FEBS Lett. 2003, 547, 101-106. (16) Lawrence, J.; Saslowsky, D.; Edwardson, J.; Henderson, R. Biophys. J. 2003, 84, 1827-1832. (17) Henderson, R.; Edwardson, J.; Geisse, N.; Saslowsky, D. News Physiol. Sci. 2004, 19, 39-43. (18) Veatch, S. L.; Keller, S. L. Biophys. J. 2003, 85, 3074-3083. (19) Keller, D.; Larsen, N. B.; Møller, I. M.; Mouritsen, O. G. Phys. ReV. Lett. 2005, 94, 025701. (20) Stottrup, B.; Veatch, S.; Keller, S. Biophys. J. 2004, 86, 2942-2950. (21) Gunton, J. D.; Miguel, M. S.; Sahni, P. S. The dynamics of first order transitions; Academic Press: London, 1983. (22) Zinke-Allmang, M. Thin Solid Films 1999, 346, 1-68. (23) Lifshitz, I.; Slyozov, V. J. Phys. Chem. Solids 1961, 19, 35-50. (24) Mouritsen, O. G. Int. J. Mod. Phys. B 1990, 4, 1925-1954. (25) Seul, M.; Morgan, N. Y.; Sire, C. Phys. ReV. Lett. 1994, 73, 2284-2287. (26) Hu, Y.; Meleson, K.; Israelachvili, J. Biophys. J. 2006, 91, 444-453. (27) Giocondi, M.; Vie, V.; Lesniewska, E.; Milhiet, P.; Zinke-Allmang, M.; Le grimellec, C. Langmuir 2001, 17, 1653-1659. (28) Taniguchi, T. Phys. ReV. Lett. 1996, 76, 4444-4447. (29) Laradji, M.; Sunil Kumar, P. Phys. ReV. Lett. 2004, 93, 198105. (30) Saeki, D.; Hamada, T.; Yoshikawa, K. J. Phys. Soc. Jpn. 2006, 75, 013602. (31) Weaire, D.; Rivier, N. Contemp. Phys. 1984, 25, 59-99. (32) Schliecker, G. AdV. Phys. 2002, 51. (33) Blodgett, K. B. J. Am. Chem. Soc. 1935, 57, 1007-1022. (34) Brian, A. A.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 6159-6163.
Jensen et al.
from previous protocols in that the formation of dehydrated, stacked bilayers takes place during the spin-coating step. The subsequent hydration and annealing step serves to equilibrate the membrane in an aqueous environment and to wash off excess bilayers. We have found this method to be applicable to all membrane compositions tested and to be robust toward the presence of salt in the aqueous phase. Full coverage of the proximal bilayer is easily obtained by appropriate adjustment of the lipid concentration in the coating solution. Patches or extended regions of double bilayers are easily prepared. The present work deals with the preparation of supported ternary model membranes displaying two fluid phases. The spincoating technique35 has been applied to prepare singly and doubly supported membranes. By a combination of AFM and fluorescence microscopy, significant differences are found between the behavior of fluid domains in the upper (distal) and lower (proximal) bilayers. The role of salt in controlling the decoupling of the membrane from the support is investigated, and quantitative measurements of domain coarsening for a decoupled membrane are demonstrated. We perform a Voronoi analysis of the spatial arrangement of membrane domains and find that Lewis’ law and the Aboav-Weaire law for cellular patterns are satisfied. Agreement with the universal behavior of equilibrated cellular patterns is observed. 2. Experimental Section 2.1. Materials. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol (Chol) were purchased from Avanti Polar Lipids and used without further purification. 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiIC18) was obtained from Molecular Probes/Invitrogen. 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES; >99.5%), 4-(2-hydroxyethyl)piperazine-1ethanesulfonic acid sodium salt (HEPES sodium salt; >99.5%), and CaCl2 (>99.5%) were all from Sigma, while NaCl (>99.5%) and ethylenediaminetetraacetic acid (EDTA; >99%) were from Merck. Solvents (hexane, methanol) were all HPLC grade quality, and ultrapure Milli-Q water (18.3 MΩ‚cm) was used in all steps involving water. HEPES buffer (10 mM HEPES, 150 mM NaCl, 30 µM CaCl2, and 10 µM EDTA) was prepared at pH 7.5 by mixing the appropriate amounts of HEPES and HEPES sodium salt. Muscovite mica (75 mm × 25 mm × 200 µm sheets) was from Plano GmbH, Germany. Mica sheets of 10 mm × 10 mm were preglued onto round (0.17 mm, 24 mm) microscope coverslips using a transparent and biocompatible silicone glue (MED-6215, Nusil Technology, Santa Barbara, CA). Immediately prior to use, the mica was cleaved with a knife, leaving a thin and highly transparent mica film on the coverslip. 2.2. Preparation of Single and Double Membrane Islands by Spin-Coating. The preparation of singly and doubly supported membranes by hydration of spin-coated lipid films has been described and validated previously.35,36 For completeness we briefly describe the procedure here. The method consists of two steps: (1) fabrication of a dry spin-coated lipid film followed by (2) controlled hydration/ annealing of the film and release of excess lipid bilayers from the surface. To prepare a dry spin-coated lipid film on mica, we used a stock solution of 10 mM lipid (total) containing 0.5% DiIC18 in hexane/ methanol (97:3 volume ratio). A droplet (30 µL) of this lipid stock solution was then applied to freshly cleaved mica and immediately thereafter spun on a Chemat Technology, KW-4A spin-coater at 3000 rpm for 40 s. This creates a dry multilamellar lipid film where lipids in the distal monolayer have their acyl chains oriented outward. The sample was then placed under vacuum in a desiccator for 10(35) Simonsen, A. C.; Bagatolli, L. A. Langmuir 2004, 20, 9720-9728. (36) Simonsen, A.; Jensen, U.; Hansen, P. J. Colloid Interface Sci. 2006, 301, 107-115.
Ternary Membrane Domain Properties
Langmuir, Vol. 23, No. 15, 2007 8137
15 h to ensure complete evaporation of solvents. The dry spincoated film was subsequently hydrated by immersing the sample in either pure water or HEPES buffer followed by heating to 55 °C for 1 h. The sample was then placed on the fluorescence microscope (see below) and flushed with 55 °C buffer/water using a pipet adjusted to 500 µL. By monitoring the response of the lipid film while washing, the removal of lipid layers can be accurately controlled. Typically, the center of the sample is flushed, and this completely removes all but the lowest bilayer in this region. Some distance away from the flushed region, lipid multilayers are present, while in the transition zone one can routinely localize regions with a second (distal) bilayer situated on top of the lowest (proximal) bilayer. After the washing procedure, the liquid volume was gently exhanged 5-10 times to remove membranes in solution and minimize background fluorescence. 2.3. Fluorescence Microscopy. For fluorescence microscopy of the lipid film the sample was placed in the fluid cell (BioCell, JPK Instruments AG, Berlin, Germany) of the atomic force microscope. A Nikon TE2000 inverted microscope with a 40× long working distance objective (Nikon, ELWD, Plan Fluor, NA ) 0.6) was used for epifluorescence observations. Fluorescence excitation was done with a halogen lamp and using a G-2A filtercube (Nikon) appropriate for the DiI probe. Images were recorded with a high-sensitivity CCD camera (Sensicam em, 1004 × 1002 pixels, PCO-imaging, Kelheim, Germany) and operated with Camware software (PCO). 2.4. Atomic Force Microscopy. Atomic force microscopy was performed using a JPK Nanowizard AFM system (JPK Instruments AG) operated in contact mode. Silicon nitride cantilevers of the triangular type (MSCT, C-lever, Veeco) were used, with a nominal spring constant of 0.01 N/m and a resonance frequency of 7 kHz. During scanning, the sample was located in the fluid cell (BioCell, JPK Instruments AG) also used for fluorescence imaging. Colocalization of structures in AFM and fluorescence images (Figure 2) requires simultaneous observation of the AFM cantilever and the sample. This is made possible because the emitted fluorescent light from the membrane scatters from the cantilever, making it visible in the fluorescence image. 2.5. Image Analysis. Image processing and analysis were performed using both the scanning probe image processor (SPIP; Image Metrology, Hørsholm, Denmark) and MATLAB (Mathworks Inc., Boston, MA). The SPIP was used for processing of AFM images and for detection of domains in the fluorescence images. Custom m-files written in MATLAB were employed for the Voronoi analysis.
3. Results and Discussion 3.1. Domain Structures in Singly Supported Membranes. We investigate the domain patterns of different length scales that arise in supported ternary membranes following hydration of the spin-coated lipid film and annealing/cooling to ambient temperature. Figure 1A shows the membrane composition (DOPC/DPPC/Chol, 2:2:1) which was chosen to be centrally located in the lo/ld coexistence region of the phase diagram.37 For membranes hydrated in pure water the typical domain pattern (T ) 20 °C) is shown in Figure 1B. The domain pattern clearly displays two distinct phases with a height difference of 6.5 ( 2 Å (line scan). This value is in agreement with previously measured height differences for the lo/ld phases38 and is smaller than the typical height difference between solid and fluid domains (so/ld) of ∼1.2 nm.35,39 We found some variation in domain sizes and to a lesser extent in domain shapes between membranes of identical compositions, hydrated in water. The domains in Figure 1 are branched and nonregular and correspond well with AFM images published by Duyl et al.15 on (37) Veatch, S. L.; Keller, S. L. Phys. ReV. Lett. 2005, 94, 148101. (38) Chiantia, S.; Kahya, N.; Schwille, P. Langmuir 2005, 21, 6317-6323. (39) Burns, A. R. Langmuir 2003, 19, 8358-8363.
Figure 1. Domain morphology of the supported membrane with the composition DOPC/DPPC/Chol (2:2:1) without added salt in the acqeous phase. The membrane state at 20 °C (black circle) corresponds to a region of the ternary phase diagram (A) where lo and ld membrane phases are found in GUVs. The boundary (gray line) of the ld/lo coexistence region was adapted from Keller et al.37 The cross-section line (bottom) displays the height difference of 6.5 ( 2 Å between domains in the lo state (bright) and domains in the ld state (dark).
membranes with a similar composition. Compared to the expected round domains found in free-standing GUVs,11 the AFM scans reveal fundamentally different shapes. The pronounced difference between supported and freestanding membranes of the same composition indicates a strong perturbation of the domain shapes by the support surface. The domains in Figure 1B are static and do not evolve over the time span of an AFM experiment (
