Effect of Line Tension on the Lateral Organization of Lipid Membranes
2007; Elsevier BV; Volume: 282; Issue: 46 Linguagem: Inglês
10.1074/jbc.m706162200
ISSN1083-351X
AutoresAna J. García‐Sáez, Salvatore Chiantia, Petra Schwille,
Tópico(s)Spectroscopy and Quantum Chemical Studies
ResumoThe principles of organization and functioning of cellular membranes are currently not well understood. The raft hypothesis suggests the existence of domains or rafts in cell membranes, which behave as protein and lipid platforms. They have a functional role in important cellular processes, like protein sorting or cell signaling, among others. Theoretical work suggests that the interfacial energy at the domain edge, also known as line tension, is a key parameter determining the distribution of domain sizes, but there is little evidence of how line tension affects membrane organization. We have investigated the effects of the line tension on the formation and stability of liquid ordered domains in model lipid bilayers with raft-like composition by means of time-lapse confocal microscopy coupled to atomic force microscopy. We varied the hydrophobic mismatch between the two phases, and consequently the line tension, by modifying the thickness of the disordered phase with phosphatidylcholines of different acyl chain length. The temperature of domain formation, the dynamics of domain growth, and the distribution of domain sizes depend strongly on the thickness difference between the domains and the surrounding membrane, which is related to line tension. When considering line tension calculated from a theoretical model, our results revealed a linear increase of the temperature of domain formation and domain growth rate with line tension. Domain budding was also shown to depend on height mismatch. Our experiments contribute significantly to our knowledge of the physical-chemical parameters that control membrane organization. Importantly, the general trends observed can be extended to cellular membranes. The principles of organization and functioning of cellular membranes are currently not well understood. The raft hypothesis suggests the existence of domains or rafts in cell membranes, which behave as protein and lipid platforms. They have a functional role in important cellular processes, like protein sorting or cell signaling, among others. Theoretical work suggests that the interfacial energy at the domain edge, also known as line tension, is a key parameter determining the distribution of domain sizes, but there is little evidence of how line tension affects membrane organization. We have investigated the effects of the line tension on the formation and stability of liquid ordered domains in model lipid bilayers with raft-like composition by means of time-lapse confocal microscopy coupled to atomic force microscopy. We varied the hydrophobic mismatch between the two phases, and consequently the line tension, by modifying the thickness of the disordered phase with phosphatidylcholines of different acyl chain length. The temperature of domain formation, the dynamics of domain growth, and the distribution of domain sizes depend strongly on the thickness difference between the domains and the surrounding membrane, which is related to line tension. When considering line tension calculated from a theoretical model, our results revealed a linear increase of the temperature of domain formation and domain growth rate with line tension. Domain budding was also shown to depend on height mismatch. Our experiments contribute significantly to our knowledge of the physical-chemical parameters that control membrane organization. Importantly, the general trends observed can be extended to cellular membranes. Cellular membranes form closed volumes that define the cell and organelle identity, although ensuring the exchange of matter, energy, and information that is required for life. They do so by means of a complex protein and lipid composition that is actively regulated in time and varies not only among the different cellular membranes but also between the two leaflets that form the membrane bilayer (1van Meer G. EMBO J. 2005; 24: 3159-3165Crossref PubMed Scopus (411) Google Scholar). During the last years, it has also become clear that the existence of lateral heterogeneities or domains in membranes is essential for a number of cellular functions. The raft theory predicts the existence of lipid assemblies that are enriched in sphingolipids and cholesterol. These membrane domains are thought to behave as protein and lipid platforms, important for protein trafficking and sorting, cell signaling, and other cellular processes (2Jacobson K. Mouritsen O.G. Anderson R.G. Nat. Cell Biol. 2007; 9: 7-14Crossref PubMed Scopus (910) Google Scholar, 3Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8157) Google Scholar, 4Simons K. Vaz W.L. Annu. Rev. Biophys. Biomol. Struct. 2004; 33: 269-295Crossref PubMed Scopus (1366) Google Scholar). Recent findings suggest that rafts are dynamic structures of transient nature and sizes in the nanometer range (5Samsonov A.V. Mihalyov I. Cohen F.S. Biophys. J. 2001; 81: 1486-1500Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). Though still debated, the widely accepted view implies a situation far from equilibrium, where signaling or sorting processes would induce the coalescence of these lipid assemblies into more stable larger platforms in the membrane (6Mayor S. Rao M. Traffic. 2004; 5: 231-240Crossref PubMed Scopus (334) Google Scholar). However, the current knowledge of membrane organization is not sufficient to fully explain the behavior and functioning of cellular membranes. Precisely because of their complex composition and dynamics, it is difficult to understand the principles that govern the lateral organization of the cell membrane in relation to its function. During the last years, some of the physical properties of the plasma membrane have been studied with model membranes that mimic the lipid composition of rafts. Model membranes are still far away from representing the intricacy found in cells, but they constitute simplistic systems that can help understanding the principles of the processes that happen in cellular membranes. In model lipid membranes with “raft-like” composition, large domains are observable by fluorescence microscopy or AFM (7Bacia K. Scherfeld D. Kahya N. Schwille P. Biophys. J. 2004; 87: 1034-1043Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 8Baumgart T. Hess S.T. Webb W.W. Nature. 2003; 425: 821-824Crossref PubMed Scopus (1305) Google Scholar, 9Chiantia S. Kahya N. Ries J. Schwille P. Biophys. J. 2006; 90: 4500-4508Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 10Kahya N. Scherfeld D. Bacia K. Poolman B. Schwille P. J. Biol. Chem. 2003; 278: 28109-28115Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 11Veatch S.L. Keller S.L. Phys. Rev. Lett. 2005; 94: 148101Crossref PubMed Scopus (461) Google Scholar). These domains are enriched in sphingolipids and cholesterol and appear as a liquid ordered (Lo) phase, coexisting with a liquid disordered (Ld) 2The abbreviations used are: Ld, liquid disordered; Lo, liquid ordered; PC, phosphatidylcholine; DPoPC, 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine; DMoPC, 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DEiPC, 1,2-dieicosenoyl-sn-glycero-3-phosphocholine; DEruPC, 1,2-dierucoyl-sn-glycero-3-phosphocholine; SM, N-stearoyl-d-erythro-sphingosylphosphocholine; Chol, cholesterol; GUV, giant unilamellar vesicle; AFM, atomic force microscopy; A/P, area to perimeter ratio; DiD, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate; CtxB-488, cholera toxin-labeled with Alexa488. phase. In such membranes, domains exhibit a circular shape, which is rapidly recovered after a mechanical distortion (5Samsonov A.V. Mihalyov I. Cohen F.S. Biophys. J. 2001; 81: 1486-1500Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 9Chiantia S. Kahya N. Ries J. Schwille P. Biophys. J. 2006; 90: 4500-4508Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). This tendency to minimize the boundary length indicates the presence of line tension at the phase interface. AFM and x-ray scattering measurements show that the Lo phase is thicker than the Ld one, giving rise to a “height mismatch” at the domain edge (13Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2065) Google Scholar, 14Maulik P.R. Shipley G.G. Biophys. J. 1996; 70: 2256-2265Abstract Full Text PDF PubMed Scopus (100) Google Scholar, 15Saslowsky D.E. Lawrence J. Ren X. Brown D.A. Henderson R.M. Edwardson J.M. J. Biol. Chem. 2002; 277: 26966-26970Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The exposure of the hydrophobic tails of the lipids to the aqueous solvent would have a very unfavorable energetic effect, and as a consequence, the membrane distorts at the boundary to avoid it (16Kuzmin P.I. Akimov S.A. Chizmadzhev Y.A. Zimmerberg J. Cohen F.S. Biophys. J. 2005; 88: 1120-1133Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). This height mismatch has an energetic cost per unit length that is probably one of the main parameters contributing to the line tension at the phase boundary. The distribution of domain sizes depends on the balance between line tension, which tends to increase size in order to reduce total boundary length and entropy and electrostatic repulsions, which oppose raft merger (16Kuzmin P.I. Akimov S.A. Chizmadzhev Y.A. Zimmerberg J. Cohen F.S. Biophys. J. 2005; 88: 1120-1133Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 17Blanchette C.D. Lin W.C. Ratto T.V. Longo M.L. Biophys. J. 2006; 90: 4466-4478Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 18Veatch S.L. Keller S.L. Biochim. Biophys. Acta. 2005; 1746: 172-185Crossref PubMed Scopus (653) Google Scholar). As a consequence, line tension is probably a major factor in the regulation of raft size. Line tension at the domain interface has been experimentally estimated in giant unilamellar vesicles with phase separation (8Baumgart T. Hess S.T. Webb W.W. Nature. 2003; 425: 821-824Crossref PubMed Scopus (1305) Google Scholar, 19Baumgart T. Das S. Webb W.W. Jenkins J.T. Biophys. J. 2005; 89: 1067-1080Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar) and very recently, in planar supported bilayers by means of nucleation rate measurements (20Blanchette C.D. Lin W.C. Orme C.A. Ratto T.V. Longo M.L. Langmuir. 2007; 23: 5875-5877Crossref PubMed Scopus (44) Google Scholar). Theoretical models have related line tension to physical properties of the membrane, like phase height mismatch, lateral tension, and spontaneous curvature (16Kuzmin P.I. Akimov S.A. Chizmadzhev Y.A. Zimmerberg J. Cohen F.S. Biophys. J. 2005; 88: 1120-1133Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 21Akimov S.A. Kuzmin P.I. Zimmerberg J. Cohen F.S. Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 2007; 75: 011919Crossref PubMed Scopus (70) Google Scholar). According to them, line tension increases quadratically with phase height mismatch. However, there is little experimental evidence about how line tension affects the lateral membrane organization and the formation of domains in terms of kinetics of domain formation, domain size and shape, and domain dynamics and stability. To address these questions, we have investigated the effects of the line tension on the formation of Lo domains in model lipid bilayers with raft-like composition. Given the link between line tension and phase height mismatch, we systematically varied the height mismatch between the two phases and consequently the line tension, by modifying the thickness of the Ld phase with PCs of different acyl chain length. Our studies involved measurements at non-equilibrium conditions. Using time-lapse confocal microscopy and AFM imaging, we analyzed the kinetics of domain formation, the domain shape and size, and the demixing temperature from Ld to Ld-Lo coexistence, as a function of the hydrophobic mismatch. Our results indicate a great influence of the line tension on the physical-chemical properties of Lo domains. We observed that at higher hydrophobic mismatch, the increased line tension led to bigger domains that formed with significantly faster kinetics to minimize the interface length. Interestingly, both the demixing temperature and the domain growth rate increased linearly with line tension, calculated from phase height mismatch measurements according to the model in (16Kuzmin P.I. Akimov S.A. Chizmadzhev Y.A. Zimmerberg J. Cohen F.S. Biophys. J. 2005; 88: 1120-1133Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). Under conditions close to equilibrium, domains were bigger and more circular at higher line tension. In addition, experiments in giant unilamellar vesicles linked height mismatch to line tension and domain budding. Preparation of Supported Bilayers—1,2-Dipalmitoleoyl-sn-glycero-3-phosphocholine (DPoPC), 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (DMoPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEiPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEruPC), N-stearoyl-d-erythro-sphingosylphosphocholine (SM), and cholesterol (Chol) were purchased from Avanti Polar Lipids (Alabaster, AL). Planar supported bilayers were prepared as described in (22Chiantia S. Ries J. Kahya N. Schwille P. Chem. Phys. Chem. 2006; 7: 2409-2418Crossref Scopus (171) Google Scholar). Briefly, lipids were dissolved in chloroform at the desired molar concentration, and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD-C18) (Molecular Probes, Eugene, OR) was added to the lipid mixtures at a 0.01% (mol/mol) concentration. The solvent was evaporated under nitrogen flux followed by vacuum for 1 h. Lipid films were rehydrated to a final concentration of 10 mg/ml in 3 mm KCl, 1.5 mm KH2PO4, 8 mm Na2HPO4, 150 mm NaCl, pH 7.2 and vortexed for 5 min. A small aliquot (10 μl) of the suspension of multilamellar vesicles was diluted in 140 μl of 3 mm CaCl2, 150 mm NaCl, 10 mm Hepes, 3 mm NaN3, pH 7.4. The suspension was then bath-solicited at 60 °C until small unilamellar vesicles were obtained and then put in contact with freshly cleaved mica substrate, previously glued to a glass coverslip. The mixture was incubated at 40 °C for 2 min and then at 68 °C for 10 min. At this temperature, the samples were rinsed several times with 150 mm NaCl, 10 mm Hepes, 3 mm NaN3, pH 7.4, to remove the non-fused vesicles. Sample temperature was controlled with a BioCell (JPK Instruments, Berlin, Germany). The lipid content per sample was ∼2 nmol, calculated assuming an average area per lipid molecule of 0.6 nm2 (4Simons K. Vaz W.L. Annu. Rev. Biophys. Biomol. Struct. 2004; 33: 269-295Crossref PubMed Scopus (1366) Google Scholar). Preparation of Giant Unilamellar Vesicles—Giant unilamellar vesicles (GUVs) of the desired lipid composition were prepared according to the electroformation method as described in (10Kahya N. Scherfeld D. Bacia K. Poolman B. Schwille P. J. Biol. Chem. 2003; 278: 28109-28115Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). Briefly, 5 μl of lipid mixture at 10 mg/ml was spread on indium tin oxide-coated coverslips at 65 °C. After solvent evaporation, the electrodes were assembled into custom-made perfusion chambers that were filled with 300 mm sucrose. Electroformation proceeded at 1.2 V and 10 Hz during ∼1 h. Samples were equilibrated to room temperature and checked for phase separation with the confocal microscope. Then, 5 μg of B subunit of cholera toxin-labeled with Alexa488 (CtxB-488) was added to the chamber, incubated for 30 min, and washed out with 300 mm sucrose solution. No apparent changes in the pattern of phase separation were observed upon CtxB-488 labeling (23Hammond A.T. Heberle F.A. Baumgart T. Holowka D. Baird B. Feigenson G.W. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6320-6325Crossref PubMed Scopus (271) Google Scholar). Confocal Microscopy—We performed confocal fluorescence microscopy of supported lipid bilayers on a LSM Meta 510 instrument (Carl Zeiss, Jena, Germany). Confocal images were taken by using the excitation light of a He-Ne laser at 633 nm, which was reflected by a dichroic mirror (HTF 488/633) and focused through a Zeiss C-Apochromat ×20, 0.75 numerical aperture objective onto the sample. The fluorescence signal was collected by the same objective, passed a 680/30-nm band pass filter and finally detected by a photomultiplier. Confocal geometry was ensured by a 100-μm pinhole in front of the photomultiplier. GUVs were imaged in a commercial ConfoCor2 system (Carl Zeiss) using multi-track mode. Light from an Ar laser at 488 nm, and a He-Ne laser at 633 nm was reflected with a HFT UV/488/543/633 dichroic. A ×40 numerical aperture 1.2 C-Apochromat water immersion objective was used, and the pinhole size was set to 90 μm in the green channel, although adjusted in the red channel for the same z thickness. Emitted fluorescence was separated with a secondary dichroic beam splitter 570 dichroic and passed through 505 nm or 650 nm long pass filters to be finally detected with a photomultiplier. Image processing and analysis was carried out with ImageJ (rsb.info.nih.gov/ij/). Atomic Force Microscopy—AFM measurements were performed using a NanoWizard system (JPK Instruments, Berlin, Germany) mounted on the same LSM Meta 510 setup used for microscopy. Contact mode topographic images were taken in the constant-deflection mode, using V-shaped silicon nitride cantilevers (Veeco, Santa Barbara, CA) with a typical spring constant of 0.08 newton/m. The force applied on the sample was maintained at the lowest possible value by continuously adjusting the set point during imaging. The scan rate was set to 1 Hz. Height and deflection were collected simultaneously in both trace and retrace directions. Images were line-fitted as required with JPK processing software (JPK Instruments, Berlin, Germany). Occasionally, isolated scan lines were removed. We performed image analysis with ImageJ and OriginPro (OriginLab, Northampton, MA). Line Tension Affects the Demixing Temperature into Ld and Lo Phases—To systematically investigate the role of line tension on the properties of raft-like domains, we modulated the height mismatch between the Ld and Lo phases by changing the thickness of the Ld phase, which is enriched in unsaturated PC (4Simons K. Vaz W.L. Annu. Rev. Biophys. Biomol. Struct. 2004; 33: 269-295Crossref PubMed Scopus (1366) Google Scholar). For this purpose, we used PC of varying acyl chain length in different samples (see Table 1). We prepared supported lipid bilayers composed by a doubly unsaturated phosphatidylcholine (PC), sphingomyelin (SM), and cholesterol (Chol) in a 2:2:1 ratio. We included 0.05% DiD, a fluorescent lipid analogue that partitions specifically to the Ld phase (7Bacia K. Scherfeld D. Kahya N. Schwille P. Biophys. J. 2004; 87: 1034-1043Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 9Chiantia S. Kahya N. Ries J. Schwille P. Biophys. J. 2006; 90: 4500-4508Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 10Kahya N. Scherfeld D. Bacia K. Poolman B. Schwille P. J. Biol. Chem. 2003; 278: 28109-28115Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). Usually, model membranes with such lipid mixture exhibit coexistence of Ld and Lo phases that can be visualized by AFM or fluorescence microscopy (7Bacia K. Scherfeld D. Kahya N. Schwille P. Biophys. J. 2004; 87: 1034-1043Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 9Chiantia S. Kahya N. Ries J. Schwille P. Biophys. J. 2006; 90: 4500-4508Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 10Kahya N. Scherfeld D. Bacia K. Poolman B. Schwille P. J. Biol. Chem. 2003; 278: 28109-28115Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar).TABLE 1Lipid composition of the supported bilayers used in this study The number of carbon atoms in the acyl chain of PC, the height difference between Ld and Lo phases, the demixing temperature, the average circularity of Lo domains and the line tension are shown.Lipid composition (2:2:1)Carbon atoms in PC acyl chainPhase height differenceDemixing temperatureAverage domain circularityaThe indicated errors correspond to the standard deviation of the mean in the case of phase height difference and average domain circularity, and the average σ of the Gaussian fittings in the height mismatch values,bCircularity was calculated as 4π (area/perimeter2) for every measured domain using the ImageJ softwareLine tensionpmaThe indicated errors correspond to the standard deviation of the mean in the case of phase height difference and average domain circularity, and the average σ of the Gaussian fittings in the height mismatch values°CaThe indicated errors correspond to the standard deviation of the mean in the case of phase height difference and average domain circularity, and the average σ of the Gaussian fittings in the height mismatch valuespNcCalculated from the model in (16), assuming a soft domain, with Br = Bs = 10 kT, and kr = ks = 40 mN/m, and no spontaneous curvature, Jr = Js = 0 (16). We considered an effective thickness of the Ld DOPC bilayer of 5.5 nm (26). Errors were estimated with Gaussian error propagationDEruPC:SM:Chol22170 ± 7038 ± 10.6 ± 0.20.06 ± 0.04DEiPC:SM:Chol20670 ± 8042 ± 10.78 ± 0.150.8 ± 0.3DOPC:SM:Chol18870 ± 10046 ± 10.78 ± 0.141.2 ± 0.5DPoPC:SM:Chol161330 ± 13058 ± 30.90 ± 0.134.1 ± 1.5DMoPC:SM:Chol141560 ± 13066 ± 30.90 ± 0.116 ± 2a The indicated errors correspond to the standard deviation of the mean in the case of phase height difference and average domain circularity, and the average σ of the Gaussian fittings in the height mismatch valuesb Circularity was calculated as 4π (area/perimeter2) for every measured domain using the ImageJ softwarec Calculated from the model in (16Kuzmin P.I. Akimov S.A. Chizmadzhev Y.A. Zimmerberg J. Cohen F.S. Biophys. J. 2005; 88: 1120-1133Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), assuming a soft domain, with Br = Bs = 10 kT, and kr = ks = 40 mN/m, and no spontaneous curvature, Jr = Js = 0 (16Kuzmin P.I. Akimov S.A. Chizmadzhev Y.A. Zimmerberg J. Cohen F.S. Biophys. J. 2005; 88: 1120-1133Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). We considered an effective thickness of the Ld DOPC bilayer of 5.5 nm (26Leonenko Z.V. Finot E. Ma H. Dahms T.E. Cramb D.T. Biophys. J. 2004; 86: 3783-3793Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Errors were estimated with Gaussian error propagation Open table in a new tab To ensure sample homogeneity, all samples had the same thermal history. After bilayer formation, samples were warmed up to 67 °C, above the transition temperature, and then cooled down to 21 °C in ∼5 min with a temperature controller. We performed this process only once for every membrane. During the cooling process Lo domains appeared when the temperature of phase demixing was achieved. We measured the formation and growth of domains with confocal microscopy. Because the fluorescent dye DiD is excluded from the Lo phase, Lo domains can be identified as dark patches in the membrane. Fig. 1 shows the first 5 min of the process. The corresponding movies can be found in the Supplemental Data and include the first 10 min of the kinetics of domain formation and growth. Interestingly, no domains were discernible in the case of the sample containing the PC with the longest acyl chain (DEruPC). For the rest of the samples, domains grew faster when membranes contained PC of shorter acyl chains (see series B to E in Fig. 1). In addition, phase demixing took place at different moments and subsequently at different temperatures depending on the acyl chain length. We define “demixing temperature” as the measured temperature at which the appearance of Lo domains was observed in our system (see Table 1). In the case of the sample composed of DEruPC:SM:Chol, we measured the demixing temperature in a different series of experiments in which the thermal history of the sample was modified to obtain observable domains (17Blanchette C.D. Lin W.C. Ratto T.V. Longo M.L. Biophys. J. 2006; 90: 4466-4478Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). When we cooled the sample just a couple of degrees below the demixing temperature, domains grew faster and bigger and could be then observed by confocal microscopy. After equilibration for 2.5 h at 21 °C, we scanned the same lipid membranes with AFM (see Fig. 2). We obtained topographical images of the sample surface at higher spatial resolution, from which we measured the height difference between the Ld and Lo phases for the different lipid compositions (see Table 1). In this case, domains with an average area of 0.056 ± 0.003 μm2 and ∼170 pm thicker than the surrounding Ld phase could be distinguished for the sample containing DEruPC. These results, contradictory to the microscopy observations, can be explained by the fact that the domain size could be below our optical resolution, or that the DiD dye would not partition specifically to the Ld phase for this lipid composition, or a combination of both. The images in Fig. 2 show that the Lo domains tend to be bigger and the difference in thickness between the two phases higher as the acyl chain length of the PC contained in the lipid bilayers decreases, in agreement with a thinner Ld phase. The height mismatch values measured for the different lipid compositions are shown in Table 1. If we combine the demixing temperatures measured for the different lipid compositions with the height mismatch that those lipid compositions exhibit between the Lo and Ld phases (see Table 1), we get an estimation of the dependence of the temperature of phase demixing with phase height mismatch. Fig. 3 depicts this relationship and shows that the demixing temperature strongly increases with height mismatch, and hence, line tension. Domain Growth, Size, and Shape Depend on the Line Tension—By image analysis, we quantified the distribution of domain sizes from the AFM images obtained for the different samples. Fig. 4 depicts the histograms of the logarithm of domain area and their corresponding fittings to Gaussian curves. There is a clear trend to domain enlargement with the increase in thickness difference between the Ld and Lo phases. Domain circularity, calculated as 4π (area/perimeter2) (ImageJ), is shown in Table 1. In agreement with the observations above, there is a tendency to increase domain circularity with phase height mismatch. As expected, phase height mismatch is related to an increased line tension and to the formation of bigger and more circular domains to minimize the energetic cost associated to the domain interface length. Though with a lower spatial resolution, we measured domain growth from the time series of domain formation obtained with the confocal microscope by image analysis. Fig. 5A shows that the average domain area increases approximately linearly with time for the different lipid mixtures. We calculated the rate of domain growth from the slope after 300 s, when the temperature of the sample could be considered constant. As observed in Fig. 5B, the rate of domain growth increased strongly with the height mismatch and thus, with the interfacial line tension, showing a similar dependence as the demixing temperature. We compared the mechanism of domain growth during the 2.5 h of membrane equilibration for the different lipid compositions. We observed that domain fusion happens mostly during the first minutes of phase separation, whereas Ostwald ripening predominates at later stages, favoring the growth of the bigger domains and the shrinkage and disappearance of the smaller ones. The latter phenomenon becomes more important for lipid mixtures that show a larger height mismatch, suggesting a higher energy barrier for domain interaction. According to Cohen and colleagues (16Kuzmin P.I. Akimov S.A. Chizmadzhev Y.A. Zimmerberg J. Cohen F.S. Biophys. J. 2005; 88: 1120-1133Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), the monolayer deformation at the raft boundary increases with height mismatch. As a consequence, the energy barrier for domains come into contact, which implies interaction of membrane deformations (16Kuzmin P.I. Akimov S.A. Chizmadzhev Y.A. Zimmerberg J. Cohen F.S. Biophys. J. 2005; 88: 1120-1133Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), is higher for larger difference in phase thickness. Membrane Curvature in GUVs Responds to Domain Line Tension—In addition to minimization of domain boundary length, line tension has been shown to induce out-of-plane membrane curvature (8Baumgart T. Hess S.T. Webb W.W. Nature. 2003; 425: 821-824Crossref PubMed Scopus (1305) Google Scholar, 19Baumgart T. Das S. Webb W.W. Jenkins J.T. Biophys. J. 2005; 89: 1067-1080Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). As a result, the shape of vesicles with line tension differs from that of vesicles without a phase separation. Because membrane curvature is constrained in supported lipid bilayers, we used GUVs, which were free-standing membranes, to investigate the role of phase height mismatch on vesicle shape. Because GUV preparation yields very heterogeneous samples, we analyzed hundreds of vesicles and attempted to extract relevant information from the ensemble measurements. Fig. 6 shows representative vesicles obtained for the different lipid compositions measured by confocal microscopy. In agreement with the observations made in planar supported bilayers, no phase separation could be distinguished in the GUVs containing DEruPC (Fig. 6, A and B), which had the longest acyl chai
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