Cholesterol Rules
2004; Elsevier BV; Volume: 279; Issue: 39 Linguagem: Inglês
10.1074/jbc.m404648200
ISSN1083-351X
AutoresJorge Bernardino de la Serna, Jesús Pérez‐Gil, Adam Cohen Simonsen, Luís A. Bagatolli,
Tópico(s)Hemoglobin structure and function
ResumoPulmonary surfactant, the lipid-protein material that stabilizes the respiratory surface of the lungs, contains approximately equimolar amounts of saturated and unsaturated phospholipid species and significant proportions of cholesterol. Such lipid composition suggests that the membranes taking part in the surfactant structures could be organized heterogeneously in the form of inplane domains, originating from particular distributions of specific proteins and lipids. Here we report novel results concerning the lateral organization of bilayer membranes made of native pulmonary surfactant where the coexistence of two distinct micrometer sized fluid phases (fluid ordered and fluid disordered-like phases) is observed at physiological temperatures by using fluorescence microscopy and atomic force microscopy. Additional experiments using fluorescent-labeled proteins SP-B and SP-C show that at physiological temperatures these hydrophobic proteins are located exclusively in the fluid disordered-like phase. Most interestingly, the microscopic coexistence of fluid phases is maintained up to 37.5 °C, where most fluid ordered phases melt. This observation suggests that the particular composition of this material is naturally designed to be at the “edge” of a lateral structure transition under physiological conditions, likely providing particular structural and dynamic properties for its mechanical function. The observed lateral structure in native pulmonary surfactant membranes is dramatically affected by the extraction of cholesterol, an effect not observed upon extraction of the surfactant proteins. Furthermore, the spreading properties of the native surfactant material at the air-liquid interface were also greatly affected by cholesterol extraction, suggesting a connection between the observed lateral structure and a physiologically relevant function of the material. We suggest that the particular lipid composition of surfactant could be finely tuned to provide, under physiological conditions, a structural scaffold for surfactant proteins to act at appropriate local densities and lipid composition. Pulmonary surfactant, the lipid-protein material that stabilizes the respiratory surface of the lungs, contains approximately equimolar amounts of saturated and unsaturated phospholipid species and significant proportions of cholesterol. Such lipid composition suggests that the membranes taking part in the surfactant structures could be organized heterogeneously in the form of inplane domains, originating from particular distributions of specific proteins and lipids. Here we report novel results concerning the lateral organization of bilayer membranes made of native pulmonary surfactant where the coexistence of two distinct micrometer sized fluid phases (fluid ordered and fluid disordered-like phases) is observed at physiological temperatures by using fluorescence microscopy and atomic force microscopy. Additional experiments using fluorescent-labeled proteins SP-B and SP-C show that at physiological temperatures these hydrophobic proteins are located exclusively in the fluid disordered-like phase. Most interestingly, the microscopic coexistence of fluid phases is maintained up to 37.5 °C, where most fluid ordered phases melt. This observation suggests that the particular composition of this material is naturally designed to be at the “edge” of a lateral structure transition under physiological conditions, likely providing particular structural and dynamic properties for its mechanical function. The observed lateral structure in native pulmonary surfactant membranes is dramatically affected by the extraction of cholesterol, an effect not observed upon extraction of the surfactant proteins. Furthermore, the spreading properties of the native surfactant material at the air-liquid interface were also greatly affected by cholesterol extraction, suggesting a connection between the observed lateral structure and a physiologically relevant function of the material. We suggest that the particular lipid composition of surfactant could be finely tuned to provide, under physiological conditions, a structural scaffold for surfactant proteins to act at appropriate local densities and lipid composition. Pulmonary surfactant is the main secretory product of the alveolar type II pneumocytes and is required to stabilize the lungs in air-breathing animals (1Perez-Gil J. Biol. Neonate. 2002; 81: 6-15Crossref PubMed Scopus (44) Google Scholar, 2Daniels C.B. Orgeig S. News Physiol. Sci. 2003; 18: 151-157PubMed Google Scholar). This surfactant material is mainly composed of lipids, mainly phospholipids, and small amounts of specifically associated proteins. Among the phospholipids significant amounts of dipalmitoylphosphatidylcholine (DPPC) 1The abbreviations used are: DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; AFM, atomic force microscopy; Bodipy-PC, 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine; DiIC18, 1,1′-dioctadecyl-3,3,3′,3′ -tetramethylindocarbocyanine perchlorate; Me2SO, dimethyl sulfoxide; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; GUV, giant unilamellar vesicle; NPSM, native pulmonary surfactant material; SP, surfactant proteins; PC, phosphatidylcholine. 1The abbreviations used are: DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; AFM, atomic force microscopy; Bodipy-PC, 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine; DiIC18, 1,1′-dioctadecyl-3,3,3′,3′ -tetramethylindocarbocyanine perchlorate; Me2SO, dimethyl sulfoxide; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; GUV, giant unilamellar vesicle; NPSM, native pulmonary surfactant material; SP, surfactant proteins; PC, phosphatidylcholine. and phosphatidylglycerol are present, both of which are unusual species in most animal membranes. Mono-unsaturated phosphatidylcholines (PC), phosphatidylinositol, and neutral lipids including cholesterol are also present in pulmonary surfactant in varying proportions. The surfactant proteins (SP) SP-A, SP-B, SP-C, and SP-D constitute about 8% of lung surfactant by weight. SP-A and SP-D are large glyco-proteins (>500 kDa) belonging to the superfamily of collectins, and they serve important functions in innate defense mechanisms of the lung (3Wright J.R. J. Clin. Investig. 2003; 111: 1453-1455Crossref PubMed Scopus (113) Google Scholar). The hydrophobic proteins SP-B and SP-C modulate the surface-active properties of surfactant lipids and are strictly required to establish an operational respiratory surface (4Whitsett J.A. Weaver T.E. N. Engl. J. Med. 2002; 347: 2141-2148Crossref PubMed Scopus (397) Google Scholar). Pulmonary surfactant lipid-protein complex is secreted into the thin aqueous alveolar lining of the lung as multilamellar assemblies, which spontaneously transform into nanotubular membrane-based structures called tubular myelin. These structures are considered reservoirs of highly surface-active components in the pathway that forms surface-active films at the lung air-water interface. Such films can reduce the surface tension to nearly 0 mn/m and in doing so prevent alveolar collapse at low lung volumes (1Perez-Gil J. Biol. Neonate. 2002; 81: 6-15Crossref PubMed Scopus (44) Google Scholar, 5Perez-Gil J. Keough K.M. Biochim. Biophys. Acta. 1998; 1408: 203-217Crossref PubMed Scopus (195) Google Scholar). Dysfunctions of the surfactant system are relevant in diseases including neonatal and acute respiratory distress syndrome, cystic fibrosis, and pneumonia (6Lewis J.F. Veldhuizen R. Annu. Rev. Physiol. 2003; 65: 613-642Crossref PubMed Scopus (134) Google Scholar). Several studies (7Discher B.M. Maloney K.M. Schief Jr., W.R. Grainger D.W. Vogel V. Hall S.B. Biophys. J. 1996; 71: 2583-2590Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 8Nag K. Perez-Gil J. Ruano M.L. Worthman L.A. Stewart J. Casals C. Keough K.M. Biophys. J. 1998; 74: 2983-2995Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) have shown that interfacial films made with the hydrophobic fraction (lipids + SP-B + SP-C) of pulmonary surfactant undergo phase separation under lateral compression. In addition, the particular phenomenon of gel/fluid phase separation induced by temperature was also reported in bilayers composed of part of the hydrophobic fraction of pulmonary surfactant at physiological temperatures (9Nag K. Pao J.S. Harbottle R.R. Possmayer F. Petersen N.O. Bagatolli L.A. Biophys. J. 2002; 82: 2041-2051Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Even though the particular composition of the lung surfactant suggests that native surfactant membrane-based structures could exhibit lateral segregation phenomena at physiological temperatures, this aspect has been merely speculative up to now. For example, the presence of high amounts of DPPC (∼40% weight of the total material) with a melting phase transition above mammalian physiological temperatures (41.5 °C) indicates the possibility of the coexistence of a solid/fluid-like phase (10Piknova B. Schram V. Hall S.B. Curr. Opin. Struct. Biol. 2002; 12: 487-494Crossref PubMed Scopus (112) Google Scholar). On the other hand, the presence of cholesterol along with other unsaturated phospholipid species in the native material suggests the possibility of a fluid ordered/fluid disordered-like phase coexistence (distinguished in part by their cholesterol content) under physiologically relevant conditions (10Piknova B. Schram V. Hall S.B. Curr. Opin. Struct. Biol. 2002; 12: 487-494Crossref PubMed Scopus (112) Google Scholar). Even though lung surfactant has cholesterol concentrations of up to 20–22 mol % (11Orgeig S. Daniels C.B. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2001; 129: 75-89Crossref PubMed Scopus (83) Google Scholar, 12Casals C. Arias-Diaz J. Valino F. Saenz A. Garcia C. Balibrea J.L. Vara E. Am. J. Physiol. 2003; 284: L466-L472Crossref PubMed Scopus (24) Google Scholar), there is no clear understanding of how this molecule impacts the lateral structure of the native material. The presence of cholesterol could thus have important consequences for the lateral organization of the native pulmonary surfactant material, illustrating another example of the fundamental role of cholesterol in modulating the lateral structure of membranes, as the raft hypothesis proposes (13Edidin M. Annu. Rev. Biophys. Biomol. Struct. 2003; 32: 257-283Crossref PubMed Scopus (1130) Google Scholar, 14Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8019) Google Scholar). Furthermore, there is presently no clear link between membrane lateral heterogeneity and the physiological function of native pulmonary surfactant material (10Piknova B. Schram V. Hall S.B. Curr. Opin. Struct. Biol. 2002; 12: 487-494Crossref PubMed Scopus (112) Google Scholar).In recent years, a new experimental strategy, based on the direct visualization of free standing lipid bilayers using giant unilamellar vesicle technology in conjunction with confocal and two photon excitation fluorescence microscopy techniques, has opened the possibility of correlating morphological and dynamical information on lipid membranes at molecular and supramolecular levels. Important novel information such as the morphology of different coexisting lipid phases (such as gel/fluid and fluid ordered/fluid disordered), mechanical properties of membranes displaying phase coexistence, local hydration, and molecular diffusion in lipid domains can be extracted directly from the fluorescence images (9Nag K. Pao J.S. Harbottle R.R. Possmayer F. Petersen N.O. Bagatolli L.A. Biophys. J. 2002; 82: 2041-2051Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 15Korlach J. Schwille P. Webb W.W. Feigenson G.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8461-8466Crossref PubMed Scopus (716) Google Scholar, 16Bagatolli L.A. Gratton E. Biophys. J. 2000; 78: 290-305Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 17Bagatolli L.A. Gratton E. Biophys. J. 2000; 79: 434-447Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 18Dietrich C. Bagatolli L.A. Volovyk Z.N. Thompson N.L. Levi M. Jacobson K. Gratton E. Biophys. J. 2001; 80: 1417-1428Abstract Full Text Full Text PDF PubMed Scopus (1187) Google Scholar, 19Feigenson G.W. Buboltz J.T. Biophys. J. 2001; 80: 2775-2788Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 20Veatch S.L. Keller S.L. Phys. Rev. Lett. 2002; 89: 268101(-1)-268101(-4)Crossref Scopus (559) Google Scholar, 21Veatch S.L. Keller S.L. Biophys. J. 2003; 85: 3074-3083Abstract Full Text Full Text PDF PubMed Scopus (1099) Google Scholar, 22Scherfeld D. Kahya N. Schwille P. Biophys. J. 2003; 85: 3758-3768Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 23Baumgart T. Hess S.T. Webb W.W. Nature. 2003; 425: 821-824Crossref PubMed Scopus (1263) Google Scholar). The present study uses the GUV technology in addition to other experimental techniques to provide evidence that a particular lateral structure occurs in native membranes of pulmonary surfactant at physiological temperatures. Additionally we demonstrate that cholesterol plays a critical role in promoting such membrane organization. On the other hand, it is important to note that preparation of GUVs was previously limited to membranes composed of artificial and natural lipid extracts (from lipid solutions in organic solvents). In this study we also present a method to prepare GUVs composed of native membranes without using organic solvents or detergents. To our knowledge, GUV experiments performed with native membrane preparations to further evaluate the concurrent effect of lipids and proteins in lateral segregation phenomena have not yet been reported.EXPERIMENTAL PROCEDURESMaterials—1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiIC18), 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (Bodipy-PC), and isothiocyanate derivatives of Alexa Fluor® 488 and Texas Red® were from Molecular Probes Inc. (Eugene, OR). Methyl-β-cyclodextrin was from Aldrich. DPPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL) and were used without further purification. Native pulmonary surfactant material was obtained from pig lungs as described previously (24Casals C. Herrera L. Miguel E. Garcia-Barreno P. Municio A.M. Biochim. Biophys. Acta. 1989; 1003: 201-203Crossref PubMed Scopus (31) Google Scholar). The cholesterol quantitation kits INFINITY™ and IVD were purchased from Sigma and Spinreact (Girona, Spain), respectively.Preparation of Surfactant and Proteins—Stock suspensions of native pulmonary surfactant material (NPSM) were in 5 mm Tris buffer, pH 7, containing 150 mm NaCl. The total concentration of phospholipid was 0.5 mg/ml, estimated by phosphorus quantitation upon phospholipid mineralization (25Rouser G. Fkeischer S. Yamamoto A. Lipids. 1970; 5: 494-496Crossref PubMed Scopus (2859) Google Scholar). For fluorescence experiments the fluorescent probes were incorporated into NPSM stock suspensions before preparing the giant vesicles. In the case of the lipophilic probes (DiIC18 and Bodipy-PC) a small aliquot (0.5 μl) of an Me2SO solution containing the fluorescent probes (0.125 mm) was added to the NPSM stock suspension (final volume 500 μl) and incubated for 20 min at room temperature. In all cases the percentage of fluorescent probes in the sample was less than 0.1 mol %.Fluorescently labeled surfactant proteins were prepared by labeling purified SP-B and SP-C solutions in organic solvent, as described previously (26Plasencia I. Cruz A. Lopez-Lacomba J.L. Casals C. Perez-Gil J. Anal. Biochem. 2001; 296: 49-56Crossref PubMed Scopus (16) Google Scholar). This procedure allows attachment of a single fluorophore to the N-terminal amine group of the protein. In our studies Alexa 488 and Texas Red were used for SP-C and SP-B, respectively. Labeled proteins were incorporated into NPSM by addition of small aliquots of fluorescent SP-B and SP-C (0.5% protein to lipid by weight) in Me2SO.Giant Vesicle Preparation—GUVs composed of NPSM organic extract, its lipid fraction, or the ternary mixture DOPC/DPPC/cholesterol were prepared as described previously (16Bagatolli L.A. Gratton E. Biophys. J. 2000; 78: 290-305Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 27Düzgünes N. Bagatolli L.A. Meers P. Oh Y. Straubinger R.M. Torchiling V.P. Weissig V. Liposomes. 2. Oxford University Press, London2003: 105-147Google Scholar) by using the electroformation method originally developed by Angelova and Dimitrov (28Angelova M.I. Dimitrov D.S. Faraday Discuss. Chem. Soc. 1986; 81: 303-311Crossref Google Scholar) and Angelova et al. (29Angelova M.I. Soléau S. Meléard P.H. Faucon J.F. Bothorel P. Colloid Polym. Sci. 1992; 89: 127-131Crossref Google Scholar). A previously described (16Bagatolli L.A. Gratton E. Biophys. J. 2000; 78: 290-305Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 27Düzgünes N. Bagatolli L.A. Meers P. Oh Y. Straubinger R.M. Torchiling V.P. Weissig V. Liposomes. 2. Oxford University Press, London2003: 105-147Google Scholar) special temperature-controlled chamber was used for this purpose. Briefly the process can be described in the following three steps. 1) ∼3 μl of the stock solution of the lipid or lipid/protein organic solution were spread on the surface of each platinum wire. The chamber was located under a stream of N2 during this procedure and then placed under vacuum overnight to remove the organic solvent. 2) Aqueous solution was added to the chamber (200 mosm sucrose solution prepared with Millipore-filtered water, 17.5 megohms/cm). The sucrose solution was previously heated to the desired temperature (above the lipid mixture phase transition, we used 45 °C), and then sufficient volume was added to cover the platinum wires (∼300 μl). 3) The platinum wires were connected immediately to a function generator (Digimess® FG 100, Germany), and a low frequency AC field (sinusoidal wave function with a frequency of 10 Hz and an amplitude of 3 V) was applied for 120 min. After vesicle formation, the AC field was turned off, and the vesicles were collected with a pipette and transferred to a plastic tube.NPSM GUVs were also prepared using the electroformation method but with some modifications in step 1 of the protocol described above. In this case NPSM GUVs were formed from NPSM suspended in buffer solution (no organic solvents in this case). Briefly, ∼3 μl of the stock suspension in buffer was spread on the surface of each platinum wire as small drops. The chamber was then placed under a stream of N2 and subsequently under low vacuum for 30 min to allow the native material to adsorb onto the platinum wire. An important point in this step is to avoid dehydration of the sample to maintain the integrity of the native membranes. Once the material was adsorbed to the Pt wire, we proceed with steps 2 and 3 as described above. As a control experiment, GUVs composed of DOPC/DPPC/cholesterol were also prepared from multilamellar suspensions in aqueous solution as described above for NPSM and then compared with those obtained from the lipid organic extract. No differences in the morphology of the final resulting GUVs were observed between the two described protocols.Observation of Giant Vesicles—Aliquots of giant vesicles suspended in sucrose were added to an equi-osmolar concentration of glucose solution. Because of the density difference between the two solutions, the vesicles precipitate at the bottom of the chamber which facilitates observation of the GUVs in the inverted confocal microscope. GUV preparations were observed in 8-well plastic chambers (Lab-Tek Brand Products, Naperville IL). The chamber was located in an inverted confocal microscope (Zeiss LSM 510 META) for observation. The excitation wavelengths were 488 (for Alexa 488 and Bodipy-PC) and 543 nm (for DiIC18 and Texas Red). The temperature was controlled from a water bath connected to a homemade device into which the 8-well plastic chamber was inserted. The temperature was measured inside the sample chamber using a digital thermocouple (model 400B, Omega Inc., Stamford, CT) with a precision of 0.1 °C.Differential Scanning Calorimetry—Experiments were performed in a Science Corp. N-DSCII calorimeter using 15 mm total lipid concentration of native pulmonary surfactant suspension. The scan rate was 0.5 K/min versus buffer. Five temperature scans were collected from each sample between 25 to 70 °C. Neither change in the total concentration nor in the scan rate gave significant changes in the obtained results.Cholesterol Extraction Experiments—Methyl-β-cyclodextrin/glucose solutions were prepared in Millipore water 17.5 megohms/cm. The osmolarity of these solutions was measured in an osmometer (The Advance Osmometer, model 3D3, Advance Instruments Inc., Norwood, MA) and adjusted to match the osmolarity of the vesicles containing sucrose solution. Vesicles were diluted in the methyl-β-cyclodextrin/glucose-containing solution and observed afterward. Alternatively, aliquots of methyl-β-cyclodextrin solutions were added to the vesicles already dispersed in glucose solution. No differences in the effect of methyl-β-cyclodextrin on the vesicles were observed between the two procedures. Cholesterol was also removed from the lipid fraction of NPSM by hydrophobic chromatography in Sephadex LH-20 (30Discher B.M. Maloney K.M. Grainger D.W. Sousa C.A. Hall S.B. Biochemistry. 1999; 38: 374-383Crossref PubMed Scopus (65) Google Scholar).Phospholipid and Cholesterol Determination—Total phospholipid concentration in the different samples was determined by phosphorus analysis as we described above (25Rouser G. Fkeischer S. Yamamoto A. Lipids. 1970; 5: 494-496Crossref PubMed Scopus (2859) Google Scholar). The amount of cholesterol in NPSM and in their different fractions was quantitatively estimated using the following: (i) a colorimetric assay based on the activity of cholesterol oxidase (IVD kit from Spinreact, Girona), and (ii) the determination of peroxide products resulting from cholesterol oxidation (INFINITY™ kit from Sigma). Cholesterol content in the samples is given as cholesterol-to-phospholipid molar ratio. Four different samples were used for total cholesterol quantitation from each of at least three different batches of each of the fractions studied.Protein Extraction from NPSM—The hydrophobic fraction of NPSM, containing all the lipid species plus the hydrophobic proteins SP-B and SP-C but lacking hydrophilic proteins SP-A and SP-D, was obtained by chloroform/methanol extraction. Separation of lipid from protein components in the organic mixture was achieved by chromatography of the surfactant organic extract through a Sephadex LH-20 column, as described previously (31Perez-Gil J. Cruz A. Casals C. Biochim. Biophys. Acta. 1993; 1168: 261-270Crossref PubMed Scopus (96) Google Scholar).Atomic Force Microscopy of NPSM Bilayers—NPSM aqueous suspensions were spread on top of an ultrapure water subphase in a Langmuir-Blodgett trough (Kibron, μ-trough) until a surface pressure of 1 mn/m was obtained. The film was allowed to equilibrate for 10 min and was subsequently compressed to 42 mn/m. While maintaining this pressure, a film was transferred to a freshly cleaved mica substrate previously immersed into the subphase by lifting the support at a constant speed of 1.5 mm/min. Following the initial deposition, a second layer was transferred onto the first by re-immersion of the coated mica into the subphase at 1.5 mm/s (also at a constant surface pressure of 42 mn/m). Consequently, the supported membranes used in the atomic force microscopy (AFM) experiments are bilayer structures formed by double transfer of the surface film (including any additional associated structure to the monolayer), which is obtained by spreading aqueous suspensions of native surfactant at the air-water interface. Topographical AFM images were obtained under aqueous conditions in a PicoSPM (Molecular Imaging) microscope operated in magnetically activated tapping mode using MAClevers.Spreading Kinetics—Surface activity of surfactant preparations was assayed by running π -t interfacial adsorption isotherms. In a typical experiment, 3 μl of surfactant suspension (10 mg/ml phospholipid concentration) were spread by direct deposition on top of a subphase 5 mm Tris, pH 7, containing 150 mm NaCl in the Teflon trough (20 cm2) of a thermostated surface balance built by Nima (Coventry, UK), and at 5 cm of a filter paper Wilhelmy plate, before monitoring surface pressure against time. Spreading experiments were done either at 25 or 37 °C. Spreading experiments in the presence of methyl-β-cyclodextrin were carried out by spreading equivalent amounts of native surfactant on top of 5 mm Tris, pH 7, subphases containing 150 mm NaCl, and different concentrations of methyl-β-cyclodextrin. Data shown are representative of three different experiments for two different batches of each assayed surfactant preparation.RESULTSFig. 1 shows images of single GUVs composed of NPSM doped with the fluorescent probes Bodipy-PC and DiIC18. Based on the particular round shape of the domains (18Dietrich C. Bagatolli L.A. Volovyk Z.N. Thompson N.L. Levi M. Jacobson K. Gratton E. Biophys. J. 2001; 80: 1417-1428Abstract Full Text Full Text PDF PubMed Scopus (1187) Google Scholar, 20Veatch S.L. Keller S.L. Phys. Rev. Lett. 2002; 89: 268101(-1)-268101(-4)Crossref Scopus (559) Google Scholar, 21Veatch S.L. Keller S.L. Biophys. J. 2003; 85: 3074-3083Abstract Full Text Full Text PDF PubMed Scopus (1099) Google Scholar) and the partition properties of the different fluorescent probes (15Korlach J. Schwille P. Webb W.W. Feigenson G.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8461-8466Crossref PubMed Scopus (716) Google Scholar, 19Feigenson G.W. Buboltz J.T. Biophys. J. 2001; 80: 2775-2788Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar), we conclude that the lateral organization of the NPSM vesicles corresponds to a fluid ordered/fluid disordered-like phase coexistence. The coexistence of two fluid phases persists from 22 to 37.5 °C. In general, when fluid domains are embedded in a fluid environment, circular domains are formed because both phases are isotropic, and the line energy (tension), which is associated with the rim of two demixing phases, is minimized by optimizing the area-to-perimeter ratio. As observed in Fig. 1C, the domains span the bilayer in agreement with previous observations done in artificial and natural lipid mixtures (15Korlach J. Schwille P. Webb W.W. Feigenson G.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8461-8466Crossref PubMed Scopus (716) Google Scholar, 16Bagatolli L.A. Gratton E. Biophys. J. 2000; 78: 290-305Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 17Bagatolli L.A. Gratton E. Biophys. J. 2000; 79: 434-447Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 18Dietrich C. Bagatolli L.A. Volovyk Z.N. Thompson N.L. Levi M. Jacobson K. Gratton E. Biophys. J. 2001; 80: 1417-1428Abstract Full Text Full Text PDF PubMed Scopus (1187) Google Scholar). NPSM frequently gave rise to multilamellar giant vesicles (see Fig. 1D), whose morphology resembles the structure of lamellar bodies that are the multilamellar assemblies storing and secreting pulmonary surfactant in the type II pneumocytes of the lung. Coexistence of two fluid phases was observed in every bilayer of these giant multilamellar assemblies. Fig. 2 shows an AFM image of an NPSM-supported membrane, without exogenous fluorescent probes. Such membranes are part of the surface-active film of pulmonary surfactant that spontaneously forms upon adsorption at the interface (32Schurch S. Green F.H. Bachofen H. Biochim. Biophys. Acta. 1998; 1408: 180-202Crossref PubMed Scopus (227) Google Scholar). It is important to emphasize that the samples visualized by AFM are not simple monolayers, as those studied previously in the literature, but bilayers as we perform a double deposition of surfactant films onto mica (see “Experimental Procedures”). AFM scanning of supported membranes demonstrates the presence of circular-shaped domains with sizes comparable with those seen in GUVs, confirming that two fluid phases coexist in the native material. This observation represents an important control experiment because it indicates that the membrane lateral structure in the GUVs is really present in the original material and is not because of the incorporation of fluorescent probes or the way the samples are prepared. AFM also reveals the coexistence of nano-scale structures inside the circular-shaped domains. Topological analysis shows that the distribution of heights observed in the round domains (i.e. higher versus lower heights) is more heterogeneous than the height distribution observed in the more homogeneous intervening background (Fig. 2). In particular, the round domains contain regions of lower heights compared to the height observed in the continuous region. This last finding suggests that 1) the round domains may correspond to a liquid disordered-like phase state and that 2) the region surrounding the round domains may display a liquid ordered-like phase state. The higher heights in the round domains may be due to the presence of surfactant proteins. The complex topography of the liquid-disordered domains is likely connected with particular organization of the many different molecular species (lipids and proteins) in the plane of the membrane. Given the compositional complexity of the material, further experiments to clarify this issue ar
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