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Surface behavior, microheterogeneity and adsorption equilibrium of myelin at the air–water interface
Chemistry and Physics of Lipids 122 (2003) 171 /176 www.elsevier.com/locate/chemphyslip
Short communication
Surface behavior, microheterogeneity and adsorption equilibrium of myelin at the air water interface /
Rafael G. Oliveira, Bruno Maggio * Facultad de Ciencias Quı´micas, Departamento de Quı´mica Biolo´gica-CIQUIBIC, Universidad Nacional de Co´rdoba, 5000 Cordoba, Argentina
Abstract Interfacial films of whole myelin membrane adsorb at the air /water interface from myelin vesicles. The films show a liquid state and their equilibrium spreading pressure is equal to the collapse pressure (about 47 mN/m). The films appear microheterogeneous as seen by epifluorescence microscopy, consisting in two liquid phases over all the adsorption isotherm, starting with rounded liquid expanded domains (low surface pressure) immersed in a cholesterol enriched phase and reaching a fractal pattern at high surface pressure similar to those previously observed by compressing the film. Vesicles adsorb to the interfacial film mainly at the lateral interfaces. The high surface pressure at equilibrium (almost equal to the collapse pressure) indicates the formation of surface multilayers, also shown by fluorescence microscopy. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Monolayers; Myelin; Epifluorescence microscopy; Critical unilamellar state; Surface pressure
1. Introduction Many lipids and membrane proteins self-associate spontaneously to form bilayer membranes. Also these components form interfacial monomolecular films, which according to Gibbs’s equation decrease the surface tension g of an aqueous solution. This drop is defined as the surface pressure, p (Gaines, 1966). Aqueous media with biomembrane and liposome suspensions invariably show spontaneously adsorbed interfacial (air / water) films. Since the film at the air /water interface is in dynamic equilibrium with the bulk * Corresponding author. Fax: /54-351-4334074. E-mail address: [email protected] (B. Maggio).
vesicle suspension, properties and processes like phase transition of the vesicle lipids can be inferred from measurements on interfacial films (MacDonald and Simon, 1987). One of such properties is the equilibrium spreading (surface) pressure of the film pesp (Gaines, 1966). This is a function of temperature T (Birdi, 1989) and generally shows a maximum at a critical temperature Tc, a few degrees over the bulk phase transition T . For liposome suspensions of pure phospholipids a maximum of pesp at Tc indicates a particular bilayer thermodynamic state, the critical unilamellar state (CUS) (Gershfeld, 1989). This is the only state at which the unilamellar bilayer is in true thermodynamic equilibrium. The criticality of this state explains why multilamellar liposomes (stable
0009-3084/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 3 0 8 4 ( 0 2 ) 0 0 1 8 7 - 1
172
R.G. Oliveira, B. Maggio / Chemistry and Physics of Lipids 122 (2003) 171 /176
below and above Tc) are more readily obtained than unilamellar bilayers. Nevertheless, most plasma membranes are stable as unilamellar structure because the mixed components are in the CUS, as indicated by a maximum in pesp at 37 8C. This maximum is considered enough criteria to ascertain the CUS (Gershfeld, 1989). In recent work we carried out a physicochemical characterization of Langmuir (air /water interface) (Oliveira et al., 1998; Oliveira and Maggio, 2000) and Langmuir /Blodgett myelin films supported over Sagiv monolayers (Oliveira and Maggio, 2002). We showed that myelin shows a liquid-expanded physical state with a single collapse point (Oliveira et al., 1998). Epifluorescence microscopy revealed liquid phase coexistence over the full compression isotherm as indicated by probe partition, domain mobility, deformability and boundary relaxation due to the line tension of surface domains (Oliveira and Maggio, 2000). These results were further confirmed by both Brewster angle microscopy of fresh films and specific labeling of individual myelin components in Langmuir /Blodgett films which showed that phase coexistence is due to compositional immiscibility (Oliveira and Maggio, 2002). Immunolabeling indicated that the major myelin proteins and GM1 ganglioside (all of them liquid expanded and electrically charged) were located in the liquid-expanded phase. The other phase showed an enrichment in the cholesterol, galactocerebroside and phosphatidylserine markers. The distribution of components was qualitatively independent on the p and was generally constituted by one phase enriched in charged components of intrinsically expanded state coexisting with another phase enriched in non charged constituents of lower compressibility. We also showed that the myelin film at room temperature shows a very high pesp of about 47 mN/m, that can not be distinguished (within experimental error) from the pcol. Thus, a film with very high p (up to the collapse point) was spontaneously formed by adsorption. Because at the pcol multilayers begin to form at the interface, and taking into account the multilamellar structure of myelin, it is very unlike that myelin could be in the CUS at physiological T because that would require that the pesp would have to be
even higher at 37 8C. The hypothesis checked in this work is that myelin should lack the CUS, in contrast to the few other natural membranes analyzed to date.
2. Experimental procedures Bovine spinal cord myelin was purified with the method that renders the purest myelin (Haley et al., 1981) and vesiculated in phosphate buffer 1 mM pH 8, Dithiothreitol 2 mM (Steck et al., 1978). The vesicles were resuspended in the same buffer to a final protein concentration of 0.24 mg/ ml, and passed five times through a 26G needle fitted to a syringe. Purity was assessed through SDS-PAGE and HPTLC. The probes N -(7-nitro2-1, 3-benzoxadiazol-4-yl) diacyl phosphatidylethanolamine (McConnell et al., 1984) and N (lissamine rhodamine B sulfonyl) diacyl phosphatidylethanolamine (RHO-PE) were from Avanti Polar Lipids (Alabaster, AL). Details about epifluorescence microscopy were as previously cited (Oliveira and Maggio, 2002). Both probes are head group labeled, with a 55% of acyl chain unsaturation, made from egg phosphatidylcholine, which favors their partition in liquid-expanded (disordered) phases, being mostly excluded from liquid condensed and cholesterol-enriched phases. The routine subphase was 10 mM Tris[hydroxymethyl aminomethane] buffer in 100 mM NaCl, 20 mM CaCl2, adjusted to pH 7.4 with HCl. The observations were carried out using a KSV Minitrough II (KSV, Helsinski, Finland) mounted on the microscope stage. Representative measurements were corroborated using N2 atmosphere to ensure no appreciable effect due to lipid peroxidation.
3. Results and discussion Fig. 1A shows a phase contrast microscopy micrograph of the myelin vesicles used in this work. They tend to self-aggregate, in agreement with the literature (Steck et al., 1978) although some free lamellar structures can be seen on the periphery of the vesicle clusters. The adsorption isotherm of myelin vesicles on a clean air-buffer
R.G. Oliveira, B. Maggio / Chemistry and Physics of Lipids 122 (2003) 171 /176
173
Fig. 1. Myelin vesicles and their adsorption to the air /water interface as a function of T . (A) Phase contrast micrograph of myelin vesicles used to measure pesp, on which the tendency to self aggregate is observed. (B) Adsorption isotherm of myelin vesicles. After p has been stabilized by a minimal of 1 h, further compression leads to a very slight increase of p which is spontaneously reverted. Further expansion leads to a transient decrease in p that returns to pesp. (C) Each point represents an independent measurement of pesp done on a individual film at constant T . (D) pesp as a function of T for a single film on which a thermal scan was performed.
interface is shown in Fig. 1B. After injecting the vesicles on the subphase, a pesp of 47 /48 mN/m is observed that only raises transiently by 1 /2 mN/m on further compression, returning to the equilibrium value after relaxation is allowed. Area expansion of the film transiently lowers the p, and the pesp recovers after a while. The convergence from low and high p to a particular value is a better criteria for ascertaining equilibrium that
the simple checking of stability for p over several hours. Fig. 1C shows the pesp as a function of T for several independent films, a constant value of pesp (within experimental error) is observed. Fig. 1D shows the pesp as a function of T for a single film over which a thermal scan was performed. Because the g of any liquid drops almost linearly with T, we discounted this variation for a clean buffer surface in order to correct the pesp values
174
R.G. Oliveira, B. Maggio / Chemistry and Physics of Lipids 122 (2003) 171 /176
obtained. A constancy of pesp is again observed. A same constancy of pesp (for over 44 mN/m) was also obtained in several experiments with different subphases, namely pure water, in the absence of Calcium and after the addition of EDTA (data not shown). Fig. 2 shows the adsorption process as seen by epifluorescence microscopy in which phase coexistence is evident. These patterns are similar to those observed by compressing a myelin film (Oliveira and Maggio, 2000, 2002), with the addition of the vesicles (very bright spots) from which the film is presumably forming. The bright rounded domains at low p correspond to liquid expanded domains. These domains are progressively distorted and percolate as p raises, given a structure of fractal dimension of about 1.7. The darker background corresponds to the cholesterol enriched phase previously reported (Oliveira and Maggio, 2002). The vesicles tend to locate at the boundaries between phases, indicating their high
line tension. Fig. 3A shows the pattern obtained by over compressing the film after the pcol. The pattern displayed by this collapsed film, in which multilayers are generated, shows rounded liquid expanded domains (superimposed on top or under the fractal structures characteristics of high p). Fig. 3B shows the same kind of pattern as in Fig. 3A, but obtained by adsorption. This is a direct visual indication that multilayers are beginning to assemble at equilibrium, which is in keeping with the fact that in myelin the pesp is the same as the pcol, at which multilayers formation occurs.
4. Conclusions Myelin is a particular membrane in many aspects, (1) it has a very specialized function isolating individual axons, (2) it is the membrane with the lowest protein content, (3) cytoplasm is
Fig. 2. Fluorescence microscopy (RHO-PE 0.5%) of myelin monolayer during the adsorption process. The numbers indicate the p (mN/m). Two liquid phases with a topography changing from rounded (low p) to fractal like structures (high p) are observed. The bright spots correspond to vesicles.
R.G. Oliveira, B. Maggio / Chemistry and Physics of Lipids 122 (2003) 171 /176
175
Fig. 3. (A) Collapsed structures generated by over compressing the film beyond pcol. Rounded collapsed structures (Ro) are observed superimposed to the fractal (Fr) pattern characteristic of high p. (B) Pattern generated after pesp has been reached in which rounded structures are evident.
squeezed out over large areas of contact between interacting cytoplasmic monolayers halves, (4) the spiral geometry on wrapping the axon permits selfassociation of the outer monolayer halves (5) in view of the intra and extra cytoplasmic interactions only 3 /5 nm of aqueous space separates the membranes, just a little more than for pure lipid bilayers, presumably due to the steric contribution of proteins (Kirschner et al., 1984). The pesp of vesicles is a function of the vesicle curvature from which the film is formed (Schindler, 1979); nevertheless, the dependence is progressively lost as the curvature of the bilayer falls. In this respect our vesicles have very low curvature (/1 mm 1), in the range of the native membranes (Fig. 1A), and dependence on curvature is negligible. The high pesp points to a very stable and cohesive film. Beside cohesion on the plane of the monolayer, the equality of pesp and pcol points to cohesion between stacked monolayers. This is reinforced by the microscopical observation of collapsed structures at equilibrium (rounded structures for collapsed trilayers were reported using Brewster angle microscopy (de Mul and Mann, 1998)). Moreover, vesicles tend to self aggregate and adsorb to the interfacial film. This is very meaningful in view of the particular multilayered (self associating) structure of myelin, and points out that this is an equilibrium structure. The
interaction between myelin layers has been long known (Kirschner et al., 1984) and is important for myelin function. We show here that the selfassociation of interfacial interacting monolayers can occur spontaneously in the film. Calcium, ionic strength and certain chemicals modulate the interactions between myelin polar interfaces, change the normal spacing of myelin in the nm scale, as observed by X-ray diffraction (Kirschner et al., 1984), but no major changes in the surface equilibrium behavior was observed in our study on different subphases. The constant value of pesp, independent on T , suggests two possibilities: (1) myelin suspensions have a unilamellar structure which is not a critical state, and (2) myelin can be thermodynamically defined as a spontaneous multilamellar structure. The first can be ruled out because fragments of stacked myelin membranes and multilamellar or oligolamellar vesicles are spontaneously obtained; special conditions of very low ionic strength and mechanical force (flushing) is needed for unilamellar smaller size vesiculation (Steck et al., 1978). The second possibility appears to be the correct one as shown in our work. Our results are different to the only report describing a CUS for a purified total lipid fraction from myelin (Ginsberg and Gershfeld, 1991) in which it was postulated that the presence of proteins would not alter the results.
176
R.G. Oliveira, B. Maggio / Chemistry and Physics of Lipids 122 (2003) 171 /176
However, we also checked the pesp for a total lipid extract from myelin and found almost the same value of about 46 mN/m for the pesp and pcol. At this moment we have no explanation for the difference. Although myelin vesicles lack the CUS, this does not represent an exception to this theory, since as was stated before myelin could be classified thermodynamically as a multilayered self associated membrane, at least in our experimental conditions, and this probably resembles the natural state in native myelin. By several techniques, the physical state of myelin has been postulated to be a liquid ‘fluid like’ state. Indeed we showed previously that the isotherm has a compressibility corresponding to a liquid state, and the epifluorescence and BAM results show coexistence between two liquid phases. In this respect, the equality of pesp and pcol should be predictable from the work of others (Birdi, 1989; Larsson and Quinn, 1994) for a discussion see (Marsh, 1996). Moreover this value should be near 49 mN/m (Feng, 1999), which is almost the same result as shown here. Lipidprotein composition in myelin, probably has evolved to accomplish stability to the multilamellar structure.
Acknowledgements This work was supported in part by CONICET, SECyT-UNC and FONCYT, Argentina.
References Birdi, K.S., 1989. Lipid and Biopolymer monolayers at liquid interfaces. Plenum Press, New York. de Mul, M.N.G., Mann, J.A., Jr., 1998. Determination of the thickness and optical properties of a Langmuir film from the domain morphology by Brewster angle microscopy. Langmuir 14, 2455 /2466.
Feng, S., 1999. Interpretation of mechanochemical properties of lipid bilayer vesicles from the equation of state or pressure-area measurement of the monolayer at the air / water or oil /water interface. Langmuir 15, 998 /1010. Gaines, G.L., Jr., 1966. Insoluble Monolayers at Liquid /Gas Interfaces. Wiley, New York. Gershfeld, N.L., 1989. The critical unilamellar lipid state: a perspective for membrane bilayer assembly. Biochim. Biophys. Acta 998, 335 /350. Ginsberg, L., Gershfeld, N.L., 1991. Membrane bilayer instability and the pathogenesis of disorders of myelin. Neurosci. Lett. 130, 133 /136. Haley, J.E., Samuels, F.G., Ledeen, R.W., 1981. Studies of myelin purity in relation to axonal contamination. Cell Mol. Neurobiol. 1, 175 /178. Kirschner, D.A., Ganser, A.L., Caspar, D.L.D., 1984. Diffraction studies of molecular organization and membrane interactions in myelin. In: Morell, P. (Ed.), Myelin, Second ed.. Plenum Press, New York, pp. 51 /95. Larsson, K., Quinn, P.J., 1994. Physical properties: structural and physical characteristics. In: Gunstone, F.D., Harwood, J.L., Padley, F.B. (Eds.), The Lipid Handbook, Second ed.. Chapman and Hall, London, pp. 401 /485. MacDonald, R.C., Simon, S.A., 1987. Lipid monolayer states and their relationships to bilayers. Proc. Natl. Acad. Sci. USA 84, 4089 /4093. Marsh, D., 1996. Lateral pressure in membranes. Biochim. Biophys. Acta 1286, 183 /223. McConnell, H.M., Tamm, L.K., Weis, R.M., 1984. Periodic structures in lipid monolayer phase transition. Proc. Natl. Acad. Sci. USA 81, 3249 /3253. Oliveira, R.G., Caldero´n, R.O., Maggio, B., 1998. Surface behavior of myelin monolayers. Biochim. Biophys. Acta 1370, 127 /137. Oliveira, R.G., Maggio, B., 2000. Epifluorescence microscopy of surface domain microheterogeneity in myelin monolayers at the air /water interface. Neurochem. Res. 25, 77 /86. Oliveira, R.G., Maggio, B., 2002. Compositional domain immiscibility in whole myelin monolayers at the air /water interface and Langmuir /Blodgett films. Biochim. Biophys. Acta 1561, 238 /250. Schindler, H., 1979. Exchange and interactions between lipid layers at the surface of a liposome solution. Biochim. Biophys. Acta 555, 316 /336. Steck, A.J., Siegrist, P., Zahler, P., Herschkowitz, N.N., Schaefer, R., 1978. Preparation of membrane vesicles from isolated myelin, studies on functional and structural properties. Biochim. Biophys. Acta 509, 397 /409.
Short communication
Surface behavior, microheterogeneity and adsorption equilibrium of myelin at the air water interface /
Rafael G. Oliveira, Bruno Maggio * Facultad de Ciencias Quı´micas, Departamento de Quı´mica Biolo´gica-CIQUIBIC, Universidad Nacional de Co´rdoba, 5000 Cordoba, Argentina
Abstract Interfacial films of whole myelin membrane adsorb at the air /water interface from myelin vesicles. The films show a liquid state and their equilibrium spreading pressure is equal to the collapse pressure (about 47 mN/m). The films appear microheterogeneous as seen by epifluorescence microscopy, consisting in two liquid phases over all the adsorption isotherm, starting with rounded liquid expanded domains (low surface pressure) immersed in a cholesterol enriched phase and reaching a fractal pattern at high surface pressure similar to those previously observed by compressing the film. Vesicles adsorb to the interfacial film mainly at the lateral interfaces. The high surface pressure at equilibrium (almost equal to the collapse pressure) indicates the formation of surface multilayers, also shown by fluorescence microscopy. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Monolayers; Myelin; Epifluorescence microscopy; Critical unilamellar state; Surface pressure
1. Introduction Many lipids and membrane proteins self-associate spontaneously to form bilayer membranes. Also these components form interfacial monomolecular films, which according to Gibbs’s equation decrease the surface tension g of an aqueous solution. This drop is defined as the surface pressure, p (Gaines, 1966). Aqueous media with biomembrane and liposome suspensions invariably show spontaneously adsorbed interfacial (air / water) films. Since the film at the air /water interface is in dynamic equilibrium with the bulk * Corresponding author. Fax: /54-351-4334074. E-mail address: [email protected] (B. Maggio).
vesicle suspension, properties and processes like phase transition of the vesicle lipids can be inferred from measurements on interfacial films (MacDonald and Simon, 1987). One of such properties is the equilibrium spreading (surface) pressure of the film pesp (Gaines, 1966). This is a function of temperature T (Birdi, 1989) and generally shows a maximum at a critical temperature Tc, a few degrees over the bulk phase transition T . For liposome suspensions of pure phospholipids a maximum of pesp at Tc indicates a particular bilayer thermodynamic state, the critical unilamellar state (CUS) (Gershfeld, 1989). This is the only state at which the unilamellar bilayer is in true thermodynamic equilibrium. The criticality of this state explains why multilamellar liposomes (stable
0009-3084/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 3 0 8 4 ( 0 2 ) 0 0 1 8 7 - 1
172
R.G. Oliveira, B. Maggio / Chemistry and Physics of Lipids 122 (2003) 171 /176
below and above Tc) are more readily obtained than unilamellar bilayers. Nevertheless, most plasma membranes are stable as unilamellar structure because the mixed components are in the CUS, as indicated by a maximum in pesp at 37 8C. This maximum is considered enough criteria to ascertain the CUS (Gershfeld, 1989). In recent work we carried out a physicochemical characterization of Langmuir (air /water interface) (Oliveira et al., 1998; Oliveira and Maggio, 2000) and Langmuir /Blodgett myelin films supported over Sagiv monolayers (Oliveira and Maggio, 2002). We showed that myelin shows a liquid-expanded physical state with a single collapse point (Oliveira et al., 1998). Epifluorescence microscopy revealed liquid phase coexistence over the full compression isotherm as indicated by probe partition, domain mobility, deformability and boundary relaxation due to the line tension of surface domains (Oliveira and Maggio, 2000). These results were further confirmed by both Brewster angle microscopy of fresh films and specific labeling of individual myelin components in Langmuir /Blodgett films which showed that phase coexistence is due to compositional immiscibility (Oliveira and Maggio, 2002). Immunolabeling indicated that the major myelin proteins and GM1 ganglioside (all of them liquid expanded and electrically charged) were located in the liquid-expanded phase. The other phase showed an enrichment in the cholesterol, galactocerebroside and phosphatidylserine markers. The distribution of components was qualitatively independent on the p and was generally constituted by one phase enriched in charged components of intrinsically expanded state coexisting with another phase enriched in non charged constituents of lower compressibility. We also showed that the myelin film at room temperature shows a very high pesp of about 47 mN/m, that can not be distinguished (within experimental error) from the pcol. Thus, a film with very high p (up to the collapse point) was spontaneously formed by adsorption. Because at the pcol multilayers begin to form at the interface, and taking into account the multilamellar structure of myelin, it is very unlike that myelin could be in the CUS at physiological T because that would require that the pesp would have to be
even higher at 37 8C. The hypothesis checked in this work is that myelin should lack the CUS, in contrast to the few other natural membranes analyzed to date.
2. Experimental procedures Bovine spinal cord myelin was purified with the method that renders the purest myelin (Haley et al., 1981) and vesiculated in phosphate buffer 1 mM pH 8, Dithiothreitol 2 mM (Steck et al., 1978). The vesicles were resuspended in the same buffer to a final protein concentration of 0.24 mg/ ml, and passed five times through a 26G needle fitted to a syringe. Purity was assessed through SDS-PAGE and HPTLC. The probes N -(7-nitro2-1, 3-benzoxadiazol-4-yl) diacyl phosphatidylethanolamine (McConnell et al., 1984) and N (lissamine rhodamine B sulfonyl) diacyl phosphatidylethanolamine (RHO-PE) were from Avanti Polar Lipids (Alabaster, AL). Details about epifluorescence microscopy were as previously cited (Oliveira and Maggio, 2002). Both probes are head group labeled, with a 55% of acyl chain unsaturation, made from egg phosphatidylcholine, which favors their partition in liquid-expanded (disordered) phases, being mostly excluded from liquid condensed and cholesterol-enriched phases. The routine subphase was 10 mM Tris[hydroxymethyl aminomethane] buffer in 100 mM NaCl, 20 mM CaCl2, adjusted to pH 7.4 with HCl. The observations were carried out using a KSV Minitrough II (KSV, Helsinski, Finland) mounted on the microscope stage. Representative measurements were corroborated using N2 atmosphere to ensure no appreciable effect due to lipid peroxidation.
3. Results and discussion Fig. 1A shows a phase contrast microscopy micrograph of the myelin vesicles used in this work. They tend to self-aggregate, in agreement with the literature (Steck et al., 1978) although some free lamellar structures can be seen on the periphery of the vesicle clusters. The adsorption isotherm of myelin vesicles on a clean air-buffer
R.G. Oliveira, B. Maggio / Chemistry and Physics of Lipids 122 (2003) 171 /176
173
Fig. 1. Myelin vesicles and their adsorption to the air /water interface as a function of T . (A) Phase contrast micrograph of myelin vesicles used to measure pesp, on which the tendency to self aggregate is observed. (B) Adsorption isotherm of myelin vesicles. After p has been stabilized by a minimal of 1 h, further compression leads to a very slight increase of p which is spontaneously reverted. Further expansion leads to a transient decrease in p that returns to pesp. (C) Each point represents an independent measurement of pesp done on a individual film at constant T . (D) pesp as a function of T for a single film on which a thermal scan was performed.
interface is shown in Fig. 1B. After injecting the vesicles on the subphase, a pesp of 47 /48 mN/m is observed that only raises transiently by 1 /2 mN/m on further compression, returning to the equilibrium value after relaxation is allowed. Area expansion of the film transiently lowers the p, and the pesp recovers after a while. The convergence from low and high p to a particular value is a better criteria for ascertaining equilibrium that
the simple checking of stability for p over several hours. Fig. 1C shows the pesp as a function of T for several independent films, a constant value of pesp (within experimental error) is observed. Fig. 1D shows the pesp as a function of T for a single film over which a thermal scan was performed. Because the g of any liquid drops almost linearly with T, we discounted this variation for a clean buffer surface in order to correct the pesp values
174
R.G. Oliveira, B. Maggio / Chemistry and Physics of Lipids 122 (2003) 171 /176
obtained. A constancy of pesp is again observed. A same constancy of pesp (for over 44 mN/m) was also obtained in several experiments with different subphases, namely pure water, in the absence of Calcium and after the addition of EDTA (data not shown). Fig. 2 shows the adsorption process as seen by epifluorescence microscopy in which phase coexistence is evident. These patterns are similar to those observed by compressing a myelin film (Oliveira and Maggio, 2000, 2002), with the addition of the vesicles (very bright spots) from which the film is presumably forming. The bright rounded domains at low p correspond to liquid expanded domains. These domains are progressively distorted and percolate as p raises, given a structure of fractal dimension of about 1.7. The darker background corresponds to the cholesterol enriched phase previously reported (Oliveira and Maggio, 2002). The vesicles tend to locate at the boundaries between phases, indicating their high
line tension. Fig. 3A shows the pattern obtained by over compressing the film after the pcol. The pattern displayed by this collapsed film, in which multilayers are generated, shows rounded liquid expanded domains (superimposed on top or under the fractal structures characteristics of high p). Fig. 3B shows the same kind of pattern as in Fig. 3A, but obtained by adsorption. This is a direct visual indication that multilayers are beginning to assemble at equilibrium, which is in keeping with the fact that in myelin the pesp is the same as the pcol, at which multilayers formation occurs.
4. Conclusions Myelin is a particular membrane in many aspects, (1) it has a very specialized function isolating individual axons, (2) it is the membrane with the lowest protein content, (3) cytoplasm is
Fig. 2. Fluorescence microscopy (RHO-PE 0.5%) of myelin monolayer during the adsorption process. The numbers indicate the p (mN/m). Two liquid phases with a topography changing from rounded (low p) to fractal like structures (high p) are observed. The bright spots correspond to vesicles.
R.G. Oliveira, B. Maggio / Chemistry and Physics of Lipids 122 (2003) 171 /176
175
Fig. 3. (A) Collapsed structures generated by over compressing the film beyond pcol. Rounded collapsed structures (Ro) are observed superimposed to the fractal (Fr) pattern characteristic of high p. (B) Pattern generated after pesp has been reached in which rounded structures are evident.
squeezed out over large areas of contact between interacting cytoplasmic monolayers halves, (4) the spiral geometry on wrapping the axon permits selfassociation of the outer monolayer halves (5) in view of the intra and extra cytoplasmic interactions only 3 /5 nm of aqueous space separates the membranes, just a little more than for pure lipid bilayers, presumably due to the steric contribution of proteins (Kirschner et al., 1984). The pesp of vesicles is a function of the vesicle curvature from which the film is formed (Schindler, 1979); nevertheless, the dependence is progressively lost as the curvature of the bilayer falls. In this respect our vesicles have very low curvature (/1 mm 1), in the range of the native membranes (Fig. 1A), and dependence on curvature is negligible. The high pesp points to a very stable and cohesive film. Beside cohesion on the plane of the monolayer, the equality of pesp and pcol points to cohesion between stacked monolayers. This is reinforced by the microscopical observation of collapsed structures at equilibrium (rounded structures for collapsed trilayers were reported using Brewster angle microscopy (de Mul and Mann, 1998)). Moreover, vesicles tend to self aggregate and adsorb to the interfacial film. This is very meaningful in view of the particular multilayered (self associating) structure of myelin, and points out that this is an equilibrium structure. The
interaction between myelin layers has been long known (Kirschner et al., 1984) and is important for myelin function. We show here that the selfassociation of interfacial interacting monolayers can occur spontaneously in the film. Calcium, ionic strength and certain chemicals modulate the interactions between myelin polar interfaces, change the normal spacing of myelin in the nm scale, as observed by X-ray diffraction (Kirschner et al., 1984), but no major changes in the surface equilibrium behavior was observed in our study on different subphases. The constant value of pesp, independent on T , suggests two possibilities: (1) myelin suspensions have a unilamellar structure which is not a critical state, and (2) myelin can be thermodynamically defined as a spontaneous multilamellar structure. The first can be ruled out because fragments of stacked myelin membranes and multilamellar or oligolamellar vesicles are spontaneously obtained; special conditions of very low ionic strength and mechanical force (flushing) is needed for unilamellar smaller size vesiculation (Steck et al., 1978). The second possibility appears to be the correct one as shown in our work. Our results are different to the only report describing a CUS for a purified total lipid fraction from myelin (Ginsberg and Gershfeld, 1991) in which it was postulated that the presence of proteins would not alter the results.
176
R.G. Oliveira, B. Maggio / Chemistry and Physics of Lipids 122 (2003) 171 /176
However, we also checked the pesp for a total lipid extract from myelin and found almost the same value of about 46 mN/m for the pesp and pcol. At this moment we have no explanation for the difference. Although myelin vesicles lack the CUS, this does not represent an exception to this theory, since as was stated before myelin could be classified thermodynamically as a multilayered self associated membrane, at least in our experimental conditions, and this probably resembles the natural state in native myelin. By several techniques, the physical state of myelin has been postulated to be a liquid ‘fluid like’ state. Indeed we showed previously that the isotherm has a compressibility corresponding to a liquid state, and the epifluorescence and BAM results show coexistence between two liquid phases. In this respect, the equality of pesp and pcol should be predictable from the work of others (Birdi, 1989; Larsson and Quinn, 1994) for a discussion see (Marsh, 1996). Moreover this value should be near 49 mN/m (Feng, 1999), which is almost the same result as shown here. Lipidprotein composition in myelin, probably has evolved to accomplish stability to the multilamellar structure.
Acknowledgements This work was supported in part by CONICET, SECyT-UNC and FONCYT, Argentina.
References Birdi, K.S., 1989. Lipid and Biopolymer monolayers at liquid interfaces. Plenum Press, New York. de Mul, M.N.G., Mann, J.A., Jr., 1998. Determination of the thickness and optical properties of a Langmuir film from the domain morphology by Brewster angle microscopy. Langmuir 14, 2455 /2466.
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