Influence of a Fluorescent Probe on the Nanostructure of Phospholipid

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Langmuir 2005, 21, 5349-5355

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Influence of a Fluorescent Probe on the Nanostructure of Phospholipid Membranes: Dipalmitoylphosphatidylcholine Interfacial Monolayers Antonio Cruz,† Luis Va´zquez,‡ Marisela Ve´lez,§ and Jesu´s Pe´rez-Gil*,† Departamento de Bioquı´mica, Facultad de Biologı´a, Universidad Complutense de Madrid, 28040 Madrid, Spain, Instituto de Ciencia de Materiales de Madrid (CSIC), C\ Sor Juana Ine´ s de la Cruz No. 3, 28049 Madrid, Spain, and Instituto Universitario de Ciencia de Materiales “Nicola´ s Cabrera”, Universidad Auto´ noma de Madrid, Spain Received December 30, 2004. In Final Form: March 7, 2005 Monolayers of dipalmitoylphosphatidylcholine (DPPC), both in the absence and in the presence of 1% (mol/mol) of a fluorescent phospholipid probe, have been spread at the air-liquid interface of a surface balance, compressed up to pressures in the liquid-expanded/liquid-condensed plateau of the isotherm, transferred onto mica supports, and analyzed by scanning force microscopy (SFM). Supported DPPC films showed micrometer-sized condensed domains with morphology and size that were entirely analogous to those observed in situ at the air-liquid interface by epifluorescence microscopy. The analysis by SFM, however, allowed the study and comparison of monolayers in the absence and in the presence of the fluorescent marker. This analysis revealed that the presence of dye reduced by 10-20% the total amount of the liquid-condensed phase in the DPPC films. The presence of the dye also decreased the mechanical stability of the film and increased the time required for the monolayer to equilibrate. The resolution of SFM permitted the determination that the structures of both the liquid-expanded and the liquid-condensed regions of DPPC films were heterogeneous at the nanometer scale. Liquid-condensed DPPC microdomains contained nanoholes covering 4-8% of their area whereas 60-80% of the surface detected as liquidexpanded by fluorescence microscopy consisted of a condensed-like framework of nanodomains. The total area, the shape of the nanodomains, and their interconnectivity were affected by the presence of the probe, suggesting that care must be taken when studying the structure, especially at the nanometer scale, and properties of model lipid films in the presence of extrinsic probes.

Introduction In the past decades interfacial monolayers have been commonly used as a model for the study of lipid structures as well as lipid-protein interactions taking place on the lipid surface, intended as models of genuine biological membranes.1,2 Direct visualization at the air-liquid interface of lipid and lipid-protein monolayers doped with fluorescent probes has proven to be a powerful tool to obtain information about potentially relevant structural transitions and lipid-protein interactions. Experimental approaches using proteins extrinsically labeled with fluorescent markers allowed the lateral distribution of some membrane-associated proteins to be resolved, as well as some of their activities on the lipid surface to be analyzed.3-5 A very attractive system that has been extensively investigated using this model is pulmonary surfactant. This system, roughly composed of equimolar amounts of saturated and unsaturated phospholipids plus specific proteins (SP-A, SP-B, SP-C), acts as a functional interfacial film in vivo, to prevent alveolar collapse during the dynamic compression-expansion cycling of the res* To whom correspondence should be addressed. Tel.: 34 91 3944994. Fax: 34 91 3944672. E-mail: [email protected]. † Universidad Complutense de Madrid. ‡ Instituto de Ciencia de Materiales de Madrid (CSIC). § Universidad Auto ´ noma de Madrid. (1) Mohwald, H. Annu. Rev. Phys. Chem. 1990, 41, 441-76. (2) Deleu, M.; Paquot, M.; Jacques, P.; Thonart, P.; Adriaensen, Y.; Dufrene, Y. F. Biophys. J. 1999, 77, 2304-10. (3) Grainger, D. W.; Reichert, A.; Ringsdorf, H.; Salesse, C. Biochim. Biophys. Acta 1990, 1023, 365-79. (4) Reichert, A.; Ringsdorf, H.; Wagenknecht, A. Biochim. Biophys. Acta 1992, 1106, 178-88. (5) Subirade, M.; Salesse, C.; Marion, D.; Pezolet, M. Biophys. J. 1995, 69, 974-88.

piratory surface.6,7 Interfacial lipid-protein model films, made from synthetic lipids plus surfactant proteins purified from natural sources, have been extensively studied in surface balances as an approach to analyze surfactant function and the role of the proteins in molecular terms.8-11 The development during the last years of new techniques such as epifluorescence microscopy to analyze the structure of lipid and lipid-protein films has revealed some significant features on the lateral organization and the plasticity of surfactant layers subjected to dynamic cycling.12-22 However, for the (6) Goerke, J. Biochim. Biophys. Acta 1998, 1408, 79-89. (7) Perez-Gil, J.; Keough, K. M. Biochim. Biophys. Acta 1998, 1408, 203-17. (8) Yu, S. H.; Possmayer, F. Biochim. Biophys. Acta 1990, 1046, 23341. (9) Possmayer, F.; Nag, K.; Rodriguez, K.; Qanbar, R.; Schurch, S. Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol. 2001, 129, 20920. (10) Veldhuizen, E. J.; Diemel, R. V.; Putz, G.; van Golde, L. M.; Batenburg, J. J.; Haagsman, H. P. Chem. Phys. Lipids 2001, 110, 4755. (11) Ross, M.; Krol, S.; Janshoff, A.; Galla, H. J. Eur. Biophys. J. 2002, 31, 52-61. (12) Perez-Gil, J.; Nag, K.; Taneva, S.; Keough, K. M. Biophys. J. 1992, 63, 197-204. (13) Discher, B. M.; Maloney, K. M.; Schief, W. R., Jr.; Grainger, D. W.; Vogel, V.; Hall, S. B. Biophys. J. 1996, 71, 2583-90. (14) Nag, K.; Perez-Gil, J.; Cruz, A.; Keough, K. M. Biophys. J. 1996, 71, 246-56. (15) Nag, K.; Taneva, S. G.; Perez-Gil, J.; Cruz, A.; Keough, K. M. Biophys. J. 1997, 72, 2638-50. (16) Nag, K.; Pe´rez-Gil, J.; Ruano, M. L. F.; Worthman, L. A. D.; Stewart, J.; Casals, C.; Keough, K. M. W. Biophys. J. 1998, 74, 298395. (17) Discher, B. M.; Schief, W. R.; Vogel, V.; Hall, S. B. Biophys. J. 1999, 77, 2051-61. (18) Kruger, P.; Schalke, M.; Wang, Z.; Notter, R. H.; Dluhy, R. A.; Losche, M. Biophys. J. 1999, 77, 903-14.

10.1021/la046759w CCC: $30.25 © 2005 American Chemical Society Published on Web 05/05/2005

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interfacial films to be analyzed by epifluorescence microscopy they must contain small traces, on the order of 0.5-1 mol % probe to phospholipid, of fluorescently labelled lipids and/or proteins. Fluorescence images inform about the lateral distribution of single or multiple molecular species during the compression-expansion cycle. The implicit assumption is that the presence of such small amounts of probe does not significantly perturb the structure and properties of the films,23-25 but few studies have been conducted to validate this assumption at all structural scales. This scarcity of studies is partially due to the lack of suitable techniques to address them. In this sense, the recent development of scanning force microscopy (SFM) is of particular significance, because it permits acquisition of images from lipid or lipid-protein films at much higher resolution than attained before by other techniques such as Brewster angle or epifluorescence microscopy.26,27 Thus, the study by SFM of supported 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers and bilayers has revealed the existence of a nonhomogeneous nanostructure.28 However, SFM has the relative disadvantage, compared with epifluorescence microscopy, of requiring the transfer of the films from the air-liquid interface onto flat solid supports to form Langmuir-Blodgett (LB) films before being scanned and visualized. Controls are required to ensure that the structure of the films is not perturbed during transfer. On the other hand, SFM has the advantage over epifluorescence or near-field scanning optical microscopy that it does not require the inclusion of traces of extrinsic probes to allow for the visualization of the films.29,30 Besides, SFM also provides the possibility to detect and characterize surface-associated three-dimensional structures occurring during compression at high pressures or formed by interaction of proteins or lipid-protein assemblies with the interfacial layer.31 In the present work we have analyzed the structure of DPPC monolayers at the micro- and nanometer scales by SFM, focusing on the effect of the presence of small proportions of a fluorescent marker (1-palmitoyl-2{12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl}phosphatidylcholine, NBD-PC) widely used to study the lateral structure of phospholipid films by epifluorescence microscopy.12-17,19,32,33 Materials and Methods DPPC and NBD-PC were provided by Avanti Polar Lipids (Pelham, Alao, U.S.A.). Chloroform and methanol solvents, HPLC grade, were from Scharlau (Barcelona, Spain). (19) Cruz, A.; Worthman, L. A.; Serrano, A. G.; Casals, C.; Keough, K. M.; Perez-Gil, J. Eur. Biophys. J. 2000, 29, 204-13. (20) Kramer, A.; Wintergalen, A.; Sieber, M.; Galla, H. J.; Amrein, M.; Guckenberger, R. Biophys. J. 2000, 78, 458-65. (21) Kruger, P.; Baatz, J. E.; Dluhy, R. A.; Losche, M. Biophys. Chem. 2002, 99, 209-28. (22) Cruz, A.; Vazquez, L.; Velez, M.; Perez-Gil, J. Biophys. J. 2004, 86, 308-20. (23) Losche, M.; Mohwald, H. Eur. Biophys. J. 1984, 11, 35-42. (24) Weis, R. M. Chem. Phys. Lipids 1991, 57, 227-39. (25) Mohwald, H. In Phospholipids Handbook; Cevc, G., Ed.; Marcel Dekker: New York, 1993; pp 579-602. (26) Dufrene, Y. F.; Lee, G. U. Biochim. Biophys. Acta 2000, 1509, 14-41. (27) Knebel, D.; Sieber, M.; Reichelt, R.; Galla, H. J.; Amrein, M. Biophys. J. 2002, 82, 474-80. (28) Hollars, C. W.; Dunn, R. C. Biophys. J. 1998, 75, 342-53. (29) Vickery, S. A.; Dunn, R. C. Biophys. J. 1999, 76, 1812-8. (30) Cordero, S. R.; Weston, K. D.; Buratto, S. K. Thin Solid Films 2000, 360, 139-44. (31) Krol, S.; Ross, M.; Sieber, M.; Kunneke, S.; Galla, H. J.; Janshoff, A. Biophys. J. 2000, 79, 904-18. (32) Ruano, M. L.; Nag, K.; Worthman, L. A.; Casals, C.; Perez-Gil, J.; Keough, K. M. Biophys. J. 1998, 74, 1101-9. (33) Piknova, B.; Schief, W. R.; Vogel, V.; Discher, B. M.; Hall, S. B. Biophys. J. 2001, 81, 2172-80.

Cruz et al. DPPC monolayers were prepared by spreading small aliquots from chloroform/methanol (3:1, v/v) solutions of the phospholipid (1 mg/mL) on top of a double-distilled water subphase at 25 ( 1 °C on a surface balance (Nima Technology, Ltd., Coventry, U.K.). After waiting 10 min to allow for solvent evaporation, monolayers were compressed to the required pressure, 11 mN/m in most of shown cases, at a compression rate of 25 cm2/min. Finally, after 10 min of equilibration, the monolayers were transferred onto mica supports or glass coverslips that were previously immersed in the subphase, to form LB films, at a transfer speed of 5 mm/min with automatic compensation of the area to prevent the pressure reduction due to the extraction of the supported lipid layer. To allow for observation of the films by epifluorescence microscopy, monolayers containing 1 mol % of the fluorescent lipid NBD-PC were transferred as described above onto glass coverslips and observed under a Zeiss Axioplan II fluorescence microscope (Carl Zeiss, Jena, D) equipped with the appropriate fluorescence filters to allow for the observation of NBD-PC fluorescence (maximum fluorescence emission at 520 nm). SFM images were obtained from mica-supported LB films using a Nanoscope IIIa scanning probe microscope (Digital Instruments, Santa Barbara, CA) operated in contact mode, using silicon nitride tips with a spring constant of 0.05 N/m. Both topography and friction images were simultaneously recorded from each sample. Images shown in the figures are representative micrographs obtained after analyzing three or more different transferred films from either DPPC or DPPC/NBD-PC monolayers, compressed to the indicated pressures. Images from both SFM and epifluorescence microscopy were quantitatively analyzed using the program Scion Image (Scion Corp., MD, U.S.A.). The data shown are averaged values with standard deviation obtained after analysis of at least five different images.

Results Figure 1 shows images taken, either by epifluorescence microscopy or SFM, from DPPC monolayers containing 1% of the phospholipid fluorescent probe NBD-PC compressed up to the liquid-expanded/liquid-condensed (LELC) coexistence regime and transferred onto solid supports. At pressures where LE-LC coexistence is expected in the compression isotherm (see the plateau around 1014 mN/m in Figure 1a), black domains excluding the fluorescent probe are observed by epifluorescence microscopy of the films (Figure 1b). At similar magnification, SFM micrographs of films transferred on top of mica substrates (Figure 1c) show a similar distribution of lipid in two different phases, with geometry similar to that of the monolayers observed under fluorescence microscopy in situ or transferred.15,19 As previously reported,22 condensed areas in the topographic images obtained by SFM are 10-13 Å higher than the lowest regions, reflecting the larger height and/or the lower deformability of the lipids as they are organized into the LC phase. SFM allows observation of the phospholipid films in the absence of the fluorescent probes required to visualize two-dimensional transitions in monolayers by epifluorescence microscopy. Therefore, it is possible to evaluate the effects of the inclusion of fluorescent probes such as NBD-PC in the compression-driven condensation of DPPC films, by comparing the morphology of films with and without the probe. Figure 2 compares the topology of both the LC domains and the liquid-expanded regions in DPPC films, with or without 1 mol % NBD-PC, compressed to 11 mN/m. No differences were ever observed when comparing the π-A isotherms from probe-containing with those from probe-free films. Probe-containing films were always more fragile to fracture upon transfer than probefree films. Films containing NBD-PC showed under SFM numerous fractures (see Figure 2b) running mostly perpendicularly to the transfer direction, especially when

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Figure 1. LE-LC phase coexistence in DPPC films. (a) Compression isotherm of a DPPC monolayer containing 1% (mol/mol) of the probe NBD-PC. (b) Epifluorescence image of a DPPC/NBD-PC monolayer transferred onto a glass support at 11 mN/m (the pressure indicated by a black dot at the isotherm of panel a). Exclusion of the probe from the condensed domains of the monolayer allows for the observation of phase coexistence in the film. (c) Contact mode topological SFM image from a pure DPPC monolayer transferred onto a freshly cleaved mica surface, showing LE-LC coexistence. In all these experiments, the monolayer was formed on top of a pure water surface at 25 ( 1 °C and transferred as indicated in Materials and Methods.

Figure 2. Effect of NBD-PC on the micro- and nanostructure of DPPC films. Contact mode SFM micrographies of DPPC monolayers in the absence (a) or presence (b) of the fluorescent probe NBD-PC (1%, mol/mol), compressed to 11 mN/m before transfer to mica supports. Magnifications of the images from condensed domain regions (a1, b1) show equivalent arrangements but differences in the height of the bright areas, around 2-3 Å taller in the monolayers lacking the probe compared with those having NBD-PC, as shown in the topographic profiles (a2, b2; obtained through the paths indicated by dotted lines in pictures a1 and b1). The effect of the probe is more significant in the distribution pattern of the condensed-like nanodomains into the expanded regions (magnified pictures a3, b3) than in the morphology of the condensed microdomains. Topographic scans from the LE phase (a4, b4) show also differences in height due to the presence of NBD-PC.

films compressed to less than 12 mN/m were transferred. DPPC films that did not contain NBD-PC were more stable

and did not tear as easily when transferred onto mica supports (see Figure 2a). Pictures a1, a3, b1, and b3 in

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Figure 3. Quantitative analysis of the effect of NBD-PC on the micro- and nanostructure of DPPC films. (a) Analysis of the percent of total area of the films occupied by condensed microdomains (left panel) and the percent of the expanded phase taken by condensed nanodomains (right panel) at different surface pressures. Data were obtained from SFM images of pure DPPC monolayers or from DPPC monolayers containing 1% (mol/mol) NBD-PC, compressed to the different pressures before transfer as LBs to mica supports. (b) Comparison of the morphology of the interconnected condensed nanodomains in the LE phase of pure DPPC (upper panel) with the isolated ones in the LE phase of DPPC/NBD-PC films (bottom panel). The panels are 4 µm in width.

Figure 2 show details of the topology of LC domains (a1, b1) and LE regions (a3, b3) in both probe-free and probecontaining DPPC films. Both films showed similar heterogeneous morphology of the two coexisting regions: abundance of nanoholes included into the condensed microdomains and a network of condensed nanodomains filling the LE areas. However, there is a remarkable difference between the structure of the expanded regions in probe-containing and that in probe-free DPPC films. Condensed nanodomains in the expanded areas of DPPC/ NBD-PC films are isolated while those of pure DPPC films are interconnected forming a sort of network (see Figure 2, a3, or images in Figure 3). Analysis of the topological profiles of these two films reveals that the difference in height between condensed and expanded regions is ∼13.0 Å in DPPC films without NBD-PC (plots a2 and a4 in Figure 2) but of only about 10.0-10.5 Å in the films containing the fluorescent probe (plots b2 and b4). Considering that the images from the two types of films were taken under the same SFM spring loads, the smaller height difference measured in NBD-PC containing films suggests that the presence of the probe in the LE regions makes this phase apparently taller, or less deformable, to the scanning cantilever. Figure 3 shows a quantitative analysis of the condensation and growth of condensed domains that occur upon compression in both types of DPPC films, containing and devoid of 1 mol % NBD-PC. To obtain the data included in Figure 3, images were corrected for drift and only frames containing nonfractured films were selected, analyzed, and averaged. Nucleation of LC microdomains starts at surface pressures a little below 8 mN/m in both probecontaining and probe-free DPPC films (Figure 3a, left panel). Further compression produced higher amounts of total condensed area occupied by the microdomains in the films without NBD-PC than in those with the probe. The difference reaches 20% at 13 mN/m, reflecting likely the effects of a progressive concentration of the fluorescent probe into the LE areas as a consequence of its progressive exclusion from the condensed domains. We also analyzed the effect of the probe in the amount of condensed nanodomains into the LE regions (Figure 3a, right panel). In the absence of NBD-PC, these nanodomains form an interconnected condensed network that take as much as around 80% of the area occupied by

the LE phase (which looks bright under epifluorescence microscopy), independently of pressure (see images of Figure 3b). The expanded regions of NBD-PC containing films were also occupied in about 80% of their surface by condensed nanodomains, at low pressures, although these nanodomains were more isolated than those observed in the absence of probe. That is to say, in the absence of probe the condensed area in the LE phase is percolated while the expanded regions constitute the percolated phase in the presence of NBD-PC. As compression increased, nanodomains accounted for a progressive lower percent of the LC regions in DPPC/NBD-PC films, pointing again to a progressive distorting effect of the probe as it is accumulated in the expanded phase. To obtain further information on the structure of the LC phase of DPPC films, and the effect of the presence of the fluorescent probe, we obtained SFM images of the films once compressed up to 30 mN/m, a pressure well above the end of the LE-LC transition plateau. Figure 4 compares the topology of the surface of LC domains of DPPC and DPPC/NBD-PC films compressed to 10 mN/m with that of the same films compressed to 30 mN/m, where the entire surface is occupied by the condensed phase and no LE-LC coexistence is ever observed. Condensed domains of both DPPC and DPPC/NBD-PC films show at 10 mN/m a similar distribution and size of the nanoholes included into the domains (see Figure 4b), in both cases accounting for about 5% of the area occupied by the condensed microdomains (Figure 4c). However, presence of the same nanoholes in the films condensed up to 30 mN/m is drastically different depending on the presence or absence of the fluorescent probe. Compression of probefree DPPC films to 30 mN/m leads to a homogeneous flat condensed layer, practically devoid of nanoholes or pores. In contrast, films containing 1 mol % NBD-PC keep showing numerous nanoholes once compressed to 30 mN/ m, still occupying 5 ( 2% of the area of microdomains. This fact strongly suggests that the presence of pores in the condensed domains could be due to incomplete LELC phase separation. The expanded phase trapped into condensed domains of pure DPPC films could probably be also condensed by further compression to higher pressures. In contrast, the expanded phase trapped into condensed domains of DPPC/NBD-PC films probably contains some

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Figure 4. Effect of NBD-PC on the total condensation of DPPC films. (a) Contact mode SFM images of condensed regions from mica supported DPPC or DPPC/NBD-PC (1% mol/mol) monolayers, transferred after compression to either 10 or 30 mN/m. The presence of nanoholes in the condensed phase depends both on the surface pressure and on the presence of the probe. Scale bars represent 200 nm. (b) Frequency analysis of the size of the expanded nanoholes in condensed microdomains of DPPC (white bars) or DPPC/NBD-PC (black bars) films compressed up to 10 (upper panel) or 30 (lower panel) mN/m. (c) Percent of total surface of the condensed phase occupied by expanded nanoholes in DPPC or NBD-PC films as a function of the surface pressure.

Figure 5. Effect of equilibration on the nanostructure of condensed domains in DPPC films. (a) Contact mode SFM images showing topography of pure DPPC films compressed to 11 mN/m and equilibrated during either 10 min or 2 h before transfer as LBs to mica substrates, and DPPC/NBD-PC films compressed to 11 mN/m and equilibrated for 2 h before transfer. Micrographies taken with different magnifications illustrate how the nanoholes from the condensed phase of pure DPPC monolayers, but not those in the condensed domains of DPPC/NBD-PC, fade away after longer equilibration times. (b) Frequency analysis of the size of the expanded nanoholes in condensed microdomains of DPPC films compressed up to 10 mN/m and equilibrated for 10 min (upper panel) or 2 h (lower panel).

amounts of probe and might be hardly condensed even after compression at considerably higher pressures. To test whether incomplete phase separation could be the origin of the pores trapped into DPPC condensed domains, we compared the nanostructure of samples equilibrated for different times before transfer. Figure 5 shows the structure and details of condensed regions in films of DPPC, without NBD-PC, compressed to 11 mN/m and equilibrated for either 10 min or 2 h before transfer to mica supports. Longer equilibration times give rise to

a more rounded shape of the condensed microdomains, probably as a consequence of a line tension driven minimization of the length of the LE-LC boundaries. Figure 5 also clearly shows that long-time equilibration reduces drastically the number and size of the pores trapped into condensed domains, indicating that heterogeneities in the condensed regions could be due to inefficiencies in phase demixing once compression of the films is stopped. In contrast, DPPC/NBD-PC monolayers still showed, after equilibration for 2 h and before transfer

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to the support, numerous holes with size and morphology similar to those observed in the films that were equilibrated for only 10 min (see also Figure 5). This result suggests that segregation of probe molecules trapped into the LC DPPC domains could require much longer times. On the other hand, we did not detect equilibrationdependent differences in the distribution or size of condensed-like nanodomains into the LE regions. Discussion The present study concludes that even the small traces of fluorescent lipid probes frequently used in epifluorescence experiments may produce significant perturbations that only can be properly analyzed and evaluated when SFM images are compared with those obtained by epifluorescence microscopy.22 Other studies have also reported significant effects of very small traces of some organic molecules on the morphology and properties of phospholipid films in the LE-LC coexistence regime.34 However, such effects usually include alterations at the π-A isotherms and important structural effects at the microscopic scale. Fluorescent phospholipid-derived probes, designed to visualize the lateral structure of lipid films, have been described to produce negligible perturbations of the isotherms and little alterations of the structure as analyzed qualitatively, at least at a microscopic scale.1,24,25 We show in the present study that the presence of 1% of one of these phospholipid probes is not enough to alter the π-A isotherm, but it does produce measurable quantitative alterations of compression-driven two-dimensional structural transitions. These perturbations seem to be especially significant at the nanoscopic structural scale of the lipid films. Caution should be taken especially when studying the films under conditions that accumulate probe in defined areas of the films. Significant effects of NBDPC have been detected in both micro- and nanodomains of transferred films. Monolayers containing NBD-PC are particularly prone to undergoing fractures, perpendicular to the transfer direction of the monolayer and affecting at a higher extent the LE areas. The perturbing effect of the probe on the morphology of the nanodomains is remarkable as well in the distribution of the condensed microdomains, as seen in Figures 2a,b and 3. However, this effect is more significant when the pressure rises above 11 mN/m, when the relative condensed area increases and the fluorescent marker is concentrated on the remnant LE regions. In addition to the effect of the probe on the morphology of the nanodomains in the LE phase, the presence of NBD-PC has also shown significant effects on the dynamics of two-dimensional phase separation that must occur during equilibration of the films, once compression is stopped. Films containing the probe retain apparent phase coexistence even after compression to pressures of 30 mN/m (Figure 4). We speculate that the nanoholes observed by SFM into the condensed domains are due to LE nanoregions trapped into a mostly LC background, which, in the case of fluorescent films, contain also trapped NBD-PC molecules. These nanoholes account for about 4-5% of the surface of the film at 30 mN/m. Considering the proportion of the probe included into the films, 1% mol/mol, LE nanoholes in DPPC/NBD-PC films compressed to 30 mN/m would contain on the order of 20-25 mol % of probe. Such proportion of NBD-PC would prevent lipid condensation and would permit a higher deformability of the film to the SFM cantilever compared (34) Caetano, W.; Ferreira, M.; Tabak, M.; Mosquera Sanchez, M. I.; Oliveira, O. N., Jr.; Kruger, P.; Schalke, M.; Losche, M. Biophys. Chem. 2001, 91, 21-35.

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with that of the probe-free condensed areas. The presence of probe into these “nano-holes” cannot be detected by epifluorescence microscopy, probably due to the low resolution of this technique compared with that of SFM. Our interpretation is that equilibration of probe-free films after compression could only require small and local lateral rearrangements of the configuration of the single molecules, while equilibration of probe-containing layers probably requires real lateral difussion of the bulky probe molecules over long distances through the rigid lowdiffusable and highly packed LC domains. This long-range diffusion would take longer time scales than those observed during our experiments. A lateral diffusion coefficient on the order of D ) 10-11 cm2 s-1 has been estimated for lipid molecules in the condensed gel phase of bilayers.35 Although extrapolation of physical parameters from bilayer to monolayer models should be taken with caution, a lateral diffusion of that order of magnitude would impose that a single lipid molecule would take about 7 h to diffuse along 10 µm, a distance in the range of the size measured for DPPC condensed microdomains. Restrictions imposed by the experimental conditions needed for monolayer formation, compression, and transfer make carrying out a detailed study such as the present one in completely equilibrated systems difficult, if not impossible. However, despite the limitations to obtain data with real thermodynamical significance, the study of nonequilibrated dynamical systems may reflect a more realistic behavior of the lipid-protein systems under physiological-like conditions. A question to be raised is whether the effects revealed in the present study would be similar for different phospholipid fluorescent probes. The NBD-PC probe studied here possesses a bulky fluorescent group attached to one of its acyl chains and cannot probably be easily accommodated into the highly packed environment of the LC DPPC domains. Furthermore, it has been proposed that the high affinity of the NBD group for the phospholipid interface can cause the NBD moiety to loop up when attached to a long enough acyl chain.36,37 The 12-NBD-PC probe studied here could then produce stronger effects than a phospholipid probe bearing the fluorophore attached at the headgroup. The perturbing effect could also be dependent on the lipid composition of the films, especially in reference to the mixture of saturated and unsaturated phospholipid species. We have tested other probes labeled at the headgroup, such as rhodamine-, fluoresceine-, or BODIPY-labeled phosphatidylethanolamine38 or DiIC18,39 in different lipid systems (results not shown). These probes always showed in our hands a qualitative behavior similar to that of 12-NBD-PC, including a preferential partition into LE regions. However, a detailed quantitative analysis of the effect of the presence of different probes on the microscopic and nanoscopic structure of films made from different lipid systems is beyond the scope of the present study. The example we have studied here is still illustrative enough to state a cautionary message when using exogenous fluorescent probes in lipid films and suggests that a careful examination should be made of the structural effects of (35) Vaz, W. L.; Clegg, R. M.; Hallmann, D. Biochemistry 1985, 24, 781-6. (36) Chattopadhyay, A.; London, E. Biochemistry 1987, 26, 39-45. (37) Huster, D.; Muller, P.; Arnold, K.; Herrmann, A. Biophys. J. 2001, 80, 822-31. (38) Saez-Cirion, A.; Nir, S.; Lorizate, M.; Agirre, A.; Cruz, A.; PerezGil, J.; Nieva, J. L. J. Biol. Chem. 2002, 277, 21776-85. (39) Bernardino de la Serna, J.; Perez-Gil, J.; Simonsen, A. C.; Bagatolli, L. A. J. Biol. Chem. 2004, 279, 40715-22.

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the particular probe of interest on the lipid system used and the conditions chosen for the experiments. In summary, the presence of the probe introduces significant effects on the structure of the lipid films at the nanoscopic level, including reduced general condensation, smaller and deformed condensed nanoregions, and more perforated solid domains, compared with probe-free monolayers. All these structural alterations correlate with higher mechanical fragility of the probe-containing films compared with those without probe. The extent to which these perturbations could introduce a deformed perception of the behavior of these models and its extrapolation to biologically relevant systems has to be carefully evaluated (40) Harder, T. Curr. Opin. Immunol. 2004, 16, 353-9. (41) Helms, J. B.; Zurzolo, C. Traffic 2004, 5, 247-54. (42) Manes, S.; del Real, G.; Martinez, A. C. Nat. Rev. Immunol. 2003, 3, 557-68.

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and taken into consideration. Processes such as the assembly of raft-associated signaling platforms,40 lipid and protein sorting in cellular membranes,41 or assembly and budding of viral particles42 have been proposed to depend on the encounter of certain protein and lipid species on the nanoscopic framework provided by membrane lipid domains. Inclusion of fluorescent probes to study different models of these systems by microscopy techniques could reveal a distorted picture of the real situation that has to be analyzed at the proper physiologically relevant scale. Acknowledgment. This work has been supported by grants from D.G.E.S.I.C. (BIO2003-09056). A.C. was recipient of a postdoctoral fellowship from C.A.M. LA046759W