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Two-Color Near-Field Fluorescence Microscopy Studies of Microdomains (“Rafts”) in Model Membranes Pierre Burgos,†,‡ Chunbo Yuan,† Marie-Laure Viriot,‡ and Linda J. Johnston*,† Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON K1A 0R6 Canada, and UMR 7630 CNRS-INPL, DCPR-GRAPP, ENSIC, 1 rue Grandville, 54000 Nancy, France Received March 31, 2003. In Final Form: May 24, 2003 The distribution of glycolipid GM1 in supported phospholipid monolayers of a ternary lipid mixture has been studied by near-field scanning optical microscopy (NSOM). Monolayers of equimolar amounts of sphingomyelin, dioleoylphosphatidylcholine, and cholesterol show clear phase separation to give large condensed domains surrounded by a fluid phase, as visualized by both atomic force microscopy (AFM) and addition of a Texas Red labeled lipid that localizes preferentially in the fluid phase. A combination of AFM and one- and two-color NSOM experiments indicates that addition of GM1-Bodipy results in small glycolipid domains within the fluid phase. This is in contrast to previous results demonstrating that GM1 is localized in the condensed phase for similar lipid mixtures and is attributed to dye-induced changes in the distribution of the glycolipid. However, NSOM experiments for GM1-containing monolayers doped with low loadings of GM1-Bodipy resulted in the observation of small fluorescent microdomains within the condensed phase. The high spatial resolution of NSOM allows the detection of small domains that have not been observed previously by fluorescence and demonstrates that similar, although not identical, distributions of glycolipid are detected by AFM and fluorescence. The relevance of these results to rafts in natural membranes is discussed.
Introduction Lateral phase separation occurs in a variety of cellular membranes and plays an important role in the aggregation of protein complexes and initiation of signal transduction pathways. The raft model recently proposed by Simons and Ikonen suggests that the plasma membrane has small, dynamic microdomains (“rafts”) that are rich in cholesterol, glycolipids, and saturated lipids such as sphingomyelin and in which specific proteins, particularly those involved in signaling cascades, are localized.1 There is increasing evidence for the biological importance of rafts, although their postulated small size is beyond the resolution of conventional optical microscopy, making their direct detection difficult.2-9 Nevertheless, methods such as extraction of detergent-insoluble membrane fragments, single-particle tracking, and fluorescence resonance energy transfer (FRET) have all provided support for the localization of a variety of proteins and glycolipids in membrane microdomains.2,7 Both the lateral size and the mechanism of formation of rafts are controversial with estimates ranging from several to hundreds of nanometers. Much of our detailed knowledge of the physical behavior of membranes comes from studies of model membranes such as phospholipid monolayers, planar lipid bilayers, * Corresponding author. E-mail:
[email protected]. Fax: 613-952-0068. † Steacie Institute for Molecular Sciences, National Research Council Canada. ‡ UMR 7630 CNRS-INPL, DCPR-GRAPP, ENSIC. (1) Simons, K.; Ikonen, E. Nature 1997, 387, 569-572. (2) Jacobson, K.; Dietrich, C. Cell Biol. 1999, 9, 87-91. (3) Brown, D. A.; London, E. J. Biol. Chem. 2000, 275, 17221-17224. (4) Brown, D. A.; London, E. Annu. Rev. Cell Dev. Biol. 1998, 14, 111-136. (5) Brown, D. A.; London, E. J. Membr. Biol. 1998, 164, 103-114. (6) Simons, K.; Ikonen, E. Science 2000, 290, 1721-1726. (7) Simons, K.; Toomre, D. Nat. Rev. Mol. Cell Biol. 2000, 1, 31-39. (8) Maxfield, F. R. Curr. Opin. Cell Biol. 2002, 14, 483-487. (9) Edidin, M. Annu. Rev. Biophys. Biomol. Struct. 2003, 32, 257283.
and vesicles, all of which are amenable for study with a variety of spectroscopic and microscopic techniques. Such models have also been used to study the formation of rafts in lipid mixtures that mimic the composition of detergentinsoluble membrane fractions. Several groups have investigated the distribution of ganglioside GM1 in model membranes since it has been used as a raft marker in cellular studies where it can be detected by the specific binding of its protein receptor cholera toxin B subunit. The ganglioside has been shown to localize in large micrometer-sized domains of an ordered sphingomyelin/ cholesterol phase in supported monolayers and bilayers and giant unilamellar vesicles prepared from lipid mixtures and from brush border membranes.10-12 Similarly, fluorescence studies of monolayers of ternary lipid mixtures at the air-water interface have shown that GM1 is localized in a condensed phase enriched in phospholipid/ cholesterol complexes.13 None of these studies provides evidence for the small rafts that have been observed using less direct methods in cellular membranes, and several explanations have been proposed to account for the apparent differences between model and real membranes. Atomic force microscopy (AFM) has been used in several recent studies to examine the distribution of GM1 in a variety of phospholipid monolayers and bilayers, including those made from ternary lipid mixtures.14-19 Our results have shown that the ganglioside is found in small (10) Dietrich, C.; Volovyk, Z. N.; Levi, M.; Thompson, N. L.; Jacobson, K. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 10642-10647. (11) Samsonov, A. V.; Mihalyov, I.; Cohen, F. S. Biophys. J. 2001, 81, 1486-1500. (12) Dietrich, C.; Bagatolli, L. A.; Volovyk, Z. N.; Thompson, N. L.; Levi, M.; Jacobson, K.; Gratton, E. Biophys. J. 2001, 80, 1417-1428. (13) Radhakrishnan, A.; Anderson, T. G.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 12422-12427. (14) Yuan, C.; Johnston, L. J. Biophys. J. 2000, 79, 2768-2781. (15) Yuan, C.; Johnston, L. J. Biophys. J. 2001, 81, 1059-1069. (16) Yuan, C.; Furlong, J.; Burgos, P.; Johnston, L. J. Biophys. J. 2002, 82, 2526-2535. (17) Vie, V.; Mau, N. V.; Lesniewska, E.; Goudonnet, J. P.; Heitz, F.; Le Grimellec, C. Langmuir 1998, 14, 4574-4583.
10.1021/la034551p CCC: $25.00 Published 2003 by the American Chemical Society Published on Web 08/16/2003
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microdomains (50-200 nm in size) that are randomly distributed in liquid ordered or fluid phosphatidylcholine (PC) bilayers.14,15 However, in ternary lipid mixtures the ganglioside is found in small microdomains within the ordered phase.16 This work suggests that model membranes can support the formation of small raftlike microdomains similar to those thought to exist in cellular membranes. Nevertheless, the apparent discrepancy between the AFM data and fluorescence results in very similar lipid mixtures is puzzling. It is possible that variations in sample preparation account for the different observations, although the fluorescence studies have used both supported and free-standing bilayer membranes, as well as monolayers. The two techniques may also report on different populations of ganglioside, and the high spatial resolution of AFM will allow one to observe small domains that may not be readily detected by conventional optical microscopy. To distinguish between these possibilities, we have used near-field scanning optical microscopy (NSOM) to probe the distribution of GM1 in the same ternary lipid mixtures examined earlier by AFM. NSOM allows combined topographic and fluorescence imaging and has resolution that is determined by the probe aperture size and is approximately an order of magnitude better than the diffraction-limited resolution (λ/2) of conventional optical microscopy.20,21 The high spatial resolution and sensitivity allow for the independent observation of molecules at physiologically relevant packing densities, and NSOM is expected to be of particular value for studies of the spatial organization of membranes.22,23 Several groups have already demonstrated its utility for studies of phaseseparated model membranes.24-28 Our NSOM experiments have utilized a dye-labeled ganglioside in combination with a dye-labeled lipid to visualize ordered versus fluid phases and two-color near-field fluorescence measurements in order to conclusively identify the complex phase separation behavior observed. The results are in good agreement with previous AFM studies in that small ganglioside domains are observed by both AFM and fluorescence. They also provide some insight into the reasons for the differences between other fluorescence measurements and the present work and demonstrate that labeling the ganglioside leads to significant changes in its localization behavior. Materials and Methods Materials. Dioleoylphosphatidylcholine (DOPC), cholesterol, and NBD-DPPE were obtained from Avanti Polar Lipids, Alabaster, AL, and were used as received. N-Palmitoyl-Dsphingomyelin (SPM) from bovine brain and monosialoganglioside GM1 from bovine brain were obtained from Sigma. Dyelabeled lipids (Texas Red-DPPE, TR-DPPE; 1,2-dipalmitoyl-snglycero-3-phosphoethanolamine-fluorescein, Fl-DPPE; 1,1-di(18) Milhiet, P. E.; Vie, V.; Giocondi, M.-C.; Le Grimellec, C. Single Mol. 2001, 2, 109-112. (19) Mou, J.; Yang, J.; Shao, Z. J. Mol. Biol. 1995, 248, 507-512. (20) Zenobi, R.; Deckert, V. Angew. Chem., Int. Ed. 2000, 39, 17461756. (21) Dunn, R. C. Chem. Rev. 1999, 99, 2891-2928. (22) de Lange, F.; Cambi, A.; Huijbens, R.; de Bakker, B.; Rensen, W.; Garica-Parajo, M.; van Hulst, N.; Figdor, C. G. J. Cell Sci. 2001, 114, 4153-4160. (23) Edidin, M. Traffic 2001, 2, 797-803. (24) Yuan, C.; Johnston, L. J. J. Microsc. 2002, 205, 136-146. (25) Hollars, C. W.; Dunn, R. C. J. Phys. Chem. B 1997, 101, 63136317. (26) Hollars, C. W.; Dunn, R. C. Biophys. J. 1998, 75, 342-353. (27) Hwang, J.; Tamm, L. K.; Bohm, C.; Ramalingam, T. S.; Betzig, E.; Edidin, M. Science 1995, 270, 610-613. (28) Tamm, L. K.; Bohm, C.; Yang, J.; Shao, Z.; Hwang, J.; Edidin, M.; Betzig, E. Thin Solid Films 1996, 284-285, 813-816.
Langmuir, Vol. 19, No. 19, 2003 8003 octadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, DiIC18) and GM1-Bodipy (Bodipy FLC5-GM1) were obtained from Molecular Probes. Monolayer Preparation. Monolayers of 1:1:1 DOPC, cholesterol, and sphingomyelin (lipid ratios in mol %) were prepared on a Langmuir-Blodgett trough (NIMA model 611, Coventry, U.K.). Milli-Q water was used as the subphase. Sample solutions of lipids with or without dye or ganglioside in chloroform or chloroform/methanol were spread on the water surface, and after solvent evaporation (10 min) the monolayer was compressed at 20 cm2/min to the required surface pressure. The surface pressure was measured with a precision of 0.1 mN/m using a Wilhelmy balance. Monolayers were annealed by at least two compression/ expansion cycles before transfer to freshly cleaved hydrophilic mica by vertical deposition with a dipping speed of 2 mm/min. All monolayers were transferred at a surface pressure of 10 ( 0.5 mN/m, and transfer ratios of unity were typical. Variations in the monolayer compression rate (10-30 cm2/min) or dipping speed (2-4 mm/min) did not affect the monolayer morphology observed by AFM. However, annealing the monolayer by several expansion/compression cycles was important for obtaining reproducible samples. Scanning Probe Microscopy. Atomic force microscopy measurements were carried out on a Multimode Nanoscope III (Digital Instruments, Santa Barbara, CA) in the repulsive mode in air using a J scanner (120 µm). Soft cantilevers (200 µm long) with integrated pyramidal tips and a spring constant of approximately 60 mN/m were used. Images were typically recorded with scan rates of 1 Hz/line and forces of 2-4 nN. Differences in height between monolayer domains were determined by the section analysis routine provided with the DI software. Near-field scanning optical microscopy experiments were carried out on a combined AFM/NSOM microscope. The microscope is based on a Digital Instruments Bioscope mounted on an inverted fluorescence microscope (Zeiss Axiovert 100) and uses a separate x-y piezo scanner (Physik Instrumente) for sample scanning for near-field measurements. A CW mixed gas argon ion laser was used for excitation purposes (488 or 568 nm). Fluorescence is collected with a 100× oil immersion objective (1.3 numerical aperture), with appropriate filters to remove residual excitation (488 or 568 nm Notch filters, Kaiser Optical Systems) and red alignment laser light (Chroma 670 nm cutoff filter), and detected using an avalanche photodiode detector (EG&G, SPCM-AQR-14). Images were recorded in tapping mode in air with typical oscillation amplitudes of ∼50 nm with continuous light collection and using a scan rate of 1 Hz and a resolution of 512 × 512. All experiments used bent doubleetched optical fiber probes with aperture sizes of 70-90 nm, probe diameters of approximately 500 nm, and estimated spring constants of 100 N/m. Full details of the probe fabrication and characterization have been reported elsewhere.29 Background noise was typically less than 10% of the optical signal.
Results Previous work has used AFM to examine the distribution of GM1 in monolayers and bilayers of a ternary lipid mixture (phosphatidylcholine, sphingomyelin, cholesterol) that mimics the composition of rafts in natural membranes.16 This work had varied the surface pressure for monolayer deposition (from 7 to 40 mN/m), the amount of GM1 (1-5%), and the fraction of cholesterol in the ternary lipid mixture. In most cases (except at a surface pressure of 40 mN/m), monolayers of the ternary lipid mixture show condensed sphingomyelin/cholesterol domains surrounded by a fluid DOPC phase. The addition of GM1 results in the formation of small microdomains that are localized preferentially in the condensed phase. Similar results were also observed for hybrid bilayers with the ternary lipid mixture in the upper leaflet. For the present comparison of AFM and NSOM results, we have selected monolayers of an equimolar mixture of the three (29) Burgos, P.; Lu, Z.; Ianoul, A.; Hnatovsky, C.; Viriot, M.-L.; Johnston, L. J.; Taylor, R. S. J. Microsc. 2003, 211, 37-47.
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Figure 1. AFM images of 1:1:1 sphingomyelin/cholesterol/DOPC monolayers in the absence (A) and presence (B, C, section analysis) of 1% GM1. The z-scales are 10 nm for (A) and (B) and 2 nm for (C).
lipids transferred at 10 mN/m as a standard sample for comparison with the earlier studies.16 Representative AFM images for a 1:1:1 sphingomyelin/ cholesterol/DOPC monolayer transferred at a surface pressure of 10 mN/m with and without 1% GM1 are shown in Figure 1. In the absence of GM1, the monolayer shows large higher domains of the condensed SPM/cholesterol phase surrounded by a fluid phase that contains a few small islands of condensed phase (Figure 1A). Addition of GM1 leads to the formation of many small microdomains and filaments that are approximately 1 nm above the condensed phase (Figure 1B,C) and that range in width/ diameter from 70 to 200 nm; based on surface coverage, these have been attributed to ganglioside-enriched microdomains rather than pure GM1 domains.14,15 Monolayers of the same ternary lipid mixture were examined by NSOM in the presence of both Fl-DPPE and TR-DPPE in order to visualize the phase separation by fluorescence. Figure 2 shows NSOM fluorescence (A) and topography (B) images for a monolayer containing 1% FlDPPE; there is good agreement between the two, although the large size of the near-field probe leads to significant broadening of the domains in the topographic image. For comparison, smaller scale NSOM fluorescence (C) and AFM (D) images of a second Fl-DPPE sample were recorded and are shown in Figure 2C,D. Interestingly, the small islands of condensed phase that are clearly evident in the fluid phase in the AFM image are not detectable by NSOM. In most cases, it is possible to obtain NSOM topographic results of similar quality to those shown in Figure 2B for monolayer samples. However, in some cases it is difficult to detect the small height difference between fluid and condensed phases using the relatively large NSOM probes; occasionally we have also observed well-resolved topographic images that probably reflect topography defined by small protrusions on the probe.
Similar experiments using TR-DPPE to visualize the condensed and fluid phases for monolayers of the ternary lipid mixture are shown in Figure 2E (NSOM) and Figure 2F (AFM). The images indicate two advantages of TRDPPE: the contrast between the two phases is slightly better than that obtained with Fl-DPPE and the small islands of condensed phase are readily observed by both AFM and NSOM, even for a relatively large scan size. The small islands are frequently aligned, a process which has been shown to occur during the transfer process in other mixed lipid monolayers.30 In addition to TR-DPPE and Fl-DPPE, we also imaged monolayers containing 1% NBD-DPPE and DiIC18. Both signal-to-noise and contrast between the two phases were much poorer for these dyes. TR-DPPE was selected for all further experiments based on both the quality of the images and the fact that Texas Red can be excited at long wavelength, thus facilitating the two-color experiments described below. The improved image quality for TR-DPPE presumably indicates that this dye-labeled lipid has a higher partition coefficient for the fluid phase. Both the shape and fractional surface area covered by the large condensed domains are sensitive to small changes in deposition pressure and conditions; this accounts for the fact that round, elliptical, or teardrop-shaped domains are observed for various samples and sometimes also for different areas of the same sample (Figures 1 and 2). The domains are more irregularly shaped than is typical for liquid ordered domains in monolayers at the air-water interface or in bilayers. This may reflect effects of the transfer process30 as well as the low mobility of condensed domains in a supported monolayer. In agreement with this, one can also occasionally observe irregular domain shapes that appear to occur from coalescence of two or (30) Moraille, P.; Badia, A. Langmuir 2002, 18, 4414-4419.
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Figure 2. Images for 1:1:1 sphingomyelin/cholesterol/DOPC monolayers containing 1% Fl-DPPE (A-D) to visualize phase separation. Images A and B show simultaneously recorded NSOM fluorescence and topography. Images C and D show NSOM fluorescence and AFM images for a second Fl-DPPE monolayer. Images E and F show NSOM fluorescence and AFM for a monolayer containing 1% TR-DPPE. All NSOM images were recorded using λexc ) 488 nm. The z-scale is 10 nm for AFM images (D,F) and 30 nm for (B); z-scales are 0.07, 0.05, and 1 MHz for optical images (A,C,E).
more adjacent domains (e.g., Figure 2F), presumably during the transfer process. The results of the addition of 1 mol % GM1-Bodipy to a 1:1:1 SPM/DOPC/cholesterol monolayer are shown in Figure 3, which gives both NSOM fluorescence (A,B) and AFM (C,D) images for the same sample. The NSOM image (A) shows some bright fluorescent islands in addition to areas with a number of curved filaments, reminiscent of the borders of elliptical-shaped domains. A smaller scale image (B) shows that many of these filaments are closely spaced small islands, the smallest of which is 150 nm in diameter (Figure 3B, section analysis). In this case, the topography obtained with the near-field probe was not of sufficient quality to ascertain whether the fluorescent areas were in the condensed or fluid phase. However, the AFM images show no evidence for microdomains within the large condensed domains, although there are many small aligned islands within the lower fluid phase and near the edges of the condensed domains. These vary in size from 70 to 200 nm (section analysis, Figure 3D) and based solely on the AFM images could be either small ganglioside domains or islands of the condensed phase. Images of a number of samples failed to provide any evidence for small domains within the condensed phase. Although the combined AFM and NSOM data suggest that the islands observed by both methods are gangliosiderich areas localized in the fluid phase, the difficulty of resolving the small islands in the topographic NSOM images makes it impossible to be certain of this. To provide more convincing evidence for the location of the labeled ganglioside, a monolayer with 1% GM1-Bodipy and 0.05% TR-DPPE was prepared and imaged using both 488 and 568 nm excitation. At 488 nm, both the Bodipy
(λmax(abs/em) ∼ 490/510 nm) and TR dyes (λmax(abs/em) ∼ 590/610 nm) are excited, whereas at 568 nm only the TR absorbs. The use of TR minimizes the possibility of energy transfer between the two dyes, although there is still a small amount of overlap between Bodipy fluorescence and TR absorption. The total emission between 500 and 670 nm was collected for 488 nm excitation (and from 575 to 670 nm for 568 nm excitation). This ensures that energy transfer between dyes in the double-labeling experiments and changes in the emission wavelength for Bodipy (monomer vs excimer) 31,32 do not affect our conclusions. The NSOM image for a monolayer with both TR-DPPE and GM1-Bodipy obtained at 488 nm (Figure 4A) shows a reasonably uniform fluorescence throughout the fluid phase with occasional much brighter small islands that are close to the borders between the two phases. These islands presumably correspond to areas of the monolayer with significant amounts of GM1-Bodipy. This is confirmed by an experiment using 568 nm excitation which excites only the TR dye and which shows a uniformly fluorescent fluid phase with no brighter islands (Figure 4B). Note that although the images at the two wavelengths were obtained sequentially, almost exactly the same area of the sample was scanned in each case. This is readily evident from the fact that the same overall domain pattern is observed in both images (see boxed regions in each). Careful examination of the small areas that show more intense fluorescence (pink areas of the image in Figure 4A) indicates that they are surrounded by the uniformly (31) Dahim, M.; Mizuno, N. K.; Li, X.-M.; Momsen, W. E.; Momsen, M. M.; Brockman, H. L. Biophys. J. 2002, 83, 1511-1524. (32) Chen, C. S.; Martin, O. C.; Pagano, R. E. Biophys. J. 1997, 72, 37-50.
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Figure 3. NSOM fluorescence (A, B, section analysis; λexc ) 488 nm) and AFM (C (z-scale 10 nm), D, section analysis) images for a 1:1:1 sphingomyelin/cholesterol/DOPC monolayer containing 1% GM1-Bodipy. The z-scales are 0.4 MHz and 10 nm for (A) and (C), respectively.
Figure 4. NSOM fluorescence images for 1:1:1 sphingomyelin/cholesterol/DOPC monolayers containing 1% GM1-Bodipy and 0.05% TR-DPPE (A,B) and 1% GM1-Bodipy (C). Images A and C were obtained using 488 nm excitation, and image B using 568 nm excitation. Note that almost the same area was scanned for images A and B, as indicated by the identical domain pattern shown in the boxed regions for each. The z-scales are 0.7, 0.2, and 0.5 MHz for (A), (B), and (C).
fluorescent fluid phase (yellow), clearly demonstrating that they are not in the condensed phase. For comparison, a sample prepared under identical conditions but containing only GM1-Bodipy and excited at 488 nm is shown in Figure 4C; excitation of the same sample at 568 nm under the conditions used to obtain the image in Figure 4B gave no fluorescence (not shown). The above results show quite conclusively that GM1Bodipy is preferentially localized in the condensed phase. These results are in contrast to those obtained with the unlabeled ganglioside which at low concentrations is found exclusively in the more ordered domains in ternary lipid monolayers as well as in simpler lipid monolayers. The change in localization can probably be attributed to the fact that the Bodipy label is attached to the C5 hydrocarbon
moiety of the glycolipid. The increased disorder thus favors preferential localization of the glycolipid in the disordered fluid phase. Since a sugar-labeled GM1 was not readily available, we explored the possibility that a small amount of labeled GM1 could be added to unlabeled glycolipid without modifying its preferential localization in the ordered phase. We reasoned that the excellent contrast obtained in the NSOM experiments would enable us to work at much lower dye levels and still clearly distinguish domain/microdomain formation. Figure 5 shows the results of such an experiment for a monolayer containing 1% GM1, 0.005% GM1-Bodipy, and 1% TR-DPPE. The NSOM image obtained using 488 nm excitation (Figure 5A) clearly shows many bright patches of fluorescence within the dark condensed do-
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Figure 5. NSOM (A, λexc ) 488 nm; B, λexc ) 568 nm) and AFM (C) images for a 1:1:1 sphingomyelin/cholesterol/DOPC monolayer containing 1% GM1, 0.005% GM1-Bodipy, and 1% TR-DPPE. (D) and (E) show small-scale NSOM and AFM images with corresponding section analyses. The z-scales are 2.0 MHz for (A) and (B) and 10 nm for (C).
mains. Interestingly, there are also several areas where a bright island is on the boundary between the fluid and condensed phases. Excitation of the sample at 568 nm shows only bright fluorescence in the fluid phase with no hint of the small fluorescent microdomains (Figure 5B). Consistent with this, the AFM image (Figure 5C) of the same sample shows that the condensed phase has some small islands of a higher phase as well as a number of thin filaments, presumably formed by condensation of the small microdomains, as observed in several other cases for GM1.14 Interestingly, the fluid phase has a number of small islands of higher phase. However, the fact that one can observe corresponding dark islands in the NSOM images indicates that in this case the islands correspond to condensed SPM/cholesterol domains, as are frequently observed for the ternary lipid mixture alone. Section analyses for both the NSOM and AFM images are shown in Figure 5D,E, respectively. The width of the narrower filaments and dots in the condensed phase varies between 150 and 200 nm for the NSOM images and between 70 and 150 nm for the AFM. The above experiments show a strong tendency for low loadings of both labeled and unlabeled GM1 to localize in small microdomains, irrespective of whether these are found in the fluid or condensed phase. We carried out several additional experiments using both higher laser power and higher glycolipid concentrations in order to look for evidence of more heterogeneous distribution of the ganglioside. Figure 6 shows NSOM topography (A) and fluorescence (B) images obtained for a monolayer with 2% GM1-Bodipy. In this case, the glycolipid seems to be
located primarily at the borders of the large condensed domains. The sizes of the domains in the topographic (A) and optical (B) images are similar; however, after taking into account the broadening of the topographic image due to the large probe diameter, it appears that GM1-Bodipy is located in both the fluid and condensed phases. The size of the GM1-rich areas is larger than for a sample with 1% GM1-Bodipy, and there is significant variation of fluorescent intensities. These results suggest that at higher concentrations the glycolipid is more heterogeneously distributed throughout the monolayer and is not exclusively found in small microdomains. Larger fluorescent areas are also detected at higher laser power. The image in Figure 6C was obtained by exciting a monolayer with 1% GM1-Bodipy using 4 times higher input power than that used for the images shown in Figures 3 and 4. Although there are some relatively small microdomains, there are also a number of large patches with less intense fluorescence, mostly in the same vicinity as the bright microdomains. The fact that both small bright islands and large, more diffuse patches of fluorescence can be detected in a single image eliminates any possible artifacts related to the probe size. An AFM image for the same sample (Figure 6D) shows only small islands in the fluid phase, although again one cannot readily distinguish if these are ganglioside domains or simply the condensed sphingomyelin/cholesterol-rich phase. These results suggest that fluorescence measurements report a somewhat different glycolipid distribution than do the AFM images. Although it is not straightforward to quantify the amounts of GM1 in the small microdomains
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Figure 6. NSOM topography (A) and fluorescence images (B, λexc ) 488 nm) for a 1:1:1 sphingomyelin/cholesterol/DOPC monolayer containing 2% GM1-Bodipy. NSOM fluorescence (C, λexc ) 488 nm) and AFM (D) images for a 1:1:1 sphingomyelin/cholesterol/DOPC monolayer containing 1% GM1-Bodipy. For image C, the excitation intensity at 488 nm was approximately 4 times higher than that used to obtain the images for GM1-Bodipy shown in Figures 3, 4, and 5. The z-scales are 0.7 and 0.5 MHz for (B) and (C) and 10 nm for (D).
and the more diffuse patches, we estimate that ∼80% of the ganglioside is in the small bright microdomains. No further changes in the fluorescence distribution were observed at higher excitations than that used for Figure 6C. Discussion The results presented above provide useful insight into the distribution of gangliosides in model supported membranes. First, the addition of a dye to the C5 hydrocarbon moiety of the ganglioside changes completely its preference for localization in the condensed “raftlike” phase. This highlights the importance of ensuring that the probe does not modify the behavior of the molecule of interest and is in agreement with a recent study in which addition of dyes to the acyl chains of sphingomyelin or cholesterol was also shown to alter its preference for localization in raft phases.11 It would obviously be of interest to compare the present results to those for a ganglioside labeled on the polar sugar headgroup. However, adding a dye to the sugar moiety is likely to interfere with protein binding (cholera toxin B subunit), a common method for monitoring ganglioside distribution. A recent study has used fluorescence microscopy to demonstrate that a sugar-labeled GM1 partitions selectively into SPM/cholesterol domains in vesicles prepared from complex lipid mixtures.11 Ganglioside localization has been used to provide evidence for the assignment of these domains to a raft phase. However, the size of the domains is large (tens of microns), in contrast both to our present results and to the estimates of raft sizes in natural membranes. Interestingly GM1-Bodipylabeled liposomes of raftlike lipid mixtures show some
Bodipy excimer emission, which is consistent with the fact that GM1-Bodipy still forms aggregates, even though its preference for fluid versus raft phases has changed.33 Despite the complications associated with using a dyelabeled ganglioside, the results obtained by adding a small amount of labeled GM1 to native GM1 do allow us to draw important conclusions concerning the formation of GM1 microdomains in model raft mixtures. Our results show quite clearly that GM1 is not homogeneously distributed throughout the condensed phase but is found in small microdomains that are typically 100-200 nm in size. This is in qualitative agreement with the AFM results obtained earlier for GM1 in a variety of lipid monolayers and bilayers, all of which show small aggregates of GM1.14-16 The average size of the microdomains measured by NSOM is slightly larger than that obtained from AFM images for the same sample (although it is not possible to image exactly the same sample area on the two instruments). This is consistent with the fact that the NSOM probe aperture is close to 100 nm in diameter; tip-sample convolution will lead to significant broadening of small features such as the ganglioside islands. The heterogeneous distribution of small microdomains in the condensed phase for this model membrane is consistent with current estimates of the size of rafts in natural membranes.2,8,9 The results also indicate that lipid properties alone can direct the formation of small microdomains. However, the presence of proteins and a means of controlling the formation and dissolution of rafts are also required for biological function in a natural membrane. (33) Kakio, A.; Nishimoto, S.; Yanagisawa, K.; Matsuzaki, K. Biochemistry 2002, 41, 7385-7390.
Microdomains in Model Membranes
The observation that both NSOM and AFM provide conclusive evidence for ganglioside microdomains within the condensed phase of ternary lipid mixtures is in contrast to previous fluorescence results indicating that GM1 is uniformly distributed in large domains.11-13 The previous fluorescence studies had examined both supported and free-standing bilayers and monolayers at the air-water interface, whereas the AFM studies have examined supported monolayers and bilayers. Given the range of samples examined by both techniques, it seems unlikely that the type of model membrane or the effects of a solid support can account for the differences between the AFM and optical results. However, our experiments with higher ganglioside concentrations and excitation intensities provide some possible explanations. The distribution of GM1-Bodipy is sensitive to concentration with larger more diffuse areas of ganglioside being detected at higher concentrations. A previous study of monolayers at the airwater interface used 7 mol % GM1,13 conditions which based on our current results are unlikely to give the small microdomains observed at low [GM1]. In addition, experiments at higher excitation intensities show significant areas of weaker fluorescence that presumably correspond to lower ganglioside concentrations. This indicates that the distribution of ganglioside observed by fluorescence is somewhat different from that obtained by AFM. The latter requires sufficient glycolipid to give a measurable height difference and does not detect relatively low GM1 concentrations that can be observed by fluorescence, particularly at higher excitation intensities. If similar effects occur for unlabeled ganglioside, then the sensitivity of fluorescence to low levels of ganglioside that are not detected by AFM may partially account for the differences between the present NSOM/AFM results and previous fluorescence microscopy studies, most of which also used 1% GM1. The increased spatial resolution available with NSOM will also facilitate the observation of small closely spaced domains that cannot be easily resolved by conventional fluorescence microscopy. Interestingly, a recent AFM study of phosphatidylcholine/ cholesterol membranes has provided direct evidence for
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nanoscopic domains that were not detected by confocal fluorescence microscopy.34 Our ongoing work is aimed at using near-field measurements in aqueous solution to assess the distribution of GM1 in bilayers using binding of a dye-labeled protein to visualize the glycolipid distribution. Finally, the present results hint at the potential of nearfield measurements for imaging cell surfaces. We have recently demonstrated that images of comparable quality to those shown here for monolayers can be obtained for two-component phase-separated supported lipid bilayers in aqueous solution,35 an important step toward realizing the potential of NSOM for interrogating cell surfaces. NSOM appears to be well-suited for studies of the aggregation of proteins in small membrane domains in the plasma membrane, a process that is now considered a key requirement for initiating signaling cascades. The ability to study such processes on a smaller length scale than that routinely attainable with confocal microscopy will facilitate detection of these small signaling domains. Further, the possibility of combining NSOM with singlemolecule sensitivity and with FRET experiments gives one the capability of observing individual protein binding events, even in cases where the membrane receptor distribution is relatively high. The resolution of NSOM is intermediate between those of confocal and FRET, and this coupled with the capabilities of single-molecule sensitivity yields a technique which is poised to make major contributions to biological imaging.22,23 Acknowledgment. Partial support of this work by a NRC-CNRS collaborative grant is gratefully acknowledged. We thank Dr. Z. Lu for probe fabrication and Dr. R. Taylor for many helpful discussions on probe fabrication and near-field microscopy. LA034551P (34) Tokumasu, F.; Jin, A. J.; Feigenson, G. W.; Dvorak, J. A. Biophys. J. 2003, 84, 2609-2618. (35) Ianoul, A.; Burgos, P.; Lu, Z.; Taylor, R. S.; Johnston, L. J. Langmuir, in press.
