Membrane Domain-Disrupting Effects of 4-Substitued Cholesterol

Jul 26, 2008 - also by the local membrane organization, including domains of specific lipid ... cholesterol and sphingolipids is an important factor f...
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Langmuir 2008, 24, 8807-8812

8807

Membrane Domain-Disrupting Effects of 4-Substitued Cholesterol Derivatives Dolores C. Carrer,† Arndt W. Schmidt,‡ Hans-Joachim Kno¨lker,‡ and Petra Schwille*,† Biophysics Group, BIOTEC, TU Dresden, Tatzberg 47-51, 01307 Dresden, Germany, and Department of Chemistry, TU Dresden, Bergstrasse 66, 01069 Dresden, Germany ReceiVed March 25, 2008. ReVised Manuscript ReceiVed June 16, 2008 A wide range of cellular functions are thought to be regulated not only by the activity of membrane proteins, but also by the local membrane organization, including domains of specific lipid composition. Thus, molecules and drugs targeting and disrupting this lipid pattern, particularly of the plasma membrane, will not only help to investigate the role of membrane domains in cell biology, but might also be interesting candidates for therapy. We have identified three 4-substituted cholesterol derivatives that are able to induce a domain-disrupting effect in model membranes. When applied to giant unilamellar vesicles displaying liquid-ordered-liquid-disordered phase coexistence, extensive reorganization of the membrane can be observed, such as the budding of membrane tubules or changes in the geometry of the domains, to the point of complete abolition of phase separation. In this case, the resulting membranes display a fluidity intermediate between those of liquid-disordered and liquid-ordered phases.

Introduction A wide range of biological processes, including signal transduction, cell adhesion, lipid trafficking, and viral and bacterial entry are made possible and regulated by the local membrane organization. The effects of the membrane state on cellular processes are mediated both by the organization of the membrane components (lipids and proteins) in different compartments and by physical parameters such as the fluidity, curvature, and lateral pressure profile of the membrane.1–3 Molecules affecting lipid membrane organization have important physiological effects. For example, the ability of the sphingolipid ceramide to disrupt lipid domains in cells has been proposed to be fundamental for their effects as inhibitors of membrane protein signaling,4 as modulators of phospholipase D1 activity,4 and as modulators of the sorting of membrane into different populations of intraluminal vesicles.5 Apart from sphingolipids, cholesterol is a major component of plasma membrane domains. Because the interaction between cholesterol and sphingolipids is an important factor for domain formation, removal of cholesterol from cells is thought to disrupt the structure of lipid rafts and caveolae and release their protein components into the bulk plasma membrane. The most common method used for acute depletion of cellular cholesterol involves the use of methyl-β-cyclodextrin. A second method is the use of filipin. Filipin is a polyene antibiotic that forms a complex with cholesterol and appears to disrupt caveolae and lipid rafts.6 An alternative approach to cholesterol depletion is the use of progesterone. Progesterone impairs cholesterol trafficking in cells and leads to a depletion of cholesterol at the plasma membrane.7 * Corresponding author. E-mail: [email protected]. † Biophysics Group, BIOTEC. ‡ Department of Chemistry.

(1) Pike, L. J. J. Lipid Res. 2006, 47, 1597–1598. (2) Simons, K.; Vaz, W. L. Annu. ReV. Biophys. Biomol. Struct. 2004, 33, 269–295. (3) Marsh, D. Biophys. J. 2007, 93, 3884–3899. (4) Gidwani, A.; Brown, H. A.; Holowka, D.; Baird, B. J. Cell Sci. 2003, 116, 3177–3187. (5) Trajkovic, K.; Hsu, C.; Chiantia, S.; Rajendran, L.; Wenzel, D.; Wieland, F.; Schwille, P.; Bru¨gger, B.; Simons, M. Science 2008, 319, 1244–1247. (6) Schnitzer, J. E.; Oh, P.; Pinney, E.; Allard, J. J. Cell Biol. 1994, 127, 1217–1232.

Many groups have reported on the effects of cholesterol depletion on membrane signaling pathways. Regarding the role of membrane organization in regulating epidermal growth factor receptor function, several significant cholesterol-dependent changes in receptor activity have been demonstrated.8 Also, cholesterol plays a significant role in organizing and promoting signaling via the insulin receptor.9 The insulin receptor clearly localizes to caveolae.10 Depletion of cholesterol with methylβ-cyclodextrin leads to the flattening of caveolae and the apparent disappearance of these invaginations from the cell surface. Whereas receptor binding and kinase activities appear to be largely retained following cholesterol depletion of caveolin-containing cells, downstream signaling of the insulin receptor is almost uniformly impaired.11,12 Also, the disruption of cholesterol domains with filipin can inhibit SHP-2, a regulator of the JAKSTAT pathway that plays important roles in cell proliferation, apoptosis, and inflammation.13 Further examples are the modulation by cholesterol of the internalization of the alpha-1a adrenergic receptor as shown by using methyl-β-cyclodextrin14 and the triggering of apoptosis by cyclodextrin in carcinoma cells, which involves mitochondrial events and is associated with activation of the death receptor Fas.15 Regarding the internalization of pathogens, it has been shown that rhinoviral and HIV infections can be prevented by treatment with β-cyclodextrin16,17 A further example of the importance of lipid membrane organization in cell function is the evidence accumulated in favor of a bilayer(7) Furuchi, T.; Anderson, R. G. W. J. Biol. Chem. 1998, 273, 21099–21104. (8) Ringerike, T.; Glystad, F. D.; Levy, F. O.; Madshus, I. H.; Stang, E. J. Cell Sci. 2002, 115, 1331–1340. (9) Pike, L. J. Biochim. Biophys. Acta 2005, 1746, 260–273. (10) Gustavsson, J.; Parpal, S.; Karlsson, M.; Ramsing, C.; Thorm, H.; Borg, M.; Lindroth, M.; Peterson, K. H.; Magnusson, K.-E.; Stralfors, P. FASEB J. 1999, 13, 1961–1971. (11) Parpal, S.; Karlsson, M.; Thorn, H.; Stralfors, P. J. Biol. Chem. 2001, 276, 9670–9678. (12) Vainio, S.; Heino, S.; Mansson, J.-E.; Fredman, P.; Kuismanen, E.; Vaarala, O.; Ikonen, E. EMBO Rep. 2002, 3, 95–100. (13) Kim, H. Y.; Park, S. J.; Joe, E. H.; Jou, I. J. Biol. Chem. 2006, 281, 11872–11878. (14) Morris, D. P.; Lei, B.; Wu, Y. X.; Michelotti, G. A.; Schwinn, D. A. J. Biol. Chem. 2008, 283, 2973–2985. (15) Bionda, C.; Athias, A.; Poncet, D.; Alphonse, G.; Guezquez, A.; Gambert, P.; Rodrı´guez-Lafrasse, C.; Ardail, D. Biochem. Pharmacol. 2008, 75, 761–772. (16) Snyers, L.; Zwickl, H.; Blaas, D. J. Virol. 2003, 77, 5360–5369. (17) Nguyen, D. H.; Taub, D. D. Mol. InterVentions 2004, 4, 318–320.

10.1021/la801471e CCC: $40.75  2008 American Chemical Society Published on Web 07/26/2008

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mediated mechanism of anesthesia:18 the anesthetic potency of long alkanols is consistent with a membrane-mediated mechanism involving the lateral pressure profile.19 The importance of lipid membrane organization for cell functioning has also prompted the search for compounds acting on the membrane as candidates for therapy. For example, alkylphospholipids have been shown to impair normal membrane function and induce apoptosis in lymphoma cells and are thus proposed as anticancer agents.20 This kind of antitumor drug has been shown to modify the plasma membrane lipid composition, which results in the displacement of an essential protein from lipid rafts.21 A further example is quercetine, a flavonoid that causes the redistribution of death receptors in membrane domains and whose activity is inhibited by nystatine, a cholesterolsequestering agent. Quercetine is thus proposed as an agent to enhance TRAIL- (tumor necrosis factor–related apoptosisinducing ligand-) based therapies of cancer.22 It has previously been shown that 3-ketocholesterol and cholesteryl-3-sulfate are not able to support phase separation when substituted for cholesterol in model membranes.23 However, we are interested in finding modified cholesterol derivatives that are able to act on pre-existing lipid domains when added from the outside. Thus, in this work, we have studied the effects of three 4-substituted cholesterol derivatives 1-3 on model membranes displaying liquid-ordered-liquid-disordered phase coexistence. Two of these molecules, the compounds 2 and 3, have previously been shown to mimic the absence of sterols in C. elegans.24 Because of their structural similarity to 3-ketocholesterol, they could act to disrupt domains in model membranes. In this work, we show that these compounds are indeed able to induce a domain-disrupting effect and to dramatically modify the membrane fluidity in model membranes.

Materials and Methods Chemicals. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (dioleoylphosphatidylcholine, DOPC), brain sphyngomyelin (BSM), brainceramide(BCer),Galβ1-3GalNAcβ1-4(NeuAcR2-3)Galβ1-4Glcβ1-1′Cer (GM1 ganglioside, GM1), and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. Dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD-C18, DiD), 3,3′-dioctadecyloxacarbocyanine perchlorate (DiOC18, DiO), and Alexa Fluor 488 cholera toxin subunit B (CTx-B) were obtained from Invitrogen (Eugene, OR). The 4-substituted cholesterol derivatives were synthesized as described in ref 24. Extra-pure glucose and sucrose were purchased from Merck (Darmstadt, Germany). Giant Unilamellar Vesicles (GUVs). Lipids and fluorescent lipid analogs were premixed in chloroform/methanol 2:1 solution at 10 mg/mL. GUVs were prepared by the method of electroswelling25 at 65 °C in a 200 mOsM sucrose solution. An aliquot of the GUVs thus formed was taken to a chambered coverglass containing 200 mOsM glucose solution. The membrane composition was DOPC/ BSM/cholesterol (Chol) ) 2:2:1 (mol/mol/mol). To image with cholera toxin, 0.1 mol % GM1 was added to the lipid mixture. Alexa 488 cholera toxin was added to the preformed GUVs in the imaging (18) Cantor, R. S. Biochemistry 2003, 42, 11891–11897. (19) Mohr, J. T.; Gribble, G. W.; Lin, S. S.; Eckenhoff, R. G.; Cantor, R. S. J. Med. Chem. 2005, 48, 4172–4176. (20) Van der Luit, A. H.; Vink, S. R.; Klarenbeek, J. B.; Perrissoud, D.; Solary, E.; Verheij, M.; van Blitterswijk, W. J. Mol. Cancer Ther. 2007, 6, 2337–2345. (21) Zaremberg, V.; Gajate, C.; Cacharro, L. M.; Mollinedo, F.; McMaster, C. R. J. Biol. Chem. 2005, 280, 38047–38058. (22) Psahoulia, F. H.; Drosopoulos, K. G.; Doubravska, L.; Andera, L.; Pintzas, A. Mol. Cancer Ther. 2007, 6, 2591–2599. (23) Bacia, K.; Schwille, P.; Kurzchalia, T. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 3272–3277. (24) Schmidt, A. W.; Doert, T.; Goutal, S.; Gruner, M.; Mende, F.; Kurzchalia, T. V.; Kno¨lker, H.-J. Eur. J. Org. Chem. 2006, 3687–3706. (25) Mathivet, L.; Cribier, S.; Devaux, P. F. Biophys. J. 1996, 70, 1112–1121.

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Figure 1. 4-Substituted cholesterol derivatives: β-keto ester 1, β-hydroxy ester 2, and β-hydroxy acid 3.

chamber. Fluorescent probes (DiO, DiD) were added to concentrations of 0.1 and 0.01 mol % for imaging and fluorescence correlation spectroscopy (FCS), respectively. The modified sterols were dissolved in dimethyl sulfoxide (DMSO) and added to the preformed GUVs in the imaging chamber. The maximum amount of DMSO in the samples was 1% v/v. Imaging and FCS. Confocal imaging was performed on a Zeiss LSM 510 Meta instrument (Carl Zeiss, Jena, Germany) using a 40X NA 1.2 water-immersion objective. FCS measurements were performed on a Zeiss LSM 510 Meta instrument using a 40X NA 1.2 UV-vis-IR C Apochromat water-immersion objective and a home-built detection unit at the fiber output channel. An appropriate band-pass filter was used behind a collimating achromat to reject the residual laser and background light. Another achromat (LINOS Photonics, Goettingen, Germany) with a shorter focal length was used to image the internal pinhole onto the aperture of the fiber connected to the avalanche photodiode (APD; Perkin-Elmer, Boston, MA). The photon arrival times were recorded in the photon mode of the hardware correlator Flex 02-01D (Correlator.com, Bridgewater, NJ). All filters and dichroic mirrors were purchased from AHF Analyze Technik, Tuebingen, Germany. The movement of the detection volume was controlled directly with the Zeiss LSM operation software. FCS curves were fitted with a software developed by Ries and analyzed by the z-scan method.26 The sample temperature was 20 °C. Chambered coverglasses were from Nunc (Rochester, NY).

Results and Discussion Figure 1 shows the chemical structures of the 4-substituted cholesterol derivatives that we studied. As a model of the outer hemilayer of the plasma membrane, we used giant unilamellar vesicles composed of a mixture of dioleoylphosphatidylcholine (DOPC), brain sphingomyelin (BSM), and cholesterol (Chol) in a molar ratio of 2:2:1. This mixture shows coexisting liquidordered and liquid-disordered domains in a wide range of temperatures.27 Figure 2a shows such phase-separating GUVs as visualized by including a fluorescent probe that partitions preferentially into the liquid-disordered phase (DiD) and Alexa 488-cholera toxin, which binds to GM1 preferentially partitioning to the liquid-ordered phase.27 Effects of the β-Keto Ester 1. Varying amounts of the β-keto ester 1 dissolved in DMSO were added to these phase-separating GUVs, and the effects were observed after 40 min of incubation. Figure 2b shows that incubation of GUVs with the β-keto ester 1 produces membranes in which phase separation cannot be (26) Humpolı´ckova´, J; Gielen, E.; Benda, A.; Faqulova, V.; Vercammen, J.; Vandeven, M.; Hof, M.; Ameloot, M.; Engelborghs, Y. Biophys. J. 2006, 91, L23–5. (27) Dietrich, C.; Bagatolli, L. A.; Volovyk, Z. N.; Thompson, N. L.; Levi, M.; Jacobson, K.; Gratton, E. Biophys. J. 2001, 80, 1417–1428.

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Figure 2. Effects of the incubation of GUVs with the β-keto ester 1. (a) GUVs composed of 2:2:1 DOPC/BSM/Chol show phase coexistence at room temperature. (b) Addition of the β-keto ester derivative at 160 µM final concentration produces the loss of domains. (c) At short incubation times or low compound concentrations, fragmentation of the liquid-ordered (Lo) phase is sometimes observed. In red, DiD partitions preferentially to the liquid-disordered (Ld) phase. In green, cholera toxin binds to GM1, partitioning preferentially to the liquid-ordered phase.

Figure 3. Effects of the 4-substitued cholesterol derivatives in GUVs as a function of concentration. The effects of the β-keto ester 1 (squares), the β-hydroxy acid 3 (circles), and the β-hydroxy ester 2 (triangles) was quantified after 40 min, 3 h, and 4 h of incubation, respectively, with the GUVs. Between 100 and 300 GUVs were inspected for phase separation for each data point.

detected by confocal laser scanning microscopy. The incubation with the modified sterol produces the loss of domains and the homogeneous mixing of the liquid-ordered-partitioning (GM1bound cholera toxin, in green) and liquid-disordered-partitioning (DiD, in red) probes. We quantified the effects of the β-keto ester 1 by incubating GUVs with different concentrations of the compound for a fixed amount of time. Figure 3 shows that the β-keto ester 1 derivative has a marginal activity at 1.6 µM concentration, whereas it is already very active at 16 µM, with more than 60% of the GUVs displaying a homogeneous membrane after 40 min of incubation. At this incubation time, a maximum effect was reached at a

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concentration of 80 µM, with 90% of the GUVs displaying a homogeneous membrane. A further increase in concentration to 160 µM showed the same amount of homogeneous GUVs after 40 min of incubation as the 80 µM concentration. To obtain information on the kinetics of the effects of the β-keto ester 1 on membranes, we studied the evolution in time of the GUVs after the addition of the β-keto ester 1. After a few minutes of incubation, most of the GUVs already looked homogeneous. Figure 4 shows that it takes approximately 8-12 min for the compound to make a phase-separating GUV look completely homogeneous. At short incubation times and/or at low concentrations of the compound, sometimes the liquidordered phase looked fragmented and dispersed into smaller domains (Figure 2c). At longer times, however, or at higher concentrations, the membrane always looked homogeneous. To obtain information about the effects of the compound on the fluidity of the membrane, we performed fluorescence correlation spectroscopy (FCS) measurements on the GUVs (containing DiO as a fluorescent membrane probe) before any treatment, after treatment with 1 vol % of DMSO, and after treatment with 160 µM concentration of the β-keto ester 1. Figure 5 and Table 1 show FCS data. Figure 5 shows the data obtained by performing z-scans on the GUVs. Diffusion times as a function of the focal volume distance from the plane of the membrane have a parabolic distribution. The fitting of these parabolas allows for the calculation of the diffusion times at the membrane without the need for an extrinsic calibration.26 Figure 5 and Table 1 show that the diffusion coefficient in the liquid-ordered phase of the GUVs (0.3 µm2/s) is 20 times slower than that in the liquiddisordered phase (6.15 µm2/s). This coincides with previous data on phase-separating GUVs.28 These values are not affected by the addition of 1% volume of DMSO (Table 1). The incubation of the GUVs with 160 µM of β-keto ester 1 for 2 h produces a homogeneous membrane with a diffusion coefficient that lies between the values of a liquid-ordered and a liquid-disordered phase, at 1.7 µm2/s. Effects of the β-Hydroxy Acid 3. The addition of the β-hydroxy acid 3 to 2:2:1 DOPC/BSM/Chol GUVs also produces a disruption in the membrane domains, with the membrane finally appearing homogeneous. Figure 6 shows confocal fluorescence images of GUVs containing DiO as a fluorescent probe. DiO partitions preferentially to the liquid-disordered phase in 2:2:1 DOPC/BSM/Chol GUVs. Figure 6a shows how, after 3 h of incubation, the GUVs display a homogeneous membrane. Control samples did not show any change after 3 h at room temperature (not shown). Budding of tubules from the vesicles was always observed upon addition of compound 3 in DMSO to the GUVs (Figure 6a,b). Vesicles showing tubules budding to the outside usually coexisted with vesicles showing tubules budding to the inside in any given sample at a particular time. We have not identified conditions upon which budding to the inside would be favored against budding to the outside. The fraction of vesicles that showed tubules attached was approximately 30% when the concentration of compound 3 was 160 µM. Figure 6c,d shows that, at short times after the addition of the β-hydroxy acid 3, a dramatic change in the shape, size, and geometric arrangement of the domains was induced, with the giant vesicles showing striped arrangements of the domains. We observed striped domains in 5% of the vesicles when the concentration of compound 3 was 160 µM and after 2 h of incubation. Under these conditions, most of the vesicles showed neither domains nor tubules attached to the membrane. The surrounding buffer, (28) Bacia, K.; Scherfeld, D.; Kahya, N.; Schwille, P. Biophys. J. 2004, 87, 1034–1043.

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Figure 4. Effects of β-keto ester 1 on phase-separating GUVs with time. From left to right, pictures taken every 2 min are shown. The fluorescent probe used was DiD.

however, showed a high amount of tubules, and many vesicles were filled with tubules that did not seem to be attached to the membrane. The occurrence of striped domains has been described in different model membrane systems containing cholesterol near a critical miscibility point, after which the membrane became homogeneous. In giant vesicles, such striped or fingered domains have been observed when the temperature is raised through the miscibility transition.29 In lipid monolayers containing cholesterol, striped domains have been observed at surface pressures near the miscibility critical point.30,31 In the present case, however, we did not observe rapid fluctuation of the striped domains, which would argue against proximity to a critical point. Striped phases can also be associated with membrane deformations/ bending and relaxation under shape changes produced, for example, by adhesion constraints, thermal expansion, or osmotic

deflation.32,33 In our case, we observed membrane tubules budding from the membrane, and this change in membrane topology coexisted with striped domains (see Figure 6d). It is therefore likely that, in our case, the striped domains were coupled to the deformation/bending of the membrane. The driving force could be the asymmetric incorporation of compound 3 between the outer and inner hemilayers of the membrane due to a slow flipflop (this compound is probably at least partially charged under the experimental conditions). This would create an excess area in the outer hemilayer that could, in turn, produce the observed membrane deformations and changes in domain geometries. In summary, the sequence of events provoked by the partition of compound 3 to the GUVs is the following: first, induction of tubules budding from the membrane, coupled with a transient change in the geometry of the domains, that adopt a striped conformation and then, a detachment of the tubules either to the inside of the vesicles or to the outer medium, along with the loss of visible domains. The result of these changes is a homogeneous membrane, where all of the molecules present are miscible in one phase (Figure 6a,b). When both the liquid-disordered and the liquid-ordered phases are labeled, a homogeneous mixing of the liquid-ordered- and liquid-disordered-preferring fluorescent probes is observed after 3 h of incubation (Figure 7a). Occasionally, and more often at shorter incubation times, it can be observed that the GM1-bound cholera toxin (in green) shows a homogeneous distribution whereas the DiD (in red) still shows the coexistence of domains (Figure 7b). The fact that GM1 is redistributed upon incubation with the compound at earlier times than DiD could indicate that GM1 partition is more sensitive to small changes in the composition and/or physical state of the membrane than DiD. We quantified the effects of the β-hydroxy acid 3 by incubating GUVs with different concentrations of the compound for a fixed amount of time (3 h). Figure 3 shows that the β-hydroxy acid 3 has no activity at 1.6 µM concentration, whereas it does show an activity at 16 µM, with more than 20% of the GUVs displaying a homogeneous membrane after 3 h of incubation. At this incubation time, a maximum effect was reached at a concentration of 80 µM, with almost 90% of the GUVs displaying a homogeneous membrane. A further increase in concentration to 160 µM resulted in almost the same amount of homogeneous GUVs after 3 h of incubation as the 80 µM concentration. The activity of the β-hydroxy acid 3 is much lower than the activity of the β-keto ester 1 as judged by the longer incubation time needed to obtain a maximum effect and by the lower activity at intermediate concentrations (16-32 µM) of the β-hydroxy acid 3 even at incubation times where the activity at higher concentrations (80-160 µM) was equal to that of the β-keto ester 1. The diffusion coefficient of a fluorescent membrane probe (DiO) as measured by FCS performed on GUVs after 3 h of

(29) Veatch, S. L.; Keller, S. L. Biophys. J. 2003, 85, 3074–3083. (30) Keller, S. L.; McConnell, H. M. Phys. ReV. Lett. 1999, 82, 1602–1605. (31) Radhakrishnan, A.; McConnell, H. M. Biophys. J. 1999, 77, 1507–1517.

(32) Baumgart, T.; Hess, S. T.; Webb, W. W. Nature 2003, 425, 821–24. (33) Rozovsky, S.; Kaizuka, Y.; Groves, J. T. J. Am. Chem. Soc. 2005, 127, 36–37.

Figure 5. FCS data of membranes treated with the cholesterol derivatives. Representative fits to FCS (z-scan) data taken in GUVs treated with the β-hydroxy acid 3 (red), with the β-keto ester 1 (green), and with the β-hydroxy ester 2 (blue) and in control GUVs, in the liquid-ordered phase (open squares) and in the liquid-disordered phase (open circles). Table 1. FCS Data on Control Samples and GUVs Incubated with the Compoundsa D (µm2/s) without compound Lo Ld with compound 1 2 3

0.3 ( 0.1 (0.3 ( 0.1) 6.1 ( 0.5 (6.5 ( 1.9) 1.7 ( 0.5 4.8 ( 1.3 1.2 ( 0.5

a Values in parentheses indicate diffusion coefficients in control samples with 1% DMSO. The fluorescent membrane probe used was DiO. The incubation times were 2 h for the β-keto ester 1, 3 h for the β-hydroxy acid 3, and 4 h for the β-hydroxy ester 2. The concentration of the compounds was 160 µM. The reported values are the mean results of at least 20 independent measurements.

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Figure 6. Confocal fluorescence images of GUVs incubated with the β-hydroxy acid 3. Effects of 3 h of incubation of the β-hydroxy acid 3 with GUVs composed of DOPC/BSM/Chol (2:2:1). Very often, tubules are detached from the membrane, being either (a) trapped inside the GUVs or (b) released to the outer medium. (c) GUV showing striped domains. (d) Series of images taken along the z axis of a GUV under the effects of the β-hydroxy acid 3. Notice the budding of the membrane. The fluorescent membrane probe used was DiO.

Figure 7. Effects of the β-hydroxy acid 3 on GUVs as visualized by the addition of cholera toxin and DiD. (a) Homogeneous distribution of cholera toxin-GM1 (green) and DiD (red). (b) Ocasionally, some GUVs show a homogeneous distribution of cholera toxin-GM1, whereas DiD can still distinguish domains.

Figure 8. Effects of the β-hydroxy ester 2 on GUVs as visualized by the addition of cholera toxin and DiD. A homogeneous distribution of cholera toxin-GM1 (green) and DiD (red) can be observed.

incubation with the β-hydroxy acid 3 lies between those measured for the liquid-ordered and liquid-disordered domains (see Table 1 and Figure 5). Effects of the β-Hydroxy Ester 2. The addition of the β-hydroxy ester 2 to the phase-separating GUVs induces a loss of phase separation. No further effects on the membrane topology were observed, apart from the loss of domain coexistence (Figure 8). We quantified the effects by incubating GUVs with different concentrations of the compound for a fixed amount of time (4 h). Figure 3 shows that the β-hydroxy ester 2 has a weaker effect than the two previous compounds regarding the percentage of GUVs modified after a given incubation time at all concentrations tested, except 1.6 µM.

Figure 9. Effect of compounds as compared to cholesterol when premixed with lipids. The percentage of GUVs that do not show visible domains is plotted as a function of the total amount of sterol (cholesterol + compound) incorporated into the lipid mixture for (b) the β-keto ester 1, (2) the β-hydroxy ester 2, (1) the β-hydroxy acid 3, and (9) cholesterol alone.

Table 1 shows the diffusion coefficient of a fluorescent membrane probe (DiO) as measured by FCS performed after 4 h incubation with the β-hydroxy ester 2. The diffusion coefficient is very similar to that of the liquid-disordered phase (see Table 1 and Figure 5) and thus indicates that this compound has a much stronger fluidizing effect than the β-hydroxy acid 3 or the β-keto ester 1. Premixing Experiments. The above experiments raise the questions of how much of each compound has to be effectively incorporated into the membrane in order to produce an effect and how the compounds are distributed among the outer and inner hemilayers. It would also be interesting to determine whether the observed deformations in the membrane can be explained by an imbalance in the amount of compound between the inner and outer hemilayers of the GUVs. To address these questions, we premixed the compounds with the lipids in solvent solution prior to the electroformation at different lipid to compound ratios. In this way, the compound should be homogeneously distributed between the inner and outer hemilayers of the GUVs. Figure 9 shows the percentages of GUVs without visible domains at different amounts of compounds premixed with the lipids. Cholesterol has also been added for comparison. The β-keto

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ester 1 shows the strongest effect, as only 9 mol % of compound needs to be incorporated in order to obtain a maximum effect. The β-hydroxy ester 2 and the β-hydroxy acid 3 show progressively weaker effects as the amount that needs to be incorporated into the membrane in order to obtain a maximum effect increases from 16.6 mol % for compound 2 to 23 mol % for compound 3. Regarding the deformation of the bilayer, in all of the premixed samples, the only observed effect of the compounds was the absence of visible domains: we observed neither tubule formation nor striped domains in the premixed samples. This indicates that the formation of tubules by the hydroxy acid 3 was probably induced by an asymmetric distribution of the compound between the inner and outer leaflets when the compound was incorporated from the surrounding buffer. It could be argued that the addition of the 4-substituted cholesterol derivatives could have been predicted by following the changes produced by an increase of sterol concentration in the DOPC/BSM/Chol phase diagram. The phase diagram allows for a quantitative description of the system as the composition changes. This is possible for ternary mixtures such as DOPC/ BSM/Chol, but not as straightforward in a four-component mixture. In the case of the addition of Chol, the amount of Lo phase increases at the expense of the Ld phase until, at concentrations of Chol above 40 mol %, all of the membrane is in the Lo state (Figure 9).34 Also, as the concentration of Chol increases, the fluidities of the two coexisting phases become increasingly similar, until at the boundary, only the Lo phase is left, with a fluididy intermediate between those of Lo and Ld phases (D ≈ 2 µm2/s).35 We did not explore the whole phase diagram for our four-component mixtures, but our results indicate roughly the position of the one-phase/two-phase line as a function of the compound concentration at a fixed lipid composition. In Figure 9, it can be seen that, for example, in the case of compound 1, we found 100% effect at a mole fraction of 9%. To interpret this result based on the DOPC/BSM/Chol phase diagram, the total amount of steroid molecules (both cholesterol and compound 1) at this amount of added compound is 27%. At this composition, if adding compound 1 were the same as adding cholesterol, phase separation should still be observed. Indeed, Figure 9 shows that the addition of cholesterol at 27 mol % abolishes the presence of domains in only 9% of the GUVs. The addition of cholesterol produces an increase in the amount of Lo phase in each GUV (Ld domains cover smaller areas of each GUV), but most GUVs still show phase coexistence up to very high cholesterol concentrations. The compounds, on the other hand, affect both the Lo and Ld phases simultaneously in such a way that the coexisting domains become very small and/or very similar to each other and merge into one phase at much lower concentrations. This confirms that the behavior of our compounds cannot be interpreted by making a simple analogy to cholesterol behavior. Regarding the fluidity of the resulting phase, the addition of compounds 1 and 3 has effects similar to those of cholesterol (34) Veatch, S.; Keller, S. L. Biochim. Biophys. Acta 2005, 1746, 172–185. (35) Kahya, N.; Scherfeld, D.; Bacia, K.; Schwille, P. J. Struct. Biol. 2004, 147, 77–89.

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in the sense that the fluidity of the GUVs after 2-3 h of incubation was intermediate between those of Lo and Ld phases (D ) 1-2 µm2/s).35 However, the addition of compound 2 has a different, strongly fluidizing effect, producing a diffusion coefficient near that of an Ld phase (5 µm2/s).

Conclusions We have identified three 4-substituted cholesterol derivatives that are able to induce a domain-disrupting effect in liquidordered-liquid-disordered phase-separating GUVs. The β-keto ester 1 exhibits the highest activity, as phase separation is abolished with high efficiency at short incubation times and the smallest amount of molecules (9 mol %) needs to be premixed with the lipids in order to obtain maximum effects. The fluidity induced by this compound lies between those of the liquidordered and liquid-disordered phases. The β-hydroxy acid 3 induces the budding of membrane tubules from the membrane when incorporated from the outside. It produces a homogeneous membrane with a diffusion coefficient lying between the values expected for liquid-ordered and liquid-disordered phases. This compound has the lowest activity in the sense that a high amount of molecules (23 mol %) needs to be incorporated into the membrane in order to obtain a maximum effect. However, even though the β-hydroxy acid 3 takes longer than compound 1 to induce a loss of phase separation, it is still faster to show maximum activity when incorporated from the outside than compound 2. The β-hydroxy ester 2, in turn, has the strongest fluidizing effect and needs an intermediate amount of molecules to become incorporated into the membrane to produce maximum effect (16.6 mol %), but it is the least active in the sense that it takes longer to show an effect when added from the outside. The three compounds studied contain groups that should exhibit strong dipole moments (ester groups) or even be partly charged at neutral pH (hydroxy acid 3). We therefore hypothesize that these compounds are able to disrupt membrane domain organization through a detergent-like effect conferred to the cholesterol molecule by the strongly polar groups introduced by the 4-substitutions that we performed. The present results provide the basis for the design of structures that interact with the lipid rafts of the cell membrane specifically by disrupting them. The biophysical method applied could be used for the development of a high-throughput assay to identify the structural requirements for the disruption of lipid rafts. Compounds targeting lipid rafts represent potential drug candidates for treatment of inflammation and infectious diseases. Thus, our findings might be of importance for the discovery of drugs based on raft targeting. Acknowledgment. D.C.C. acknowledges a Postdoctoral Fellowship from the Alexander von Humboldt Foundation. This work was supported by grants from the Alexander von Humboldt Foundation to D.C.C. and from the European Fonds for regional development and the State of Saxony (EFRE Project 4212/0608) to P.S. and H.-J.K. We also thank JADO Technologies GmbH, Dresden, Germany, for support. Salvatore Chiantia is gratefully acknowledged for help with FCS data acquisition and analysis. LA801471E