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Langmuir 2006, 22, 5786-5791
Membrane Resistance to Triton X-100 Explored by Real-Time Atomic Force Microscopy Sandrine Morandat† and Karim El Kirat*,‡ Laboratoire de Ge´ nie Enzymatique et Cellulaire, UniVersite´ de Technologie de Compie` gne, UMR-CNRS 6022, BP 20529, 60205 Compie` gne Cedex, France, and Laboratoire de Biome´ canique et Ge´ nie Biome´ dical, UniVersite´ de Technologie de Compie` gne, UMR-CNRS 6600, BP 20529, 60205 Compie` gne Cedex, France ReceiVed February 14, 2006. In Final Form: April 8, 2006 Lateral segregation of lipids and proteins in biological membranes leads to the formation of detergent-resistant domains, also called “rafts”. Understanding the mechanisms governing the biomembrane’s resistance to solubilization by detergents is crucial in biochemical research. Here, we used real-time atomic force microscopy (AFM) imaging to visualize the behavior of a model supported lipid bilayer in the presence of different Triton X-100 (TX-100) concentrations. Mixed dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) supported bilayers were prepared by vesicle fusion. Real-time AFM imaging revealed that, at concentrations below the critical micelle concentration (CMC), TX-100 did not solubilize the bilayer, but the DPPC domains were eroded in a time-dependent manner. This effect was attributed to the DPPC molecular packing disorganization by the detergent starting from the DOPC/DPPC interface. Just above the CMC, the detergent led to a complete solubilization of the DOPC matrix, leaving the DPPC domains unaltered. At higher TX-100 concentrations, the DOPC was also immediately removed just after detergent addition, and the DPPC domains remaining on the mica surface appeared to be more swollen and were gradually solubilized. This progressive solubilization of the DPPC remaining phase did not start at the edge of the domains but from holes appearing and expanding at the center of DPPC patches. The swelling of the DPPC domains was directly correlated with TX-100 concentration above the CMC and with detergent intercalation between DPPC molecules. We are convinced that this approach will provide a key system to elucidate the physical mechanisms of membrane solubilization by nonionic detergents.
Introduction Detergents are most commonly used in biology to disrupt the lipid membrane of cells in order to solubilize membrane-bound proteins.1,2 They are also used to crystallize, stabilize, or denature proteins.3 Other important applications include the prevention of nonspecific binding in immunoassays, cell lysis, and liposome preparation. Nonionic detergents are often nondenaturing, making them suitable for membrane protein purification. Triton X-100 (TX100) is a nonionic detergent with a low critical micelle concentration (CMC) of 0.24 mM, which is especially useful for the purification and reconstitution of integral or lipid-modified proteins in lipid membranes.1,4 Furthermore, TX-100 finds a remarkable application in membrane fractioning at low temperature (4 °C) for the preparation of detergent-resistant membranes (DRMs), also called “rafts”.5 The resistance to solubilization is directly related to the melting temperature (Tm) of the lipids, which tend to be tightly packed and form gel-phase bilayers. These DRMs are particularly enriched in cholesterol (Chol), sphingolipids, and phospholipids bearing long and saturated acyl chains.6-9 To date, a wide body of literature * Corresponding author. Phone: 33 (0)3 44 23 79 43. Fax: 33 (0)3 44 23 79 42. E-mail:
[email protected]. † Laboratoire de Ge ´ nie Enzymatique et Cellulaire. ‡ Laboratoire de Biome ´ canique et Ge´nie Biome´dical. (1) Morandat, S.; Bortolato, M.; Roux, B. Biochim. Biophys. Acta 2002, 1564, 473-8. (2) Ronzon, F.; Morandat, S.; Roux, B.; Bortolato, M. J. Membr. Biol. 2004, 197, 169-77. (3) le Maire, M.; Champeil, P.; Moller, J. V. Biochim. Biophys. Acta 2000, 1508, 86-111. (4) Rigaud, J. L.; Paternostre, M. T.; Bluzat, A. Biochemistry 1988, 27, 267788. (5) Brown, D. A.; Rose, J. K. Cell 1992, 68, 533-44.
describes DRMs in model membranes such as liposomes, Langmuir monolayers, or supported bilayers.1,10-14 Although, in the latter case, the solid support may stabilize the bilayer compared with nonsupported lipid systems. A three-stage model has been proposed to describe lipiddetergent interaction during solubilization.4,15 When increasing amounts of detergent are added to liposomes, they are incorporated into the membrane, leading to a higher suspension turbidity characteristic of the vesicle size increase. When liposomes are saturated with detergent molecules, a gradual disintegration of the membrane can take place, and lipid-detergent mixed micelles appear in solution. The solubilization process finally ends in a third step: when there are no more intact liposomes left, all the lipids are associated with detergent into mixed micelles. Atomic force microscopy (AFM) is a powerful technique that allows one to image biological specimens at high resolution and under aqueous conditions.16 Therefore, AFM is widely used to investigate phase separations in membranes17 and the interaction (6) Pike, L. J.; Han, X.; Chung, K. N.; Gross, R. W. Biochemistry 2002, 41, 2075-88. (7) Brown, D. A.; London, E. J. Biol. Chem. 2000, 275, 17221-4. (8) Schroeder, R. J.; London, E.; Brown, D. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 12130-4. (9) Simons, K.; Ikonen, E. Nature 1997, 387, 569-72. (10) Ahmed, S. N.; Brown, D. A.; London, E. Biochemistry 1997, 36, 1094453. (11) Morandat, S.; Bortolato, M.; Roux, B. J. Membr. Biol. 2003, 191, 21521. (12) Schroeder, R. J.; Ahmed, S. N.; Zhu, Y.; London, E.; Brown, D. A. J. Biol. Chem. 1998, 273, 1150-7. (13) Nyholm, T.; Slotte, J. P. Langmuir 2001, 17, 4724-30. (14) Rinia, H. A.; Snel, M. M.; van der Eerden, J. P.; de Kruijff, B. FEBS Lett. 2001, 501, 92-6. (15) Paternostre, M. T.; Roux, M.; Rigaud, J. L. Biochemistry 1988, 27, 266877. (16) Muller, D. J.; Janovjak, H.; Lehto, T.; Kuerschner, L.; Anderson, K. Prog. Biophys. Mol. Biol. 2002, 79, 1-43.
10.1021/la0604228 CCC: $33.50 © 2006 American Chemical Society Published on Web 05/24/2006
Membrane Resistance to TX-100
Langmuir, Vol. 22, No. 13, 2006 5787
Figure 1. TX-100 interaction with supported lipid membranes at CMC/2 (0.12 mM). An AFM height image (20 × 20 µm; Z-scale: 10 nm) of a mixed DOPC/DPPC (1:1) bilayer was first recorded in Tris buffer before TX-100 addition (A). After addition of 0.12 mM TX-100, images of the same zone were acquired at different incubation times: (B) 10, (C) 30, (D) 60, (E) 90, and (F) after 120 min.
of supported lipid bilayers with peptides,18,19 proteins,20 drugs,21 or solvents.22 So far, only a few articles have described detergent interaction with membranes by AFM. Giocondi et al.23 imaged, by intermittent contact mode, the effect of a TX-100 treatment on living cells. The images revealed the presence of the cytoskeleton presumably associated with DRMs. With supported lipid membranes, Rinia et al.14 showed the resistance of sphingomyelin (SM)/Chol gel domains against 10% (v/v) TX100 in cold conditions, whereas the surrounding fluid dioleoylphosphatidylcholine (DOPC) bilayer was completely removed. Here, AFM was used to follow, in real time, the TX-100 solubilization activity on bilayers composed of DOPC/dipalmitoylphosphatidylcholine (DPPC) (1:1 mol/mol). This lipid composition is known to produce fluid and gel phases at room temperature and can be considered as a simple model to study membrane resistance to detergents. Different TX-100 concentrations were tested, and the behavior of each phase in terms of covering area, shape, and height was characterized by timelapse AFM. Experimental Section Materials. L-R-Dioleoylphosphatidylcholine (DOPC), L-R-dipalmitoylphosphatidylcholine (DPPC) and TX-100 were purchased from Sigma (St. Louis, MO) and used without any further purification. Other chemicals were purchased from Merck (Darmstadt, Germany). For all experiments, the distilled water was purified with a Millipore filtering system (Bedford, MA), yielding an ultrapure water with a resistivity of 18.2 MΩ‚cm. Preparation of Supported Lipid Bilayers. Supported DOPC/ DPPC (1:1 mol/mol) bilayers were prepared using the vesicle fusion method.24-26 To this end, lipids were dissolved in chloroform at 1 (17) Giocondi, M. C.; Pacheco, L.; Milhiet, P. E.; Le Grimellec, C. Ultramicroscopy 2001, 86, 151-7. (18) El Kirat, K.; Lins, L.; Brasseur, R.; Dufrene, Y. F. Langmuir 2005, 21, 3116-21. (19) El Kirat, K.; Lins, L.; Brasseur, R.; Dufrene, Y. F. J. Biomed. Nanotechnol. 2005, 1, 39-46. (20) Milhiet, P. E.; Giocondi, M. C.; Baghdadi, O.; Ronzon, F.; Roux, B.; Le Grimellec, C. EMBO Rep. 2002, 3, 485-90. (21) Berquand, A.; Mingeot-Leclercq, M. P.; Dufrene, Y. F. Biochim. Biophys. Acta 2004, 1664, 198-205. (22) Mou, J.; Yang, J.; Huang, C.; Shao, Z. Biochemistry 1994, 33, 9981-5. (23) Giocondi, M. C.; Vie, V.; Lesniewska, E.; Goudonnet, J. P.; Le Grimellec, C. J. Struct. Biol. 2000, 131, 38-43. (24) Reviakine, I.; Brisson, A. Langmuir 2000, 16, 1806-1815.
mM final concentration. The mixture of these lipids was then evaporated under nitrogen and dried in a desiccator under vacuum for 2 h. Multilamellar vesicles (MLV) were obtained by resuspending the lipidic dried film at room temperature in calcium containing buffer (10 mM Tris, 150 mM NaCl, 3 mM CaCl2, pH 7.4; Tris/ calcium buffer) at a 1 mM final lipid concentration. To obtain small unilamellar vesicles (SUV), the suspension was sonicated to clarity (three cycles of 2 min 30 s) using a 500 W probe sonicator (Fisher Bioblock Scientific, France; 35% of the maximal power; 13 mm probe diameter) while being kept in an ice bath. The liposomal suspension was then filtered on a 0.2 µm Acrodisc (Pall Life Sciences, USA) to eliminate titanium particles. Freshly cleaved mica squares (16 mm2) were glued onto steel sample disks (Agar Scientific, England) using Epotek 377 (Polytec, France). A 150 µL portion of the SUV suspension was then deposited onto the mica samples, and the SUVs were allowed to adsorb and fuse on the solid surface for 1 h at 60 °C. Subsequently, samples were rinsed with 3 mL of buffer (10 mM Tris, 150 mM NaCl, pH 7.4; Tris buffer) and slowly cooled to room temperature. Atomic Force Microscopy. Supported bilayers were investigated using a commercial AFM (NanoScope III MultiMode AFM, Veeco Metrology LLC, Santa Barbara, CA) equipped with a 125 × 125 × 5 µm scanner (J-scanner). A quartz fluid cell was used without the O-ring. Topographic images were recorded in contact mode using oxide-sharpened microfabricated Si3N4 cantilevers (Microlevers, Veeco Metrology LLC, Santa Barbara, CA) with a spring constant of 0.01 N/m (manufacturer specified), with a minimal applied force (
