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Adhesion between Giant Vesicles and Supported Bilayers Decorated with Chelated E-Cadherin Fragments P.-H. Puech,*,† H. Feracci,‡ and F. Brochard-Wyart† Laboratoire PCC/UMR 168, Institut Curie, 11 rue P. & M.Curie, 75005 Paris, France, and Laboratoire C. Burg/UMR 144, Institut Curie, 26 rue d’Ulm, 75005 Paris, France Received May 28, 2004. In Final Form: August 9, 2004 Here, we present a study of adhesion between cadherin fragments using giant unilamellar vesicles and supported bilayers. These objects are partially made of nickel chelating lipids and are subsequently decorated with proteins bearing a 6His tag. Initially, we observed their fixation and correct orientation by using a fluorescent protein, the green fluorescent protein (GFP)-6His. The adhesive behavior of E-cadherin functionalized giant vesicles and supported bilayers was studied as a function of the calcium concentration and of the protein functionality by reflection interference microscopy. We show that such a system retains specific cadherin-mediated adhesion and could be used to study the statics and dynamics of adhesive plaques as well as to gain insight into the fundamental mechanisms of cellular adhesion at the mesoscopic scale.
1. Introduction Adhesion is an essential phenomenon in biology finely regulated and governed by highly specific molecules. Unlike colloidal aggregation, which is mediated through generic interactions, cells use optimized sets of interacting proteins which interact as molecular keys and locks.1 In recent years, it has become clear that the function of adhesion molecules on the cell surface goes beyond the simple task of tethering cells to a specific location within the organism. Adhesive interactions are involved in the formation and the cohesion of tissues.2 It has been shown that cell adhesion events can provide critical information about environmental conditions and can affect many central aspects of cell behavior. When a cell responds to a chemotaxis signal, it moves by exerting tractions via lamellipodia that adhere on the substrate.1 Adhesion is also involved in many pathologies such as cancer proliferation: malignant cells can detach after modification of their adhesive properties and crawl through tissues to reach the vascular system and subsequently invade other tissues.3 This adhesive system is also used in certain conditions to trigger cell death. Many classes of cellular adhesion molecules (CAMs) coexist on the cell surface and contribute to recognition and adhesive events. As an example, if we consider a simple epithelium, the cells adhere to one another through junctional complexes which are specialized regions of the lateral cell surface. These complexes, namely, zonula occludens, zonula adherens, and desmosomes, have unique morphologies, molecular compositions, and physiological roles. Moreover, these cells bind selectively to extracellular matrix components present through integrins. Here, we report the adhesive response of model systems decorated with E (epithelial)-cadherin fragments. Classical cadherins were the first family of calciumdependent adhesion molecules found in the adherens * Corresponding author. E-mail:
[email protected]. † Laboratoire PCC/UMR 168. ‡ Laboratoire C. Burg/UMR 144. (1) Alberts, B.; Johnson, A.; Lewis, J.; Ralf, M.; Roberts, K.; Walter, P. Molecular biology of the cell; Garland Publishing: 2002. (2) Gumbiner, B. Cell 1996, 84, 345. (3) Thiery, J.-P. C. R. Acad. Sci. 2003, 4.
junctions. These single-pass transmembrane glycoproteins establish homophilic interactions; that is, cadherins are both the key and the lock. They play a major role in morphogenesis.4 The deregulation of their expression has been shown to correlate with the invasive capabilities of tumoral cells.3 Their structure can be roughly divided into three parts: (i) a fingerlike extramembrane domain that protrudes over several tens of nanometers from the cell surface and establishes adhesive contacts with cadherins expressed on neighboring cells, (ii) a transmembrane part that anchors the protein in the membrane, and (iii) a cytoplasmic tail which can interact with the cytoskeleton (via several proteins such as catenins). For the so-called “classical cadherins”, such as E-cadherin, the extramembrane part is comprised of five similar immunoglobulinlike domains and is ∼25 nm long.5 The two outermost domains have been intensively studied by crystallography to define the nature of the adhesion interface. On the basis of these studies, several models of the molecular organization within the homophilic adhesion plaque have been proposed.6,7 Due to their central role in cell-cell adhesion, a large number of studies have been performed on cadherins using either biological methods involving cell8,9 or protein10,11 modifications or physical techniques such as force measurements using atomic force microscopy,12,13 surface force apparatus,14,15 or the BioForce probe16 to localize the (4) Takeichi, M. Curr. Opin. Cell Biol. 1995, 7, 619. (5) Boggon, T. J.; Murray, J.; Chappuis-Flament, S.; Wong, E.; Gumbiner, B. M.; Shapiro, L. Science 2002, 296, 1312. (6) Nagar, B.; Overduin, M.; Ikura, M.; Rini, J. M. Nature 1996, 380, 360. (7) Shapiro, L.; Fannon, A. M.; Kwong, P. D.; Thompson, A.; Lehmann, M. S.; Grube, L. G.; Legrand, J. F.; Als-Nielsen, J.; Colman, D. R.; Hendrickson, W. A. Nature 1995, 374, 327. (8) Renaud-Young, M.; Gallin, W. J. J. Biol. Chem. 2002, 277, 39609. (9) Rothen-Rutishauser, B.; Riesen, F. K.; Braun, A.; Gunthert, M.; Wunderli-Allenspach, H. J. Membr. Biol. 2002, 188, 151. (10) Chappuis-Flament, S.; Wong, E.; Hicks, L. D.; Kay, C. M.; Gumbiner, B. M. J. Cell Biol. 2001, 154, 231. (11) Tomschy, A.; Fauser, C.; Landwehr, R.; Engel, J. EMBO J. 1996, 15, 3507. (12) Baumgartner, W.; Hinterdorfer, P.; Ness, W.; Raab, A.; Vestweber, D.; Schindler, H.; Drenckhahn, D. PNAS 2000, 97, 4005. (13) Baumgartner, W.; Gruber, H. J.; Hinterdorfer, P.; Drenckhahn, D. Single Mol. 2000, 1, 119. (14) Sivasankar, S.; Brieher, W.; Lavrik, N.; Gumbiner, B.; Leckband, D. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11820.
10.1021/la048682h CCC: $27.50 © 2004 American Chemical Society Published on Web 09/28/2004
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adhesive sites along the protein structure and characterize their dynamic properties. The dynamics of the binding/ unbinding have also been addressed by flow chamber experiments.17,18 Here, we introduce a new model system to further investigate these specific cadherin-mediated interactions by characterizing the adhesion of cadherin decorated giant vesicles on suitably decorated surfaces. Giant vesicles are 10-100 µm (in diameter) “baggies”, composed of a single lipid bilayer (∼5 nm thick) that separates an inner aqueous compartment from the surrounding medium. They have been extensively used for reproducing and studying certain properties of the plasma membranes of cells, including thermal undulations, incorporation and function of membrane proteins,19 tubular structure extrusion,20,21 and pore formation.22-24 Coupled to decorated surfaces, these vesicles allow the study of the cooperative action of linker couples with or without a steric repulsion mimicking the glycocalyx.25,26 Here, we extend this approach, usually proposed for strongly adhesive couples such as biotin/ streptavidin, to a low affinity homophilic system. 2. Materials and General Methods 2.1. Chemicals. The lipids, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-{[N(5-amino1-carboxypentyl)iminodiacetic acid]succinyl} (NTA lipids), were obtained in powder form from Avanti Polar Lipids Inc. (Alabaster, AL). The NTA lipids were preloaded with nickel salt. Solutions of 10 mg/mL lipids in chloroform were used as stock solutions and preserved for months under argon at -20 °C. From the mother solutions, a 1 mg/mL spreading solution in a 2:1 v/v chloroform/methanol mixture was prepared with the desired composition of NTA lipid (0-30 mol %). These solutions were stored under the same conditions as described above. Sugars, salts, and buffers (as preset tablets) were obtained from Sigma-Aldrich and made using fresh Millipore water. Solvents of analytical grade were obtained from ABCR. 2.2. Proteins. Green fluorescent protein construct with a 6 histidine tag (6His) was received as a gift from F. Amblard (UMR 168, Institut Curie). The orientation of the tagged protein is shown in Figure 1. 6His-tagged E-cadherin fragments (namely, E/EC12 for the wild type fragment and E/W2A fragments for the mutant type) were obtained as previously described18 (see Figure 1 for their geometry). Substituting the tryptophan 2 (Trp2) with an alanine (Ala) in the E/EC12 fragments reduces the adhesion efficiency of the proteins.18 The fragments were stored on NTA(Ni) beads (Qiagen) at 4 °C, eluted just prior to use with a 250 mM imidazole buffer, and dialyzed by centrifugation in Vivaspin tubes (MWCO 5 kDa) against 5 mM Tris (pH 8.1)/glucose (310 mOsm) at 4 °C. Finally, the protein fragments were concentrated to ∼20 µM, as (15) Sivasankar, S.; Gumbiner, B.; Leckband, D. Biophys. J. 2001, 80, 1758. (16) Perret, E.; Leung, A.; Feracci, H.; Evans, E. Proc. Natl. Acad. Sci., in revision, 2004. (17) Pierres, A.; Feracci, H.; Delmas, V.; Benoliel, A.-M.; Thiery, J.-P.; Bongrand, P. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 9256. (18) Perret, E.; Benoliel, A.-M.; Nassoy, P.; Pierres, A.; Delmas, V.; Thiery, J.-P.; Bongrand, P.; Feracci, H. EMBO J. 2002, 21, 2537. (19) Manneville, J.-B.; Bassereau, P.; Ramaswamy, S.; Prost, J. Phys. Rev. E 2001, 69, 021908. (20) Roux, A.; Cappello, G.; Cartaud, J.; Prost, J.; Goud, B.; Bassereau, P. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5394. (21) Rossier, O.; Cuvelier, D.; Borghi, N.; Puech, P. H.; Dere´nyi, I.; Buguin, A.; Nassoy, P.; Brochard-Wyart, F. Langmuir 2003, 19, 575. (22) Sandre, O.; Moreaux, L.; Brochard-Wyart, F. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10591. (23) Karatekin, E.; Sandre, O.; Guitouni, H.; Borghi, N.; Puech, P.H.; Brochard-Wyart, F. Biophys. J. 2003, 84, 1734. (24) Puech, P.-H.; Borghi, N.; Karatekin, E.; Brochard-Wyart, F. Phys. Rev. Lett. 2003, 90, 128304. (25) Albersdo¨rfer, A.; Feder, T.; Sackmann, E. Biophys. J. 1997, 73, 245. (26) Kloboucek, A.; Behrisch, A.; Faix, J.; Sackmann, E. Biophys. J. 1999, 77, 2311.
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Figure 1. Schematic representation of the proteins used in this study. The GFP bears its histidine tag in one of the barrel extremities, leading to positioning its chromophore parallel to the membrane. To mimic the in vivo configuration, the E-cadherin fragments are hexahistidine-tagged at the end of EC2, so that the EC1 domain (bearing a hydrophobic pocket) is directed toward the exterior. For the E/W2A mutant, the tryptophan 2 (Trp2) has been replaced by an alanine (Ala). The global orientation of the proteins should be perpendicular to the membrane as they are chelated on an NTA(Ni) lipid. determined by measuring the optical density (OD) (at λ ) 280 nm) with a spectrometer. The protein was then stored at 4 °C and used for no more than 2 days. 2.3. Giant Unilamellar Vesicles (GUVs). GUVs were generated using the “electroformation” method developed by Angelova.27 Lipids (150 µg) from the dilute solutions were cast onto the two faces of a home-built “electroformation cell", made of conducting indium tin oxide (ITO)-covered glass. The solvents were evaporated overnight in vacuo. Following closure of the cell using an inert mastic (Critoseal, Sherwood AG) and a 1 mm thick Teflon spacer, the lipid film was swollen in an aqueous buffer (1:1 v/v of 300 mOsm sucrose and 300 mOsm glucose solutions as measured with a fusion point osmometer) under an alternating currect (ac) field of 1.2 V at 10 Hz for ∼2 h at room temperature (25 °C). Vesicles with 10-100 µm diameters were obtained and were extracted with great care from the electroformation cell after at least 1 h of rest at 4 °C. Preliminary tests had shown that one can obtain high densities of giant unilamellar vesicles for binary mixtures of lipids containing e10% NTA(Ni) lipids. For higher percentages, few aggregates and few GUVs formed. This does not ensure the homogeneity of the reconstitution of the deposited lipid mixture in the GUVs. We chose to use the 10% composition in order to obtain a dense layer of proteins on the surface for the adhesion experiments. 2.4. Small Unilamellar Vesicles (SUVs). To obtain SUVs, the lipid mixture was evaporated under argon and the residual solvents were allowed to evaporate overnight in a small glass tube under vacuum. A solution of 300 mM sucrose/5 mM Tris (pH 8.1)/50 mM NaCl was then introduced and the tube vortexed for 1 min. The lipids were swollen for at least 1 h at 4 °C before an intense 15 min probe sonication was conducted on ice. As for preparing the GUVs, we used a 10% mixture of NTA lipids in DOPC. 2.5. Fluorescent Labeling. The membranes of our small and giant vesicles are fluid at room temperature and can be labeled for fluorescence imaging by perfusion of a lipophilic fluorescent dye, N-(4-sulfobutyl)-4-(4-(dihexylamino) styryl) pyridinium (kindly provided by M. Blanchard-Desce, U. de Rennes, France). This dye inserts spontaneously into the membrane.22,24 Alternatively, 1% of DOPE-NBD lipid (Sigma) was added before the formation processes.23 2.6. Supported Bilayers (SBs). SBs were obtained by the fusion of SUVs on glass (see, for instance, refs 28-30). They were generated in situ in 20 µL chambers that were made by molding stacks of common adhesive paper using poly(dimethylsiloxane) (PDMS, Sylgard 184, Dow Corning).21 Glass coverslides (from ESCO) were cured in a dilute surfactant solution (Micro90 or Helmanex) at 60 °C for 30 min. After careful and (27) Luisi, P. L., Walde, P., Eds. Giant Vesicles; Wiley: New York, 1999. (28) Cremer, P. S.; Boxer, S. G. J. Phys. Chem. B 1999, 103, 2554. (29) Yang, T.; Jung, S.-Y.; Mao, H.; Cremer, P. S. Anal. Chem. 2001, 73, 165. (30) Kam, L.; Boxer, S. G. Langmuir 2003, 19, 1624.
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Figure 2. (A) FM picture of a giant vesicle labeled with Di6ASPBS. (B) Its contact zone with the observation chamber surface as seen in RIM. intensive rinsing with fresh Milli-Q water, they were dried under an argon flux and stored in a Petri dish to avoid contamination of the surface. Before the cell was assembled, a PDMS replica and a clean coverslide were oxidized with a residual air plasma cleaner for 1 min and then pressed on the other to ensure a good sealing. Within 30 min, the SUVs were allowed to fuse in a dark environment for 20 min and the cell was carefully rinsed five times with 100 µL of sucrose/5 mM Tris (pH 8.1, 310 mOsm). 2.7. Observation Techniques. 2.7.1. Fluorescence Microscopy (FM) and Reflection Interference Microscopy (RIM). An upright Reichert-Jung MET microscope (Vienna) was used to acquire images with these two techniques (see Figure 2), by switching between two filter sets (FM, λex ) 455-490 nm and λem ) 580 nm; RIM, λR ) 546.1 nm, without further add-in in the setup). We used a 60× water immersion objective (NA 0.9, Olympus). The microscope was equipped with a charge-coupled device (CCD) camera, coupled with a real time VCR (Sony). Images were digitized for subsequent analysis using Scion Image software (Scion Corporation). All experiments were performed at 25 °C. 2.7.2. Two-Photon Fluorescence Microscopy (TPEF). TPEF images were obtained using a home-built facility as described elsewhere.31 All of the dye molecules or fluorescent molecules previously described can be excited and observed by tuning the wavelength of the laser. The main advantage of such a technique is an enhancement in the contrast due to strong confinement of the excitation. 2.7.3. Atomic Force Microscopy (AFM). A Nanoscope E microscope (Digital Instruments), equipped with a piezoelectric actuator having a range of 150 µm (J-scanner) and a fluid cell, was used in contact mode. Scanning frequencies were ∼5 Hz. Silicon nitride cantilevers (100 µm long) with a nominal spring constant of 0.1 N/m (Olympus) were used. Imaging was done at constant low force in 10 mM Tris HCl (pH 7.5)/150 mM KCl.
3. Experiments and Results This section is organized as follows: In the first part, fluorescence microscopy and AFM studies of the chelation capabilities and resistance of both GUVs and SBs are reported. In the second part, observations for adhesion tests using fragments of E-cadherin fragments (wild type or mutated) are presented and discussed. 3.1. Protein Chelation. 3.1.1. Protein Chelation on Vesicles or Supported Bilayers. GUVs were sedimented in a 5 mM Tris (pH 8.1)/glucose (310 mOsm) solution for at least 30 min, and the supernatant was gently removed. The tagged protein was added to a final concentration of 10 µM and incubated in the dark for 45 min at 25 °C. In the case of supported bilayers, they were gently rinsed first with a sufficient volume of 5 mM Tris (pH 8.1)/glucose (310 mOsm) solution to ensure complete exchange of the medium and finally with 50 µL of a 10 µM protein/5 mM Tris (pH 8.1)/glucose (310 mOsm) solution. The incubation step followed the same procedure as that for the GUVs. Figure 3A shows that the tagged protein could be chelated on NTA(Ni) containing membranes. In the absence of nickel ion, almost no difference between the membrane signal and the background was observed. Moreover, a (31) Moreaux, L.; Sandre, O.; Charpak, S.; Blanchard-Desce, M.; Mertz, J. Opt. Lett. 2000, 25, 183.
Figure 3. (A) Fluorescence picture of a giant vesicle containing 10% of chelating lipid decorated with GFP-6His. Inset: Intensity profile at the equator. (B) GUVs decorated with Alexa 546-labeled E/EC12-6His fragments of E-cadherin observed with an intensified camera.
polarization analysis of the emitted fluorescence light in the two-photon experiments showed qualitatively that the protein is mainly oriented perpendicularly to the membrane. When the same experiment was performed on a supported bilayer, a uniform fluorescence from the surface was observed within the observation chamber limits and was strongly localized in the vicinity of the glass surface (as seen with z-scans of several micrometers). When large defects (such as air bubbles, usually sticking to the cell walls) were present, they appeared as black zones surrounded by homogeneous fluorescence. By coupling a dye to the NH2 groups of cadherin fragments (Alexa 546, Molecular Probes), we observed that this protein can also be chelated by NTA(Ni) lipids (see Figure 3B). 3.1.2. Stability of Chelation to Dilution. Green fluorescent protein (GFP)-6His labeled vesicles were diluted to a bulk protein concentration of
