pubs.acs.org/Langmuir © 2010 American Chemical Society
Concentrated Diffusing Colloidal Probes of Ca2þ-Dependent Cadherin Interactions W. Neil Everett,† Daniel J. Beltran-Villegas,‡ and Michael A. Bevan*,‡ †
‡
Chemical Engineering, Texas A&M University, College Station, Texas 77843, United States, and Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United States Received September 24, 2010. Revised Manuscript Received November 7, 2010
We report video microscopy measurements and inverse simulation analyses of specific Ca2þ-dependent interactions between N-cadherin fragments attached to supported lipid bilayer-coated silica colloids in quasi-2D concentrated configurations. Our results include characterization of the bilayer formation and fluidity and the attachment of active extracellular cadherin fragments on bilayers. Direct measurements of interaction potentials show nonspecific macromolecular repulsion between cadherin fragments in the absence of Ca2þ and irreversible bilayer fusion via cadherin-mediated attraction at >100 μM Ca2þ. Analysis of Ca2þ-dependent N-cadherin bond formation in quasi-2D concentrated configurations using inverse Monte Carlo and Brownian Dynamics simulations show measurable attraction starting at 0.1 μM Ca2þ, a concentration significantly below previously reported values.
Introduction Transmembrane protein interactions play a critical role in determining cell behavior. One important example is cadherins,1 which are a superfamily of Ca2þ-dependent cell adhesion proteins involved in cellular processes including, for example, tissue morphogenesis, synaptic plasticity,2,3 apoptosis,4 and cancer metastasis.5 Over 100 distinct cadherins have been identified, and they are generally associated with different tissue types, as captured by their conventional nomenclature (e.g., N-cadherins in neural tissue, E-cadherins in the epithelium). Varying relative affinities and expression levels of different cadherin types are considered to control cell sorting leading to distinct tissue morphologies, which has been supported by point mutation6,7 and gene regulation8-11 studies. Cadherins are transmembrane proteins, and their binding is mediated by Ca2þ binding sites at junctions between five repeat domains in their extracellular portion. Homophilic and heterophilic interactions at fixed [Ca2þ] are very weak (e.g., KD = 0.7 mM at 1 mM Ca2þ 12 and KD = 0.17 mM at 10 mM Ca2þ 13), but their specificity gives rise to manifold cellular responses. In addition, the [Ca2þ] dependence of homophilic cadherin interactions is *To whom correspondence should be addressed. E-mail: mabevan@ jhu.edu. (1) Takeichi, M. Science 1991, 251, 1451–1455. (2) Luthi, A.; Laurent, J. P.; Figurov, A.; Muller, D.; Schachner, M. Nature 1994, 372, 777–779. (3) Bozdagi, O.; Shan, W.; Tanaka, H.; Benson, D. L.; Huntley, G. W. Neuron 2000, 28, 245–259. (4) Tsujii, M.; Dubois, R. N. Cell 1995, 83, 493–501. (5) Egeblad, M.; Werb, Z. Nat. Rev. Cancer 2002, 2, 161–174. (6) Ozawa, M.; Engel, J.; Kemler, R. Cell 1990, 63, 1033–1038. (7) Handschuh, G.; Candidus, S.; Luber, B.; Reich, U.; Schott, C.; Oswald, S.; Becke, H.; Hutzler, P.; Birchmeier, W.; Hofler, H.; Becker, K. F. Oncogene 1999, 18, 4301–4312. (8) Steinberg, M. S.; Takeichi, M. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 206–209. (9) Islam, S.; Carey, T. E.; Wolf, G. T.; Wheelock, M. J.; Johnson, K. R. J. Cell Biol. 1996, 135, 1643–1654. (10) Duguay, D.; Foty, R. A.; Steinberg, M. S. Dev. Biol. 2003, 253, 309–323. (11) Halbleib, J. M.; Nelson, W. J. Genes Dev. 2006, 20, 3199–3214. (12) Haussinger, D.; Ahrens, T.; Aberle, T.; Engel, J.; Stetefeld, J.; Grzesiek, S. EMBO J. 2004, 23, 1699–1708. (13) Alattia, J. R.; Ames, J. B.; Porumb, T.; Tong, K. I.; Heng, Y. M.; Ottensmeyer, P.; Kay, C. M.; Ikura, M. FEBS Lett. 1997, 417, 405–408.
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important to cellular behavior. For example, differential affinities of cadherins as a function of [Ca2þ] give rise to synaptic remodeling14 and intracellular signaling15 with N-cadherins binding at ∼0.3 mM [Ca2þ]16 and cadherin-11 binding at ∼0.1 mM [Ca2þ].14 These [Ca2þ] are significantly below the 1.5-2.0 mM average physiological range in the mammalian brain17 and much greater than the 0.1 μM cytosolic neuron concentration.18 Attempts to measure cadherin interactions have primarily involved direct interrogation of immobilized extracellular fragments19,20 or cadherins on cell surfaces21 using “mechanical probes”. Mechanical probe methods (e.g., surface forces apparatus,22 atomic force microscopy (AFM),20,21 biomembrane force probes,23 optical tweezers (OT)14,16) quantify forces via deflection of actual or virtual cantilevers with known spring constants (i.e., mechanical force transducers).24,25 Measurements of cadherin interactions with these methods are appealing because they are direct, interrogate small numbers of molecules, and resolve spatial information. However, they also have inherent lower force limits based on their spring constant,26 which has been demonstrated to bias unbinding processes.27 At fixed [Ca2þ] in the millimolar range, these types of direct measurements have shown that heterophilic (14) Heupel, W. M.; Baumgartner, W.; Laymann, B.; Drenckhahn, D.; Golenhofen, N. Mol. Cell. Neurosci. 2008, 37, 548–558. (15) Murase, S.; Mosser, E.; Schuman, E. M. Neuron 2002, 35, 91–105. (16) Baumgartner, W.; Golenhofen, N.; Grundhofer, N.; Wiegand, J.; Drenckhahn, D. J. Neurosci. 2003, 23, 11008–11014. (17) Tai, C. Y.; Kim, S. A.; Schuman, E. M. Curr. Opin. Cell Biol. 2008, 20, 567–575. (18) Ghosh, A.; Greenberg, M. E. Science 1995, 268, 239–247. (19) Sivasankar, S.; Brieher, W.; Lavrik, N.; Gumbiner, B.; Leckband, D. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11820–11824. (20) Baumgartner, W.; Hinterdorfer, P.; Ness, W.; Raab, A.; Vestweber, D.; Schindler, H.; Drenckhahn, D. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 4005–4010. (21) Panorchan, P.; Thompson, M. S.; Davis, K. J.; Tseng, Y.; Konstantopoulos, K.; Wirtz, D. J. Cell Sci. 2006, 119, 66–74. (22) Prakasm, A. K.; Maruthamutha, V.; Leckband, D. E. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15434–15439. (23) Bayas, M. V.; Leung, A.; Evans, E.; Leckband, D. Biophys. J. 2006, 90, 1385–1395. (24) Israelachvili, J. N. Intermolecular and Surface Forces, 2nd ed.; Academic Press: New York, 1992. (25) Leckband, D.; Israelachvili, J. Q. Rev. Biophys. 2001, 34, 105–267. (26) Nassoy, P. Biophys. J. 2007, 93, 361–362. (27) Tshiprut, Z.; Klafter, J.; Urbakh, M. Biophys. J. 2008, 95, L42–L44.
Published on Web 11/18/2010
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and homophilic binding properties do not correlate with a mechanism by which cadherin subtypes control cell sorting via differences in relative binding affinities.28 Cadherin interactions have been interrogated indirectly by other methods. Multidimensional nuclear magnetic resonance has been used to measure trans dimerization of extracellular E-cadherin fragments in 1 mM Ca2þ,12 providing an important nonintrusive measure of weak cadherin interactions. Electron microscopy has been used to estimate that a [Ca2þ] > 50 μM is required to maintain a rodlike structure thought to be necessary for dimer formation.29 While such methods measure weak interactions in bulk ensembles, they do not directly measure spatially dependent interactions between small numbers of surface-immobilized fragments as occurs between cells. Other approaches such as flow cytometry measurements of cadherin-mediated colloidal aggregation30-32 have produced results more similar to mechanical probe measurements than cell sorting behavior, probably due to the role of shear forces in perturbing very weak bonds. In this work, we investigate a new approach to directly and sensitively measure [Ca2þ]-dependent homophilic cadherin interactions. We report results of nonintrusive microscopy measurements of diffusing colloidal particles with supported lipid bilayers (SLBs)33-35 bearing immobilized cadherin fragments (i.e., extracellular fragments conjugated to lipids). The goal of such measurements is to be both direct like mechanical probe measurements and nonintrusive like spectroscopic measurements. Our results first characterize the fluidity, stability, and nonspecific interactions of supported lipid bilayers on silica colloids using total internal reflection microscopy (TIRM) and confocal scanning laser microscopy (CSLM). Characterization of nonspecific van der Waals and macromolecular interactions between SLBcoated colloids and surfaces provides a basis to unambiguously monitor specific [Ca2þ]-dependent N-cadherin interactions. We then demonstrate N-cadherin fragments are active on SLBs and produce only nonspecific repulsion in the absence of Ca2þ. To quantify [Ca2þ]-dependent interactions, we obtain static and dynamic information from video microscopy (VM) measurements of concentrated configurations of N-cadherin-decorated SLB-coated colloids as a function of [Ca2þ], which we analyze with inverse simulations.
Interaction Potentials. For macromolecule-coated colloids interacting with each other and an underlying macromoleculecoated planar surface in physiological ionic strength media (where electrostatic double-layer interactions are negligible), the net potential energy of each particle, unet i (r,h), is given by ¼
pw uG ðhÞ þ upw macro ðhÞ þ uvdW ðhÞ þ
X ðupp macro ðri, j Þ
pp þ upp vdW ðri, j Þ þ uNcad ðri, j ÞÞ
j6¼i
ð1Þ
(28) Shi, Q. M.; Chien, Y. H.; Leckband, D. J. Biol. Chem. 2008, 283, 28454– 28463. (29) Pertz, O.; Bozic, D.; Koch, A. W.; Fauser, C.; Brancaccio, A.; Engel, J. EMBO J. 1999, 18, 1738–1747. (30) Grumet, M.; Friedlander, D. R.; Edelman, G. M. Cell Adhes. Commun. 1993, 1, 177–190. (31) Lambert, M.; Padilla, F.; Mege, R. M. J. Cell Sci. 2000, 113, 2207–2219. (32) Chappuis-Flament, S.; Wong, E.; Hicks, L. D.; Kay, C. M.; Gumbiner, B. M. J. Cell Biol. 2001, 154, 231–243. (33) Brian, A. A.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 6159–6163. (34) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105–113. (35) Sackmann, E. Science 1996, 271, 43–48.
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uG ðhÞ ¼ Gh ¼ mgh ¼ ð4=3Þπai 3 ðFp - Ff Þgh
ð2Þ
where m is buoyant mass, g is acceleration due to gravity, and Fp and Ff are the particle and fluid densities. Nonspecific osmotic repulsion due to interpenetration and compression of interacting macromolecules on adjacent surfaces is effectively captured by short-range exponentials as upw macro ðhÞ ¼ B exp½ - Kðh - ai - δHW Þ
ð3Þ
upp macro ðri, j Þ ¼ B exp½ - Kðr - ai - aj - δHW Þ
ð4Þ
where B is the intercept at the separation δHW, κ is the inverse decay length, and δHW = δi þ δj - 4κ-1 where δi þ δj are near hard wall dimensions of adsorbed macromolecules on adjacent surfaces. van der Waals attraction is computed for macromolecule coated colloids and surfaces to account for retardation36 and spatially varying dielectric properties37 as described elsewhere.38 For convenience, van der Waals potentials are modeled with inverse power laws as p upw vdW ðhÞ ¼ - Apw ai h
ð5Þ
p upp vdW ðri, j Þ ¼ - App 0:5ðai þ aj Þðri, j - ai - aj Þ
ð6Þ
where p is an noninteger power and Apw, App are effective Hamaker constants over a separation range of interest, which are constrained as Apw = 2App for identical particle and wall materials and based on the Derjaguin geometric correction.39,40 Multiple, parallel, specific interactions between N-cadherins fragments on supported lipid bilayers on colloids are shown to form essentially irreversible particle-particle bonds represented by harmonic potentials as 2 upp Ncad ðri, j Þ ¼ Kh ðri, j - ai - aj - δM Þ
Theory
unet i ðr, hÞ
where r is particle center-particle center separation, h is the particle surface-wall surface separation, and ai is the particle radius. The gravitational potential energy of each particle depends on its height, h, relative to the wall, multiplied by its buoyant weight, G, as given by
ð7Þ
where Kh is the effective spring constant and δM corresponds to the equilibrium most probable separation between particles.
Materials and Methods Chemicals, Lipids, and Proteins. Polystyrene (250 kDa), NaCl (e0.002% Ca2þ), CaCl2, MgSO4, nitric acid, and toluene were obtained from Sigma-Aldrich (St. Louis, MO). Nominal 2.34 μm SiO2 colloids were purchased from Bangs Laboratories (Fishers, IN). 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (PEGPE), and 1,2-dioleoyl-sn-glycero3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] with nickel salt (DGS-NiNTA) were obtained from Avanti Polar Lipids (Alabaster, AL). The fluorescent lipid 4-(4-(didecylamino)styryl)-N-methylpyridinium iodide (DiA) was purchased from Invitrogen (Carlsbad, CA). Disulfide-linked homodimers of (36) Dzyaloshinskii, I. E.; Lifshitz, E. M.; Pitaevskii, L. P. Adv. Phys. 1961, 10, 165–209. (37) Parsegian, V. A. J. Colloid Interface Sci. 1975, 51, 543–546. (38) Bevan, M. A.; Petris, S. N.; Chan, D. Y. C. Langmuir 2002, 18, 7845–7852. (39) Bevan, M. A.; Prieve, D. C. Langmuir 1999, 15, 7925–7936. (40) Wu, H. J.; Bevan, M. A. Langmuir 2005, 21, 1244–1254.
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Article monoclonal antihuman E-cadherin-allophycocyanin, recombinant human E-cadherin/Fc chimera, and recombinant human N-cadherin/ Fc chimera were purchased from R&D Systems (Minneapolis, MN). Cadherin fragment characterization included manufacturer supplied N-terminal sequence analysis, SDS-PAGE molecular weight and purity measurements, and activity confirmation via a cell adhesion assay and studies41-45 using the same lot numbers. Poly(ethylene glycol)/poly(propylene oxide)/poly(ethylene glycol) block copolymer (5.4/3.3/5.4 kDa) was donated by BASF (Wyandotte, MI). Imidazole was purchased from Fisher Scientific (Pittsburgh, PA). Particle and Surface Preparation. SiO2 colloids were sedimentation fractionated four times and then suspended in a 1:4 solution of 7X:DI water under sonication for 30 min and then washed six times (i.e., centrifugation, removal of supernatant, and sonication/redispersion for 5 min in DI water). The particles were then added to a 2 mL vial of nitric acid at 70 °C for 15 min, diluted to one-quarter the concentration of nitric acid with DI water, and then washed ten times. Coverslips were sonicated in acetone for 5 min, rinsed with DI water, submerged in 7X detergent, sonicated for an additional 30 min, and heated to 70 °C for 30 min, after which they were removed and rinsed thoroughly with DI water and transferred to a 3:1 H2SO4:H2O2 solution for 10 min.41 Coverslips to be modified with adsorbed F108 were heated on a hot plate at 120 °C for 10 min, spin-coated with a 3% polystyrene/ toluene solution at 3000 rpm for 30 s, and placed in a vacuum oven for 30 min at 100 °C to remove residual solvent. Supported Lipid Bilayer Formation. Vesicle fusion and rupture was used to form SLBs on all surfaces.42 Each lipid composition was dissolved in chloroform followed by evaporation with a N2 stream and drying in a vacuum desiccator for 4 h to remove residual solvent. Dried lipids were reconstituted in DI water at 1.0 mg/mL. After ten freeze-thaw cycles in liquid N2, small unilamellar vesicles42 were prepared via extrusion (10 passes) of the lipid mixtures at room temperature in a 10 mL thermobarrel vesicle extruder from Lipex Biomembranes Inc. (Vancouver, Canada) using a nominal 50 nm pore size polycarbonate track etch membrane from Structure Probe, Inc. (West Chester, PA). The average vesicle size was determined by dynamic light scattering as ∼80 nm. Vesicles were stored in polystyrene tubes, kept in the dark at room temperature, and used in fewer than 2 days. Vesicles were deposited on coverslips and particles at a lipid concentration of 0.25 mg/mL, and SLBs were allowed to form for 1.5 h. For 0.1% DiA in POPC, vesicles were adsorbed to particle and coverslip surfaces in DI water.43 Vesicles containing PEGPE were adsorbed in 0.15 M NaCl/0.7 M MgSO4 to collapse the brush layer via specific ion-mediated desolvation44 to aid vesicle adsorption, rupture, and fusion. When forming PEGPE bilayers,45,46 vesicles in DI water were added to the colloids in DI water to allow for homogenization, adsorption, and stabilization before suppression of electrostatic repulsion at physiological ionic strengths. Dispersions were then diluted with DI water to 0.1 the salt concentration used during fusion, allowing PEG layers to resolvate into brushes. Particles were kept on a shaker without sonication or centrifugation, which irreversibly damages SLBs. Excess vesicles were removed by rinsing with DI water in the flow cell following sedimentation. Microscopy Experiments. Fluorescence recovery after photobleaching (FRAP)47 experiments were conducted with a Leica TCS SP5 CSLM using an oil-immersion 100 objective (Leica, (41) Seu, K. J.; Pandey, A. P.; Haque, F.; Proctor, E. A.; Ribbe, A. E.; Hovis, J. S. Biophys. J. 2007, 92, 2445–2450. (42) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307–316. (43) Cremer, P. S.; Boxer, S. G. J. Phys. Chem. B 1999, 103, 2554–2559. (44) Fernandes, G. E.; Bevan, M. A. Langmuir 2007, 23, 1500–1506. (45) Kuhl, T. L.; Leckband, D. E.; Lasic, D. D.; Israelachvili, J. N. Biophys. J. 1994, 66, 1479–1488. (46) Needham, D.; McIntosh, T. J.; Lasic, D. D. Biochim. Biophys. Acta 1992, 1108, 40–48. (47) Axelrod, D.; Koppel, D. E.; Schlessinger, J.; Elson, E.; Webb, W. W. Biophys. J. 1976, 16, 1055–1069.
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Everett et al. NA = 1.4) with 458 nm Ar or 632 nm HeNe lasers. For SLBs on coverslips, the imaging field of view was 35 35 μm2, and circular photobleaching spot sizes of either 8 or 5 μm were used at a laser intensity 200 higher than the imaging intensity. Diffusion coefficients were obtained by fitting the time-dependent intensity data with an exponential.47 Qualitative FRAP experiments were performed on particles by focusing the beam at the equator of the particle and scanning a single window double the size of the particle at an intensity 50 higher than the imaging intensity. All TIRM data were collected with a 12-bit CCD camera (ORCA-ER Hamamatsu, Japan) on an upright optical microscope (Axioplan 2, Zeiss, Germany) using a 40 air objective (Achroplan, NA = 0.6). A 15 mW, 632 nm HeNe laser (Melles Griot, Carlsbad, CA) was used to generate an evanescent wave in a batch sedimentation cell optically coupled to a 68° dovetail prism (Reynard Corp., San Clemente, CA). Details of the ensemble TIRM experiment and analysis methods are described in extensive detail elsewhere.40,48 In VM experiments, the flow cell channel was machined in a 675 μm Si wafer, which was attached to a microscope slide with a thin polydimethylsiloxane layer cured for 30 min at 100 °C. Holes were drilled through the microscope slide, and fluidic ports (NanoPort Assembly, Upchurch Scientific, Oak Harbor, WA) mounted above each hole were connected to Teflon tubing at the flow cell inlet and outlet. A polystyrene-coated coverslip was attached to the bottom side of the machined Si wafer with a thin layer of quick-dry epoxy. 2000 ppm F108 Pluronic in DI water was passed through the assembled flow cell and absorbed to the polystyrene substrate for 4 h to prevent nonspecific particle deposition on the wall at high ionic strengths. After flowing in and allowing the SLB-modified SiO2 particles to sediment, the cell was thoroughly flushed with (i) 2000 ppm F108 in DI water, (ii) followed by 2000 ppm F108, 0.15 M NaCl, and 50 μg/mL N-cadherin fragments, which were allowed to bind for 1 h, (iii) 2000 ppm F108 in DI water for 30 min, and (iv) finally 0.15 M NaCl. The flow cell was then tilted ∼0.5° to produce slow particle migration to the stagnation zone, producing a quasi-2D concentrated configuration of SLB-coated silica colloids for VM measurements. All flow cell VM data were collected on an inverted microscope using a 12-bit monochromatic CCD camera (ORCA-ER Hamamatsu, Japan) operated at 1 binning with a 100 oil-immersion objective (Zeiss, NA = 1.4), yielding a lateral resolution of 60 nm/pixel.
Monte Carlo and Brownian Dynamics Simulations. Inverse simulation methods included NVT Monte Carlo (MC) and BD simulations of the 0 μM Ca2þ case using particle-particle potentials (eqs 1-7) with parameters gathered from direct TIRM measurements (Figure 3, Table 1). Static (i.e., radial distribution function, g(r)’s) and dynamic (i.e., mean-square displacements, MSDs) information were used to match experimental microscopy and simulation results. For increasing [Ca2þ], irreversible bonds (eq 7) were populated among particle pairs from the shortest to longest center-to-center separations with the total number of bonds being the only adjustable parameter. Simulation methods and associated analyses are described in extensive detail in previous papers on inverse methods,48-50 dynamic simulations,51,52 and using MC and BD together in a consistent fashion to analyze VM data.53,54 Experimentally measured particle coordinates were used as starting configurations, and multiple runs were performed with different random number generator seeds. Colloids at 0.69 area fraction were allowed to (48) Wu, H.-J.; Pangburn, T. O.; Beckham, R. E.; Bevan, M. A. Langmuir 2005, 21, 9879–9888. (49) Pangburn, T. O.; Bevan, M. A. J. Chem. Phys. 2005, 123, 174904. (50) Pangburn, T. O.; Bevan, M. A. J. Chem. Phys. 2006, 124, 054712. (51) Anekal, S.; Bevan, M. A. J. Chem. Phys. 2005, 122, 034903. (52) Anekal, S.; Bevan, M. A. J. Chem. Phys. 2006, 125, 034906. (53) Fernandes, G. E.; Beltran-Villegas, D. J.; Bevan, M. A. Langmuir 2008, 24, 10776–10785. (54) Fernandes, G. E.; Beltran-Villegas, D. J.; Bevan, M. A. J. Chem. Phys. 2009, 131, 134705.
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Table 1. Parameters in Potentials Used in MC/BD Inverse Simulationsa variable
equation
value
2 2100 2a (nm)a 2 1.00 Ff (g/cm3)b 3 b 2 1.96 Fp (g/cm ) 3, 4 10 Β (kT)c 3, 4 3 κ-1 (nm)c c 3, 4 28.0 δHW (nm) -1.154 d ) 6 2.10 Apw (kT nm 6 1.05 App (kT nm-1.154)d 5, 6 2.15 pd -2 e 7 2.55 10-2 Kh (kT nm ) e δM (nm) 7 28.0 0.69 φf 8.90 10-4 μ (kg/ms)g a Parameters obtained from (a) fits to TIRM gravitational potentials in Figure 3, (b) standard literature values in agreement with previous TIRM measurements on the same SiO2 particles,40,48 (c) previous TIRM measurements of PEG brushes,90 (d) power law approximations of retarded van der Waals potentials previously shown to agree with TIRM measurements,38,39,90 (e) fit to MSD plateau value and first peak in g(r) in Figure 5 for 100 μM Ca2þ, (f) measured directly from VM results in Figure 4, and (g) standard value.
relax for 106 MC steps before extracting static information or using MC-generated configurations to initiate subsequent BD simulations. BD simulations were performed for 24 106 time steps with an integration time of 2.5 μs, which is both larger than the momentum relaxation time and shorter than the diffusive time. In BD simulations, the total simulated time was set as the same VM acquisition period. All simulations included the independently measured particle size polydispersity and limited image resolution.49,50
Results and Discussion Bilayer and Cadherin Assembly. Figure 1 depicts the experimental configurations used in this work for measuring Ca2þdependent homophilic interactions between N-cadherin fragments. The basic probe in these measurements consists of supported lipid bilayer modified SiO2 colloids (LBCs) to which N-cadherin fragments are attached (i.e., extracellular fragments conjugated to lipids). Figure 1A depicts a sedimentation flow cell designed to (i) collect LBCs into quasi-2D concentrated configurations via a weak sedimentation equilibrium profile54,55 parallel to the microscope slide surface, (ii) remove defective particles through irreversible deposition in the main channel before collection in a stagnant region, and (iii) efficiently exchange media without disturbing56 the quasi-2D concentrated configurations. Figure 1B depicts the SLBs on adjacent micrometer-scale colloidal particles, which is primarily POPC but also includes sufficient PEGPE to produce brush structures57 and DGS-NiNTA lipids conjugated to N-cadherin fragments. Before presenting results for the experiment in Figure 1, we present results characterizing SLBs and nonspecific interactions on adjacent surfaces. Figure 2 shows results from several control experiments to characterize SLBs on planar substrates and SiO2 colloids as a function of bilayer composition. Figure 2A shows FRAP measurements of predominantly POPC bilayers without (99.9% POPC, 0.1% DiA) and with PEGPE (94.9% POPC, 5% PEGPE, 0.1% DiA) in 0.15 M NaCl. The inset in Figure 2A shows a series of confocal images used to construct FRAP data for the case without PEGPE. Results in Figure 2A clearly show SLB fluidity (55) Beckham, R. E.; Bevan, M. A. J. Chem. Phys. 2007, 127, 164708. (56) Bevan, M. A.; Lewis, J. A.; Braun, P. V.; Wiltzius, P. Langmuir 2004, 20, 7045–7052. (57) Marsh, D.; Bartucci, R.; Sportelli, L. Biochim. Biophys. Acta, Biomembr. 2003, 1615, 33–59.
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Figure 1. (top, A) Schematic views of VM flow cell. Gray arrow indicates direction for lateral sedimentation and concentration of LBCs, and blue arrows indicate flow direction when changing [Ca2þ]. (bottom, B) Schematic cross-sectional view of SLB on SiO2 colloid surfaces showing (i) primarily 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC) lipids, (ii) lipids with grafted 2 kDa poly(ethylene glycol), 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (PEGPE), (iii) fluorescent lipids, 4-(4-(didecylamino)styryl)-N-methylpyridinium iodide (DiA), for visualization and FRAP studies, (iv) 1,2-dioleoylsn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] with nickel salt (DGS-NiNTA), and (v) disulfide-linked homodimers of extracellular cadherin fragments attached to DGSNiNTA lipids through hexahistidine-terminated Fc domains. Approximate layer dimensions are 4 nm inner PEG layer,57 5 nm lipid bilayer,87 4 nm outer PEG layer,57 ∼1.5 nm hexahistidine tags (estimate based on chemical structure), 4.5 nm Fc domain,88 and 22.5 nm N-cadherin fragment.73,74,89
both with and without PEGPE. The higher diffusivity of the fluorescent lipids within the PEG/POPC bilayer (DSLB = 7.27 μm2/s) compared to POPC alone (DSLB = 6.1 μm2/s) is consistent with previous observations that attribute this difference to hydrodynamic and electrostatic decoupling of SLBs by the bottom leaflet PEG brush.58,59 The lower percentage FRAP recovery in the PEG/POPC bilayer (85%) compared to the POPC alone (94%) is ascribed to the hindered long-time self-diffusion of the fluorescent lipids among randomly distributed, less mobile PEGPE that act as diffusion obstacles.60-63 Figure 2B characterizes the fluidity of the assembled SLB including E-cadherin fragments attached to DGS-NiNTA lipids and fluorescent E-cadherin antibodies bound to E-cadherin fragments (fluorescent N-cadherin antibodies were not available). The spatial FRAP data in Figure 2B show limited fluorescence recovery indicating slow lateral diffusion of antibody-protein conjugates within the SLB, which could be further slowed by antibody induced clustering of adjacent E-cadherins. Rinsing with 200 mM imidazole (a competitive displacer of hexahistidine64) produced a significant reduction in bound fluorescent antibodycadherin complexes, which also demonstrated the specificity of the interactions. Additionally, performing the same binding protocol either in the absence of DGS-NiNTA lipids or without (58) Evans, E.; Sackmann, E. J. Fluid Mech. 1988, 194, 553–561. (59) Sonnleitner, A.; Schutz, G. J.; Schmidt, T. Biophys. J. 1999, 77, 2638–2642. (60) Saxton, M. J. Biophys. J. 1987, 52, 989–997. (61) Saxton, M. J. Biophys. J. 1994, 66, 394–401. (62) Schutz, G. J.; Schindler, H.; Schmidt, T. Biophys. J. 1997, 73, 1073–1080. (63) Horton, M. R.; Reich, C.; Gast, A. P.; Radler, J. O.; Nickel, B. Langmuir 2007, 23, 6263–6269. (64) Schmitt, L.; Dietrich, C.; Tampe, R. J. Am. Chem. Soc. 1994, 116, 8485– 8491.
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Figure 2. Characterization of SLBs on microscope slides and 2.1 μm SiO2 colloids. (top, A) Temporal FRAP data for predominantly POPC SLBs (Δ) without PEGPE (99.9% POPC, 0.1% DiA) and (O) with PEGPE (94.9% POPC, 5% PEGPE, 0.1% DiA). Fits indicate diffusivities of 6.10 μm2/s for (Δ) and 7.27 μm2/s for (O). Inset images are shown at 5 s intervals, and the scale bar is 10 μm. (middle, B) Spatially averaged radial FRAP data for fluorescent E-cadherin antibody bound to E-cadherin fragments on a SLB (94.4% POPC, 5% PEGPE, 0.5% DGS-NiNTA, 0.1% DiA) at (O) t = 0 and (Δ) t = 10 min. The time-dependent shift in radial intensity indicates recovery. Inset images (confocal and associated intensity map) of the two data sets have a 10 μm scale bar. (bottom, C) Qualitative FRAP images of POPC SLBs with PEGPE (94.9% POPC, 5% PEGPE, 0.1% DiA) on a single SiO2 colloid, where a continuous SLB on the particle and wall enables fluorescence recovery. Scale bar is 2 μm.
the addition of E-cadherin led to no observable antibody binding to SLBs. Considering the results in Figure 2A,B, cadherin fragments appear to be active within SLBs composed of a predominant and highly mobile POPC/DiA component, while PEGPE and cadherin-conjugated lipids are much less mobile. Figure 2C shows a qualitative FRAP experiment verifying SLB fluidity on SiO2 colloids via a series of images of a PEGPE/POPC/ DiA bilayer on a single LBC deposited onto a microscope slide. A quantitative FRAP analysis is not possible, but the results in Figure 2C are qualitatively consistent with trends observed for the same bilayers on the coverslip surfaces in Figure 2A. Again, tracer recovery is partial and can be attributed to relatively immobile 18980 DOI: 10.1021/la1038443
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Figure 3. Ensemble TIRM measurements of interactions between dilute ensembles of LBCs and SLBs on planar microscope slide surfaces. (top, A) Ensemble TIRM profiles of nominal ∼2.1 μm SiO2 colloids functionalized with POPC SLBs with PEGPE (94.4% POPC, 5% PEGPE, 0.5% DGS-NiNTA, 0.1% DiA) levitated above a microscope slide with an SLB of identical composition before (blue circles) and after (red circles) N-cadherin binding. Main plot shows gravitational potential subtracted to clarify the attractive potential, and the inset plot shows the same ensemble average data (red) as well as individual particle profiles (black) with their gravitational potentials. The line (green) is the fit to the ensemble average potential (red) using eqs 1-3 and 5. (middle, B) Attractive portion of ensemble TIRM potential in the top plot with fit (green) and Lifshitz theory with (solid black line) and without (dashed line) the SLB contribution considered. (bottom, C) Ensemble profiles for (O) bare SiO2 on bare microscope slides in 0.15 M NaCl and for particles and surfaces with N-cadherins immobilized on SLB (same as in top plot) in 0.15 M NaCl with 10 μM CaCl2 (Δ) and 100 μM CaCl2 (0). Inset shows normalized single particle intensity vs time data for the two cases with same colors as main plot.
PEGPE acting as an effective obstacle course for mobile fluorescent lipids.60-63 Bilayer and Cadherin Nonspecific Interactions. To resolve the onset of specific [Ca2þ]-dependent interactions between N-cadherin fragments on LBCs, we first quantified the underlying Langmuir 2010, 26(24), 18976–18984
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nonspecific colloidal and macromolecular interactions. Ensemble TIRM40,48,65 was used to measure colloid-surface interactions to quantify potential energy profiles with kT-scale energy vs nanometer-scale separation. These colloid-surface interactions can be readily interpreted as colloid-colloid interactions using the Derjaguin approximation.24 Figure 3A shows interactions of LBCs (94.4% POPC, 5% PEGPE, 0.5% DGS-NiNTA, 0.1% DiA, for 2.1 μm SiO2) and an underlying coverslip having the same SLB composition both with and without N-cadherins and in the absence of Ca2þ. The profiles in Figure 3A are similar in the absence of Ca2þ, with the N-cadherin case showing slightly softer repulsion and less attraction than the PEG-only case possibly due to a thicker macromolecular layer. Figures 3A,B show curve fits to analytical potentials (eqs 3 and 5) that include macromolecular osmotic repulsion and van der Waals attraction (after subtracting the buoyant particle weight). These curve fits provide analytical representations of nonspecific interactions for input in subsequent inverse analyses. To begin probing Ca2þ-dependent N-cadherin interactions, Figure 3C shows TIRM measured potentials and temporal height excursions for deposited LBCs at 10 and 100 μM [Ca2þ]. Below a threshold of 10 μM [Ca2þ], the interaction potentials were identical to the nonspecific N-cadherin profiles in Figure 3A. Height fluctuations in the inset of Figure 3C show transient temporal sampling of two states at 10 μM [Ca2þ], while fluctuations at a single elevation are observed for 100 μM [Ca2þ]. LBCs deposited at 10 and 100 μM [Ca2þ] were irreversibly bound, even following the removal of [Ca2þ] and exposure to 200 mM imidazole (an attempt to displace N-cadherin fragments). Both conditions appear to cause fusion of SLBs on adjacent surfaces. The two- and single-state wells at the different [Ca2þ] are not understood, but the two profiles together suggest a fusion process that gradually overcomes PEG repulsion with increasing [Ca2þ] possibly with a ratcheting effect by the N-cadherins.66 It should be noted that the SLBs are stabilized by PEG brushes that prevent Ca2þ induced fusion between adjacent bilayers. To confirm this, control experiments were performed to show that SLB fusion does not occur in the [Ca2þ] range investigated unless N-cadherin fragments are present. This control also demonstrates that Ca2þ-dependent specific ion effects (i.e., Hofmeister effects67) do not cause the PEG brushes to become attractive toward each other.44,68 LBC Concentrated Configurations vs [Ca2þ]. With an understanding of LBC interactions with surfaces in Figure 3, Figure 4 shows VM results for the [Ca2þ]-dependent behavior of LBCs within quasi-2D concentrated configurations. Figure 4 shows microscopy and BD simulation results for the evolution of the static microstructure and dynamics of quasi-2D concentrated configurations of N-cadherin LBCs for stepwise increases in [Ca2þ]. The experimental arrangement (Figure 1) consists of LBCs levitated over a PEG copolymer-coated substrate within a fluidic channel tilted at ∼0.5° to produce a small component of gravity parallel to the substrate surface. Equilibration of sedimentation and lateral diffusion produces a sedimentation equilibrium profile55 with concentrated phases present in a stagnant corner region, which is where VM image data are collected. Figure 4 shows as a function of [Ca2þ] representative static images and trajectories for 60s from VM movies of concentrated LBCs. (65) Everett, W. N.; Wu, H.-J.; Anekal, S. G.; Sue, H.-J.; Bevan, M. A. Biophys. J. 2007, 92, 1005–1013. (66) Sivasankar, S.; Gumbiner, B.; Leckband, D. Biophys. J. 2001, 80, 1758– 1768. (67) Hofmeister, F. Arch. Exp. Pathol. Pharmakol. 1888, 24, 247–260. (68) Hwang, K.; Wu, H.-J.; Bevan, M. A. Langmuir 2004, 20, 11393–11401.
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Figure 4. Colloidal particle static images and trajectories from VM measurements and inverse BD simulations. Columns from left to right are (A) VM static images, (B) experimental particle trajectories for 60 s, (C) rendered particle configurations from inverse BD simulations, and (D) simulated particle trajectories for 60 s. Rows from top to bottom correspond to [Ca 2þ] (in 150 mM NaCl media) of (i) 0.0, (ii) 0.1, (iii) 1.0, (iv) 10.0, and (v) 100.0 μM.
Figure 4 also contains rendered images and particle trajectories obtained from BD simulations, which were performed as part of an inverse analysis to extract interactions by matching static and dynamic features with experiments. Trajectory plots visually demonstrate the affect of bond formation on particle dynamics. Structures in Figure 4 are not disturbed during Ca2þ exchange in the flow cell because the constant 0.15 M NaCl medium dominates ∼μM Ca2þ gradients.56 To quantify the LBC static structure and dynamics with changing [Ca2þ], Figures 5A,B show MSDs and radial distribution functions obtained from the VM experiments reported in Figure 4. The initial slopes of MSD curves in Figure 5A display a monotonic decrease with increasing [Ca2þ] whereas slopes at longer times increase first at 0.1 μM Ca2þ and then decrease monotonically with increasing [Ca2þ]. These trends show (i) a gradual drop in short-time self-diffusion due to increasing pair attraction, (ii) an initial increase in self-diffusion at intermediate times due to increased free-volume in the presence of attraction (i.e., unbound particles diffuse faster in voids), and (iii) slowing at long times due to dynamic arrest in the presence of increasing attraction. Consistent with the images in Figure 4, radial distribution functions in Figure 5B also display a loss of long-range order. Figures 4 and 5 also show inverse BD analysis results to interpret the changes in effective LBC pair interactions in the presence of Ca2þ. Several combinations of particle attraction and simulation conditions were investigated including (i) isotropic interparticle attraction of several functional forms (i.e., square well, power law), (ii) irreversible bond formation modeled by harmonic potentials that could be populated amongst particles in several different ways, and (iii) different ensembles including NVT (i.e., constant density) and NPT (i.e., constant pressure). Only one model produced satisfactory agreement with both static DOI: 10.1021/la1038443
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Figure 5. Data from analyses of experimental VM and converged inverse BD simulations of Ca2þ-dependent behavior shown in Figure 4. In each plot, the corresponding [Ca2þ] is given as (red circles) 0 μM, (orange downward triangles) 0.1 μM, (lime squares) 1.0 μM, (forest diamonds) 10.0 μM, and (blue triangles) 100.0 μM. (top, A) MSDs from experiments (points) and simulations (lines). (middle, B) Radial distribution functions, g(r), from experiments (points) and simulations (lines). (bottom, C) Number of irreversible bonds between LBCs due to N-cadherin bonds and SLB fusion (left, closed circles) and percent reduction in LBC diffusion (right, open triangles) vs [Ca2þ]. Three Hill plot curve fits are shown: (i) for no bonds at 0.01 μM Ca2þ with KD = 0.086 μM and nH = 1.6 (right-most dashed line), (ii) for no bonds at 0.001 μM Ca2þ with KD = 0.080 μM and nH = 0.89 (solid line), and (iii) without making any assumptions about the zero bond limit [Ca2þ] to give KD = 0.078 μM and nH = 0.078 (left-most dashed line). Approximate error estimates are (10 bonds based on inverse analysis convergence criteria and (0.1 μM [Ca2þ] based on solution preparation methods.
and dynamic data; the final model includes nonspecific van der Waals and macromolecular interactions in combination with Ca2þ-mediated specific, irreversible bonds between LBCs at constant density. The nonspecific portion of the potential (eqs 2-6, Table 1) is obtained from TIRM measurements in Figure 3 with no adjustable parameters. Specific interactions are included by 18982 DOI: 10.1021/la1038443
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(i) populating irreversible bonds among initial LBC configurations (from VM images) starting from the smallest interparticle gaps and (ii) using a harmonic well form to allow for particle thermal motion along the line of centers with a spring constant and equilibrium position obtained by fitting both the MSD plateau in Figure 5A and the first peak in g(r)’s in Figure 5B for the 100 μM Ca2þ case. The only adjustable parameter is the total number of irreversible bonds. Figure 5C shows the number of irreversible bonds between particle pairs vs [Ca2þ] from the inverse BD analysis. Figure 5C also shows the percent reduction in LBC diffusion vs [Ca2þ] is correlated with the number of irreversible bonds. Several “Hill plot” curve fits with different assumptions about the low binding data point in Figure 5C all suggest ∼KD = 0.08 μM. TIRM particle-wall measurements in Figure 3 show SLB fusion occurs between 10 and 100 μM [Ca2þ], which means the interactions become irreversible somewhere above 10 μM [Ca2þ]. As a result, the binding data in Figure 5C are not strictly for equilibrium interactions, but rather only suggests a KD where the number of bonds starts to increase in a sigmoidal fashion. Nevertheless, this indicates interactions of extracellular N-cadherin fragments across interparticle gaps are producing LBC bonds at ∼0.1 μM Ca2þ, which indicates N-cadherin homophilic attraction at a lower [Ca2þ] range than any other reported measurements.69,70 Past studies69,70 have measured KD = 0.72 mM using an AFM with N-cadherin fragments attached to the AFM tip and a flat surface and KD = 0.65 mM using an OT to pull-off N-cadherin coated 3 μm polystyrene beads from rat neurons. Because AFM and OT use mechanically manipulated probes, they are limited to measuring pN forces based on the minimum force required to cause detectable probe deflection. In contrast, nonintrusive observation of “diffusing probes” in the present work allows for direct measurement of fN forces with a statistical mechanical analysis (rather than intrusive manipulation of “mechanical probes” with a deterministic mechanical analysis). As a result, the KD ≈ 0.1 μM for N-cadherin obtained using a fN resolution measurement method in this work is not at odds with the previous measurements of KD ≈ 0.1 mM obtained with pN resolution forces measurements. The results in Figure 5C simply show the onset of Ca2þ-dependent N-cadherin fragment interactions at a higher force and energy resolution. In other words, cadherin adhesion may have not vanished at the lowest [Ca2þ] investigated in previous studies, but was less than the ∼10 pN lower limit of mechanical probe methods. In this sense, the approach described here is complementary to other methods; while diffusing probes are not well-suited to measuring strong adhesive interactions, their enhanced sensitivity to low-energy bond formation may enable access to biomolecular interactions that have been overlooked in the past due to the force/energy limits of existing methods. Relating LBC Bonding to N-Cadherin Interactions. The irreversible LBC bonds (i.e., infinite lifetime) reported in Figure 5C likely include many N-cadherin dimers acting together. As such, the results in Figure 5C probe Ca2þ-dependent interactions either for (i) parallel, independent N-cadherin interactions all having single N-cadherin pair affinity, (ii) parallel, interdependent N-cadherin interactions having a net interaction different than the sum of pair affinities (i.e., avidity), or (iii) a net interaction influenced by immobilization via changes to configurational entropy on translational degrees of freedom (69) Baumgartner, W.; Golenhofen, N.; Grundh€ofer, N.; Wiegand, J.; Drenckhahn, D. J. Neurosci. 2003, 23, 11008–11014. (70) Heupel, W. M.; Baumgartner, W.; Laymann, B.; Drenckhahn, D.; Golenhofen, N. Mol. Cell. Neurosci. 2008, 37, 548–558.
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(e.g., rotation effects,71 “matricity” via fluidity72). It is likely that Ca2þ-dependent N-cadherin dimer formation causes fusion of SLBs on adjacent particles based on measurements in Figure 3C. Although the dynamic nature of such a fusion process complicates the analysis of effective LBC pair interactions, the number of N-cadherin pairs per irreversible bond between LBCs can be estimated based on some limiting cases and assuming a net threshold attraction causes fusion. An upper estimate of ∼104 N-cadherin bonds per irreversible LBC bond is obtained assuming (i) 100% of 0.5% NiNTAterminated lipids are occupied by single N-cadherin fragments, (ii) there is extensive overlap of ∼22.5 nm (from X-ray73 and electron microscopy74) between all EC domains75 to produce the maximum interaction area (i.e., ∼0.1 μm2) between adjacent spheres,24 and (iii) lipid conjugated N-cadherins remain sufficiently mobile such that all of them diffuse into the interaction areas and are evenly distributed among six nearest neighbors. A lower estimate of ∼101 N-cadherin bonds per irreversible LBC bond is obtained assuming (i) ∼10% of 0.5% NiNTA-terminated lipids are occupied by single N-cadherin fragments,76 (ii) there is overlap of ∼22.5/5 nm between single terminal subunits on interdigitating trans dimers75 to produce a minimum interaction area (i.e., ∼0.01 μm2), and (iii) lipid conjugated N-cadherins are immobile so that only those already present in the gap contribute to bond formation. Assumptions associated with predicting the lower bound on the number of N-cadherin pair interactions are more likely to be valid, including (i) the FRAP results in Figure 2C suggest that N-cadherin fragments are relatively immobile, (ii) bioconjugation of hexahistidine-terminated protein fragments to NiNTA-terminated lipids is generally incomplete,76 and (iii) weak, equilibrium interactions of N-cadherin may only produce overlap between terminal domains (in contrast to active compression of N-cadherin-modified surfaces22,77). While we cannot eliminate the possibility of cis dimer formation between N-cadherins on adjacent particles due to the highly flexible nature of cadherins at low [Ca2þ],29 assuming trans dimer formation does not significantly alter the estimate of bond density across interparticle gaps. In future studies, interactions could be biased toward trans dimer formation through the use of thicker PEG brush layers that would act to sterically orient cadherins into a rodlike state in the absence of Ca2þ. To form single, irreversible bonds between LBCs on the time scales of the measurements in Figure 5C, the effective pair attraction must be >10kT.78 Although six weaker bonds with adjacent particles could produce dynamic arrest of a single Brownian colloid, the LBC bonds in Figure 4 were observed to form irreversibly in a pairwise manner, suggesting an effective pair attraction >10kT. Using 10kT as the LBC irreversible bond strength and the previous estimates of cadherin pairs per bond, N-cadherin trans or cis dimer interactions could be as weak as E/N = 10-3-1kT. This analysis neglects avidity, which could change these estimates and may even be enhanced by disulfide linked Fc domains of N-cadherin constructs (existing between pairs of “as-purchased” protein fragments from the manufacturer). (71) Biancaniello, P. L.; Kim, A. J.; Crocker, J. C. Phys. Rev. Lett. 2005, 94, 058302. (72) Schmid, E. M.; Ford, M. G. J.; Burtey, A.; Praefcke, G. J. K.; Peak-Chew, S.-Y.; Mills, I. G.; Benmerah, A.; McMahon, H. T. PLoS Biol. 2006, 4, e262. (73) Nagar, B.; Overduin, M.; Ikura, M.; Rini, J. M. Nature 1996, 380, 360–364. (74) Pokutta, S.; Herrenknecht, K.; Kemler, R.; Engel, J. Eur. J. Biochem. 1994, 223, 1019–1026. (75) Gumbiner, B. M. Nat. Rev. Mol. Cell Biol. 2005, 6, 622–634. (76) Nye, J. A.; Groves, J. T. Langmuir 2008, 24, 4145–4149. (77) Gumbiner, B. M. Nat. Rev. Mol. Cell Biol. 2005, 6, 622–634. (78) Russel, W. B.; Saville, D. A.; Schowalter, W. R. Colloidal Dispersions; Cambridge University Press: New York, 1989.
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Although this analysis does not benefit from any independent direct measurements of the number of trans dimers, it suggests the magnitude of interactions at ∼0.1 μM Ca2þ and their collective role on the dynamics and structure of LBCs. Although tissue morphogenesis clearly involves much more complex dynamic physicochemical and biological processes, the role of adhesive N-cadherin interactions at ∼0.1 μM Ca2þ in suppressing Brownian motion of micrometer-sized LBCs suggests the potential importance of such weak interactions to cadherinmediated tissue morphogenesis.79 For example, catenin phosphorylation77 and actin depolymerization by intracellular calcium69 have both been implicated in attenuating N-cadherin mediated cell adhesion and tissue morphogenic behavior. However, the extracellular N-cadherin fragment has an important role as one end of a cascade of mechanisms that mediate Ca2þdependent cell adhesion. The [Ca2þ] range in which the extracellular N-cadherin fragment is active in this work is also much lower than the average physiological [Ca2þ] of around 1.5-2 mM in the brain.17 However, such weak interactions could become important in determining cell adhesion and cell-cell communication in a variety of tissues and for other cadherin subtypes when, for example, Ca2þ is deficient (e.g., breast cancer80) or actively regulated (e.g., synaptic plasticity69). In addition, the importance of subplasmalemmal [Ca2þ] in the ∼0.2 μM range has been shown to dictate local growth cone dynamics of developing neuronal cells,81 and voltagegated Ca2þ channels have been found to influence axonal outgrowth and path-finding processes.82 Ultimately, how local and global spatial and temporal [Ca2þ] changes regulate numerous cellular processes is still the subject of research,83 so that the physiological significance of the weak cadherin interactions reported here might have to be incorporated into emerging [Ca2þ]-dependent mechanisms. Understanding the Ca2þ dependence of weak homophilic cadherin interactions provides a basis for future investigations of heterophilic cadherin interactions using LBCs, which could lend insights into discrepancies between direct force measurements,22 bead assays,30,31,84 and cell sorting experiments.85 Although LBCs have previously been used to detect soluble proteins binding to lipid conjugated proteins,86 our results are also the first to demonstrate quantitative analysis of LBC interactions to measure protein-protein interactions between extracellular fragments of transmmembrane proteins on adjacent surfaces.
Conclusions Our results demonstrate the successful attachment and orientation of cadherin fragments to fluid supported lipid bilayers on colloidal particles, which has allowed us to measure how [Ca2þ] controls interactions between N-cadherin-modified LBCs in (79) Chen, C. P.; Posy, S.; Ben-Shaul, A.; Shapiro, L.; Honig, B. H. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 8531–8536. (80) Sergeev, I. N.; Rhoten, W. B. Endocrine 1998, 9, 321–327. (81) Chadborn, N.; Eickholt, B.; Doherty, P.; Bolsover, S. Eur. J. Neurosci. 2002, 15, 1891–1898. (82) Archer, F. R.; Doherty, P.; Collins, D.; Bolsover, S. R. Eur. J. Neurosci. 1999, 11, 3565–3573. (83) Berridge, M. J.; Bootman, M. D.; Lipp, P. Nature 1998, 395, 645–648. (84) Chappuis-Flament, S.; Wong, E.; Hicks, L. D.; Kay, C. M.; Gumbiner, B. M. J. Cell Biol. 2001, 154, 231–243. (85) Duguay, D.; Foty, R. A.; Steinberg, M. S. Dev. Biol. 2003, 253, 309–323. (86) Baksh, M. M.; Jaros, M.; Groves, J. T. Nature 2004, 427, 139–141. (87) Lewis, B. A.; Engelman, D. M. J. Mol. Biol. 1983, 166, 211–217. (88) Boggon, T. J.; Murray, J.; Chappuis-Flament, S.; Wong, E.; Gumbiner, B. M.; Shapiro, L. Science 2002, 296, 1308–1313. (89) Prakasam, A. K.; Maruthamuthu, V.; Leckband, D. E. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15434–15439. (90) Bevan, M. A.; Prieve, D. C. Langmuir 2000, 16, 9274–9281.
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quasi-2D concentrated configurations. In physiological ionic strength media in the absence of Ca2þ, nonspecific osmotic repulsion between PEG brushes and cadherin fragments, balanced by weak van der Waals attraction between SLBs and SiO2 colloids, produces thermodynamically stable concentrated configurations in a weak lateral gravitational field. Both the timeaveraged microstructure and short-time self-diffusion of LBCs on lattice positions are in excellent agreement with MC and BD simulations and demonstrate the absence of any specific or anisotropic attraction between N-cadherin fragments in the absence of Ca2þ. This baseline measurement provides a basis to unambiguously monitor the evolution of specific N-cadherin interactions for [Ca2þ] in the micromolar range. With increasing [Ca2þ] in the micromolar range, specific N-cadherin-mediated attraction alters the microstructure and causes dynamic arrest of LBCs, which is accurately captured in
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BD simulations by the addition of irreversible bonds between particles. Because this attraction is not reversible upon reducing Ca2þ or imidazole addition, these findings suggest adjacent SLBs become fused as part of N-cadherin binding at higher [Ca2þ]. The observed N-cadherin attraction at micromolar Ca2þ concentrations is much lower than previously reported measurements of the Ca2þ dependence of N-cadherin interactions. Future measurements using similar diffusing probe configurations could provide access to other tunable, low-energy biomolecular interactions, including Ca2þ-dependent heterophilic cadherin interactions, that fall below the resolution limits of existing force measurement techniques. Acknowledgment. Financial support was provided by the NSF (CTS-0346473, CBET-0834125) and the Robert A. Welch Foundation (A-1567).
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