pubs.acs.org/Langmuir © 2010 American Chemical Society
Ion-Specific Effects on the Interaction between Fibronectin and Negatively Charged Mica Surfaces Matthew D. Heath,†,§ Brian Henderson,‡ and Susan Perkin*,† †
Department of Chemistry, University College London, 20 Gordon Street, London WC1H 0AJ, United Kingdom, ‡Division of Microbial Diseases, UCL Eastman Dental Institute, University College London, 256 Gray’s Inn Road, London WC1X 8LD, United Kingdom, and §Department of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom Received February 15, 2010. Revised Manuscript Received March 16, 2010 Atomic force microscopy (AFM) imaging and subsequent image analysis have been used to measure the ion-specific and ionic strength effects on the adsorption of fibronectin to mica surfaces in buffer solution. Increasing the concentration of monovalent Naþ salt solutions is shown to cause a transition from tightly aggregated and “stringof-beads” structures on the mica surface to well dispersed single-molecule adsorption. Studying the effect of two divalent salts, Ni2þ and Ca2þ, reveals a dramatic enhancement of fibronectin adsorption to mica in buffer solutions containing Ni2þ, but not for Ca2þ. The origin of this ion-specific effect is discussed.
Introduction Fibronectin (Fn) is a large and essential1 glycoprotein that is found in all body fluids, in the extracellular matrix (ECM), and on the surface of cells. In this latter site, it functions as a bridge between cells and their contiguous ECM2 by binding to a number of cell surface receptors termed integrins.3 The Fn protein is a dimer composed of three distinct modules (Figure 1) known as the type I module (FnI), type II module (FnII), and type III module (FnIII).4 Each module contains sites that allow Fn to bind to other host molecules such as heparin, collagen, DNA, fibrin, syndecan, and fibulin and to Fn itself.5 On the surface of cells, the β1-integrin family contains a number of members that bind to Fn, through the RGD triplet peptide.3 Such binding is part of the association of cells with the ECM and functions as a sensor for cells to probe their associated ECM.6 In addition, Fn is also involved in the blood clotting process7 and in wound healing,8 and is an acute phase *To whom correspondence should be addressed. E-mail: susan.perkin@ ucl.ac.uk. (1) George, E. L.; Georges-Labounesse, E. N.; Patel-King, R. S.; Rayburn, H.; Hynes, R. O. Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin. Development 1993, 119, 1079-1091. (2) Wierzbicka-Patynowski, I.; Schwarzbauer, J. E. The ins and outs or fibronectin matrix assembly. J. Cell Sci. 2003, 116, 3269-3276. (3) Hynes, R. O. The emergence of integrins: A personal and historical perspective. Matrix Biol. 2004, 23, 333-340. (4) Staunton, D.; Millard, C. J.; Aricescu, A. R.; Campbell, I. D. Preparation of recombinant fibronectin fragments for functional and structural studies. Methods Mol. Biol. 2009, 522, 1-27. (5) Pankov, R.; Yamada, K. M. Fibronectin at a glance. J. Cell Sci. 2002, 115, 3861-3863. (6) Morgan, M. R.; Byron, A.; Humphries, M. J.; Bass, M. D. Giving off mixed signals;distinct functions of alpha5beta1 and alphavbeta3 integrins in regulating cell behaviour. IUBMB Life 2009, 61, 731-738. (7) Cho, J.; Mosher, D. F. Role of fibronectin assembly in platelet thrombus formation. J. Thromb. Haemostasis 2006, 4, 1461-1469. (8) Midwood, K. S.; Mao, Y.; Hsia, H. C.; Valenick, L. V.; Schwarzbauer, J. E. Modulation of cell-fibronectin matrix interactions during tissue repair. J. Invest. Dermatol. Symp. Proc. 2006, 11, 73-78. (9) Pick-Kober, K. H.; Munker, D.; Gressner, A. M. Fibronectin is synthesised as an acute phase reactant in rat hepatocytes. J. Clin. Chem. Clin. Biochem. 1986, 24, 521-528. (10) Ding, L.; Guo, D.; Homandberg, G. A. Fibronectin fragments mediate matrix metalloproteinase upregulation and cartilage damage through proline rich tyrosine kinase, c-src, NF-kappaB and protein kinase Cdelta. Osteoarthritis Cartilage 2009, 17, 1385-1392.
5304 DOI: 10.1021/la100678n
reactant.9 There is also growing evidence that fragmentation of Fn can produce a wide range of signaling protein species.10 The growing number of biological roles for Fn and, in particular, its participation in controlling cell shape, polarity, and function has meant that it has potential in therapy, particularly in tissue engineering.11,12 There is also an increasing role for this protein in controlling other conditions such as preterm labor.13 Thus, Fn is a key homeostatic protein involved in a wide range of cellular processes. It is also recognized as being a very important target for the adhesion of prokaryotic and eukaryotic pathogens. Bacteria, in particular, have evolved a large number of Fn binding proteins which can interact with a range of sites in the three different Fn modules. Such binding is now recognized to be of major importance in the virulence of different bacterial pathogens.14 The enormous importance of Fn in biological systems and the increasing realization of its medical importance as a material and as a target of pathogens means that it is important to understand the behavior and conformation of Fn at surfaces, including nonbiological surfaces,15 and the conditions affecting the adsorption of the molecule on these surfaces. A number of studies have sought to investigate the conformation of Fn on various surfaces using XPS, ToF-SIMS,16 FTIR-ATR,17 electron microscopy,18-21 and atomic force microscopy (AFM).22,23 Previous (11) Bloom, L; Calabro, V. FN3: a new protein scaffold reaches the clinic. Drug Discovery Today 2009, 14, 949-955. (12) von der Mark, K.; Park, J.; Bauer, S.; Schmuki, P. Nanoscale engineering of biomimetic surfaces: cues from the extracellular matrix. Cell Tissue Res. 2010, 339, 131-153. (13) Siassakos, D.; O’Brien, K.; Draycott, T. Healthcare evaluation of the use of atosiban and fibronectin for the management of pre-term labour. J. Obstet. Gynaecol. 2009, 29, 507-511. (14) Schwarz-Linek, U.; Hook, M.; Potts, J. R. The molecular basis of fibronectin-mediated bacterial adherence to host cells. Mol. Microbiol. 2004, 52, 631-641. (15) Petrie, T. A.; Reyes, C. D.; Burns, K. L.; Garcı´ a, A. J. Simple application of fibronectin-mimetic coating enhances osseointegration of titanium implants. J. Cell. Mol. Med. 2009, 13 (8), 2602-2612. (16) Lhoest, J. B.; Detrait, E.; van den Bosch de Aguilar, P.; Bertrand, P. J. Fibronectin adsorption, conformation, and orientation on polystyrene substrates studied by radiolabeling, XPS, and ToF SIMS. Biomed. Mater. Res. 1998, 41, 95-103. (17) Baujard-Lamotte, L.; Noinville, S.; Goubard, F.; Marque, P.; Pauthe, E. Kinetics of conformational changes of fibronectin adsorbed onto model surfaces. Colloids Surf., B 2008, 63, 129-137.
Published on Web 03/26/2010
Langmuir 2010, 26(8), 5304–5308
Heath et al.
Letter
Figure 1. Schematic representation of one unit of a Fn molecule. Fn contains three modules/repeats: FnI (type I), FnII (type II), and FnIII (type III). Disulfide bridges in the C-terminal connect a second almost identical chain.
investigations suggest that the nature of Fn adsorption on any artificial surface will depend upon a number of different factors as a result of the type of surface used and its chemical and physical properties, such as roughness, wettability, and charge.17,22,24 Hydrophilic and charged surfaces are thought to tease apart the ionic interactions that stabilize Fn in a compact conformation in solution. Other reports indicate that, on increasing the ionic concentration of a solution, Fn will adopt more elongated structures as a result of interchain disruption of ionic interactions that stabilize its compact form.18,23,25-28 Recently, Cheung and Walker used immuno-AFM to detect Fn-antibody complexes enabling them to infer available heparin binding sites when Fn is adsorbed to a mica surface.22 AFM has been used extensively to study both DNA and proteins on surfaces under ambient solution conditions in order to investigate the structure or conformation of adsorbed molecules with high resolution. For example, studies of another ligand host protein of the ECM (fibrinogen) showed more protein was adsorbed when the ionic concentration of the buffer was increased, while further increases of salt concentration up to 500 mM resulted in decreased adsorption.29 In a separate AFM study, it was demonstrated that the DNA binding to mica correlates with cationic radius.30 Freshly cleaved mica is a particularly useful substrate for these surface studies because it has a uniform high negative surface charge in water, has a well characterized surface structure, and is atomically smooth. In order to understand intermolecular and intersurface interactions involved in Fn adsorption, it is necessary to study ion-specific and ion-valency (18) Erickson, H. P.; Carrell, N. A. Fibronectin in extended and compact conformations. J. Biol. Chem. 1983, 258, 14539-14544. (19) Engel, J.; Odermatt, E.; Engel, A. Shapes, domain organizations and flexibility of laminin and fibronectin: two multifunctional proteins of the extracellular matrix. J. Mol. Biol. 1981, 150, 97-120. (20) Odermatt, E.; Engel, J. Shape, conformation and stability of fibronectin fragments determined by electron microscopy.Circular dichroism and ultracentrifugation. J. Mol. Biol. 1981, 159, 109-123. (21) Price, T. M.; Rudee, M.; Pierschbacherm, M.; Rouslahti, E. Structure of fibronectin and its fragments in electron microscopy. Eur. J. Biochem. 1982, 129, 359-363. (22) Cheung, J. W. C.; Walker, G. C. Immuno-atomic force microscopy characterization of adsorbed fibronectin. Langmuir 2008, 24, 13842-13849. (23) Bergkvist, M.; Carlsson, J.; Oscarsson, S. Surface-dependent conformations of human plasma fibronectin adsorbed to silica, mica, and hydrophobic surfaces, studied with use of atomic force microscopy. J. Biomed. Mater. Res., Part A 2003, 64, 349-356. (24) Horbett, T. A. The role of adsorbed proteins in animal cell adhesion. Colloids Surf., B 1994, 2, 225-240. (25) Grant, R. P.; Spitzfaden, C; Altroff, H; Campbell, I. D.; Mardon, H. J. Structural requirements for biological activity of the ninth and tenth FIII domains of human fibronectin. J. Biol. Chem. 1997, 272, 6159-6166. (26) Williams, E. C.; Janmey, P. A.; Ferry, J. D.; Mosher, D. F. Conformational states of fibronectin. J. Biol. Chem. 1982, 257, 14973-14978. (27) Rocco, M.; Carson, M.; Hantgan, R.; McDonagh, J.; Hermans, J. Dependance of the shape of the plasma fibronectin molecule on solvent composition. J. Biol. Chem. 1983, 258, 14545-14549. (28) Markovic, Z.; Lustig, A.; Engel, J. Shape and stability of fibronectin in solutions of different pH and ionic strength. Hoppe-Seyler’s Z. Physiol. Chem. 1983, 364, 1795-1804. (29) Tsapikouni, T. S.; Missirlis, Y. F. pH and ionic strength effect on single fibrinogen molecule adsorption on mica studied with AFM. Colloids Surf., B 2007, 57 (1), 89-96 (30) Hansma, H. G.; Laney, D. E. DNA binding to mica correlates with cationic radius: Assay by Atomic Force Microscopy. Biophys. J. 1996, 70, 1933-1939.
Langmuir 2010, 26(8), 5304–5308
effects on Fn surface structure. Here, we describe our initial investigations into the parameters affecting the adsorption and binding of Fn to mica. Ionic strength was varied for a selection of mono- and divalent cations (Naþ, Ni2þ, Ca2þ) in the adsorption buffer solution, under ambient conditions and pH. In particular, Ni2þ was selected due to its known ability to bind other biomolecules to mica,30 and for its charge-reversal of mica surfaces at low concentrations.34 The resulting AFM topographic images were used to determine surface coverage and to reveal the effect of different ionic conditions on Fn adsorption to mica.
Materials and Methods Soluble human plasma fibronectin (Fn) (Sigma) was used without further purification. Solutions were prepared at a concentration of 1 mg/mL in 10 mM ammonium acetate buffer (pH ∼7.2) and stored at 4 °C. Different adsorption buffers (in 10 mM ammonium acetate) were prepared, each with one of the following electrolytes: 15, 150, or 500 mM NaCl, 15 mM CaCl2, or 15 mM NiCl2. The Fn concentrations in these buffers were either 1 or 0.1 μg/mL (see figure captions). A 100 μL droplet of the Fn in buffer was deposited on freshly cleaved mica, with a deposition time of 10 min. The substrate was then rinsed with 3 mL of 10 mM ammonium acetate buffer and left to dry in a dustfree laminar flow cabinet. All procedures were carried out under a laminar flow cabinet, and all buffers were filtered using 0.2 μm pore filters prior to use. Square sheets (10 10 mm) of muscovite mica were freshly cleaved before each experiment to obtain an atomically clean and smooth surface. AFM images were conducted with a JPK Nanowizard system. All images were obtained in ambient conditions using intermittent-contact (IC) mode. Feedback controls were adjusted accordingly, in order to keep the oscillation amplitude at a fixed value. The set point was typically chosen to be 70% of the free amplitude. Silicon tips (Mikromasch, series NSC15) with a resonance frequency of ∼325 kHz and a force constant of 40 N/m were used. According to the manufacturer, the tips had a radius of curvature of
