Letter pubs.acs.org/NanoLett
Nanoscale E-Cadherin Ligand Patterns Show Threshold Size for Cellular Adhesion and Adherence Junction Formation Stine H. Kristensen,† Gitte A. Pedersen,‡ Lene N. Nejsum,†,‡ and Duncan S. Sutherland*,† †
Interdisciplinary Nanoscience Center (iNANO) and ‡Department of Molecular Biology and Genetics, Aarhus University, Denmark S Supporting Information *
ABSTRACT: The role of ligand spatial distribution on the formation of cadherin mediated cell−cell contacts is studied utilizing nanopatterns of E-cadherin ligands. Protein patches ranging in size from 100 to 800 nm prepared by colloidal lithography critically influence adhesion, spreading, and formation of adherence junctions in epithelial cells. Cells at 100 nm patterns show poor adhesion, while larger pattern sizes show good adhesion, significant spreading, and defined cortical actin. We estimate a threshold of 0.03 μm2 for epithelial cellular attachment via E-Cadherin. KEYWORDS: Protein nanopatterns, adherens junctions, E-cadherin, cell adhesion, sparse colloidal lithography
E
E-cadherin from opposing cells. Even though both AJs and desmosomes are cadherin-based junctions, a clear difference in cadherin density is observed in the two types of junctions. Desmosomal cadherins (Desmocollins and Desmogleins) form a densely packed layer with 17 500 cadherins per μm2,15 whereas the density of the cadherin molecules at AJs is much lower and have been estimated to 700 or 1200 per μm.214 Upon initial cell−cell contact formation, actin is reorganized from actin networks into bundled actin. The formation of AJs is thought to be force dependent through the recruitment of vinculin under applied force.16 The actin cytoskeleton and AJs are proposed as centers of force sensing in cell−cell contacts in analogy to focal adhesions in cell-ECM contacts.16 Nanopattern surfaces offer a number of advantages for studying the properties of specific cellular adhesion processes. Surface patterning can be achieved through various approaches such as microcontact printing, electron beam lithography, and colloidal lithography. However, surface patterns at the nanometer scale are often limited by the fabrication speed, pattern flexibility, and the patterned area. Sparse colloidal lithography permits rapid fabrication of large areas of defined size of protein patches from nanometer to micrometer scale.17 We have previously applied nanopatterning of ECM proteins to study development of focal adhesions.8,18 Here, we perform nanopatterning of the extracellular domain of E-cadherin (E-cad/Fc) to study E-cadherin organization in epithelial cell−cell contact formation. Here, we report a model system with surfaces presenting functional nanoscale domains of the ectodomain of E-cadherin with systematically varied area. The patterned substrates were biofunctionalized with E-cad/Fc in various sizes of smaller
pithelial cellular adhesion complexes with the surrounding extracellular matrix (ECM) and neighboring cells provide critical structures for the transfer of mechanical forces implicated in tissue integrity. Such adhesion complexes are proposed to be regulators of cellular responsivity to their mechanical surroundings and transduction of mechanobiological signals.1,2 Rapid recent developments in understanding of the composition and structure of the ECM contacts through focal adhesion complexes and cell−cell contacts through desmosomes, adherens junctions, and tight junctions have opened a broader view of the function of cellular adhesion complexes.2 They are now thought to be more than mere mechanical contacts but also with functions regulating processes such as proliferation, migration, differentiation,3 and tissue morphogenesis.4 The size of cellular adhesion complexes spans from several tens of nanometers up to micrometers in size. Structural organization of proteins at this length scale is expected to play a crucial role in formation and function of adhesion complexes.5−7 While cell adhesion to the ECM and cellular organization have been studied widely as a function of ligand spacing,7 available adhesive area,8,9 surface rigidity, and surface topography,10,11 fewer studies have looked at the effect of nanoscale ligand presentation in the formation of cell−cell contacts.12 Adherens junctions (AJs) are cell−cell adhesion complexes13,14 and are implicated as key regulators of embryogenesis. One prominent AJ is the zonula adherens, which forms a band just below the tight junctions. A core element of AJs is the cadherin molecules that engage in homophilic binding with cadherin molecules from adjacent cells thereby facilitating cell− cell adhesion. During expansion and completion of cell−cell contacts, cadherin accumulates along the contact and actin is transformed from a network to bundled actin, thereby dampening lamellipodia activity.13 E-cadherin clustering is promoted by lateral interaction between the extracellular domains of © 2012 American Chemical Society
Received: February 7, 2012 Published: March 5, 2012 2129
dx.doi.org/10.1021/nl300514v | Nano Lett. 2012, 12, 2129−2133
Nano Letters
Letter
Sweden, data shown in Supporting Information Figure S1 and Table S2) and quartz crystal microbalance with dissipation (QCM-D) (Q-sense E4, Q-sense AB, Sweden, data and modeling shown in Supporting Information Figure S1 and Table S2). Combination of those two techniques allowed determination of both the adsorbed mass and the effective density of the adsorbed layers.24 Modeling of the data show that the protein layers are highly hydrated (density 1030 kg/m3 corresponding to >90% water) with a characteristic thickness of the combined protein layers of 22 nm. The characteristic thickness of the sequentially deposited layers shows the expected layer-by-layer buildup indicating a correct geometry. To study how the size of E-cadherin substrates affects epithelial cell adhesion and spreading, the epithelial cell line Madin-Darby Canine Kidney (MDCK) stably expressing GFPtagged E-cadherin were seeded onto substrates of nanopatterned or homogeneous E-cad/Fc. After 16 h, cells were fixed and additionally stained to visualize the nucleus and bundled actin, followed by fluorescent microscopy imaging. We utilize an extremely soft rinsing protocol (see method section in Supporting Information) to probe the weak interactions that are likely to occur when the area of adhesion is limited. Large area patterned surfaces were used to enable quantification of cell adhesion and cell area from whole cell populations. Figure 2A shows representative fluorescence microscopy images of cells on 100, 200, 300, and 800 nm protein patches and homogeneous Au surfaces stained with Hoechst (nuclei staining, blue) and phalloidin (actin filaments, red). Very few cells adhered to the SiO2 surfaces, compared to nanopatterns and the homogeneous surfaces. The separate channels for Hoechst and phalloidin are shown in the Supporting Information (Figure S3). To evaluate the ability of cells to adhere to the substrates, total numbers of cells, average cell sizes, and number of cells in clusters were quantified (Figure 2B). A similar number of cells adhered to all the protein patterns, except for the 100 nm patterns, where much fewer cells adhered (104 ± 22 mm−2 for 100 nm vs 329 ± 25 mm−2 for 200 nm), however, the number of cells adhering to 100 nm patterns was significantly higher than cells adhering to the negative control homogeneous PLL-g-PEG coated SiO2 surface (104 ± 22 mm−2 vs 4 ± 3 mm−2 SiO2) A clear difference in cell number is observed between the nanopatterned surfaces and the homogeneous E-cad/Fc surfaces (Figure 2B), which might be due to substantially more E-cad/Fc on the homogeneous surface and/or continuously distributed across the surface. The data presented in Figure 2 are from one experiment done in quadruplicates showing randomly selected regions from five different images. The experiment was repeated three times. Data from cell adhesion for the three repeats is shown in the Supporting Information (Figure S4) The same number of cells were seeded in each experiment although the total amount of cells adhering on the substrates varied somewhat between experiments; however, the clear trend that fewer cells adhered to 100 nm compared to larger size patterns was observed (Supporting Information Figure S2). On 100 nm patterns, approximately 10% of the cells were single cells (Figure 2C). The percentage of single cells decreased linearly with increasing pattern size (Figure 2C) with a concomitant increase of cells in large clusters (more than 4 cells) perhaps reflecting a higher motility of cells and increased incidence of cell−cell interaction. Analysis of size and morphology of single cells revealed changes in cell morphology from homogeneous surfaces (Figure 3A) compared to cells on different size patterns.
patches and the intervening space was passivated through polyethylene glycol chemistry to prevent protein binding and cell attachment.19−21 E-cad/Fc has previously been used in various studies of epithelial cell−cell adhesion and homophilic E-cadherin interaction.4,22,23 To generate dense, short-range ordered arrays of gold holes in a silica film, we utilized sparse colloidal lithography by a process reported elsewhere.12 We combined this with site-specific chemical modification to generate a series of E-cadherin nanopatterns with pattern sizes of 100, 200, 300, and 800 nm in diameter (Figure 1a) as
Figure 1. (A) SEM images of Au patches on a SiO2 background (100, 200, 300, and 800 nm). Scale bars are 1 μm. (B) Schematic of the produced protein pattern. SAMs of alkanethiol (red) provide a hydrophobic chemistry giving strong, nonspecific and irreversible binding of a neutravidin layer (blue), followed by specific binding of biotinylated protein A (green), and finally E-cad/Fc (orange). Preadsorption of Pll-g-PEG prevents protein binding to SiO2 while allowing neutravidin binding to the hydrophobically modified gold surface.
well as homogeneous controls. The Au regions of the surfaces were modified by an alkaneethiol monolayer to have a hydrophobic chemistry and the SiO2 regions were made protein repellent by adsorption of Pll-g-PEG. Neutravidin, biotinylated Protein A, and E-cad/Fc were then sequentially deposited onto the hydrophobic regions of patterns or onto homogeneous hydrophobic surfaces. Figure 1a shows scanning electron microscopy images of 100, 200, 300, and 800 nm Au patches before deposition of protein. Fluorescently labeled E-cad/Fc deposited on 300 and 800 nm patterns are shown in Supporting Information Figure S1. Schematic representation of the produced protein pattern is shown in Figure 1b. Quantified characteristics of the patterns are displayed in the Supporting Information (Table S1). To quantify the adsorption of macromolecules onto the alkanethiol chemistry, we used surface plasmon resonance (SPR) (Biacore X, Biacore AB, 2130
dx.doi.org/10.1021/nl300514v | Nano Lett. 2012, 12, 2129−2133
Nano Letters
Letter
Figure 2. (A) Fluorescent microscopy images of actin filaments stained by phalloidin (red) and nuclei stained by Hoechst (blue). The protein patch size is shown for each sample type. Scale bars are 20 μm. (B) Mean cell number (per mm2). (*) The mean difference is significantly different from homogeneous Au at the 0.05 level. (†) The mean difference is significantly different from 200 nm at the 0.05 level. (C) Mean cluster size of different sample types. Error bars indicate standard error of mean.
retraction fibers localized over the protein patches and the ends of fibers were primarily observed on protein patches. We utilized an apotome imaging system to display optical sections of the adherent cells allowing imaging of those sections closest to the substrate. Figure 4 shows representative fluorescence sections of the cellular E-cadherin-GFP and actin in single cells adherent to different surfaces. In cells on homogeneous surfaces as well as 800 and 300 nm patch sizes, both E-cadherin-GFP and actin were concentrated in well-defined cortical rings. At smaller patch sizes (200 and 100 nm) E-cadherin, and to some extent cortical actin, were observed but with substantially reduced intensity. E-cadherinGFP colocalized with patches of patterned E-cadherin/Fc, however, we were only able to observe this for patch sizes of 300 nm and larger, due to the limited resolution of the microscope. Cells on the smaller patterns (200 and 100 nm) show almost no membrane extension and weak staining for actin. The formation of discrete protein patches separated by protein-free regions means that the global density of E-cad/Fc is substantially lower on the nanostructured surfaces compared to the homogeneous control, however the amounts of protein presented at the different surfaces are relatively constant allowing a direct comparison. For cells adhering to extracellular matrix proteins, it is clear that the local protein concentration is much more important than the global protein concentration in determining cellular responses.9 In our study, we present protein patterns of varying sizes where the global densities of adhesive ligands (800−1300 per um2) are comparable with the density of cadherins found in adherens junction (700− 1200 per um2).14 The protein patterns consist of smaller subclusters of ∼40−2000 E-cadherin molecules (Supporting Information Table S1). Wolfram et al. have showed that there is a minimum ligand spacing of ECM components for formation of cell adhesion complexes, whereas a threshold minimum ligand spacing was not observed for cell−cell adhesive molecules.25 In this study, we focused on the area/number of clustering cadherin molecules required to form stable adhesions. The distance between individual cadherin molecules within each cluster is comparable with that found in adherens junctions, while the distance between each cluster is large compared with the size range investigated by Wolfram et al.25
Figure 3. (A) Fluorescent microscopy images of E-cadherin-GFP expressing cells on different pattern sizes and Au. Patch size is displayed for each image. Scale bars are 20 μm. (B) Calculated mean area of single cells for different pattern sizes and Au surfaces. Error bars indicate standard error of mean. The means difference is significant at the 0.05 level.
Thirty to fifty cells per sample were quantified for single cells on 100, 800 nm, and homogeneous hydrophobic surfaces (Figure 3B). Cells on homogeneous surfaces were spread with large lamellipodia indicating that the cells interact strongly with the surface. Cells seeded onto 800 nm nanostructured surfaces (Figure 3A) were also spread, although to a lesser extent than cells on the homogeneous E-cad/Fc surfaces. The overall morphology of the cells adhering to the 800 nm protein patterned surfaces was more asymmetric compared to the homogeneous surfaces and cells on 800 nm patterns more often had a crescent arc morphology indicating motile cells. Cells adhering to 100 nm E-cadherin patches (Figure 3A) had a spherical morphology, covered a smaller area, and had smaller lamellipodia compared to the cells on both homogeneous surfaces and 800 nm surfaces. Some cells that attached to the protein nanopatterns showed long retraction fibers when imaged by SEM (see Supporting Information Figure S5). The 2131
dx.doi.org/10.1021/nl300514v | Nano Lett. 2012, 12, 2129−2133
Nano Letters
Letter
Figure 4. Fluorescent microscopy images of cellular E-cadherin-GFP (top), bundled actin (middle), and merged actin (red) and E-cadherin-GFP (green) (bottom). The patch size is displayed for each image. Scale bars are 10 μm.
but only allow formation of poorly defined cortical bundled actin. It should be noted that cells in clusters formed clear adherens junctions between the cells. Pairs of cells adherent at the surfaces showed qualitatively similar behavior to single cells albeit with enhanced formation of cortical actin and membrane spreading at the 100 and 200 nm patterns (see Supporting Information Figure S6). We have studied the effect of variation in E-cadherin ligand patch size on epithelial cell behavior and demonstrate a systematic change of cell attachment and morphology with the immobilized ligands. A low level of adhesion and a lack of cell spreading were seen on 100 nm E-cad/Fc patches. For larger patch sizes from 200 nm and up, adhesion was supported and cell spreading with the development of cortical actin was being systematically observed for increased patch sizes. These results point to a critical role of ligand patch size in the formation of cell−cell contacts. We demonstrate a control of cadherinmediated adhesion by materials interfaces with nanopatterned E-cadherin ligands. Homogeneous surfaces gave both higher adhesion and larger spreading indicating that both the local distribution of ligands and the global ligand availability play a role. This experimental system that limits the area/number of cadherins that can form in a single adherens-type junction has the potential to limit the force that can be applied by the cell to that mechanical contact and potentially modify the mechanotransduction events within the adhesion.
We observed a threshold behavior in cellular adhesion with a significantly reduced cell number at 100 nm E-cad/Fc patches compared to larger patch sizes (Figure 2B). We estimate a lower limit of area/number cadherins within an adhesion to give stable attachment at 200 nm patches (corresponding area 0.03 μm2 or ∼17 Cadherin molecules for an adherens type junction). On the homogeneous E-cad/Fc surfaces actin and E-cadherin colocalized in a cortical ring but not in the membrane extension. In this model system, the cells do not have access to adhesion mechanisms based on integrin mediated binding to extracellular matrix proteins via focal adhesions during the initial attachment but the substrates provide a high density of E-cadherins for binding. Adam et al. have shown that E-cadherin localizes along cell− cell contacts in spatial discrete microdomains and these microdomains are associated with a bundle of actin.26,27 They proposed that the strength of the contact was increased by spacing multiple puncta along the length of contact. The size of adherens junctions has previously been used to estimate the level of force that can be applied by cells.23 The formation of adherens junctions has also been shown to be force dependent and they were observed to grow in response to mechanical force.23 We observed well-defined cortical actin in single cells at homogeneous surfaces. Such actin structures while also present in the larger (300 and 800 nm) pattern sizes were less organized and were only poorly stained at the smallest patch sizes (200 and 100 nm). For cells adherent at our surfaces, the patterning of the E-cad/Fc limits the size of the regions of the substrate where direct mechanical contacts can be formed. We estimate an upper limit number of E-cadherins that could form in an adherens type junction above a single patch. The 100 and 200 nm patches could support AJs with 6 or 17 cadherins, respectively. The 200 nm patterns with up to 17 cadherins in AJs apparently can support sufficiently strong mechanical contacts to the substrate to allow cell attachment (see Figure 2b)
■
ASSOCIATED CONTENT
S Supporting Information *
Detailed experimental methods, SPR data, QCM data, characteristic data for substrates, fluorescence images of 800 nm E-cadherin-Fc pattern, data and from full repeats, and SEM images of cells interacting with 800 nm nanopatches. This material is available free of charge via the Internet at http://pubs.acs.org. 2132
dx.doi.org/10.1021/nl300514v | Nano Lett. 2012, 12, 2129−2133
Nano Letters
■
Letter
(19) Michel, R.; Pasche, S.; Textor, M.; Castner, D. G. Influence of PEG architecture on protein adsorption and conformation. Langmuir 2005, 21, 12327−12332. (20) Pasche, S.; De Paul, S. M.; Voros, J.; Spencer, N. D.; Textor, M. Poly(L-lysine)-graft-poly(ethylene glycol) assembled monolayers on niobium oxide surfaces: A quantitative study of the influence of polymer interfacial architecture on resistance to protein adsorption by ToF-SIMS and in situ OWLS. Langmuir 2003, 19, 9216−9225. (21) Raynor, J. E.; Capadona, J. R.; Collard, D. M.; Petrie, T. A.; Garcia, A. J. Polymer brushes and self-assembled monolayers: Versatile platforms to control cell adhesion to biomaterials(Review). Biointerphases 2009, 4, FA3−FA16. (22) Cavey, M.; Rauzi, M.; Lenne, P.-F.; Lecuit, T. A two-tiered mechanism for stabilization and immobilization of E-cadherin. Nature 2008, 453, 751−756. (23) Yamada, S.; Pokutta, S.; Drees, F.; Weis, W. I.; Nelson, W. J. Deconstructing the cadherin-catenin-actin complex. Cell 2005, 123, 889−901. (24) Reimhult, E.; Larsson, C.; Kasemo, B.; Hook, F. Simultaneous surface plasmon resonance and quartz crystal microbalance with dissipation monitoring measurements of biomolecular adsorption events involving structural transformations and variations in coupled water. Anal. Chem. 2004, 76, 7211−7220. (25) Wolfram, T.; Spatz, J. P.; Burgess, R. W. Cell adhesion to agrin presented as a nanopatterned substrate is consistent with an interaction with the extracellular matrix and not transmembrane adhesion molecules. BMC Cell Biol. 2008, 9. (26) Adams, C. L.; Chen, Y. T.; Smith, S. J.; Nelson, W. J. Mechanisms of epithelial cell-cell adhesion and cell compaction revealed by high-resolution tracking of E-cadherin-green fluorescent protein. J. Cell Biol. 1998, 142, 1105−1119. (27) Adams, C. L.; Nelson, W. J.; Smith, S. J. Quantitative analysis of cadherin-catenin-actin reorganization during development of cell-cell adhesion. J. Cell Biol. 1996, 135, 1899−1911.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We thank Jacques Chevallier and Folmer Lyckegaard for performing sputtering and evaporation. This work was funded through the Danish research council (Sags no. 09-065900) to D.S.S. and a Lundbeck Foundation Junior Group Leader Fellowship to L.N.N..
■
REFERENCES
(1) Geiger, B.; Spatz, J. P.; Bershadsky, A. D. Environmental sensing through focal adhesions. Nat. Rev. Mol. Cell Biol. 2009, 10, 21−33. (2) Leckband, D. E.; le Duc, Q.; Wang, N.; de Rooij, J. Mechanotransduction at cadherin-mediated adhesions. Curr. Opin. Cell Biol. 2011, 23, 523−530. (3) Geiger, B.; Yamada, K. M. Molecular Architecture and Function of Matrix Adhesions. Cold Spring Harbor Perspect. Biol. 2011, 3. (4) Zhang, Y. X.; Sivasankar, S.; Nelson, W. J.; Chu, S. Resolving cadherin interactions and binding cooperativity at the single-molecule level. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 109−114. (5) Castner, D. G.; Ratner, B. D. Biomedical surface science: Foundations to frontiers. Surf. Sci. 2002, 500, 28−60. (6) Kasemo, B. Biological surface science. Surf. Sci. 2002, 500, 656− 677. (7) Wilson, C. J.; Clegg, R. E.; Leavesley, D. I.; Pearcy, M. J. Mediation of biomaterial-cell interactions by adsorbed proteins: A review. Tissue Eng. 2005, 11, 1−18. (8) Malmstrom, J.; Lovmand, J.; Kristensen, S.; Sundh, M.; Duch, M.; Sutherland, D. S. Focal Complex Maturation and Bridging on 200 nm Vitronectin but Not Fibronectin Patches Reveal Different Mechanisms of Focal Adhesion Formation. Nano Lett. 2011, 11, 2264−2271. (9) Deeg, J. A.; Louban, I.; Aydin, D.; Selhuber-Unkel, C.; Kessler, H.; Spatz, J. P. Impact of Local versus Global Ligand Density on Cellular Adhesion. Nano Lett. 2011, 11, 1469−1476. (10) Teixeira, A. I.; Abrams, G. A.; Bertics, P. J.; Murphy, C. J.; Nealey, P. F. Epithelial contact guidance on well-defined micro- and nanostructured substrates. J. Cell Sci. 2003, 116, 1881−1892. (11) Dalby, M. J.; Riehle, M. O.; Johnstone, H.; Affrossman, S.; Curtis, A. S. G. In vitro reaction of endothelial cells to polymer demixed nanotopography. Biomaterials 2002, 23, 2945−2954. (12) Denis, F. A.; Hanarp, P.; Sutherland, D. S.; Dufrêne, Y. F. Nanoscale Chemical Patterns Fabricated by Using Colloidal Lithography and Self-Assembled Monolayers. Langmuir 2004, 20, 9335−9339. (13) Halbleib, J. M.; Nelson, W. J. Cadherins in development: cell adhesion, sorting, and tissue morphogenesis. Genes Dev. 2006, 20, 3199−3214. (14) Harris, T. J. C.; Tepass, U. Adherens junctions: from molecules to morphogenesis. Nat. Rev. Mol. Cell Biol. 2010, 11, 502−514. (15) Al-Amoudi, A.; Diez, D. C.; Betts, M. J.; Frangakis, A. S. The molecular architecture of cadherins in native epidermal desmosomes. Nature 2007, 7171. (16) Yonemura, S.; Wada, Y.; Watanabe, T.; Nagafuchi, A.; Shibata, M. alpha-Catenin as a tension transducer that induces adherens junction development. Nat. Cell Biol. 2010, 12, 533−U35. (17) Hanarp, P.; Sutherland, D. S.; Gold, J.; Kasemo, B. Control of nanoparticle film structure for colloidal lithography. Colloids Surf., A 2003, 214, 23−36. (18) Malmstrom, J.; Christensen, B.; Jakobsen, H. P.; Lovmand, J.; Foldbjerg, R.; Sorensen, E. S.; Sutherland, D. S. Large Area Protein Patterning Reveals Nanoscale Control of Focal Adhesion Development. Nano Lett. 2010, 10, 686−694. 2133
dx.doi.org/10.1021/nl300514v | Nano Lett. 2012, 12, 2129−2133
