Invited Feature Article pubs.acs.org/Langmuir

Molecularly Engineered Surfaces for Cell Biology: From Static to Dynamic Surfaces J. Justin Gooding,*,†,‡ Stephen G. Parker,†,‡ Yong Lu,†,‡ and Katharina Gaus†,§ †

The Australian Centre for NanoMedicine, ‡School of Chemistry, and §Centre for Vascular Research, The University of New South Wales, Sydney 2052, Australia ABSTRACT: Surfaces with a well-defined presentation of ligands for receptors on the cell membrane can serve as models of the extracellular matrix for studying cell adhesion or as model cell surfaces for exploring cell−cell contacts. Because such surfaces can provide exquisite control over, for example, the density of these ligands or when the ligands are presented to the cell, they provide a very precise strategy for understanding the mechanisms by which cells respond to external adhesive cues. In the present feature article, we present an overview of the basic biology of cell adhesion before discussing surfaces that have a static presentation of immobile ligands. We outline the biological information that such surfaces have given us, before progressing to recently developed switchable surfaces and surfaces that mimic the lipid bilayer, having adhesive ligands that can move around the membrane and be remodeled by the cell. Finally, the feature article closes with some of the biological information that these new types of surfaces could provide.

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INTRODUCTION Cells respond to their environment, and how they react often determines their fate.1,2 Hence there is great interest in understanding how cells sense their surroundings and identifying which as well as how; environmental cues govern cell-fate decisions. Cell responses can be elicited by soluble chemicals (soluble cues) or the physical properties of surface-bound cues (physical cues). The latter are derived from the arrangement of proteins and other biomolecules of the extracellular matrix (ECM), the topography and rigidity of the substratum, and applied mechanical forces. The consequence of these physical cues can be changes in gene expression, phenotype, morphology, cell mobility, and eventually cell fate. Almost all cell types, including prokaryotes, respond in such a manner, but both the physical cues and cellular responses are incredibly complex. A consequence of this complexity is the difficulty in deconvoluting the roles of different cues present in natural materials and assigning specific cues to specific cellular outcomes. Hence researchers have turned to model surfaces and thus gain much greater control over the presentation of environmental cues.3 Being able to design surfaces that can give us insight into how cells perceive various physical cues requires precise surface engineering.3 For example, for cell adhesion, a surface must be designed with cell-adhesive ligands on a cell-inert background.4 The cell-inert background is typically achieved with an oligo- or poly(ethylene glycol) layer to which adhesive ligands can be attached. However, the spacing of these ligands must be tightly defined because, as we will discuss later, changes in ligand spacing of a few nanometers can influence the cell adhesion quite dramatically.2 Work on model surfaces initially concentrated on © XXXX American Chemical Society

protein adsorption or polymer-modified surfaces where the celladhesive ligands were attached to or incorporated within the adsorbed protein or polymer.3 However, the conformational complexity of polymers creates some ambiguity regarding whether all attached adhesive ligands are present on the cell and hence an ambiguity regarding ligand spacing.3 As a consequence of this ambiguity, Whitesides and coworkers5 turned to self-assembled monolayer (SAM)-modified gold surfaces to provide better-defined model surfaces for studying cell−surface interactions. The key development here was to show that components in a monolayer containing oligo(ethylene glycol) moieties resist the nonspecific adsorption of proteins and cells.4,6 Ligands that promote cell adhesion can be attached to such moieties, the most common of which is the tripeptide RGD that binds cell−surface receptors called integrins.7 In this way, a very well defined presentation of celladhesive ligands can be fabricated. Because self-assembled monolayers can be formed with multiple components, monolayers could be prepared in which the cell-adhesive ligands were diluted with another antifouling component so that the average density and spacing of the adhesive ligands could be controlled.8 Hence, the impact of the spacing of cell-adhesive ligands can be evaluated. Similarly, by changing the type of adhesive ligand, their accessibility or the topography of the underlying surface can allow the effect of other parameters to be explored. Received: September 30, 2013 Revised: November 13, 2013

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Figure 1. Adhesion dynamics. (A) Nascent adhesions initially form in the lamellipodium, in which the ARP2/3 complex creates a dendritic actin network. Nascent adhesions either disassemble or elongate in the transition zone between the lamellipodium and lamellum. Adhesion maturation to focal complexes and focal adhesions is accompanied by the bundling of actin filaments (e.g., by α-actinin) and actomyosin contractility (e.g., myosin II activity). (B) Models of adhesion nucleation, assembly, maturation, and signal regulation.

decreases to allow contact with the surface.10 This contact typically involves the engagement of surface ligands with receptors on the cell surface. The best known and most studied cell−surface interactions are between specific peptide ligands, such as the RGD peptide sequence7 and the PHSRN synergy peptide1 found, for example, in fibronectin or the YIGSR and IKVAV sequences of laminin and integrins. Integrins are a diverse family of transmembrane heterodimeric receptors composed of two high-molecular-weight polypeptide chains, the α and β subunits. Many α and β subunits have been identified, and different combinations give rise to 24 different receptors, many of which bind to the RGD peptide motif. However, integrins lack enzymatic activity so that the engagement of ligands is transmitted to intracellular signaling processes via conformational changes (outside-in signaling) that reveal a short tail in the cytoplasm side of the integrin that allows the formation of multimolecular complexes and linkage to the actin cytoskeleton.2 Alternatively, intracellular rearrangements can prime integrins for engagement with extracellular ligands (insideout signaling). Both processes facilitate the anchorage of the intracellular cytoskeleton to the extracellular substrate and allow integrins to transduce mechanical forces. Although the integrin−actin linkage has been intensely studied,11 the hierarchy of the integrin network and the multiple interactions within the structure are highly complex and probably functionally diverse.12 The connection between the actin and the integrins involves a number of other proteins2 such as talin,13 which binds to the cytoplasmic tail of integrins (and causes their activation through conformational changes), and vinculin,14 which forms a bridge between talin on one end (N terminus of talin) and filamentous actin on the other. Adhesion proteins are intimately linked to actin-regulating proteins, such as α-actinin that cross-links actin filaments and ARP2/3 that initiates the growth of new actin filaments from an existing filament at an angle of 70°, thus creating a branched actin network. The engagement of a single integrin does not typically anchor the cell to a surface, and in fact, other integrins are needed to cluster together to form an adhesion site, thus triggering the recruitment of a range of scaffold and signaling proteins. Two relevant proteins in this regard are paxillin,15 which is a scaffold protein, and the signaling protein, focal adhesion kinase (FAK).16 Signals from both newly formed and more stable adhesions regulate Rho

It is this surface construct of covalently attached cell−adhesive ligands, on an otherwise inert monolayer, that is the foundation upon which molecularly engineered surfaces for cell biology are built.9 This feature article focuses on biological information that such surface constructs can provide. There are in fact two demarcations made in the article: one between static and dynamic surfaces and the other between immobile and mobile cell−adhesive ligands. The adjective static here is used to refer to surfaces where the molecules by which the surface is modified do not change their position or accessibility, whereas in dynamic surfaces external stimuli such as light and electrical potential are used to allow the accessibility of the cell-adhesive ligands to change or the adhesive ligands to be completely removed such that surfaces can capture or release cells. With regard to mobile and immobile ligands, immobile ligands here refers to the situation where the molecules of the cell-adhesive ligands are anchored to the surface such that the ligands are immobile in space relative to each other. Surfaces with immobile ligands can be thought of as analogous to cell adhesion to the ECM. With mobile ligands, the ligands are attached to molecules that are free to move across the surface such that the cell can alter the spatial organization of the ligands. The most common surfaces with this feature are lipid bilayers, to which cell-adhesive ligands are attached to one leaflet. The biological analogy here is the interaction of receptors and ligands during cell−cell contacts. Because the emphasis is on very well defined surfaces, this feature article is limited to monolayers and bilayer systems. We will first provide an overview of the basic biology of cell adhesion, followed by a summary of what static molecularly engineered surfaces have taught us about cell adhesion. We will then discuss dynamic surfaces that first control the accessibility of cell− surface ligands and second can be used for the capture and release of cells. Then we will discuss the current state of research in developing surfaces with mobile ligands before providing a guide to future studies in which biological questions are integrated into the design of dynamic surface and surfaces with mobile ligands to advance this trans-disciplinary area of research.

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BRIEF OVERVIEW OF CELL ADHESION When a cell approaches an adhesive surface, it is initially held about 50 nm from the surface because of the glycosylation of the plasma membrane. Within a few minutes, however, the spacing B

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GTPase activation17 that in turn regulates cytoskeletal and adhesion assembly and organization.11,18 The complexity does not stop here. Further signaling and adaptor proteins are recruited, activated, and reorganized. In fact, there are more than 180 in the so-called adhesome, and an understanding of how specific functions such as signaling and migration emerge from the interactions between these proteins is still a long way from being fully understood.11 Hence, investigating how cells sense and respond to adhesive ligands may also provide key insights into how the adhesome is organized. The clustering of proteins at the site of integrin engagement with the ECM is the beginning of the formation of a focal adhesion. Focal adhesions are important both in anchoring cells to the ECM and in cell migration. Hence adhesion formation, maturation, and disassembly is a continuous and well-regulated process.11 In fibroblasts, the process begins with small, shortlived nascent adhesions in the lamellipodium close to the leading edge that either turn over rapidly (∼60 s) or mature to larger dotlike focal complexes at the lamellipodium−lamellum interface that persist for several minutes and can elongate into dashlike 3− 10-μm-long focal adhesions that reside at the ends of large actin bundles or stress fibers (Figure 1A).19 ATP-dependent molecular motors, typically myosin II, bind across actin filaments to shorten the bundles and create tension in the cytoskeleton of the cell. This is referred to as actomyosin contractility. Because of the incredible complexity of focal adhesion formation, key questions about adhesion nucleation, assembly, maturation, and signal regulation have remained unanswered (Figure 1). For example, it is unclear what determines the localization and lifetimes of adhesions. Are adhesion complexes partially preformed? In the literature, there are currently two models for nascent adhesion nucleation.11 In the first model, nucleation of adhesion is initiated when ligand-bound integrins cluster together. This model is supported by the observation that integrin ligands, or anti-integrin antibodies, coupled to beads induce the clustering of adhesion components around the bead.20 In the second model, assembly is initiated by actin polymerization and uses dendritic actin as the template for the nucleation of adhesion complexes. Evidence for this model comes from reports showing that before adhesion formation vinculin and FAK bind directly to ARP2/3 complexes that are responsible for dendritic actin meshwork in the lamellipodia.21 These fundamental questions in adhesion biology have remained unanswered because the traditional tools of overexpression and underexpression of specific proteins can indicate only whether a protein is required; they can rarely reveal the intricacy of dynamic processes. Here model surfaces with molecularly controlled engineering of the adhesive environment have a distinct advantage. This is especially true for dynamic surfaces that provide temporal control of ligand presentation and thus a clear start signal for the biological reactions that result in cell spreading and migration.

of cell-adhesive ligands may be known, it is less certain whether all ligands are accessible to the cell and present in the same orientation. Roberts et al. introduced the basic surface construct for model surfaces to explore cell adhesion in 1998.5 The surface was modified with a mixed self-assembled monolayer (SAM) of a protein- and cell-resistant layer, oligo(ethylene oxide), and an oligo(ethylene oxide) containing a cell-adhesive ligand (the RGD peptide) on its distal end (Figure 2A). By varying the ratio of these two components, the number of cell-adhesive ligands on the surface is varied such that the impact of the density of cell− surface ligands on cell adhesion can be explored. Importantly, the

Figure 2. Ligand spacing. (A) Examples of surface chemistry and respective cell images of surfaces that provide control over the spacing of cell-adhesive ligands that are presented to cells. (A) Schematic of the surfaces used by Mrksich and colleagues8 where cell-adhesive ligands are attached to an otherwise cell-inert oligo(ethylene oxide) layer. Spacing is controlled by altering the concentration of two components incorporated into the SAM. (B) Schematic of the surface adapted by Spatz and colleagues22 from del Campo and colleagues23 for the control of ligand density presented to cells. A glass surface was modified with gold nanoparticles to which RGD ligands were attached. By controlling the density of gold nanoparticles, the density of ligands is controlled. The space between the nanoparticles was modified with oligo(ethylene oxide) molecules as a cell-inert layer. (C) 3T3 fibroblast cell with the staining of actin stress filaments adhered to a surface as shown in part A. (D) MC3T3-E1 osteoblast cultured for 24 h on a surface as shown in part B. Parts A and C are reprinted from ref 8. Copyright 2001, with permission from Elsevier. Parts B and D are reprinted (adapted) with permission from ref 35. Copyright 2009, American Chemical Society.

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SELF-ASSEMBLED MONOLAYER-MODIFIED SURFACES WITH WELL-DEFINED PRESENTATION OF IMMOBILIZED STATIC LIGANDS Surface Considerations. The basic principle of surface design for studies of cell adhesion is to create a cell-inert surface with a well-defined number of cell-adhesive ligands in an unambiguous manner. This latter differentiates what we are referring to as molecularly engineered surfaces over similar surfaces that employ polymers or protein absorption. In the case of protein absorption and polymers, although the average density C

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scopes. However, although gold is probably the easiest surface on which to produce a well-defined presentation of cell-adhesive ligands, it is not particularly compatible with high-resolution fluorescence microscopy because of its quenching of fluorescence at up to 10 nm from the surface. Kandere-Grzybowska et al.33 partially addressed this issue to enable TIRF microscopy by forming gold islands on a glass surface. These surfaces are rather similar to the surfaces of the Spatz group, who have also used TIRF microscopy.31 The quenching issue of gold is one of the reasons that we turned to silicon surfaces,24,25 but this substrate is not transparent and thus cannot be used for TIRF or superresolution techniques. Hence, well-defined surfaces on glass or other transparent surfaces would be ideal, and here indium tin oxide may be a suitable alternative because well-defined SAMs can be formed from organophosphonates.34 What Have Surfaces with Static, Immobile Ligand Presentation Taught Us about Cell Adhesion? One of the important discoveries made with molecularly engineered surfaces is that fibroblasts, osteoblasts, and B16-melanocytes spread well on surfaces with 70 nm spacing where cell spreading and adhesion are highly restricted. This was attributed to insufficient integrin clustering to form focal adhesions. The spacing is consistent with the hypothesis that the cellular adhesion apparatus has its own internal periodicity of 67 nm.35 It is also consistent with the periodicity of extracellular matrix protein fibronectin and collagen fibers.22 More recently, Wind and coworkers36 used surfaces with RGD peptides on nanoparticles that clustered four engaged integrins within ∼60 nm and facilitated cell spreading. Because talin has four integrin binding sites, it is possible that at least four integrins are needed to engage talin. Importantly, with a ligand spacing of less than 70 nm, it did not matter whether the ligands were spaced in an ordered array or in a random fashion, but at greater average spacing, the ligand arrangement did matter. Presumably on surfaces with a random distribution of ligands, cells can adhere in locations where there is a sufficiently high local density of ligands, although this is not the case in an ordered ligand arrangement. Similarly, Arnold et al.37 varied the number of gold nanoparticles and hence the number of adhesive ligands per cell to ask how many adhesive sites for individual integrins are necessary for a stable cell-adhesion site and hence the inducement of outside-in signaling. The answer was six because MC3T3 osteoblasts plated onto these surfaces elongated and had a tendency to migrate toward higher ligand densities. What these studies show is the amazing sensitivity of cells to changes in spacing of integrin coupling points. Precisely how sensitive was mapped by a later study by Spatz and coworkers.38 Surfaces were fabricated with a gradient of nanoparticle coupling points across the surface. The shallowest gradient had a variation in ligand spacing of ∼15 nm per mm (including interparticle spacings of 58−73 nm, in the physiologically relevant range), which equates to a change in ligand spacing of 1 nm across a cell 60 μm long, and yet the cells could still sense the gradient. Kato and Mrksich29 compared linear RGD to cyclic RGD that bound more strongly and gave twice as many focal adhesions per cell for 3T3 Swiss fibroblasts than linear RGD although the size of the focal adhesions was smaller. One of the biological points of interest in this study is that with fewer RGD peptides (a decrease in the percentage of the cyclic RGD from 1 to 0.1%) or loweraffinity ligands (linear RGD vs cyclic RGD) there were fewer focal adhesions and they were found mostly toward the cell interior. On the basis of their results, Kato and Mrksich29

surfaces give a random distribution of cell-adhesive ligands and hence only an average ligand density or spacing. There are variants of this construct formed on surfaces other than gold, such as silicon,24,25 but the same basic features are retained. Some subtleties in surface design that should be considered are worth pointing out. For example, Houseman and Mrksich8 have shown that the number of cells that adhere and the area over which they spread are dependent on the microenvironment of the immobilized RGD ligands. This was achieved by using the same cell-adhesive molecule in Figure 2A but with the oligo(ethylene oxide) portion of the cell-inert component changing in length from three ethylene oxides to four, five, and six. As the oligo(ethylene oxide) increased in length, the number of adherent cells and their spreading area decreased. How far the RGD ligand protrudes above the surface can also be important as indicated by Beer et al.,26 who showed that increasing the number of glycines between the RGD ligand and a polyacrylonitrile bead to which a cell attaches (i.e., increasing the linker length to up to nine glycine residues) increased the cell binding. An important negative control in such cell-adhesion measurements is the addition of peptides that do not facilitate cell binding. The most commonly used peptide for this purpose is RGE, for which integrins have no affinity but carries the same charge as RGD.27 Although the original aim of the mixed SAMs strategy was to assemble inert and adhesive molecules on the surface in a simple manner, differences between the two types of molecules, such as their relative solubilities, means the ratio in solution may not exactly reflect the ratio on the surface.28 Hence, it is more common that the surfaces are prepared by assembling two very similar oligo(ethylene oxide) molecules on the surface followed by coupling of the cell-adhesive ligand to one oligo(ethylene oxide) species24,25,29 to give a more reliable estimate of the ligand density. An alternative strategy for preparing highly engineered surfaces for cell-adhesion studies was introduced by Spatz and colleagues.22,30,31 They use diblock copolymer micelles that contain gold nanoparticles that are deposited on glass coverslips. Removal of the polymer with hydrogen plasma leaves the gold nanoparticles on the surface, which can then be modified with RGD peptides. The bare glass between the gold nanoparticles is filled with poly(ethylene glycol) polymer as an inert surface (Figure 2B). The size of the copolymer then dictates the spacing of gold nanoparticles and hence cell-adhesive ligands in the range of 10−200 nm.31 The power of this approach is that imaging allows the experimentalist to know exactly where the RGD ligands are because they are located only on the nanoparticles. In addition, because the nanoparticles can be arranged in an ordered array on the surface or in a random fashion,22 gradients can be formed, and because the size of the gold nanoparticles can easily be altered, the surface can be tuned to have from one RGD ligand per particle up to a cluster of ligands. Referring to the discussion above, as integrins cluster in a focal adhesion, having several RGD ligands on a single particle can be used to explore the biology of integrin cross-linking. It is prudent to make a statement regarding gold as the surface material. In most, if not all, studies of cell−surface interactions, fluorescence microscopy is the tool of choice because the cell phenotype, cytoskeleton organization, and focal adhesions are easily examined. The advent of total internal reflectance (TIRF) microscopy and super-resolution fluorescence microscopy techniques can provide much greater detail, including quantification at the single-protein level32 that far exceeds the information on traditional confocal and epi-fluorescence microD

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modified silicon surfaces with well-defined surface chemistry but with different roughnesses.24 The roughness was generated by etching the silicon with potassium hydroxide, which created pyramids on the surface. In this way, we could keep the same surface density of RGD ligands to explore whether cell adhesion was due to the topography or more adhesion points. Our results pointed to fewer BAECs adhering to rougher surfaces, whereas the biphasic trend in cell adhesion observed for flat surfaces, with optimal signaling and cell adhesion with 44 nm ligand spacing, remained regardless of the topography. The results are important from two perspectives. The influence of ligand spacing regardless of topography means that the knowledge gained from model surfaces regarding ligand density can be translated to any type of surface independently of its roughness. Second, the results suggest a dual mechanism of cell adhesion where the initial contact of the cells with the surface may be controlled by the topography but the engagement of cell surface receptors is solely determined by the surface chemistry.

proposed a two-step model for cell adhesion based on the nucleation and growth of focal adhesions. They suggested that nucleation occurs when two or more mobile receptors in the cell membrane cluster together and are prevented from disassociation by the recruitment of proteins such as talin and vinculin and the binding of actin filaments. Growth is facilitated by the recruitment of other receptors and cytoskeletal elements to form a mature focal adhesion. In this model, stronger-binding ligands cause a higher frequency of nucleation sites and hence more, but smaller, focal adhesions. Importantly, in this paper the authors also presented evidence of the functional importance of cells adhering to model substrates using Western blotting to quantify the phosphorylation of FAK. The results suggested that the initiation and rate of FAK signaling were approximately the same for both ligands, suggesting that the mechanisms of outside-in signaling may be independent of the conditions of adhesion nucleation. Our own work25 continues on from these studies but extends the importance of the distribution of cell-adhesive ligands to specialized cell functions. Using silicon surfaces modified with RGD peptides attached to a cell-inert surface, we explored the effect of ligand density on the adhesion, migration, and signaling activity of bovine aortic endothelial cells (BAECs) to gain insight into the transformation of endothelial cells into a proliferative and invasive phenotype that is characteristic of angiogenesis. We saw a biphasic response for all parameters measured, where with increasing average ligand density, more cells adhered, there was greater spreading, more focal adhesions and longer focal adhesions were observed until an optimum was reached, and with higher ligand densities all parameters decreased again. The interesting aspect was that different ligand densities were optimal for different cellular functions. We observed more spread cells, with more focal adhesions and longer focal adhesions, on surfaces with an average ligand spacing of 44 nm, consistent with the observations of Spatz and co-workers.31 Similarly, focal adhesions were most ordered (quantified using a high-resolution fluorescence microscopy technique39 that investigated the fluidity of the cell membrane) and signaling induced by the vascular endothelial growth factor (VEGF) was most efficient at this average ligand density. In contrast, migration speeds were highest at much larger average ligand spacings. However, stimulation of the cells from quiescent to invasive/migratory with VEGF shifted the optimal ligand spacing for migration to 44 nm, where signaling was optimal. Taken together, the data suggest that although adhesive cues and growth factors induce endothelial cell migration, for this cell type the addition of soluble growth factor dominates the ligand presentation on the surface. Hence in this study, even with a relatively simple static ligand surface architecture, the model surfaces allowed us to delineate between growth factor and integrin signaling even though the two processes depend on each other and employ many of the same proteins. Apart from the ligand density, there is a large body of literature that looks at topographical effects on cell adhesion primarily concerned with guiding cell growth and the design of implants. This literature in itself is quite contradictory, with some studies suggesting that cell adhesion is promoted by topographical features and others suggesting the opposite.40 It is certainly clear that the observations are dependent to some extent on the cell type, type of topographical feature, size of features, and substrate materials.40 However, few of these studies employ controlled surface chemistry, so a rougher surface also has more adhesive points. In an attempt to add clarity to this debate, we prepared

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IMMOBILE LIGANDS AND DYNAMIC SURFACES Dynamic biomimetic substrates have the potential to mimic not only cell−ECM interactions but also the time-dependent variations in the ECM. These are surfaces whose properties can be altered upon application of a stimulus so that they can be used as a “starting gun” to monitor the processes that lead to cellular responses, such as the formation of focal adhesions, ruffling of the cell membrane, organelle realignment, and polarization as cells adhere, migrate, and detach from a surface. The application of an external stimulus such as temperature, light, or electrochemistry typically controls the presentation of a surface cue that enables, or prevents, cell adhesion. This section of the review will focus on how current approaches to monitoring the behavior of cells on dynamic surfaces has the potential to help explain how cells respond to changes in surfaces upon which they interact. Dynamic Surfaces to Capture and Release Cells. Some of the initial applications of dynamic surfaces were to capture and release cells controllably. The capturing of the cells was optimized by using the knowledge discussed in the previous section with regard to the type of cell-adhesive ligand and its spacing. Revzin and co-workers41 developed a simple surface strategy for capturing and releasing cells based on adopting the use of antibodies to promote cell adhesion. Gold microelectrodes were first fabricated on glass slides using typical photolithography methods. The exposed glass regions were coated with poly(ethylene glycol) (PEG) polymer to resist cell adhesion, and the gold electrode surfaces were modified with mercaptoundecanoic acid (MUA), which allowed the T-cell specific antibody, antihuman CD4 antibody, to be conjugated to the surface via EDC/NHS coupling chemistry. Molt-3 cells (CD4 positive) were then incubated on the surface to bind to the antihuman CD4 antibody tethered to the Au microelectrodes while the PEG hydrogel restricted nonspecific adsorption. By applying a sufficiently reducing potential, it was possible to desorb the SAM electrochemically, leading to the detachment of the cells. Because each microelectrode was independently addressable, cell release for individual microelectrodes could be achieved independently of the other electrodes (Figure 3). This independence generates multiple defined time points in which to monitor the shape of the cells as they are detached from the surface or the time scales of activation of the T cells by the antibodies. E

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adsorption. The differently modified regions, as shown in Figure 4A, were responsive to an applied potential. The monolayers within the red regions in Figure 4A underwent cleavage and subsequent cell release at −650 mV whereas cleavage in the blue region occurred at 650 mV. The central gray region contained a noncleavable unit and acted as a control to show that cells are being released as a result of the cleavage of the underlying monolayer, not as a direct result of the applied potentials. Being able to detach a subset of cells in different regions at different potentials allows for the controlled temporal release of smaller regions of cells. The images in Figure 4B were recorded only 5 min after the application of the potentials with no postcleavage washing procedure to assist in the detachment of the cells, apart from exchanging the media in which to record the images. The rapid nature of cell detachment from the surface suggests that the destruction of formed focal adhesions is potentially an instantaneous event as the cell-adhesive RGD ligand is removed. To learn more about the time frame in which other events such as surface ruffling and cell conformation (including organelle realignment and polarization) occur, surfaces that not only can control the release of cells from a surface temporally but also can control the migration of cells across it with precise time definition are required. Dynamic Surfaces that Temporally Control the Migration of Cells. Irreversible Switching. Surfaces similar to the capture-and-release surfaces described above can be used to initiate cell migration with a high degree of temporal control. Whitesides and co-workers45 used the same strategy as Revzin for the reductive desorption of alkanethiols but for initiating cell migration. In this procedure, cell-adherent regions of HS(CH2)17CH3 (C18) molecules were patterned onto a gold surface using microcontact printing (μCP) with cell-resistant HS(CH2)11(OCH2OCH2)3OH(C11EG3) molecules being used to cover the remaining surface. Bovine capillary endothelial (BCE) cells were incubated on the surface and were confined to the C18 regions with no cells on the C11EG3 regions. Cell-resistant C11EG3 was reductively desorbed from the gold surface at lessnegative potentials than in the cell-adherent regions and hence could be selectively removed at a defined time to initiate BCE cell migration across the newly cell-adherent regions. These surfaces were used to compare the effectiveness of various drugs at inhibiting the migration of BCE cells (Figure 5B). An

Figure 3. Molt-3 cells (CD4+) were plated on the entire ABfunctionalized surface. The top left and bottom right electrodes were activated, resulting in the release of the cells while the central electrode did not have −1.2 V applied to it and so it remained fouled by the cells. Reprinted from ref 41. Copyright 2008, with permission from Elsevier.

There have been several more sophisticated strategies developed to capture and release cells. Most of these employ cleavable molecules in the monolayers rather than the entire monolayer as in the Revzin case.23,42−44 Of these, the surfaces of most value are those that allow the release of cells in defined regions with well-defined time scales of release. Mrksich and coworkers42 developed a gold surface that contained patterns of differently modified regions. The predetermined regions were generated by placing a thin PDMS membrane with holes (500 μm in diameter) on top of a gold-coated glass slide to form wells. Three different solutions of maleimide-containing disulfides (used instead of thiols to avoid intermolecular maleimide−thiol interactions), mixed with tri(ethylene glycol)-terminated disulfides, in a ratio of 1:200 were applied to the wells (Figure 4A). The PDMS membrane was then removed, and the surface was immersed in an ethanolic solution of tri(ethylene glycol)terminated disulfide to passivate the regions between the holes, completing the assembly of the monolayer. RGD-containing peptides were then immobilized on this monolayer by treatment with a solution of CGRGDS peptide, where the thiol within the cysteine attached to the maleimide groups present within the circles. Swiss 3T3 fibroblast cells were allowed to attach to these circles by adhering to the RGD ligand, and the tri(ethylene glycol) patterned between the circles prevented nonspecific

Figure 4. (A) Schematic showing how Mrksich’s patterned surface was fabricated with the surface constructs within each region. (B) Optical micrographs showing the confinement of Swiss 3T3 fibroblast cells to predetermined regions and their subsequent release as a result of a specific applied potential. Reprinted (adapted) from ref 42. Copyright 2006, American Chemical Society. F

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Figure 5. (A) Migration of BCE cells from cell-adherent C18 regions into surrounding regions after the cell-repellent C11EG3 molecules have been electrochemically desorbed from the surface. (B) Effect of various migration-inhibiting drugs on BCE motility after C11EG3 has been electrochemically desorbed. Reprinted (adapted) from ref 56. Copyright 2003, American Chemical Society.

Figure 6. (A) UV light used to photocleave a 2-nitrobenzyl group controllably from a predetermined region of a surface to eliminate BSA from that region and replace it with fibronectin to change the surface from a state the repels cell adhesion to a state the promotes it. (B) UV light (λ = 365 nm, 1 W/ cm2, 10 s) was applied to an initial region (right circle) to remove the BSA and promote cell adhesion. The UV light was then applied to an adjacent region (left circle) to remove the BSA and allow cells to migrate to that region from the initial region. Reprinted from ref 48. Copyright 2006, with permission from Elsevier.

glycol) monolayer containing a thiol-coupling agent that when oxidized converted from hydroquinone-terminated to benzoquinone-terminated. Oxidation of the monolayer was used to (1) characterize gradients of adhesive ligands, generated by the flow of the monolayer solution over a gold substrate in a microfluidic channel, with the remaining regions backfilled with tetra(ethylene glycol) to prevent nonspecific adsorption, and (2) provide a way to tether RGD ligands to the gradient through an oxyamine reaction. Swiss Albino3T3 fibroblasts were then seeded on the surface and initially adhered to the high-RGDdensity regions only, before slowly proliferating and migrating down the gradient. The actin, nucleus, and Golgi were stained to determine that cells polarized toward the direction of migration. Nakanishi et al.48,49 developed a different strategy for initiating cell migration using light. In this strategy, a glass surface was modified with a silane-coupling agent that contained a photocleavable 2-nitrobenzyl group, 1-(2-nitrophenyl)ethyl 5trichlorosilylpentanoate (Figure 6Aa). The sterilized glass substrate was then coated with bovine serum albumin (BSA) by placing it in a BSA solution for 1 h (Figure 6Ab). UV light was then applied to a predetermined region to cleave the 2nitrobenzyl group to form a carboxyl group on that region (Figure 6Ac). The negative charge repulsion between the carboxylic acid group and the BSA present within the illuminated region led to localized BSA detachment. The substrates were then incubated in a fibronectin solution for 30 min to modify the

investigation of the morphology of the cells shows that the potential used to remove the C11EG3 regions did not directly affect the cells. This work showed that surfaces can be fabricated that have the ability to control how cells adhere and migrate across surfaces on a whole-surface scale. It has served as a good stepping stone in the development of surfaces that will enable further information to be gained about the adhesion and migration of cells on a more local level. As can be seen in Figure 5A, the cells move randomly across the surface. Further delineation of the time scale of events will lead to the further knowledge of the events that take place within a cell as it interacts with its environment. As with the capture-and-release surfaces, Mrksich and coworkers46 have used patterned surfaces with electrochemically cleavable units to release cells in defined regions. The chemistry that remains after the cleavage is cell-resistant, and hence the migration of remaining cells is confined. Attachment of new RGD ligands to the electrochemically cleaved regions allowed Swiss 3T3 fibroblast cells to again migrate onto these regions. Such a surface has a similar capability to that by Whitesides shown in Figure 5, except that the actual number of adhesive ligands in the regions in which the cells migrate can be controlled such that the impact of ligand density in these initial stages of cell migration could be studied. Similar ideas to those of Mrksich and co-workers were explored by Yousaf and co-workers,47 who used a tetra(ethylene G

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Figure 7. (A) Schematic showing how the surface switches its orientation as a response to the applied positive or negative potential. (B) Schematic of how the patterned surface that contains a central region of negatively charged sulfonate-terminal species that surround the RGD ligands and the exterior region of positively charged ammonium-terminal species surrounding the RGD ligands responds to an externally applied potential. HL60 cells were expected to be confined to the exterior region when a negative potential was applied. Cells were expected to migrate toward the central region upon application of +300 mV. (C) Fluorescent images of adhered HL60 (Celltracker staining) showed that the cells adhered to the outer region (outside the dashed line) when −300 mV was applied to the surface. The cells then migrated into the circular region upon switching the potential to +300 mV. Scale bar = 100 μm. Reprinted from ref 51. Copyright 2012, with permission from Wiley-VCH Verlag GmbH & Co. KGaA.

illuminated regions with fibronectin whereas BSA prevented fibronectin from binding to nonilluminated regions (Figure 6Ad). Incubating HEK293 cells on the surfaces led to cell adhesion only on the fibronectin regions (Figure 6Ba). UV light was then applied to a region adjacent to the initial region containing cells. This led to the photocleavage of the SAM within this adjacent region and subsequent localized removal of the antifouling BSA layer. The removal of the antifouling layer from the adjacent region allowed for the migration of cells into this newly created region from the initial site (Figure 6B). This technique allows for cells to be moved repeatedly into new regions on the surface by photocleaving regions adjacent to the cluster of cells and provides a good platform to learn about the initial steps in cell migration. To gain this extra information, a way to examine these cells within the initial moments of migration (between Figure 6Ba,b) would be required, including the ability to image a single cell at this initial stage of migration. A possible weakness of this surface is that there is no controlled presentation of adhesive ligands on the newly exposed celladhesive surface that the cells migrate onto. Surfaces that instantaneously reveal cell adhesion points at a well-defined density would provide fine temporal control of cell migration and adhesion that may be required. Such temporal control can be achieved with some reversibly switchable surfaces. Reversibly Switching. Reversibly switchable surfaces can repeatedly change their cell-adhesive character on application of external stimuli. Their precise control over the timing of the “starting gun” for cell migration, coupled with new imaging techniques that can track a single cell with high resolution, will enable a detailed examination of the sequential events that take place as a cell interacts with a surface.

We have developed a reversibly switchable surface for cell adhesion based on the seminal idea of Langer and co-workers50 to use the electrostatic potential to change the conformation of molecules on a surface. Langer and co-workers used conformation changes to reversibly switch a surface from hydrophilic to hydrophobic.50 We subsequently extended this concept to control cell adhesion reversibly.51 Figure 7A illustrates how patterned surfaces were fabricated that could direct the adhesion or repulsion of cells. A modified silicon surface was fabricated with a mixed base monolayer with two different coupling points in a ratio of 1:1000. The minor coupling point was a carboxylic acid to which cell-adhesive RGD peptides could be attached. The other coupling point was an alkyne to which azido-oligo(ethylene oxide) species (OEG) could be bound using the copper -catalyzed Huisgen 1,3-dipolar cycloaddition “click” reaction. Importantly, the OEG species possessed either a sulfonate or an ammonium charged moiety on the distal end. Hence if the same potential as the charged moiety was applied to the silicon surface, then the OEG molecules were fully extended and the RGD ligands were concealed such that bovine aortic endothelial cells (BAECs) or HL60 cells did not adhere. If the potential was switched to oppositely charged to the distal moiety, then the OEG molecules would collapse toward the surface, revealing that the RGD ligands and cells would adhere (Figure 7B). Mendes and co-workers52 have also used electrostatic interactions to expose or conceal binding motifs to control the adhesion and repulsion of biomolecules to a surface. However, our reversibly switchable surface drew inspiration from a study by Kessler and co-workers,53 who employed an optically switchable surface. The surface contained RGD peptides attached to a molecule containing an azobenzene that can be reversibly H

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Figure 8. (A) Continuous cell membrane (bottom panel) and compartmentalized membrane (top panel) models used to trigger and manipulate receptors on the surface of living cells. Diffusion barriers on a glass support are fabricated by electron-beam lithography and provide lateral confinement of membrane components. (B) Fluorescent images show that the bull’s-eye protein pattern of the immunological synapse in live T cell (d) is altered by the presence of diffusion barriers on supported lipid bilayers (b). Scale bar = 5 μm. The images in part B are reprinted from ref 66. Copyright 2005, with permission from the American Association for the Advancement of Science.

dependent on a number of variables including how the proteins are anchored. For example, GPI-anchored proteins diffuse freely, but transmembrane proteins typically do not.60 In addition, diffusion for GPI-anchored proteins is also concentrationdependent as molecular crowding becomes an issue at high concentrations.54 It is this mobility that is particularly attractive for the use of lipid bilayers as model surfaces for cell biology,58 but other features of importance include the ease of incorporation of multiple chemical components and the rich array of coupling schemes to attach even larger proteins to the lipid bilayer. Initially, black lipid membranes were formed, as used by electrophysiologists, where a bilayer is suspended over an aperture. In the 1980s,61 the trend emerged to support the bilayer on a glass substrate such that it was continuous on the macroscopic level. In some ways, a continuous membrane reflected the view of the cell membrane at the time of a homogeneous passive layer around the cell. As a greater understanding of cell membranes emerged with concepts such as the lipid raft hypothesis and other models of compartmentalized cell membranes, strategies for fabricating compartmentalized artificial bilayers also emerged.55 In this context, the term compartmentalized lipid bilayers is used to refer to bilayers where the diffusion of lipids and proteins is confined to a small region of the bilayer (Figure 8). There have been a number of strategies developed to achieve this compartmentalization. Groves and colleagues58 have pioneered lithographic methods of fabricating compartmentalized lipid bilayers where structures patterned on a glass surface are used such that the bilayer can form only on specific regions of the glass.58,59 For example, a raised metal structure made of alumina or gold can be used as a barrier. Deposition of the bilayer is achieved from unilamellar vesicles, and because the bilayer forms only on the glass, the bilayer is partitioned or corralled such that lateral diffusion is confined by the barriers.58 A related strategy has been used by Craighead and Baird62 to form nanodomains of bilayers to allow the systematic exploration of signaling of receptor clusters by

switched between trans (E) and cis (Z) using light at different wavelengths to promote cell adhesion or repulsion via altering the accessibility of the RGD ligands to the cells. In this case, however, the rate of switching was quite slow, on the order of an hour, but the electrostatic switching was virtually instantaneous. In summary, the current motivation in adhesion and migration biology is the pursuit of temporal information about interactions leading to the adhesion and migration of cells across a surface. Thus far, a promising way to acquire such knowledge is through the use of reversibly switchable surfaces that can alternate between two regions that will control the movement of cells back and forth across the top of them with precise temporal control to initiate migration. To take advantage of recent developments in microscopy that now allow us to track individual cells and image the localization of single proteins, substrates that are compatible with these microscopes are required to be used as the platform on which to form these reversibly switchable surfaces.

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MOBILE LIGANDS: USING LIPID BILAYERS AS THE BASE LAYER Although model surfaces with immobile ligands allow us to interrogate cell adhesion to and migration over solid substrata, they are not particularly good models for signaling and adhesion processes between cells such as cell−cell contacts. Lipid-bilayermodified surfaces have the potential to be model surfaces for this type of situation but have mainly been applied to immune and cancer cells.54,55 Because the application of bilayers for cell biology has been reviewed recently,54,55 we will simply provide an overview for completeness. Supported lipid bilayers have been made by chemists for several decades as models of cell membranes56,57 but have recently begun to be used extensively in immunology to study molecular interactions at interfaces as a model for cell−cell interaction.54,55,58 The important feature of lipid bilayers is that lipids and proteins that are coupled to the bilayer can diffuse over large distances. The diffusion coefficients are typically around 1 μm2/s.59 Importantly, however, the diffusion of proteins is I

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engineered surfaces has already informed us that VEGF signaling dominates the optimal ligand spacing in endothelial cell migration although VEGF and integrin signaling use the same signaling proteins. Similarly, the use of lipid bilayers for T-cell activation clearly demonstrated that the spatial arrangement of receptors and adhesion proteins is a key regulator of T-cell activation, thus providing a greater appreciation of the immunological synapse. Biologists have spent considerable effort, time, and money to map and characterize proteins involved in adhesion and describe the adhesome as an integrated network. Although this “parts list” of molecules and interactions is an extremely useful resource, a functional understanding of how an adhesion is formed can only come about by placing these molecules into a cellular context. And if there is anything that model surfaces have taught us, it is that cellular processes take place on local length and time scales. In other words, whether an integrin engages a ligand depends on whether a ligand is present in the integrin within the area and time scales that the unbound integrin covers by diffusing across the cell surface. This also implies that there is an inherent stochasticity in every biological process, and only by measuring the likelihood that a particular process takes place, in this case, the engagement of the integrin, can we understand the network in its entirety. This, however, requires that we study cell behavior on the molecular scale. Monitoring adhesion from the perspective of a single integrin requires two things: molecular control over the extracellular environment and a molecular readout of the cellular machinery. Although the former has been the motivation for most of the work reviewed here, the latter is possible with advanced and super-resolution fluorescence microscopy. Integrin movement and clustering has for example been recently described with single-molecular localization microscopy.68 However, even this elegant study could not explain why adhesions were formed in particular sites within the cell simply because there was no control over ligand placement. Inversely, molecularly engineered surfaces have revealed a correlation between small and dynamic adhesions with fast migration speeds versus large and stable adhesions with slow migration speeds. However, whether large adhesions are simply many small adhesions clustered together or whether there are fundamental structural differences, such as the presence of additional scaffolding proteins or an alteration in actin linkages between large and small adhesions, is unknown. This lack of knowledge exists because no comprehensive structural analysis, either microscopically or biochemically, has yet been done on model surfaces with various degrees of ligand spacing. Similarly, most of the studies on dynamic surfaces are at the proof-of-concept stage so that the obvious advantage of following the dynamics of adhesion assembly and disassembly has not yet been exploited. Dynamic surfaces in particular hold promise for resolving controversial issues in adhesion biology including whether nascent adhesions are preformed or induced by integrin clustering. An interesting case is the recently reported Arp2/3-deficient cell line that senses soluble cues but not a gradient of fibronectin,69 suggesting that the motility apparatus in these cells is uncoupled from sensing adhesive cues. Here model surfaces would be extremely useful in investigating how actin linkage to adhesion is facilitated and to what extent this linkage is dependent on ligand and integrin arrangements. The marriage of molecularly engineered surfaces and advanced fluorescence microscopy places additional constraints on surfaces and their chemistry (such as optical transparency), but would allow chemistry to reach deep inside the biological

controlling the position and size of receptor clusters in mast cells. An alternative strategy is to use curvature to organize phaseseparated membrane domains spatially.63 So what sorts of biological questions have been addressed using a lipid bilayer as the base layer for engineered surfaces? Unlike dynamic surfaces with immobile ligands discussed above, where progress has perhaps been driven mainly by advances in developing surfaces, the applications of lipid bilayers have mostly been motivated by specific biological questions, most notably, understanding the immunological synapse of T-cells. Model surfaces using lipid bilayers are ideal for the characterization of immunological synapses because with the T cell the lateral redistribution of receptors and signaling proteins occurs during the formation of the synapse. Hence lipid bilayers are an ideal platform for observing this large-scale redistribution and restricting it by placing diffusion barriers into the lipid bilayer. The immunological synapse forms between T cells and socalled antigen-presenting cells (APCs). The APCs literally present the ligands of the T cell receptor (TCR) on their cell surfaces as part of the major histocompatibility complex (MHC). When T cells engage the peptide ligand on the MHC, large-scale segregation occurs at the position of contact where activated TCRs accumulate in the center of the T-cell−APC contact zone and adhesion receptors form a ring around the center (referred to as the central supramolecular activation complex or cSMAC). This bulls-eye pattern is referred to as the immunological synapse.64 Lipid bilayers modified with peptide MHCs and adhesion proteins have been essential in studying how the immunological synapse is formed and what cellular machinery is essential for the segregation of TCR from adhesion receptors55 after it was first demonstrated in the 1980s that lipid bilayers with MHC molecules could be uses as model APCs and activate T cells.65 Model surfaces with the nanoscale confinement of lipid bilayers have shown that the spatial organization, not simply the engagement of TCRs and adhesion receptors, is critical for the regulation of T-cell signaling and activation.66 These discoveries had a major impact beyond the immunology community and influenced concepts and lines of inquiry in signal transduction in general because they demonstrate that the spatial organization that is superimposed onto the biochemical signaling network controls receptor signaling and cellular outcomes.67 This, of course, provides additional motivation to design more sophisticated model surfaces that impose the specific organization of receptors onto the cells by presenting ligands in a given arrangement in time and space, thus achieving the desired cellular outcome. Hence, in addition to fundamental insights into cell function, molecularly engineered model surfaces may also find applications in translational studies to enhance T-cell response to a vaccine.

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CONCLUDING REMARKS Model surfaces with a high degree of control over the molecular architecture, particularly static surfaces with immobile ligands, have been shown to provide astonishing insights into how cells sense and respond to their environment. The revelation that cells can sense nanometer variations in ligand spacing was a major breakthrough in adhesion biology and solely driven by the advances in surface chemistry. Too often, however, the cells are treated as a “black box” by chemists who simply read out the cell number, size, and location. The complexity inside that box seems to be too great to be examined with such a simple device as a monolayer- or bilayer-modified surface. However, the exact opposite is true. The nuanced behavior of cells on molecularly J

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toolbox. There are a number of imaging techniques that provide higher spatial and temporal readouts than traditional epi- and confocal microscopy. These include FRET sensors that can monitor kinase activity such as Src70 or specific protein interactions, number & brightness (N&B), other fluctuation analyses to quantify protein oligomerization and diffusion, and of course super-resolution imaging,68 all of which have been applied to adhesion. Given the relative ease with which cells can be transfected and the wide availability of confocal and TIRF microscopes, it can be expected that chemists who can engineer model surfaces with molecular control over ligand presentation will make the greatest contribution to a functional understanding of cell adhesion and migration.

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AUTHOR INFORMATION

Yong Lu graduated with a B.Sc. in chemistry from Jilin University, China, in 2011. He joined the Biosensors and Biointerfaces Research Group at the University of New South Wales as a Ph.D. student in 2012, where he has been ever since.

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies

Professor Katharina Gaus is an NHMRC Senior Research Fellow at the University of New South Wales. She received her Ph.D. from the University of Cambridge in 1999 and has led the Cell Membrane Biology Group since 2005. Her group investigates signal transduction processes with advanced fluorescence microscopy approaches. She was awarded the Young Investigator Award from the Australia and New Zealand Society for Cell and Developmental Biology (2010), the Gottschalk Medal from the Australian Academy of Science (2012), and the New South Wales Science and Engineering Award for Excellence in Biological Sciences (2013).

Scientia Professor Justin Gooding is the leader of the Biosensor and Biointerfaces Research Group at the University of New South Wales. He obtained a D.Phil. from Oxford University and undertook postdoctoral training from the Institute of Biotechnology at Cambridge University. In 1997, he returned to his native Australia, joining the University of New South Wales. He was promoted to full professor in 2005 and is currently an Australian Research Council Professorial Fellow. His research interests lie in biosensors, biointerfaces, and surface chemistry.

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REFERENCES

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dx.doi.org/10.1021/la4037919 | Langmuir XXXX, XXX, XXX−XXX