Focal Complex Maturation and Bridging on 200 nm Vitronectin but Not

May 20, 2011 - The effects of protein type and pattern size on cell adhesion, spreading, and focal adhesion development are studied. Fibronectin and v...
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Focal Complex Maturation and Bridging on 200 nm Vitronectin but Not Fibronectin Patches Reveal Different Mechanisms of Focal Adhesion Formation Jenny Malmstr€om,†,‡ Jette Lovmand,† Stine Kristensen,† Maria Sundh,† Mogens Duch,§ and Duncan S Sutherland*,† †

Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus, Denmark Polymer Electronics Research Centre, Department of Chemistry, University of Auckland, Auckland 1142, New Zealand § Department of Molecular Biology, Aarhus University, DK-8000 Aarhus, Denmark ‡

bS Supporting Information ABSTRACT: The effects of protein type and pattern size on cell adhesion, spreading, and focal adhesion development are studied. Fibronectin and vitronectin patterns from 0.1 to 3μm produced by colloidal lithography reveal important differences in how cells adhere to and bridge focal adhesions across protein nanopatterns versus micropatterns. Vinculin and zyxin in focal adhesions but not integrins are seen to bridge ligand gaps. Differences in protein mechanical properties are implicated as important factors in focal adhesion development. KEYWORDS: Protein patterns, fibronectin, vitronectin, cell adhesion, focal adhesion

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ell adhesion is an important process in many biological phenomena serving both as a mechanical anchor and as a signal transduction pathway for extracellular signals.1,2 Focal adhesions (FAs) regulate cell binding to the extracellular matrix (ECM).1,2 They consist of assembled transmembrane integrin receptors bound to ECM ligands on the extracellular side and to a cluster of proteins on the intracellular side connecting to the actin filaments of the cell. Focal adhesions have been reported to be of the size scale from 1 to 10 μm1,3 and it has been shown that integrin ligands (RGD-motif containing oligopeptides) with an individual lateral spacing of 73 nm and above do not support focal adhesion formation and cell spreading.4,5 On a larger scale, nano- and micropatterning of topography,6,7 chemistry,8 or ligands4,9 have been shown to influence cell survival,8 cell shape and alignment,7,8,10,11 adhesion strength,12 and differentiation.6,10,13 15 In 2D culture systems, cell shape is widely correlated to differentiation while similar correlations are not routinely reported in 3D culture,16 where instead integrin receptor mediated adhesion to the ECM is proposed as one mechanism for cellular sensitivity to the local mechanical environment and associated stem cell differentiation.16 A number of molecular level mechanisms for mechanotransduction have been proposed including force modulated integrin ligand catch bonds and ECM or intracellular protein unfolding revealing cryptic adhesion or phosphorylation sites.17 The adhesive strength of integrin ECM interactions is known to be significantly enhanced by clustering of integrins.2 Initial integrin clustering into so-called nascent adhesions or focal complexes (FCs) is linked to the actin matrix through multiple proteins (talin, filamin, actinin, and tensin). Developing adhesions r 2011 American Chemical Society

proceed to recruit vinculin, paxillin, and zyxin as additional links to actin. The nascent adhesions move back from the cell’s leading edge in connection with an actin retrograde motion and connect to actin stress fibers and on rigid surfaces elongate into mature focal adhesions.2 The process of focal adhesion development and its function remains incompletely understood; however, the focal adhesion is now thought to provide, in addition to a direct mechanical contact with the ECM, also a signaling complex integrating mechanical and biochemical signals.18,19 We and others have shown that cells have reduced attachment, spreading and focal adhesion development on ligand patterns in the submicrometer range.9,20 22 We proposed that the size of fibronectin (FN) patches regulates the conversion of focal complexes to focal adhesions, by limiting the force that can be applied, particularly for the patch sizes in the 200 500 nm range. In contrast, Arnold et al.20 produced adhesive patches of RGD peptides with side lengths ranging from 100 to 3000 nm where cells showed a clear behavior for patterns below 500 nm to bridge multiple adjacent ligand domains via individual actin fibers in order to adhere. In this study, we investigate cell adhesion and spreading at protein patterns of either FN or vitronectin (VN) in the range of 100 3000 nm, revealing important differences in focal adhesion development at nanopatterns of the two proteins. These proteins were chosen as they are important components of the ECM with different mechanical properties and different Received: February 7, 2011 Revised: May 7, 2011 Published: May 20, 2011 2264

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Figure 1. (A) SEM images of gold domains within SiO2 films. Nominal diameters (nm) are presented in each top left corner. Scale bars represent 1 μm. (B) Fluorescence microscopy image of FN patterned at 0.5 μm holes stained with anti-FN. Scale bar represents 20 μm. (C) Fluorescence microscopy image of VN patterned at 0.8 μm holes and stained with anti-VN. Scale bar represents 20 μm.

involvement in mechanotransduction. Fibronectin is known to unfold and reveal cryptic binding sites upon force pulling,23 whereas VN provides a mechanically less pliable protein due to its structure and smaller size. Fibrillation of released cellular fibronectin proceeds by a force induced process and has been observed to occur during the transition and translocation of focal adhesions at the periphery to the central portion of the cell in 2D cell culture. The distinct long fibrillar adhesions formed in this process are bound to the newly generated fibrillar forms of fibronectin.24 Fibrillar adhesions, as well as a form of matrix adhesions recently observed in 3D cultures, show differences in their composition from focal adhesions or focal complexes.25 We have utilized colloidal lithography26 combined with site specific chemical modification27 to generate a series of protein patterns of FN, VN, or bovine serum albumin (BSA). In brief, dense short-range ordered arrays of circular gold holes in SiO2 films were produced using dispersed colloidal monolayer masks. Hole diameters were systematically varied by utilizing a series of particle sizes. The characteristics of the arrays are shown in Table S1 (Supporting Information). Figure 1a shows scanning electron microscopy (SEM) images of a selection of the hole sizes. The Au regions of the surfaces were modified with alkanethiols to have a hydrophobic chemistry and the SiO2 regions with PLL-g-PEG to be protein repellent.28 FN, VN, or BSA was then defined into patterns or onto homogeneous surfaces by adhesion to the hydrophobically modified gold. Panels B and C of Figure 1 show fluorescence microscope images of FN adsorbed into 0.5 μm holes stained by fluorescently labeled anti-FN and VN adsorbed in a 0.8 μm pattern stained by fluorescently labeled anti-VN, respectively. More images of different size FN patterns are available in Figure S1 in the Supporting Information. These images demonstrate the high-quality protein patterns of both FN and VN and are further supporting our previous evidence for a robust protein patterning method.21,27 Quantification of Cell Adhesion and Area. The substrates displaying protein patterns of FN, VN, or BSA (as a negative control) and homogeneous protein films were used as cell culture substrates. The adhesion of C2C12 myoblasts was studied after 20 h postseeding. We utilize the large area patterned samples to enable quantification of cell adhesion and area from whole cell populations. These data are presented as bar graphs in Figure 2. The data in Figure 2 are collected from one experiment with n = 4

Figure 2. (A) Mean cell number (per mm2) on the different sample types. (B) Mean spread cell area for the different sample types. Standard errors of means are displayed. The data are derived from four images per sample using a 10 objective and four samples per condition. Significant differences (t < 0.05) between each sample and the flat control (Au) are denoted with a star, and differences between FN and VN on the same sample type with a cross.

samples and using images of four randomly selected regions from each sample. Another full repeated experiment and several smaller experiments focused on different immunostains were run (see Figures S2, S4, S5, and S7 in the Supporting Information). These repeated experiments confirm the data and conclusions presented here unless specifically stated. 2265

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Figure 3. Fluorescent microscopy images using 40 objective with a 1.6 optical zoom: blue stain, the nucleus; red, the actin cytoskeleton; green, vinculin. The patch size is displayed for each pair of images (FN and VN). Scale bar 30 μm.

In all cases, more cells adhere to the fibronectin and vitronectin coated surfaces than on the BSA coated negative controls or the homogeneous PLL-g-PEG coated SiO2 surfaces exposed to FN or VN (negative controls with as expected low or no cell adhesion). Fewer cells adhere to the smaller protein patterns with very impaired cell adhesion to the 0.1 μm patterns in particular (Figure 2a). Similar levels of cell adhesion are observed for FN and VN for the smaller pattern sizes of 0.1 0.2 μm, while above this size there are consistently more cells adhering to the FN coated surfaces. At around 0.8 1 μm patterns, the increase in cell adhesion with increasing pattern size levels off and the cell numbers reach a plateau value. The slightly higher cell number at 0.8 μm patterns compared to the larger structures was not seen to be significant in the repeated experiments, and although the total number of cells was seen to vary slightly between experiments, the general trends were otherwise very similar (Figure S4 in the Supporting Information). We have quantified the mean spread area per cell (Figure 2b) and have chosen to calculate only the area for those cells that show significant spreading and interaction with the substrate and have excluded a minority of features below a given threshold in size (typically 320 μm2 for smaller patterns and 430 μm2 for patterns above 300 nm). This approach robustly excluded the noninteracting rounded cells. The total number of cells involved in the analysis is typically between 750 and 2000 cells per sample type. The 100 nm patterns with the low cell binding are from around 250 cells. The cells at BSA coated

surfaces were almost exclusively rounded up (data not shown). The cell spreading clearly increases with increasing structure size over the whole range for cells adhering on FN, in accordance both to cell adhesion and to our previously published data for a breast cancer cell line adhering on FN patterns.21 For cells adhering on VN, a somewhat different pattern is observed especially for the smaller patch sizes. Already at 200 nm patches significant spreading is observed (>55% of that at homogeneous VN) which is not increased until at 500 nm patches. By contrast cell spreading at 200 nm FN patterns is only ∼30% of that at homogeneous FN, which increases already at 300 nm FN patterns. The spread area on larger VN patterns appears to level off and is significantly lower at homogeneous VN than at homogeneous FN. Interestingly, while the absolute spread area is greater at large patterns/homogeneous FN compared to large patterns/ homogeneous VN, at smaller patterns the opposite is observed. The results point toward differences in the adhesion and spreading mechanism of cells at the different protein patterns. Cell Morphology and Focal Adhesions. The clear trends in quantified cell adhesion and cell area are also observed as clear changes in the morphology of the cells adherent at the different proteins and with changing pattern size. Figure 3 shows representative examples of cells at the different substrates. The images are overlays of blue staining of the nucleus, green staining of vinculin, and red staining of actin (for zoom in at interesting regions see Figure S3, Supporting Information). For cells on FN, 2266

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Nano Letters a long and narrow morphology is seen at the smaller structures. From 0.5 μm and up, the cells spread well on FN with a stellate morphology. On VN, the cells already interact with and spread significantly on the 0.2 μm patterns. With increasing structure sizes the cells spread further on VN, until a threshold area is reached as seen in Figure 2B. The cells on VN exhibit a round or fanlike morphology indicating that the cells are mobile on the surfaces.29 The cells adhering to the smallest pattern size (0.1 μm) for both proteins are relatively few and small. As the protein patches become larger, the vinculin spots become more prominent. Also the actin cytoskeleton is more distinct for the cells on the homogeneous control and the larger patterns. The cells were also stained for zyxin, a protein proposed in the literature30 to locate to FAs but not to FCs. In our case, within the limits of our optical microscope, vinculin stained structures were routinely also stained positive for zyxin. Interestingly, it was possible to clearly distinguish singular structures down to 0.5 μm in diameter stained for zyxin (see Figure S6 in Supporting Information). Traditionally FAs are typically a few micrometers and above in length,31 but here we show structures of half that length distinctly staining positive for both zyxin and vinculin, as also described in a recent article utilizing single molecule imaging supporting our data.32 A careful examination of the vinculin/zyxin stained adhesions reveals clear differences in the number, size, and distribution at the different proteins and pattern sizes (Figure S5 in Supporting Information for zyxin stained images). In general, the size of the FAs correlate with the spreading of the cells. At the 0.2 μm VN patterns the vinculin spots are observed as very long and thin lines and must bridge several VN patches. By contrast, in cells on FN patterns of the same size, no defined vinculin stained structures are observed at all. This bridging of several adjacent protein patches as seen by long stretched vinculin stained structures becomes less prominent for the larger structures. At the largest structures each patch is apparently sufficiently large to on occasion even accommodate multiple adhesions. Generally, there are fewer but more distinct FAs for cells on VN than on FN, and also the FAs are generally less evenly distributed around the periphery of the cells on VN. This is not surprising seeing the polarized fan-shaped morphology often observed for the cells on VN. Also, the actin cytoskeleton appears altered in cells on VN with a more cortical appearance as compared to in cells on FN where the actin fibers are straighter and cross-sectional. For cells on FN, the FAs appear to be more plentiful for protein patches of 0.5 μm and above and evenly distributed around the perimeter of the cells. Additionally, distinct adhesions can also be seen in the center of the cells on the FN coated substrates in particular. We interpret these as fibrillar adhesions which can be generated by cells adhering on FN by an R5 integrin and a force-dependent process of induced fibrillation of released cellular fibronectin.33 However, we will refer to this population of adhesions as “mature central adhesions” throughout the paper as we cannot prove in each instance that they are strictly fibrillar adhesions. Additionaly, the use of the term fibrillar adhesion in vitro is debated. Cells adhering both on larger VN and FN patterns exhibit some of these centrally located adhesions, but these are far more pronounced on FN coated substrates. For cells on VN coated substrates, these mature central adhesions are interpreted as being on top of fibrillated cellular deposited FN fibers. Staining against FN confirms this as seen in Figure S8 in the Supporting Information. A higher magnification image of vinculin and actin stained cells on FN/VN as 0.2 and 0.8 μm patches can be seen in

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Figure 4A. The long narrow bridged FAs for cells on small VN patterns are evident here as compared to the lack of FAs for cells on the small FN patches. For the 0.8 μm patches both single well separated circular FAs and some FAs that appear to bridge adjacent patches and form long elongated structures can be seen. In this figure some central adhesions are visible in the cell on the VN coated 0.8 μm surface. We interpret these as mature central adhesions at cellular deposited FN fibers. Although these are not evident for the cell on FN shown in Figure 4, generally speaking this behavior is more prevalent in cells on FN coated substrates (see, e.g., Figures S7 and S8 in the Supporting Information). A clearly defined actin cytoskeleton can be seen in the cells on VN structures from a small size and for the larger FN structures in Figure 3. The actin staining is not very well-defined in the outer regions of the cells (possibly due to some interference with our Percoll lift off protocol and penetration of phalloidin into the cell) so it is difficult to determine whether individual FAs are connected to actin fibers. It is evident in Figure 4A, however, that at least some of the less peripherally located adhesions are located at the end of actin fibers. Integrin Involvement and Adhesion Maturation. To shed some more light on the processes occurring at these different interfaces, we co-stained the cells against either R5 or Rv integrins and vinculin. Cells on either FN or VN coated as 0.2, 0.5, or 0.8 μm patches or on the homogeneous control were investigated (images shown in Figure S7 in the Supporting Information). In general the central part of the cells shows a speckled appearance when staining for both R5 and Rv, probably reflecting integrins that are not involved in the adhesion processes. However, clear differences are observed in the display of integrins at the cellular periphery or in elongated central adhesions. The homogeneous surfaces show that the cells grown on FN as expected utilize both R5 and Rv integrins to adhere to the substrate, whereas cells on VN almost exclusively utilize Rv integrins. While there is a limited R5 involvement for cells on VN, these integrins are in such cases only present in the central portion of the cell and are interpreted as mature central adhesions adhering to cell deposited fibronectin fibers. In particular on the homogeneous surface with VN, these mature central adhesions exhibit only weak vinculin staining. This is in line with literature that suggests that fibrillar adhesions contain only low levels of vinculin.34 The trend is slightly different on the nanopatterned surfaces. At 0.8 μm patterns, and with high magnification, it is possible to clearly distinguish the protein pattern (see Figure 1C). Here it is clear that cells on FN mainly use R5 particularly in the central parts of the cells, whereas some Rv involvement in the periphery of the cells can be seen. This differs from the behavior on the homogeneous control in that R5 was in that case also clearly localized at the periphery as well as the central areas (Figure S7, Supporting Information). Also, in the FN case, the R5 rich centrally located elongated adhesions appear to stain more for vinculin than in the VN case. Cells on 0.8 μm VN patterns stain exclusively for Rv integrins at their periphery, while centrally in addition to Rv integrins only few well-defined mature central adhesions staining for R5 are observed. This may be an effect of reduced fibrillar deposition at nanostructured surfaces as compared to the homogeneous control. The Rv integrins are clearly aligned to the underlying VN pattern and it is evident that they are left behind at the substrate as the cells move. This further points to the cells being motile at the VN surfaces. The cells are not well spread at 0.2 μm FN patterns, and therefore it is not surprising that the lack of defined vinculin stain also presents as lack of 2267

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Figure 4. (A) Enlargement of fluorescence images showing cells on 0.2 and 0.8 μm surfaces coated with FN or VN: green, vinculin; red, actin. Scale bar 10 μm. (B) Fluorescence images of the periphery of a cell on 0.8 μm FN pattern: green, vinculin; red, fibronectin. Scale bar 5 μm. (C) Fluorescence images of the periphery of a cell on 0.5 μm VN pattern: green, vinculin; red, Rv integrin. Scale bar 5 μm.

defined integrin staining. On these small VN patterns however, the cells are generally better spread with long but narrow vinculin stained adhesions. Although vinculin is nicely defined there, we observe almost no defined structures stained with Rv integrins. In some cases however, some defined R5 stained structures can be seen in the central portion of the cells (which we interpret as mature central adhesions located at FN fibers). Focal Adhesion Bridging. Mature central adhesions are observed in the cells on the larger structures. These mature central adhesions, which stain for R5 both on VN and FN patterns, appear to form on fibers of cellular deposited fibronectin. On the VN patterns, such fibers are observed to a limited extent, and only in the central zone of the cells. The mechanism by which these extended mature central adhesions are created can be understood in terms of the R5 mediated fibrillation of cellular fibronectin during the translocation and transformation of focal adhesions from the cellular periphery to the cell center. The bridging of focal adhesions between VN patches at the periphery of the cell is the most pronounced for the 0.2 μm patterns but is seen also for larger VN patterns. The mechanism behind this bridging appears to be different from the fibrillar adhesion mediated extension of adhesions at the center of

the cell. Figure 4b shows separate and merged images of FN (red) and vinculin (green) stained channels from a region at the periphery of a cell adsorbed to 0.8 μm FN patches. The vinculin stained regions show multiple elongated FAs which bridge over several FN patches. This type of bridging while more prevalent at VN patterns, is observed also at larger FN patterns (0.5 μm and greater). The lack of fibronectin between the patches confirms that this is not related to adhesion to fibrillated fibronectin. In recent work on another cell type (MDA-MB-435 human breast cancer cell line) we showed a similar lack of bridging at small (0.2 μm) FN patterns, but also no significant bridging at larger patterns (0.5 and 1 μm)21 in contrast to this work. It is not surprising that the behavior of different cells types is different. Here, we observe that for sufficiently large FN patches, focal adhesions can form across two or more fibronectin patches. Bridging between FAs at nanoscale patches of RGD presenting oligopeptides has been observed recently by Arnold et al.20 In that work, bridging occurred between multiple FAs each located at discrete ligand patches by individual actin fibers for patches from 0.1 to 1 μm. The FAs did not stain for paxillin between the ligand patches being bridged by actin. Here, we observe such bridging regularly with multiple FAs strung together connected 2268

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Figure 5. SEM images of the outer region of cells adhering to (A) 0.8 μm FN patterns, (B) 0.6 μm VN patterns or (C, D) homogeneous controls of FN (C) and VN (D). Scale bars in (A) and (B) represent 2 μm and in (C) and (D) represent 5 μm. Images (B) and (C) are acquired with a 30° tilt of the stage while (A) and (D) are without any tilt.

by a single actin fiber at FN, but only for patterns from 500 nm and up. However, we also observe a different type of bridging where a single focal adhesion bridges across several FN patches across gaps of more than 0.2 μm (see arrow in Figure 4B). To the best of our knowledge this is the first observation of such bridging events of single FAs across ligand gaps. The bridging is not explained by fibrillated fibronectin since counterstaining for FN shows no FN between the patches under the FA. Similar bridging events occur at VN patterns with single FAs forming across several VN patches. Interestingly, such bridging occurs for the larger patterns similarly to FN but also for the smaller patch sizes down to 0.2 μm. For the 200 nm patterns individual protein patches cannot be resolved in our microscope but clear long focal adhesions are seen. The length and aspect ratio of focal adhesions have been quantified for 200 and 800 nm patterns and shows a clear trend with the VN patterns showing a similar length and aspect ratio of the “bridging” focal adhesions. The few bridging events observed on 200 nm FN surfaces showed only short and low aspect ratio vinvulin domains (see Table S2 in the Supporting Information for details). When stained for R5, or particularly clearly against Rv integrins, such long FAs only show integrins at the VN or FN patches and not in between (see arrows in Figure 4C). Figure 4C shows simultaneous immunostaining for Rv integrins and vinculin. Thus, while the FA bridges the ligand patches, it appears to be only some of the components that bridge (vinculin and zyxin, but not the integrins). This is an important observation and indicates that a focal adhesion does not require integrin ligand interactions across the whole adhesion complex. A body of work studying the force exerted per integrin ligand pair, where substantial differences are reported, has led to the suggestion that only a proportion (which can vary)

of the integrins within each FA may be bound to the ECM at any one time.17 Our data open the possibility that the whole area of a specific focal adhesion may not have integrins between it and the ECM, which could provide an alternative explanation. Recent work utilizing iPALM has demonstrated that FAs have a relatively complex 3D structure and show that for cells adhering to homogeneously distributed FN, the vinculin present in the focal adhesions is spaced at least 25 nm above the integrins with zyxin located even further up.32 We observe (Figure 4B, right panel, Figure S9 in the Supporting Information) that the vinculin stained regions of a FA do not typically lie entirely above the ECM protein patches but often half of the vinculin stained area lies off the patch toward the cell center. This indicates that FA proteins are also laterally organized in this forced situation and on significantly longer length scales (we observe vinculin several hundreds of nanometers from the underlying ligand patterns). It is clear that the spatial distribution of the different proteins within a focal adhesion may give important information about their role in signal transduction and an ability to decouple FA proteins lateral distribution from the ligand integrin interactions supports the current idea that they play a broader role than transmitting force to and from the ECM. Importantly though, the width of the focal adhesions (whether located on a single protein patch, bridging across several protein patches or with several FAs connected together by a single actin fiber) is limited by the width of the protein patches. We have utilized SEM and a modified sample preparation protocol to provide images of the few 10 100 nm of cellular material closest to the surfaces. Figure 5 shows peripheral regions of cells adherent at homogeneous and patterned surfaces, observed via SEM microscopy. The images show denser regions (darker as a result of low secondary electron 2269

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Nano Letters yield from organic material) of the cell in proximity to the FN/ VN patches which we attribute to the intracellular focal adhesions proteins. The position of these cellular structures is, as seen by the fluorescence microscopy (for enlarged fluorescence microscopy image demonstrating this see Figure S9 in the Supporting Information), not directly correlated to the ECM protein patch but extend out from the VN/FN patch in the pulling direction of the actin fiber toward the cellular center. It was observed that some focal adhesions (seen by vinculin staining) bridge across non-ECM-coated regions and join multiple integrin binding patches together (Figure 4B). Rv integrin and R5 integrin stained images at similar pattern sizes do not show elongated integrin stained patches at the cellular periphery (Figure 4C). Such FA bridging was seen to occur across at least 200 nm gaps in ECM proteins and at patch sizes from 0.5 μm for FN and from 0.2 μm for VN. The mechanical properties of the two proteins are significantly different and offer one explanation for this behavior. Fibronectin, as a large multidomain protein, has been studied in terms of its ability to alter conformation under force, revealing cryptic sites. It is widely implicated as a mechanotransductive element and shows a systematic unravelling in vitro when pulled.35 The main mechanical contact between the cellular actin cytoskeleton and the ECM are the integrin ECM protein interactions. Pioneering work with laterally spaced RGD oligopeptides as ligands for integrins has established a minimum required spacing of around 60 nm between ligands to allow integrin clustering, with little difference with further reduced spacing.36 This allows an estimate for the density of integrins (∼300 per μm2) in FAs.17 There is roughly 4 times more ligands than integrins on the FN surface (estimate from QCM-D, see Supporting Information), and an even higher number of ligands present on the VN surface. This indicates that the protein ligand density is not a limiting factor for integrin binding to the surfaces. We estimate the number of integrins that could cluster onto each patch from their area (see Table S1 in Supporting Information). For the smallest patches (0.1 μm), where we see little adhesion and almost no spreading of cells, we estimate approximately three integrins and this is below the limit believed to be needed for formation of a focal complex. Pentameric integrin complexes have been shown to support a 7 times larger force per integrin ligand interactions compared to individual integrins.37 For 0.2 μm structures we estimate that approximately nine integrins can fit at each patch. We observe FAs (zyxin staining) bridging across several VN patches but not across similarly sized FN patches. Defined FAs are not routinely observed on FN patches until they reach 0.5 μm in size, where we estimate that 50 integrins can fit. The mechanism of bridging across small VN patches is not completely understood but may indicate that at these surfaces the force that can be exerted on a limited number of integrin ligand interactions may be larger than at similar FN patches. This may relate to observations for FN that force can induce unraveling of the protein. A number of reports suggest that for migrating cells the force exerted by focal complexes may in fact be larger than by focal adhesions and having an important role in motility and spreading.38 Thus, an ability of VN to support a larger force on a smaller number of integrins than FN could implicate it as an ECM component supporting motility which may partly explain its proposed role in, for example, tumor metastasis. We have utilized protein nanopatterns prepared by colloidal lithography to study cell adhesion, spreading, and focal adhesion formation and development at ligand patterns of VN and FN. We observe similar cell numbers but very different cell spread area,

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cell morphology, and focal adhesions at small VN patches (0.2 and 0.3 μm) compared to small FN patches (0.2 and 0.3 μm). Importantly, we observe single focal adhesions (as identified by staining for vinculin and zyxin) bridging across gaps between ligand patches, while integrins (Rv) are observed localized to the ligand patches and do not appear to extend in between. Such bridging occurs at VN patches from 0.2 μm but not until 0.5 μm for FN patches. Larger patch sizes show this type of bridging to a smaller extent and, in addition, show a bridging of multiple FAs by single actin stress fibers. FN patches below 0.5 μm do not show defined FAs at all. There appears to be a differential ability of FN but not VN to prevent bridging of FAs across gaps between the small patch sizes. FN appears to be able to modulate the force exerted at these small patch sizes which may relate to its ability to alter conformation in response to mechanical force. For both proteins, the width of FAs that form is limited by the size of the protein patches suggesting a route to regulate cell mechanics through nanoscale protein patterns.

’ ASSOCIATED CONTENT

bS

Supporting Information. Detailed experimental methods, characteristic data for substrates, data and images from full repeat, and images from staining against FN and integrins. This material is available free of charge via the Internet at http://pubs. acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT We thank Jacques Chevallier and Folmer Lyckegaard for kindly performing sputtering and evaporation. This work was funded through the Danish research council (645-05-0016 and 274-08-0464). ’ REFERENCES (1) Geiger, B.; Bershadsky, A.; Pankov, R.; Yamada, K. M. Nat. Rev. Mol. Cell Biol. 2001, 2 (11), 793–805. (2) Geiger, B.; Spatz, J. P.; Bershadsky, A. D. Nat. Rev. Mol. Cell Biol. 2009, 10 (1), 21–33. (3) Zimerman, B.; Volberg, T.; Geiger, B. Cell Motil. Cytoskeleton 2004, 58 (3), 143–159. (4) Arnold, M.; Cavalcanti-Adam, E. A.; Glass, R.; Blummel, J.; Eck, W.; Kantlehner, M.; Kessler, H.; Spatz, J. P. ChemPhysChem 2004, 5 (3), 383–388. (5) Cavalcanti-Adam, E. A.; Micoulet, A.; Blummel, J.; Auernheimer, J.; Kessler, H.; Spatz, J. P. Eur. J. Cell Biol. 2006, 85 (3 4), 219–224. (6) Dalby, M. J.; McCloy, D.; Robertson, M.; Agheli, H.; Sutherland, D.; Affrossman, S.; Oreffo, R. O. C. Biomaterials 2006, 27 (15), 2980–2987. (7) Flemming, R. G.; Murphy, C. J.; Abrams, G. A.; Goodman, S. L.; Nealey, P. F. Biomaterials 1999, 20 (6), 573–588. (8) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276 (5317), 1425–1428. (9) Lehnert, D.; Wehrle-Haller, B.; David, C.; Weiland, U.; Ballestrem, C.; Imhof, B. A.; Bastmeyer, M. J. Cell Sci. 2004, 117 (1), 41–52. (10) Brunette, D. M.; Chehroudi, B. J. Biomech. Eng. 1999, 121 (1), 49–57. 2270

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dx.doi.org/10.1021/nl200447q |Nano Lett. 2011, 11, 2264–2271