Probing the Role of Integrins in Keratinocyte Migration Using

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Probing the role of integrins in keratinocyte migration using bio-engineered ECM mimics Wilhelm Weihan Chen, Monica Suryana Tjin, Alvin Wen Choong Chua, Seng Teik Lee, Chor Yong Tay, and Eileen Fong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06959 • Publication Date (Web): 02 Oct 2017 Downloaded from http://pubs.acs.org on October 3, 2017

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ACS Applied Materials & Interfaces

Probing the role of integrins in keratinocyte migration using bio-engineered ECM mimics ξ

ξ

Wilhelm W. Chen†‡, Monica S. Tjin §, Alvin W. C. Chua , Seng Teik Lee , Chor Yong Tay†∇*, Eileen Fong †* †

School of Materials Science and Engineering, Nanyang Technological University, N4.1, 50

Nanyang Avenue, Singapore 639798, Singapore ‡

Mechanobiology Institute, National University of Singapore, 5A Engineering Drive 1, 117411,

Singapore §

Duke-NUS Medical School, Program in Cardiovascular and Metabolic Disorder, 8 College

Road, Singapore 169857, Singapore ξ

Department of Plastic Reconstructive & Aesthetic Surgery, Singapore General Hospital,

Outram Road, Singapore 169608, Singapore ∇

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive,

Singapore 637551, Singapore

KEYWORDS：： biomimetic materials, engineered protein, surface display, keratinocyte, cell migration, integrin

*Corresponding authors: [email protected], [email protected]

1 Environment ACS Paragon Plus


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Abstract

Bio-engineered extracellular matrix (ECM) mimetic materials have tunable properties and can be engineered to elicit desirable cellular responses for wound repair and tissue regeneration. By incorporating relevant cell-instructive domains, bioengineered ECM mimics can be designed to provide well-defined ECM-specific cues to influence cell motility and differentiation. More importantly, bio-engineered ECM surfaces are ideal platforms for studying cell-material interactions without the need to genetically alter the cells. Here, we showed that bioengineered ECM mimics can be employed to clarify the role of integrins in keratinocyte migration. Particularly, the role of α5β1 and α3β1 in keratinocytes were examined, given their known importance in keratinocyte motility. Two recombinant proteins were constructed — each protein contains a functional domain taken from fibronectin (FN-mimic) and laminin-332 (LN-mimic), designed to bind α5β1 and α3β1 respectively. We examined how patient-derived primary human keratinocytes migrate when sparsely seeded as well as when allowed to move collectively. We found consistently, that FN-mimic promoted cell migration while the LN-mimic did not support cell motility. We showed that when keratinocytes utilize α5β1 integrins on FN-mimics, they were able to form stable focal adhesion plaques and stabilized lamellipodia. On the other hand, keratinocytes on LN-mimic utilized primarily α3β1 integrins for migration and strikingly, cells were unable to activate Rac1 and form stable focal adhesion plaques. Taken together, employment of our bioengineered mimics has allowed us to clarify the roles of α5β1 and α3β1 integrins in keratinocyte migration, as well as further provided a mechanistic explanation for their differences.


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1. Introduction It is desirable to tailor the microenvironment of a cell to direct cellular behavior for proper tissue repair and regeneration. For example, biomaterials have been designed to incorporate native extracellular matrix (ECM) proteins so as to provide the relevant biological, mechanical or topographical cues to influence cellular decisions. With advances of recombinant technologies, it is now possible to engineer simple biomolecules that contain selected functional motifs (e.g. RGDS, IKVAV) derived from native ECM proteins. Bioengineered ECM mimics can be designed to have well-defined properties, and can be produced in large-scale using synthetic means. For instance, a variety of bioengineered ECM mimics have been used to regulate cell phenotypes1, study the synergistic effects of various ECM motif combinations on cell proliferation2, and elucidate the various functions of ECM domains in cell adhesion3. Bioengineered ECM mimics can also be designed to include ligands that elicit a desired cellular behavior. For example, Garcia and co-workers have reported the use of collagen-based peptides to control the manner in which cells interact with the surface4. They showed that when fibroblast-like cells preferentially utilize the α2β1 integrins for binding, cells can be guided to selectively differentiate into osteoblasts instead of other lineages. Similarly, the full-length fibronectin 9th and 10th domains has also been shown to increase wound healing rates compared to short RGD peptides5. Hence, there are tremendous interests to use ECM mimics as outside-in approaches to guide cellular behaviors, or as platforms to study how cellular behavior in their unaltered states.



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It is known that both fibronectin and laminin-332 (formerly known as laminin-5) are upregulated during wounding6-7. In particular, laminin-332 is a major ligand for keratinocyte adhesion in the epidermis, and is generally thought to contribute to wound healing by supporting cell adhesion, spreading and proliferation. Keratinocytes are thought to utilize the α5β1 integrins for binding to fibronectin, while the α3β1 integrin is used for interaction with laminins. Yet, the role of these integrin-mediated ECM engagements in keratinocyte migration during skin wound healing remained controversial to date. Several groups showed that engagement with fibronectin or laminin-332 enhances keratinocyte migration8-11. Conversely, there are reports showing the anti-migratory properties of fibronectin12 and laminin-33213-16. Traditionally, integrin-specific studies rely on the genetic ablation of target integrins in the cell and observing their behavior on native ECM substrates. However, genetic ablation of α3 in keratinocytes (Itga3-/-) can lead to unwanted consequences such as uncharacteristic laminin332 remodeling17, enhanced activities of fibronectin and collagen receptors18, or triggering of TGF-β-mediated Smad7 which decreases TGF-β receptor 1 and Smad2/Smad3 activities.19 In addition, cells can make multiple interactions with their underlying native ECMs20; these interactions may directly or indirectly influence cell motility. Hence, artefacts rising from genetic manipulation of the cells, as well as complex interactions with the ECMs, could have contributed to contradictory conclusions about the role of integrins in cell motility. In this work, bioengineered ECM mimics were employed to study the role of integrins in cell migration. We incorporated motifs with high selectivity towards α5β1 and α3β1 integrins within our ECM mimics, and examined how primary normal human skin keratinocytes migrated on these surfaces. Figure 1 illustrates our design approach as well as the steps involved in the construction and purification of the ECM-mimetic recombinant proteins. The first recombinant


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protein contains the fibronectin domains 9th and 10th, designed to preferentially engage α5β1 integrins21 (named FN-mimic). The second variant bears the PPFLMLLKGSTR sequence, taken from laminin-332 (named LN-mimic). Several studies in the literature have reported the ability of PPFLMLLKGSTR to promote wound healing22-24, even though its working mechanism is not clarified. Nonetheless, the PPFLMLLKGSTR sequence was previously shown to bind α3β1 integrins25, and is ideal for investigating the role of α3β1 integrins in cell motility. Elastin-like polypeptide sequences were added to both proteins to aid protein expression and purification. We cultured primary normal human skin keratinocytes (NHKs) on both surfaces and examined their migratory behaviors using time-lapse microscopy. We found that FN-mimic substrates promoted keratinocyte migration while the LN-mimic does not. Similar trends were also observed in collective migration of keratinocytes, where “wounds” on LN-mimic surfaces did not advance as far compared to on FN-mimic substrates. We also found that keratinocytes cultured on FN-mimic substrates exhibited “fan-shaped” morphologies, while cells on LN-mimic surfaces adopted primarily bipolar or multipolar morphologies. In addition, on LN-mimic substrates, keratinocytes failed to form stress fibers, focal adhesion plaques or activate Rac1. Taken together, our findings show that α5β1 integrin engagement promotes keratinocyte migration whereas α3β1 integrin engagement alone is not sufficient to promote keratinocyte migration.

2. Experimental section Construction of FN- and LN-mimic recombinant proteins: Genes encoding for both recombinant proteins were separately ligated into the pET-19b expression vector (Novagen) using standard cloning techniques24 via NcoI and NotI restriction sites (New England Biolabs,



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MA). The recombinant plasmids were transformed into Escherichia coli XL10-Gold competent cells (Agilent Technologies, CA) using heat shock with 2xYT media and plates with ampicillin. Transformed colonies were screened by DNA sequencing (1st BASE, Singapore). Protein Expression and Purification: Transformed colonies in BL21(DE3)pLysS were cultured in Terrific Broth at 37 °C overnight with shaking at 225 rpm. Then, 10 mL culture was transferred to 1 L 2xYT and incubated at 37 °C for 3 h. When the optical density at 600 nm (OD600) reached 0.6 to 0.8, protein expression was induced by adding IPTG (1 mM, Gold Biotechnology, St. Louis, MO) for 4 h. Cells were harvested by centrifugation at 4 °C, 9000 rpm for 30 min, and resuspended in TEN buffer at a concentration of 0.5 g/mL. A series of freeze/thaw cycles at –80 °C and RT was performed to lyze the bacteria cells. Phenylmethylsulfonyl fluoride (50 µg/mL) was added to inhibit proteases. Complete cell lysis was achieved by sonication on ice for 30 min with 30% amplitude and 5 s pulses. The sonicated mixture was subjected to inverse transition cycling, exploiting the thermoresponsive properties of elastin. First, NaOH was added dropwise to the sonicated mixture to achieve pH 9, and stirred for 2 h to achieve a homogenous solution. The solution was then centrifuged at 40,000 g for 2 h at 4 °C (cold cycle). The supernate was collected before adding NaCl (to final concentration of 1 M). The supernate was then warmed to 37 °C to trigger protein aggregation. The solution was again centrifuged at 40,000 g at 37 °C for 2 h (warm cycle). The pellet was harvested and resuspended in pH 9 water at 4 °C overnight. The cold/warm cycles were repeated 3 – 4 times to increase the purity of the target proteins. The final solution was dialyzed against pure water and lyophilized. The molecular weights and purities of the as-obtained proteins were verified by SDS-PAGE and Western blot.


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Keratinocyte Isolation and Cell Culture: Primary normal human epidermal keratinocytes (NHK) were isolated from skin biopsies of four adult skin donors at Singapore General Hospital. NHKs were cultured with a feeder layer of gamma-irradiated 3T3-J2 fibroblasts in Gibco DMEM and Ham’s F12 media (3:1 mixture) with 10% FBS, 5 µg/mL insulin, 0.18 × 10-3 M adenine, 0.4 µg/mL hydrocortisone, 0.1 × 10-9 M cholera toxin, 2 × 10-9 M triiodothyronine, 10 ng/mL epidermal growth factor and 100 µg/mL penicillin-streptomycin as Green’s method.26 Subsequently, NHKs were cultured in serum-free media EpiLife (Thermo Fisher Scientific, MA) supplemented with EpiLife Defined Growth Supplement and 1% penicillin/streptomycin. All experiments were performed with NHKs at passage three to avoid interference of serum and differentiation. Keratinocyte Migration Characterization on Protein-coated Surfaces: Native FN/LN (0.01 mg/mL) and FN-/LN-mimic proteins (1 mg/mL) were adsorbed on 24-well plates or glass slide (Lab-Tek Chamber Slide, Thermo Fisher Scientific) overnight at 4 °C. Subsequently, surfaces were rinsed thrice with DPBS, and blocked with 1% heat-denatured BSA for 30 min at room temperature and washed thrice with DPBS. For single cell migration analysis, NHKs were sparsely seeded at a density of 3 × 103 cells/cm2 and allowed to adhere to the surfaces for 2 h before subjected to time lapse microscopy (Olympus IX81) in serum-free EpiLife media at 37 °C and 5% CO2/95% air atmosphere. Images at various positions were acquired at 10 min intervals for 12 h. Only isolated cells were analyzed (n > 100) for each condition. For collective cell migration studies, glass slides were first incubated with various proteins overnight at 4 °C. PDMS blocks (10 x 10 mm) were then placed in the center of each well to create cell-free zones. Keratinocytes were then cultured on various surfaces and grown to



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confluence. Upon confluence, the PDMS blocks were removed and the blocked area was recoated with the respective proteins for 30 min at 37 °C. The wound edge was captured at 30 min time interval for 48 h. Migrating NHKs (n > 50) were manually tracked using MtrackJ27. At least three independent experiments from NHKs of three different donors were performed.

Keratinocyte

Morphological

Characterization

on

Protein-coated

Surfaces:

For

morphological characterization, cells were traced manually from immunostaining data using ImageJ. Keratinocytes were regarded as “fan-shaped” cells if their morphologies resembled that shown in Figure 5a (top image). Percent “fan-shaped cells” was calculated by dividing the number of “fan-shaped cells” by the total number of cells analyzed. Cell areas and solidity indices were further calculated using the shape descriptor function of ImageJ28. The solidity index is calculated by dividing image area by the convex hull area (see Figure S3a). Typically, a concentric circle has a solidity index of 1.

Pharmacological and Antibodies Treatments: Anti-integrin antibodies (anti-α5β1, JBS5; anti-α3, P1B5; anti-α6, MAb 1378; Thermo Fisher Scientific) were added at 0.2 µg/ml prior to cell seeding. RhoA inhibitor, Rhosin and Rac1 inhibitor (Merck) were directly added 1 h before cell-seeding at final concentrations of 30 µM and 50 µM respectively. Immunofluorescence staining: After 5 h of culture in serum free media, NHKs were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature. The cells were permeabilized and blocked with 0.3% Triton-X 100, 5% goat serum for 1 h at ambient temperature. The samples were then incubated with antibodies (monoclonal rabbit anti-paxillin, Abcam; monoclonal mouse anti-human vinculin, Sigma; monoclonal rabbit Phospho-anti-FAK (Tyr397), Thermo Fisher Scientific; monoclonal mouse anti-α-Tubulin, Sigma) diluted in the blocking


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buffer overnight at 4 °C. On the following day, the samples were washed trice with PBS and incubated with anti-rabbit Alexa Fluor 488 secondary antibody or anti-mouse Alexa Fluor 568 secondary antibody (Abcam) and Alexa Fluor 647 Phalloidin (Thermo Fisher Scientific) for 1 h at room temperature in the dark. After incubation, samples were washed thrice with PBS and subsequently mounted using the ProLong gold antifade reagent with DAPI (Thermo Fisher Scientific). Fluorescence images were acquired using confocal spinning disk microscopy (Nikon CSU-W1). Representative images were selected from more than 50 cells observed. Statistical analysis: Experiments were performed in triplicates with NHKs from three different donors. The one-way analysis of variance (ANOVA) and the Student two-tailed t test were used for data analysis. Data are means ± standard errors of the mean.



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3. Results and Discussion 3.1. Employment of ECM mimics to reveal the relative importance of engaged integrins for single and collective keratinocyte migration Cell migration is an integral part of the wound healing process. Upon wounding, keratinocytes at the wound edge disassemble from neighboring cells, and are required to migrate over the wound bed. Since keratinocytes express both α5β1 and α3β1 in response to wounding20, it is necessary to clarify how each integrin influence in cell motility. To eliminate contributions due to cell-cell interactions, we seeded NHKs sparsely on surfaces adsorbed with various proteins and only stringently analyzed cells that were isolated from one another. Trajectories of 100 cells were tracked over a period of 12 h for each surface and their average velocities were calculated. Cells on native FN and FN-mimic surfaces were able to migrate freely, moving with distances of up to 200 µm from their starting positions (Figure 2a). We noted that keratinocytes on FN-mimic substrates adopted similar trajectories, even though cells travelled shorter distances compared to native FN. Interestingly, keratinocytes on native LN did not migrate as far as that of native FN surfaces. In addition, we also found that keratinocyte motility was severely impaired on LN-mimic protein substrates. When we tabulated the average cell speeds for each surface, we found that keratinocytes on native FN had the highest speeds (68.6 ± 6.4 µm/h), consistent with literature values29 reported for fibronectin (Figure 2b). Significantly, lower cell speeds were recorded for FNmimic (48.8 ± 5.8 µm/h) and native LN surfaces (47.2 ± 4.7 µm/h). In contrast, keratinocytes on LN-mimics displayed an almost 5-fold decrease in cell speeds (10.2 ± 2.3 µm/h).


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We wondered if similar trends could be observed in collective migration of keratinocytes. We performed an in vitro “wounding” assay where cells were allowed migrate over a denuded area coated with various proteins of interest (Figure 3a). This assay simulates physiologicallyrelevant wound repair through biochemical cues30-31. We quantified the overall rates of “wound closure” by following the “wound edge” over 48 h (Figure 3b, d). Indeed, all surfaces apart from LN-mimic substrates promoted similar extents of wound closure. To compare with our single cell migration data, we tracked cells at the “wound” edge that has migrated into the “wound area”. Figures 3c and e show the trajectories and speeds of tracked cells (n > 50 for each surface). Consistent with our single cell migration observations, we found that keratinocyte migration on LN-mimic surfaces was significantly impaired. Cell trajectories were markedly shorter, with average cell speeds that were 2-folds lower than on all other surfaces (Figure 3e). Taken together, surfaces coated with LN-mimics did not promote wound closure, likely attributed to hampered cell motility.

3.2. Bio-engineered ECM mimics exhibit high selectivity towards respective integrins in keratinocyte migration Both single and collective migration data clearly show that FN-mimic proteins promoted keratinocyte migration while LN-mimics did not. We hypothesized that the striking difference was likely due to the type of cell–ECM interactions present. To clarify this, cells were treated with integrin-blocking antibodies to determine if the respective integrins were indeed responsible for cell migration on various surfaces. Figure 4 show the trajectories and speeds of cells on various surfaces, treated with anti-α3, anti-α6 and anti-α5β1 antibodies.



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On both native FN and FN-mimic substrates, cell motility was significantly impaired in the presence of anti-α5β1 antibodies. We also noted a dramatic change in their cell trajectories and reduction in their average cell speeds, suggesting that the α5β1 integrin was indeed utilized for migration on native FN and FN-mimic surfaces (Figure 4a). Addition of anti-α3 antibodies severely reduced cell motility on both native LN and LNmimic surfaces, suggesting that keratinocytes utilized α3β1 for migration on both surfaces. Interestingly, addition of anti-α6 integrins only affected cell migration on native LN, but not on LN-mimics (Figure 4b). This result confirmed that LN-mimic proteins bind exclusively to the α3β1 integrins via the PPFLMLLKGSTR motif. We also confirmed that keratinocytes indeed expressed the respective integrins regardless of the substrates they were cultured on (Figure S2). Hence, we conclude that FN-mimics selectively engaged α5β1 integrins, whereas the LN-mimics preferentially engaged α3β1 integrins for cell motility. We also noted disparities in the cell speeds presented in Figure 2b and the respective controls in Figures 4c and d. We attributed these differences to significantly fewer cells analyzed in Figures 4c and d (i.e., n < 20 per condition), compared to n < 100 per condition in Figure 2b. Nonetheless, we were still able to observe similar trends, where cell speeds on LN-mimics were significantly lower than on all other surfaces.

3.3. Keratinocyte morphologies and immunostaining We observed that keratinocytes exhibited distinct morphologies in response to the type of proteins presented. On native FN and FN-mimic surfaces, cells often adopt a “fan-shaped” appearance (Figure 5a, top image), which is a hallmark of polarization32. In addition, polarized


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cells are also known to be highly migratory in nature32. We found that 50 – 60% of the cells adopted the “fan-shaped” morphology on all native FN and FN-mimic surfaces (Figure 5b). In contrast, the number of “fan-shaped” cells found on LN-mimic surfaces was only about 5%. In fact, most of the cells examined were found to have either bipolar or multipolar morphologies (Figure 5a, bottom image). We have also further quantified cell shapes by determining their solidity indices (Figure S3). In particular, the mean solidity index for LN-mimics was found to be the lowest among the surfaces. This data supports our observations where keratinocytes on LN-mimics typically adopt elongated appearances and not “fan-shaped” morphologies. We further followed cells on various surfaces for 12 h, and tracked the positions of their nuclei. Cells on both of these surfaces were also found to move with larger displacements (up to 40 µm) within a 12-h duration (Figure 5c). Further, it was also clear from Supporting Movie S4, that cells on LN-mimic surfaces were frequently making protrusions, but were unable to form stable lamellipodia. As a result, there were minimal displacements in their positions throughout the 12-h duration (Figure 5d). Given the distinct difference in cell morphologies on FN- and LN-mimic substrates, we wondered if there are any involved in physical connections between ECMs and the cytoskeleton through integrins, all of significant differences in the distribution of focal adhesion proteins and cytoskeletal organizations. Focal adhesion proteins are which mediate cell functions between the cell and the environment, in particular, cell migration33. Figure 6 shows immunofluorescence images of keratinocytes adhered on various proteins surfaces. On native FN and FN-mimic substrates, large elongated focal adhesion (FA) plaques that were rich in paxillin (Figures 6a – h), vinculin and pFAK (Figure S4) were



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observed. These FA plaques were located at the terminals of actin stress fibers. Cells on both surfaces have well-organized α-tubulins and actin bundles; these features are characteristics of highly motile keratinocytes32. In contrast, cells grown on native laminin have stress fibers and contiguous FA plaques at the cell peripheries (Figures 6i – l). In comparison, cells adhered on LN-mimic surfaces barely form FA plaques and transverse arcs stress fibers (Figures 6m − p). This finding is in line with the previous study showing that integrin α3β1-deficient mouse keratinocytes increase stress fiber formation and concentration of FA proteins at focal contacts.18 Establishment of stable lamellipodia requires the transport of actin monomers and concerted recruitment and activation of FA proteins. It is well known that paxillin is the first component in the FA formation34 and its phosphorylation is required for the recruitment of vinculin with pFAK at immature adhesion sites.35 The maturation of FAs then reinforces the traction force exerted by cells to migrate.36 In addition, we found that a large proportion of cells on LN-mimic surfaces have disrupted and loose microtubule networks (high magnification image of the cytoskeleton can be found in Figure S5). Taken together, our data suggest that engagement of the α3β1 integrin did not promote FA formation and actin bundles; both events are necessary in order for cells to establish stable lamellipodia and make translocations.

3.4. Cellular pathways affected by integrin engagement Finally, we subjected cells to various GTPase inhibitors to study how cellular pathways were affected by the choice of integrin engagement. Both Rac1 and Rho are widely known to regulate the actin dynamics contributing to cell migration.37 During cell migration, Rac1 involves


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in the recruitment of actin monomers and focal complex formation at protrusion sites. Rho partakes in the late process to mature FA and regulate actomyosin-mediated contraction.37 We treated cells seeded on various substrates with inhibitors of Rac1 (Rac1 Inhibitor, Merck) and Rho (Rosin), and analyzed their migratory behaviors (Figure 7). Note that the values of the respective controls for Figures 7a and b were taken from Figures 2a and 5b respectively. Here, inhibition of Rho resulted in reduction in cell speeds, only for native FN and FN-mimic surfaces, but had no significant effects on native LN or LN-mimics. However, inhibition of Rac1 affected cell speeds across all surfaces (Figure 7a). Importantly, blocking Rac1 had more severe morphological effects on native LN, compared to LN-mimics and fibronectin-based surfaces (Figure 7b). It has been shown on laminin that Rac1 is required for the formation of stable lamellipodia mediated by α3β1 integrin.32 Indeed, we found that on native LN, the percent of cells with fan-shaped lamellipodia had decreased by almost 9-folds, resulting in a significantly lower number of solidity index (Figure S6). Instead, almost all of the cells adopted the bipolar or multipolar appearance that frequently make unstable protrusions, and became non-migratory, displaying similar features of cells cultured on LN-mimic surfaces. This observation is consistent with previous findings where α6β4 engagement was shown to be necessary for Rac1 activation.38



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4. Conclusion In this work, we utilized bioengineered ECM mimics to examine the role of integrins in keratinocyte motility. We designed two variants of recombinant proteins containing key ligands derived from fibronectin and laminins. We found that keratinocytes preferentially engage α5β1 integrins on FN-mimic (bearing PHSRN and RGD motifs), but α3β1 integrins on LN-mimic substrates (bearing PPFLMLLKGSTR). Despite no differences in cell attachment rates on FNand LN-mimetic substrates24, FN-mimic proteins promoted keratinocyte migration, but LNmimics did not. Hence, we conclude that α5β1 engagement is critical for proper FA formation to support cytoskeletal organization and keratinocyte migration (Figure 8a). On native LN, α3β1 integrins as well as both α6 integrins (i.e., α6β1 and α6β4) were used for migration38-39. However, keratinocytes utilized only α3β1 integrins for binding to LN-mimic. Hence, engagement of α3β1 integrins alone was insufficient to result in the recruitment of FA proteins and in turn, activate Rac1 for cell migration (Figure 8b)38, 40. In summary, we showed that an outside-in approach can be used to probe the role of integrins in cell motility without genetically altering the cells. The observations in this work are in agreement with previous reports that demonstrated the anti-migratory role of α3β1 integrin. Here, we further provide a mechanistic explanation of how α3β1 integrins regulate cell motility. Hence, there is great promise to use bioengineered ECM mimics as useful tools to study cell– ECM interactions in order to provide guidelines for future design of biomaterials that can be used to regulate cell motility in vivo.


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ASSOCIATE CONTENT Supporting Information. This material is available free of charge via the Internet at ACS Publications website at DOI: Methods Figure S1｜Genetic maps and full sequence of FN-mimic and LN-mimic proteins. Figure S2｜ RT-PCR analysis of integrin expression levels on cells cultured on various protein substrates. Figure S3｜Cell shape analysis of keratinocytes. Figure S4｜Immunostaining for pFAK and vinculin of cells cultured on various substrates. Figure S5｜Magnified images of microtubules networks of keratinocytes cultured on FN-mimic (Left) and LN-mimic (Right) substrates shown in Figure 6g, and 6o. Figure S6｜Solidity indices of cells on various surfaces subject to Rac1 or Rho inhibitors. Movie S1｜Keratinocyte migration on native fibronectin. Movie S2｜Keratinocyte migration on FN-mimic. Movie S3｜Keratinocyte migration on native laminin. Movie S4｜Keratinocyte migration on LN-mimic. Movie S5｜Keratinocyte collective migration on native fibronectin. Movie S6｜Keratinocyte collective migration on FN-mimic. Movie S7｜Keratinocyte collective migration on native laminin. Movie S8｜Keratinocyte collective migration on LN-mimic.

AUTHOR INFORMATION Corresponding Authors Eileen Fong, [email protected] Chor Yong Tay, [email protected]



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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors acknowledge funding from Ministry of Education, Singapore (RG41/10) and National Research Foundation, Competitive Research Program (NRF-CRP11-2012-02). Skin biopsies were taken after obtaining approval by the Centralised Institutional Review Board (CIRB) of Singapore General Hospital (SGH). W. Chen is supported by Taiwan’s Ministry of Education and the Singapore International Graduate Award (SINGA) from A*STAR. We thank Associate Professor Virgile Viasnoff (NUS) and Céline Stoecklin for microscopy assistance.


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Figure 1｜Design and construction of ECM-mimetic recombinant proteins bearing fibronectin and laminin-332 domains. Five steps involved in the construction of FN-mimic and LN-mimic proteins are illustrated. After purification, proteins were physically adsorbed on cell culture petri dishes. The “outside-in” approach is also illustrated graphically. Complete amino acid sequences of both recombinant proteins can be found in Figure S1. 130x104mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Figure 2｜Bioengineered ECM-mimics and their native compartments modulate keratinocytes migration. (a) Plots showing trajectories of cells (n > 100/condition) sparsely seeded on various protein substrates. (b) Average cell speeds of cells tracked on various protein substrates in (a). **P < 0.01, ***P < 0.001. 96x56mm (300 x 300 DPI)



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Figure 3｜Collective cell migration studies using an in vitro “wounding” assay. a) Schematic diagram of ‘wounding’ assay. b) Phase contrast images of “wounding assay” on time points and substrates. c) Migration plots of wound edge keratinocytes on various substrates. Distances are measured in µm. d) Wound closures rates on various protein surfaces. e) Average cell speed for individual cells migrating at the “wound” edge for first 8 h. Error bars represent ±SEM from five independent experiments. ***P < 0.001. 209x274mm (300 x 300 DPI)


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Figure 4｜Bio-engineered proteins exhibit high selectivity towards respective integrins in NHK motility. Trajectories and cell speeds of NHKs seeded on native FN/FN-mimic substrates (a, c) and native LN/ LNmimic substrates (b, d) with various integrin blocking treatments (n < 20 per condition). *P 100 per surface). c - d) Comparing displacements of NHKs between native FN/FNmimic substrates (c) and native LN/LN-mimic substrates (d). ***P < 0.001. 183x212mm (300 x 300 DPI)


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Figure 6｜Focal adhesion and cytoskeleton formation on native fibronectins (a-d), fibronectin-mimics (e-h), native laminin (i-l) and LN-mimics (m-p). Scale bars are 10µm. Keratinocytes form focal adhesion plaques (stained for paxillin, yellow), trans-arc stress fibers (stained for actin with phalloidin, red) and microtubule networks (stained for α-tubulin, green) on ECM surfaces except LN-mimics. Nuclei were stained with DAPI. Immunofluorescence images were acquired by confocal Spinning-disc microscopy and then pseudocolors were added by ImageJ. 173x173mm (300 x 300 DPI)



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Figure 7｜Treatment of Rac1 and Rho inhibitors affects (a) cell motility and severely interfered with the (b) ability of keratinocytes to form lamellipodia on native laminin. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 69x29mm (300 x 300 DPI)


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Figure 8｜Interactions between bio-engineered proteins and keratinocytes lead to differences in keratinocyte motility. a) FN-mimics engaged α5β1 integrins promoted focal adhesion and lamellipodia formation. Both events resulted in high cell motility, comparable to native fibronectin. b) LN-mimics engaged α3β1 integrins where cells were unable to maintain stable membrane protrusions necessary for migration. 92x73mm (300 x 300 DPI)



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Table of Content 111x59mm (300 x 300 DPI)


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Probing the Role of Integrins in Keratinocyte Migration Using

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