Integrin α5β1 nano-presentation regulates collective keratinocyte migration independent of substrate rigidity

Nanometer-scale properties of the extracellular matrix influence many biological processes, including cell motility. While much information is available for single-cell migration, to date, no knowledge exists on how the nanoscale presentation of extracellular matrix receptors influences collective cell migration. In wound healing, basal keratinocytes collectively migrate on a fibronectin-rich provisional basement membrane to re-epithelialize the injured skin. Among other receptors, the fibronectin receptor integrin α5β1 plays a pivotal role in this process. Using a highly specific integrin α5β1 peptidomimetic combined with nanopatterned hydrogels, we show that keratinocyte sheets regulate their migration ability at an optimal integrin α5β1 nanospacing. This efficiency relies on the effective propagation of stresses within the cell monolayer independent of substrate stiffness. For the first time, this work highlights the importance of extracellular matrix receptor nanoscale organization required for efficient tissue regeneration.


Introduction
Collective cell migration is a fundamental biological process characterized by the coordinated movement of interconnected cells to achieve specific functions [1][2][3] . Basal keratinocytes collectively migrate to re-epithelialize a wound in the skin as the first step to re-establish tissue integrity 4,5 . At the onset of migration, keratinocytes deposit a provisional basement membrane that provides the necessary support for adhesion required for cell locomotion 6 . Among other components of this provisional matrix, fibronectin plays a pivotal role in supporting keratinocyte migration via the adhesion of a5b1 and avb1 integrins that are upregulated in re-epithelialization [7][8][9] . In contrast to avb1, which plays only a minor role in migration, a5b1 is required for efficient reepithelialization, partially through the regulation of cellular traction forces 10,11 . In epithelial collective migration, each cell needs to coordinate cell-matrix traction forces with cell-cell stresses in order to propagate directed migratory cues to surrounding neighbours 2,12-14 . Previous work from our group and others have elucidated the many aspects of intercellular stress coordination governing collective cell migration 12,13,15-17 , including the overall stress heterogeneity present in the migratory cell sheets, as well as the single molecular players involved in the mechanotransduction process 13,16 .
Nevertheless, the role of extracellular matrix (ECM) adhesive sites in regulating such migratory coordination remains largely unknown.
Over the past two decades, it has become clear that nanometer-scale properties of the ECM strongly influence cell motility, in particular at the single cell level 18,19 . Previous work from our lab has highlighted the critical role of integrin lateral nanospacing in controlling single cell spreading, migration speed, and persistence 20,21 . Additionally, lateral adhesion spacing has been shown to regulate intracellular force generation in a manner consistent with the molecular clutch model 19 . In order to understand the role of ECM nanoscale organization in collective cell migration, we took a bottom-up approach by synthesizing nanopatterned hydrogels of discrete spacings, such that adhesive site organization could be precisely controlled. In order to investigate integrin a5b1 regulation of keratinocyte re-epithelialization, we utilized an engineered a5b1 integrin-specific peptidomimetic as the adhesive ligand (Fig. S1A). This specific peptide has a high binding affinity for a5b1 integrin (IC50 = 1.5nM), but orders of magnitude lower affinity for the other RGDrecognizing integrins, including av-containing isoforms 22 . The advantage of using synthetic hydrogels lies in their ability to better mimic the physical properties of the ECM in the wound, as well as in their protein-repellent nature. With this approach, we could systematically address the role of cell-ECM interactions in cell monolayers by controlling both ECM stiffness and ligand density with nanometer precision. We were able to show that keratinocytes require an optimal integrin a5b1 spacing in order to efficiently coordinate their collective movement independent from ECM stiffness.

Results
Integrin a5b1 surface density controls keratinocyte migration efficiency. To investigate the role of integrin a5b1 in regulating keratinocyte collective migration, we fabricated gold nanopatterns on glass surfaces via block-copolymer micelle nanolithography (BCMN). BCMN allows for the precise modulation of ligand density at the nanometer level. The nanopatterns were covalently transferred onto polyacrylamide (PAA) hydrogels of ~ 23 kPa stiffness (approximately the stiffness of freshly wounded skin) 23,24 . The use of PAA hydrogels as culture substrate allows us to provide relevant substrate rigidity to cells and systematically control surface adhesion biochemistry due to the protein-repellent nature of PAA. Therefore, cells will only interact at nanoparticle sites, where highly specific integrin a5b1 peptidomimetics are linked, thereby allowing cell-surface adhesion via integrin a5b1 in a precisely controlled manner 25,26 (Fig. 1A). In continuity with previous studies indicating cell-ligand length scale relevance for focal adhesion formation and cell motility, we modulated ligand density using three discrete inter-ligand distances: 35, 50, and 70 nm 20,21 .
Confluent monolayers of human immortalized keratinocytes (HaCaT) were cultured within a lateral confinement using a polydimethylsiloxane (PDMS) stencil placed onto the nanopatterned PAA hydrogels (Fig. 1B). Cell monolayer density was comparable among all three inter-ligand distances (Fig. S2). After PDMS stencil removal, cell sheet migration was triggered onto the empty regions of the gel, mimicking the collective migration process of basal keratinocytes post-skin wounding 4,5 . Quantifying cell sheet migration speed over culture time revealed a differential migration efficiency on the three conditions, with the highest speed observed at 50 nm (~ 20 µm/h) vs. slower speeds at 35 and 70 nm (~10 µm/h) (Fig. 1C, E, sup. movie 1, 2 and 3).
Next, we examined the extent to which ECM nano-presentation affects the directional migration of individual cells within the monolayer. Each cell in the monolayer coordinates its movement with its neighbours, and the length of such coordination can be quantified by comparing the lateral components (orthogonal to the direction of sheet migration) of the individual cell velocity vectors in the monolayer 13 . We thus used particle image velocimetry (PIV) to measure the velocity fields of migrating keratinocytes after 16 hours and calculated the lateral correlation length, C(r) (Fig. 1C). This revealed that keratinocytes migrating on 50 nm inter-ligand spacing most efficiently coordinate their movements, with an average correlation which extended over ~ 20 cells (~ 400 µm) ( Fig. 1D, F). In contrast, keratinocytes on both 35 nm and 70 nm inter-ligand spacing were less able to coordinate their movements, only propagating their movements across a few cells (~ 200 µm) (Fig. 1D, F). The density of integrin-ligand interactions alters cell spreading and migratory persistence through the regulation of focal adhesion maturation and dynamics in fibroblasts 18,20 . Since the correlation length of epithelial cell migration can be regarded as single cell migratory persistence, we analysed how integrin a5b1 lateral spacing influences focal adhesion dynamics (Fig. 2). After transient transfection of HaCaT cells with a mCherry-a-paxillin construct, we tracked focal adhesions and quantified their lifetime (sup. movies 4, 5, 6).
Cells exhibited the highest ability to remodel their focal adhesions when migrating on 50 nm-spaced a5b1 ligands, resulting in the fastest turnover, or lowest lifetime. In contrast, on 35 and 70 nm inter-ligand spacing, focal adhesions exhibited longer lifetimes and therefore slower remodelling ( Fig. 2A, B).
Yet, a5b1 is not the sole ligand involved in keratinocyte migration. During wound healing, basal keratinocytes also interact with their provisional basement membrane using avcontaining integrin isoforms. We therefore examined whether migration was regulated in the same manner when engaging other integrins by using the c(RGDfK) peptide for cell attachment (Fig. S3A). c(RGDfK) allows for cell adhesion through a broader range of integrins, including avb1 and avb6, which are expressed by basal keratinocytes in the wound 8,22,27,28 . While c(RGDfK) still allows for a5b1 interaction, the affinity for this integrin subtype is lower compared to av-containing isoforms (IC50 = 200nM) 22 (Fig. S1B).
Interestingly, on c(RGDfK), keratinocytes exhibit faster migration on 35 nm inter-ligand spacing (vs. 50 nm on a5b1-presenting substrates), and correlation length does not change with receptor densities, which we showed to be biphasic for a5b1 (Fig. S3B, C).
All together, these data reveal the existence of a specific optimal integrin a5b1 density that promotes faster focal adhesion dynamics and more efficient keratinocyte coordination, thereby leading to the most effective re-epithelialization process.

Integrin a5b1 nanospacing controls force propagation in keratinocyte monolayers
Epithelial cells coordinate their movements as intercellular stress polarization increases, leading to collective cell migration through mechanotransductive processes 12,13,29 .
Integrin nano-presentation in single cells has been shown to regulate not only the persistence of migration but also traction force generation 19,20 . Therefore, we sought to understand how integrin a5b1 density controls the amount of surface traction forces, and consequently, the intercellular stresses in the monolayer. To do this, we incorporated fluorescent beads into nanopatterned PAA hydrogels to perform traction force microscopy (TFM) on migrating monolayers. Surprisingly, this revealed a significant increase in monolayer tractions with decreasing integrin a5b1 ligand density (Fig. 3C, D), which did not follow the optimum migratory behaviour (velocity, correlation length) that we observed on 50 nm inter-ligand spacing (Fig. 1). We previously showed that the distance of stress propagation within the monolayer is crucial for collective cell migration, and that this is related to the levels of actomyosin contraction in individual cells 13,17 .
Therefore, we were curious if integrin a5b1 lateral spacing differentially regulates the length scale of stress propagation in keratinocyte sheets, independent from the amount of traction forces generated.
To investigate this, we calculated the stress vectors using a force balance algorithm (monolayer stress microscopy -MSM) 12,17 from the measured traction forces. We did this within confluent monolayers themselves in order to avoid any bias resulting from migration of the cell sheets (Fig. 3A). While the comparison of the absolute values of stress was in line with the traction forces, the spatial correlation length of the stress vectors was the largest on 50 nm inter-ligand distance (C(r) ~ 200µm) versus on 35 nm or 70 nm (C(r) ~ 50 µm) (Fig. 3A, B). To understand if this stress correlation length relationship was specific to a5b1 integrin, we performed MSM on c(RGDfK)-functionalized substrates.
The results show no difference between the force correlation length on the three interligand densities, underlining the specific role of a5b1 in controlling the length of force propagation in keratinocyte monolayers (Fig. S3D).
In conclusion, the monolayer force correlation length was in line with the velocity correlation length in migrating keratinocyte sheets, suggesting that keratinocytes have an optimum integrin a5b1 density that best dictates the direction of intercellular stresses, and thereby the extent of force propagation in the monolayer. Representative phasecontrast images, monolayer stress plots and profiles, and spatial correlation curves from keratinocyte monolayers at the different integrin a5b1 ligand lateral spacings (35, 50, 70 nm). Stress correlation can be identified by inter-peak distance of the stress profile as schematically represented by the red arrows. Quantification of stress correlation length (B), traction forces (C) and stresses (D) in the three spacing conditions. Scatter and box and whiskers plots show values and mean ± s.e.m from at least 4 independent experiments. n.s. = not significant; ** p < 0.01; *** p < 0.001; **** p < 0.0001 using a Mann-Whitney test.

Integrin a5b1 lateral spacing overrides substrate stiffness and controls Ecadherin-mediated collective cell migration efficiency.
It has been shown both in vivo and in vitro that collective cell migration is affected by substrate rigidity, with stiffer substrates generally enhancing migration efficiency [30][31][32] .
Furthermore, it has been observed that focal adhesion dynamics increase with increasing substrate stiffness 33 . Therefore, we sought to understand the influence of substrate stiffness on the integrin a5b1 lateral spacing effect previously described on 23 kPa Finally, we sought to understand how integrin a5b1 ligand spacing directly regulates intercellular force transmission efficiency in keratinocytes. In epithelial sheets, the actin cytoskeleton physically connects cell-ECM adhesion structures with cell-cell adhesions (Fig. 1A). Between the latter, adherens junctions are responsible for maintaining the intercellular force continuum via E-cadherin-based homophilic interactions 16,30,34 . This mechanical continuum is crucial for coordinating collective migration by regulating intercellular pulling forces necessary for mechanotransduction pathways upstream of cell polarization 13 . We hypothesised that outside of the optimal 50 nm inter-ligand spacing of a5b1 peptide we observed, single keratinocytes would move counterproductively, thereby inhibiting monolayer motion. Thus, we inhibited E-cadherin interactions with the addition of a specific blocking antibody (DECMA-1) as previously shown 13 . When E- cadherin was inhibited, keratinocyte migration was enhanced on both 35 and 70 nm interligand spacing vs. 50 nm, as indicated by the distance of migration over culture time (Fig. 5A, B). Furthermore, when quantifying the change in sheet speed, Δv, after inhibition of Ecadherin vs. untreated cells, we observed migration speed was reduced on 50 nm vs. enhanced on 35 and 70 nm, indicating that the previously observed migration efficiency on 50 nm requires precise coordination between cell-cell and cell-ECM structures.
All together, these data indicate a vital role of integrin a5b1 lateral spacing in regulating keratinocyte collective migration. The optimal 50 nm lateral spacing outweighs the effect of substrate rigidity, as well as and regulates E-cadherin-mediated transmission of intercellular forces.

Discussion
We show here that ligand nanospacing is integral to collective migration in keratinocytes, even outweighing the effects of substrate stiffness. Specifically, we found that 50 nm interligand distance is optimal for integrin a5b1, whereby collective cell migratory speed  (Fig. 1D, F, Fig. 2).
Quantification of the monolayer stress distribution revelated that this ideal inter-ligand spacing is not dependent on the absolute traction forces generated on the surface, but rather by the correlation length of stress vectors in the monolayer (Fig. 3A, B). This was also confirmed on hydrogels of varying Young's moduli , with keratinocytes exhibiting enhanced migratory behaviour (faster and larger velocity and stress correlation lengths) at 50 nm spacing regardless of substrate stiffness (Fig. 4). This was particularly interesting because higher substrate rigidity is generally known to induce greater cellular traction forces, resulting in faster epithelial sheet migration 15,30,31 .
Each epithelial cell within a monolayer migrates in the direction of the maximal normal stress in a phenomenon called plithotaxis 12,29 . In cell collectives, adherens junctions, which are connected to the actin cytoskeleton, are required for intercellular force transmission 16,30 . As the actin cytoskeleton is also connected to the ECM, this explains why we observed disrupted keratinocyte migration on 50 nm integrin a5b1 ligand lateral spacing when E-cadherin interactions were inhibited by addition of DECMA-1 antibody (Fig. 5). In contrast, at higher and lower ligand spacing, keratinocytes were able to migrate faster when we disturbed intercellular force transmission (Fig. 5). Therefore, we can conclude that outside of the optimal integrin a5b1 inter-receptor spacing of 50 nm, uncorrelated intercellular stresses slow keratinocyte sheet migration and result in inefficient collective movement (Fig. 6).
It has previously been shown in single cells that integrin a5b1 regulates the generation of traction forces upon adhesion, whereas av-class integrins mediate ECM rigidity sensing 11 .
While we do observe faster collective migration with increasing substrate stiffness, as has been previously reported (Fig. 4A) 15,30,31 , a5b1 inter-receptor spacing outweighs stiffness effects alone. This could be attributed to integrin a5b1's role in regulating traction force generation but not rigidity sensing. Indeed, higher substrate rigidity causes higher traction forces at the sheet edge at the outset of migration (removal of PDMS stencil), thereby transmitting higher net stress to the cell followers, ultimately resulting in more efficient collective migration 15 . This validates our data that 50 nm lateral spacing of integrin a5b1 is required for efficient force transmission independent from the net force exerted on the cells.
The severe phenotype acquired by murine animal models genetically lacking a5 or b1 integrin subunits has prevented our deep understanding into the roles specific integrin subtypes and organization play in wound healing 35,36 . With our bottom-up approach, we systematically identified the importance of integrin a5b1 density in coordinating force propagation within keratinocyte monolayers. How this inter-ligand optimal spacing can Figure 6: Keratinocytes require an optimum integrin a5b1 density to efficiently collectively migrate. In contrast to the optimal integrin a5b1 lateral spacing (green), lower and higher spacings (red) lead to uncorrelated single-cell movement and stress propagation. This results in inefficient collective behaviour, significantly slowing keratinocyte sheet migration. be understood in the in vivo context still requires further exploration, but our findings suggest that an integrin subtype-specific arrangement may be present in specific biological contexts that function through mechanosensitive pathways mediated by focal adhesions. Future studies will be required to address this phenomenon in order to more closely dissect the role of integrins and associated signalling pathways in the mechanobiology of wound closure. The following ratio of acrylamide/bis-acrylamide were employed to reach different hydrogel Young's moduli: 10%/0.07% for 11 kPa; 7.5%/0.2% for 23 kPa; 12%/0.6% for 55 kPa; 12%/0.3% for 90 kPa 37 . Hydrogels were allowed to swell for at least 72 hours in PBS to facilitate the detachment of the coverslips and the transfer efficiency was evaluated by SEM of both the glass and hydrogels surface. The obtained hydrogels were sterilized with 30 minutes ultraviolet light irradiation before their employment as cell culture substrates. Traction force and monolayer stress microscopy. Traction force and monolayer stress microscopy (TFM, MSM) were performed as previously described 13,17 . 1 μm fluorescent carboxylate-modified polystyrene beads (Sigma) were mixed in the polyacrylamide solution before polymerization and allowed to reach the hydrogel surface by gravity during gelation. Cell-induced bead displacement vectors were calculated by comparing the picture with relaxed beads (cell-free gel after trypsinization) from the picture acquired with cells using the particle image velocimetry (PIV) plugin of ImageJ. Traction forces were calculated from these vectors using the Fourier transform traction cytometry plugin (FTCC). Average normal stress vectors within the monolayer were calculated using the traction force information using a force balance algorithm in MATLAB (MathWorks) as formulated elsewhere 17 . The force propagation within the monolayer is characterized by the force correlation length, which is the length-scale of the following spatial autocorrelation function: where Vmean is the mean lateral velocity. The lateral velocity correlation function was formulated as following: where 〈… 〉 symbolizes the average, r is the vector of coordinate (i,j) and r is the norm of r defined as r = ||r||. Similar to the force correlation length, the lateral correlation length was determined at the point where the function was equal to 0.01 13 .
Quantification of cell density. To quantify keratinocyte cell density on the different substrates, the monolayers were fixed for 10 minutes using 4% paraformaldehyde in PBS for the same conditions as in migration experiments. Upon permeabilization using 0.3% Triton X-100 for 2 minutes, the monolayers were stained for DNA using DAPI. The number of nuclei were counted using ImageJ software (NIH) in immunofluorescent imaged obtained at 10x magnification.
Statistics. Statistical tests and graphics were performed using GraphPad Prism software, choosing parametric or not parametric tests after evaluating the normality distribution of the data. Sample conditions and the specific test used for each data set are indicated in corresponding figure captions.