Integrin α5β1 nano-presentation regulates collective keratinocyte migration independent of substrate rigidity
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Curated by eLife
Evaluation Summary:
This paper is of great interest for researchers working in the filed of cell adhesion and cell migration. The authors show for the first time that alpha5beta1 ligand spacing profoundly influences the collective migration behaviour of keratinocytes. They demonstrate that this parameter can outcompete the well-known and studied influence of substrate stiffness.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
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.
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Evaluation Summary:
This paper is of great interest for researchers working in the filed of cell adhesion and cell migration. The authors show for the first time that alpha5beta1 ligand spacing profoundly influences the collective migration behaviour of keratinocytes. They demonstrate that this parameter can outcompete the well-known and studied influence of substrate stiffness.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
Collective cell motion plays important roles in embryogenesis, wound healing and for cancer progression. Speed and directionality is modulated by numerous parameters including gradients of soluble biochemicals and ECM substrates, substrate rigidity, etc. In the present study, Russo and colleagues investigated how collective migration of a keratinocyte cell line is influenced by integrin ligand nanospacing at different substrate rigidities. The authors perform the studies with gold particles conjugated with alpha5beta1-binding peptidomimetica in PAA hydrogels. They find that the nanospacing of integrin alpha5beta1 promotes the collective movement of keratinocytes (faster focal adhesion dynamics, better keratinocyte co-ordination) independent of substrate stiffness. In addition, the authors showed the …
Reviewer #1 (Public Review):
Collective cell motion plays important roles in embryogenesis, wound healing and for cancer progression. Speed and directionality is modulated by numerous parameters including gradients of soluble biochemicals and ECM substrates, substrate rigidity, etc. In the present study, Russo and colleagues investigated how collective migration of a keratinocyte cell line is influenced by integrin ligand nanospacing at different substrate rigidities. The authors perform the studies with gold particles conjugated with alpha5beta1-binding peptidomimetica in PAA hydrogels. They find that the nanospacing of integrin alpha5beta1 promotes the collective movement of keratinocytes (faster focal adhesion dynamics, better keratinocyte co-ordination) independent of substrate stiffness. In addition, the authors showed the migration efficiency by optimal ligand spacing depends on effective propagation of stresses and intercellular stress propagation/co-ordination mediated by E-cadherin.
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Reviewer #2 (Public Review):
This manuscript investigates the mechanism of collective keratinocyte migration in vitro. The study focuses on the manipulation of the precise distribution of the main integrin adhesion receptor, alpha5-beta1 integrin, required for migration. It finds that the nano spacing of alpha5-beta1 integrin ligands in the extracellular matrix is key to control collective cell migration. These findings are interesting as they go significantly beyond previous studies that largely focused on the stiffness of the extracellular matrix only and do not take their spatial arrangement into account to regulate collective cell migration. It is likely that similar principles are also relevant in vivo, although this manuscript is not directly testing this in in vivo models.
Strengths:
In collective cell migration, the cell sheet - …
Reviewer #2 (Public Review):
This manuscript investigates the mechanism of collective keratinocyte migration in vitro. The study focuses on the manipulation of the precise distribution of the main integrin adhesion receptor, alpha5-beta1 integrin, required for migration. It finds that the nano spacing of alpha5-beta1 integrin ligands in the extracellular matrix is key to control collective cell migration. These findings are interesting as they go significantly beyond previous studies that largely focused on the stiffness of the extracellular matrix only and do not take their spatial arrangement into account to regulate collective cell migration. It is likely that similar principles are also relevant in vivo, although this manuscript is not directly testing this in in vivo models.
Strengths:
In collective cell migration, the cell sheet - extracellular traction forces need to be well coordinated to enable coordinated migration of the entire sheet. How this is precisely achieved and which are the underlaying mechanisms is only partially known to date. In particular, the role of the extracellular matrix is unclear. Using state of the art nano-patterning technology Spatz and colleagues have produced nanopatterned hydrogels decorated with artificial highly specific alpha5-beta1 integrin ligands, mimicking a fibronectin matrix with a defined geometry. Hence, not only the stiffness of the matrix but also the spacing of the integrin ligand could be precisely controlled.
Using this technique, the authors find that optimal integrin ligand spacing (50 nm) strongly impacts sheet migration speed, similar as has been shown before in single cell migration assays. Optimal spacing (50 nm) is also needed for optimal coordination of the migrating cell sheet therefore explaining how it results in faster net migration of the entire sheet. This is supported by faster turnover of integrin adhesions, as assayed by tracking paxillin foci turnover.
To quantitate traction forces present in cell sheets, the authors incorporated fluorescent beads into the hydrogels and performed traction force microscopy. The results are intriguing. While the forces are largest at lowest ligand density (70 nm), the stress correlation, which is a measure of force coordination in the sheet, is longest at the intermediate density (50 nm) that also resulted in fastest sheet migration speed.
By varying the stiffness of the hydrogels, the authors make the interesting finding that not only the stiffness but to a larger extend the optimal density (50 nm) of the integrin ligands result in optimal migration speed of the cell sheets. The authors further suggest that the cells need Cadherin to coordinate the forces in the sheets effectively in order to migrate at maximal speed at the optimal ECM ligand density.
Together, these data support that optimal ECM protein distribution can strongly impact how well tissues can regenerate if collective migration is involved as is the case for wound healing. This is summarized in a nice model in Figure 6.
Weakness:
Traction forces were not measured in migrating sheets but only in stationary sheets. Hence, if the force regime on the leading edge of the migrating sheets similarly depends on ligand density as in stationary sheets is not known.
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Reviewer #3 (Public Review):
The manuscript by Di Russo et al., "Integrin a5b1 nano-presentation regulates collective keratinocyte migration independent of substrate rigidity" deals with the fundamental question of how the nanoscale organization and mechanical properties of the extracellular matrix control and regulate collective cell migration. In particular, the authors studied how the combined effects of integrin ligands nanoscale spacing and substrate stiffness control the collective migration of keratocytes.
The manuscript of Di Russo et al. builds on previous articles from the same group, in particular a study studying collective cell migration of keratocytes but focusing on the emergence of leader cells (Vishwakarma et al., Nat. Comm. 2018). Importantly, the authors developed their analysis pipeline in this previous article. The …
Reviewer #3 (Public Review):
The manuscript by Di Russo et al., "Integrin a5b1 nano-presentation regulates collective keratinocyte migration independent of substrate rigidity" deals with the fundamental question of how the nanoscale organization and mechanical properties of the extracellular matrix control and regulate collective cell migration. In particular, the authors studied how the combined effects of integrin ligands nanoscale spacing and substrate stiffness control the collective migration of keratocytes.
The manuscript of Di Russo et al. builds on previous articles from the same group, in particular a study studying collective cell migration of keratocytes but focusing on the emergence of leader cells (Vishwakarma et al., Nat. Comm. 2018). Importantly, the authors developed their analysis pipeline in this previous article. The current manuscript also uses integrin-specific ligands designed in a previous article (Rechenmacher et al., Angewandte Chemie 2013).
The main message of the manuscript is that the nanoscale spacing of a5b1 integrin ligands is a crucial parameter that controls collective cell migration. Importantly, this parameter prevails over the effects of substrate stiffness. This is particularly interesting since in isolated cells, it was recently demonstrated, that both the nanoscale spacing of integrin ligands and substrate stiffness control the formation on integrin-based adhesions. The authors used as a model system of collective cell migration, keratinocytes monolayers during wound healing.
The authors used nano-patterned hydrogels to control both integrin ligand spacing and substrate stiffness. The nano-patterned hydrogels were functionalized with a ligand highly specific for a5b1 integrin or a cyclic RGD (c(RGDfK)) more selective for avb1 integrin and avb6 integrin of keratocytes. They started with a stiffness of 23 kPa, which corresponds to the stiffness of wounded skin. The authors compared inter-ligand distances of 35 nm, 50 nm, 70 nm and showed that 50 nm is the most effective distance for a5b1 integrin to promote collective migration of keratocytes.
At 50 nm inter-distance, the migration speed and coordination of cell migration within the monolayer were optimal compared to 35 nm and 70 nm. The authors then explored the dynamics of integrin adhesion under the different experimental conditions. Their results suggest that the turnover of integrin-dependent adhesions was faster for cells migrating on 50 nm inter-distance compared to 35 nm and 70 nm. In contrast to a5b1 integrin-specific ligands, keratinocytes on cRGD-functionalized nanopatterns exhibited faster migration on 35-nm inter-ligand spacing (versus 50 nm on a5b1-presenting substrates), and the correlation length did not change with integrin ligand densities. Thus, at 50 nm integrin ligand spacing, integrin a5b1 fosters the maximal migration speed and the most efficient coordination of cell migration within the monolayer.
The authors then tested whether a5b1 integrin ligand density regulates integrin-generated traction and intercellular stress in the keratocyte monolayer. For this, the authors used traction force microscopy on nanopatterned hydrogels. The traction increased with decreasing integrin ligand density, but did not show an optimal value at 50 nm inter-distance. However, the stress correlation length follows the bell-shaped curves observed for migration speed and coordination of cell migration within the monolayer. The authors then calculated the stress vectors within the monolayers using an analysis algorithm developed previously called monolayer stress microscopy (MSM)(Vishwakarma et al., Nat. Comm. 2018). Using this MSM analysis on substrate of different rigidities: 11, 23, 55, and 90 kPa, the authors also found that the spatial correlation length of stress vectors were optimal on 50 nm ligand spacing. Thus, the optimal ligand spacing is independent of the traction forces exerted on the substrate but depends on the correlation length of stress vectors in the monolayer, and this independently of the substrate stiffness.
Since E-cadherin cell-cell interactions are responsible for the mechanical connections crucial for the coordination of collective cell migration, the authors explored whether E-cadherins are participating with integrin ligand spacing in defining the optimal speed of collective cell migration. They inhibited E-cadherins homophilic interactions using a blocking antibody against E-cadherins. This treatment increased collective migration on ligand spacing of 35 nm and 70 nm, while decreasing collective migration on 50 nm inter-distance. These results indicate that cell-cell interactions are also important for optimizing a5b1 integrin-mediated collective cell migration.
The conclusions of the manuscript are, in most cases, convincingly supported by the results. The authors have performed a very comprehensive characterization of the physical parameters (ligand spacing, substrates stiffness) at the base of collective epithelial cell migration. The study represents a huge amount of state-of-the-art biophysical experiments that nicely support their findings. In particular, they have a body of evidence showing that optimal stress propagation within monolayers, which promotes efficient collective cell migration, depends on both integrin ligand spacing and cadherin-mediated cell-cell interactions. However, the results found concerning the dynamics of integrin-based adhesions are more preliminary and need to be further analyzed to extract potentially interesting parameters of cell-ECM interactions important to control collective cell migration. In addition, it would be very interesting to unveil the relative contribution of b1-class integrins versus av-class integrins in collective cell migration. Indeed, it is not clear if the findings of the manuscript, which focus on a5b1 integrin, are relevant during collective cell migration on permissive substrates for b1-class and av-class integrins (e.g. fibronectin).
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