Periodic Reporting for period 4 - MolCellTissMech (Molecular and cellular determinants of cell monolayer mechanics)
Reporting period: 2020-03-01 to 2021-02-28
Epithelial monolayers are amongst the simplest tissues in the body, yet they play fundamental roles in adult organisms where they separate the internal environment from the external environment and in development when the intrinsic forces generated by cells within the monolayer drive tissue morphogenesis. The mechanics of these simple tissues is dictated by the cytoskeletal and adhesive proteins that interface cells into a tissue-scale mechanical entity. However, a quantitative understanding of how subcellular structures govern monolayer mechanics, how cells sense their mechanical environment and what mechanical forces participate in tissue morphogenesis is lacking.
To overcome these challenges, we devised a new technique to study the mechanics of load-bearing monolayers under well-controlled mechanical conditions while allowing imaging at subcellular, cellular and tissue resolutions. With this, we aimed to understand the molecular determinants of monolayer mechanics.
To overcome these challenges, we devised a new technique to study the mechanics of load-bearing monolayers under well-controlled mechanical conditions while allowing imaging at subcellular, cellular and tissue resolutions. With this, we aimed to understand the molecular determinants of monolayer mechanics.
In Aim 1, we characterised the response of cell monolayers to changes in length. This revealed that monolayers are surprisingly dynamic and can accommodate reductions in their length of up to ~40% in less than a minute. Cell monolayers are naturally under intrinsic tension generated by the actomyosin cytoskeleton. This tension allows them to accommodate reductions in length while remaining planar, thereby minimizing the risk of delaminating from their substrate (Wyatt et al, Nature Materials, 2020).
We observed that suspended monolayers form curls at their free edges. This spontaneous curvature is due to basal polarization of myosin motors. Curling results from a competition between the elastic energy in the plane of the monolayer and the energy of curvature transversal to the plane (Fouchard et al, PNAS, 2020; Recho et al, Phys Rev E, 2020).
Finally, we examined how fracture arises in monolayers. We found that monolayers can withstand a tripling in length before failing. Failure occurs through unzipping of junctions rather than cell ruptures. Stress can arise due to extrinsic forces or because of intrinsic contractility within the tissue and stresses arising from these two sources are additive (manuscript in preparation).
In Objective 2, we examined how cell monolayers dissipate stresses to avoid fracture. By comparing stress relaxation in monolayers and single cells, we found that the submembranous actin cortex controls stress relaxation. Thus, monolayers comprising tens of thousands of cells behave rheologically as a single cell (Khalilgharibi et al, Nature Physics, 2019).
Based on the results of aims 1 and 2, we worked with Dr Alexandre Kabla at the University of Cambridge to develop a unified framework based on springpots to model the mechanical response of tissues and single cells (Bonfanti et al, RSOS, 2020; Bonfanti et al, Soft Matter, 2020).
One key challenge in morphogenesis is to understand how tissue shape emerges from differences in cell mechanics. As a simplified model system, we have been using early C elegans embryos. In the 4-cell embryo, we characterized cortical tensions for each cell using AFM and junctional tension by measuring contact angles between cells. Cortical tension in cells derived from AB (ABx) was ~3-fold higher than in cells derived from P1 (EMS, P2). Using numerical simulations, we showed that changes in aggregate shape can be predicted from measured cortical and junctional tensions (manuscript in preparation).
Aim 3 investigated mechanotransduction in monolayers. We examined the recruitment of vinculin, EPLIN, alpha-catenin, Par3, and RhoA in response to a variety of mechanical stimuli with different frequencies, aimplitudes, and durations. However, no recruitment was apparent. We hypothesise that this is due to overexpression of the mechanosensitive proteins. We also fluorescently tagged YAP, a key mechanosensor, using CRISPR. When we subjected monolayers grown from these cells to 30% stretch for 1-2h, we did not observe nuclear relocalisation of YAP – contrary to observations in adherent monolayers. Thus, YAP mechanotransduction may be dominated by signals from integrins.
Aim 4 examined how cell division orientation responds to mechanical stress. When monolayers are compressed, cell division becomes poorly oriented with many cells dividing out of plane. When contractility is inhibited without change in shape, cell division is also poorly oriented, indicating that tissue tension is necessary for correct orientation of division in plane. Consistent with this, stretch applied to monolayers in the presence of inhibitors of contractility rescued orientation (manuscript in preparation).
Finally, we examined how a local change in tissue stress affects spindle orientation in metaphase cells. Using optogenetic actuators to locally increase stress, we asked how local/global changes in tissue stress affect spindle orientation. When we increase stress globally, spindle orientation remains unchanged; whereas when stress is increased at the cell poles, spindles rotate away from this region. This may represent a mechanism to allow cells to orient their division to relieve stress (manuscript in preparation).
We observed that suspended monolayers form curls at their free edges. This spontaneous curvature is due to basal polarization of myosin motors. Curling results from a competition between the elastic energy in the plane of the monolayer and the energy of curvature transversal to the plane (Fouchard et al, PNAS, 2020; Recho et al, Phys Rev E, 2020).
Finally, we examined how fracture arises in monolayers. We found that monolayers can withstand a tripling in length before failing. Failure occurs through unzipping of junctions rather than cell ruptures. Stress can arise due to extrinsic forces or because of intrinsic contractility within the tissue and stresses arising from these two sources are additive (manuscript in preparation).
In Objective 2, we examined how cell monolayers dissipate stresses to avoid fracture. By comparing stress relaxation in monolayers and single cells, we found that the submembranous actin cortex controls stress relaxation. Thus, monolayers comprising tens of thousands of cells behave rheologically as a single cell (Khalilgharibi et al, Nature Physics, 2019).
Based on the results of aims 1 and 2, we worked with Dr Alexandre Kabla at the University of Cambridge to develop a unified framework based on springpots to model the mechanical response of tissues and single cells (Bonfanti et al, RSOS, 2020; Bonfanti et al, Soft Matter, 2020).
One key challenge in morphogenesis is to understand how tissue shape emerges from differences in cell mechanics. As a simplified model system, we have been using early C elegans embryos. In the 4-cell embryo, we characterized cortical tensions for each cell using AFM and junctional tension by measuring contact angles between cells. Cortical tension in cells derived from AB (ABx) was ~3-fold higher than in cells derived from P1 (EMS, P2). Using numerical simulations, we showed that changes in aggregate shape can be predicted from measured cortical and junctional tensions (manuscript in preparation).
Aim 3 investigated mechanotransduction in monolayers. We examined the recruitment of vinculin, EPLIN, alpha-catenin, Par3, and RhoA in response to a variety of mechanical stimuli with different frequencies, aimplitudes, and durations. However, no recruitment was apparent. We hypothesise that this is due to overexpression of the mechanosensitive proteins. We also fluorescently tagged YAP, a key mechanosensor, using CRISPR. When we subjected monolayers grown from these cells to 30% stretch for 1-2h, we did not observe nuclear relocalisation of YAP – contrary to observations in adherent monolayers. Thus, YAP mechanotransduction may be dominated by signals from integrins.
Aim 4 examined how cell division orientation responds to mechanical stress. When monolayers are compressed, cell division becomes poorly oriented with many cells dividing out of plane. When contractility is inhibited without change in shape, cell division is also poorly oriented, indicating that tissue tension is necessary for correct orientation of division in plane. Consistent with this, stretch applied to monolayers in the presence of inhibitors of contractility rescued orientation (manuscript in preparation).
Finally, we examined how a local change in tissue stress affects spindle orientation in metaphase cells. Using optogenetic actuators to locally increase stress, we asked how local/global changes in tissue stress affect spindle orientation. When we increase stress globally, spindle orientation remains unchanged; whereas when stress is increased at the cell poles, spindles rotate away from this region. This may represent a mechanism to allow cells to orient their division to relieve stress (manuscript in preparation).
This project has resulted in many advances beyond the state of the art. Some results are already published, while others are still in preparation.
These include:
1) An in depth characterisation of stress relaxation in monolayers demonstrating that it is governed by the actomyosin cortex.
2) Discovering that epithelial tissues behave as pretensed viscoelastic sheets that can buffer against compression and rapidly recover from buckling.
3) Characterising active torques in living tissues arising from apico-basal gradients in myosin. .
4) A new framework for describing the rheology of living tissues and living cells.
5) Characterising the fracture of soft tissues.
6) Discovering that accurate orientation of cell division in the plane depends on the presence of tension in the tissue.
These include:
1) An in depth characterisation of stress relaxation in monolayers demonstrating that it is governed by the actomyosin cortex.
2) Discovering that epithelial tissues behave as pretensed viscoelastic sheets that can buffer against compression and rapidly recover from buckling.
3) Characterising active torques in living tissues arising from apico-basal gradients in myosin. .
4) A new framework for describing the rheology of living tissues and living cells.
5) Characterising the fracture of soft tissues.
6) Discovering that accurate orientation of cell division in the plane depends on the presence of tension in the tissue.