Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - MolCellTissMech (Molecular and cellular determinants of cell monolayer mechanics)

Teaser

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...

Summary

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 the constituent cells into a tissue-scale mechanical entity. Mutations in these proteins lead to diseases with fragilised epithelia. 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 have 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. Using this instrument, we aim to understand the molecular determinants of monolayer mechanics as well as the cellular behaviours that drive tissue morphogenesis. We are focusing our efforts on four objectives: 1) discover the molecular determinants of monolayer mechanics, 2) characterise monolayer mechanics, 3) dissect how tension is sensed by monolayers, and 4) investigate the biophysics of individual cell behaviours participating in tissue morphogenesis.

Together these studies will enable us to understand how monolayer mechanics is affected by changes in single cell behaviour, subcellular organisation, and molecular turnover. This multi-scale characterisation of monolayer mechanics will set the stage for new theoretical descriptions of living tissues involving both molecular-scale phenomena (cytoskeletal turnover, contractility, and protein unfolding) operating on short time-scales and rearrangements due to cell-scale phenomena (cell intercalation, cell division) acting on longer times.

Work performed

On Objective 1, we have been characterising the dynamic responses 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. This is a surprising and novel result whose molecular and physical basis is currently being investigated. For this, we are collaborating with theoretical physicists to design a minimal rheological model to guide our thinking. Our current experiments show that cell monolayers are naturally under intrinsic tension generated by the actomyosin cytoskeleton. This tension allows monolayers to accommodate reductions in length while remaining planar, thereby minimizing the risk of delaminating from their substrate. These data are currently being finalized for publication.

On objective 2, we have been examining how cell monolayers can dissipate stresses after being deformed to avoid fracture. Stress relaxation comprises two phases with different functional forms: a power law phase at short time-scales and an exponential phase at long time-scales. The first phase is ATP-independent while the second depends upon ATP, indicating that it stems from an active biological phenomenon. We are currently investigating the molecular mechanisms underlying these behaviours and are in the process of designing a computational model to guide interpretation of the data.

Objective 4 has been the focus of most of the work so far. We have designed instrumentation to allow for continuous imaging of cells within monolayers as monolayers change length. Using this method, we have been investigating the sensitivity of the different phases of the cell cycle to mechanical stresses as well as the stiffness of the cell in each phase of the cell cycle. Our preliminary results show that there is a clear threshold strain above which division fails and that cells are sensitive to strain only during prophase and metaphase but not during anaphase or telophase. We are currently exploring the molecular mechanisms underlying this sensitivity. To control the shape of individual cells and understand how individual shape changes affect the shape of cell monolayers, we have been adapting existing optogenetic constructs to control contractility in epithelial cells. In collaboration with Dr Xavier Trepat’s laboratory in Barcelona, we have shown that constructs based on the relocalisation of GEF domains to the cell membrane allow control of cell and tissue mechanics (Valon et al, Nature Communications, 2017). With this proof of principle, we are now designing new optogenetic probes to control other important regulators of cell and monolayer mechanics.

Final results

This action has the following objectives:
(1) Discover the molecular determinants of monolayer mechanics - Although the molecular mechanisms necessary to form and maintain intercellular junctions are now well understood, little is known about what proteins in these pathways participate in monolayer mechanics. We will systematically investigate the relationship between protein function and monolayer mechanics using a targeted siRNA screen. This will provide candidate proteins for objectives 2-4.
(2) Characterise monolayer rheology and stress transmission within monolayers - We will characterise monolayer rheology at all the time-scales relevant to physiology and development and determine what physical and biological phenomena underlie it. In particular, we will investigate the role of protein turnover in setting monolayer rheological time-scales. Combined with laser ablation techniques, we will determine what structures are load-bearing in epithelial monolayers and characterise stress transmission at multicellular junctions. By exploring these questions, this study will challenge our current assumptions on monolayer mechanics and provide valuable insight for interpretation of results in objectives 3-4.
(3) Identify mechanisms of mechanotransduction - Individual cells and tissues can detect changes in their mechanical environment. However, current assays for studying mechanotransduction are effected under poorly controlled mechanical conditions making a quantitative understanding of mechanotransduction impossible. We will examine the molecular mechanisms of mechanotransduction in well-controlled mechanical conditions to investigate the sensitivity, kinetics, and specific site of force detection.
(4) Investigate feedback control and the restoration of homeostasis driven by individual cellular behaviours - In response to prolonged stretch, monolayers seek to return to homeostasis through cellular-scale behaviours akin to those observed in developmental morphogenesis. Though the molecular bases of these behaviours are progressively better understood, little attention has focused on the mechanical forces operating at the single cell level and the processes of feedback governing return to homeostasis.