What are epithelial monolayers?
One-cell thick monolayers are the simplest tissues in multicellular organisms, yet they fulfil critical roles in development and normal physiology. In early development, embryonic morphogenesis results largely from monolayer rearrangement and deformation due to internally generated forces. Later, monolayers act as physical barriers separating the internal environment from the exterior and must withstand externally applied forces. Though resisting and generating mechanical forces is an essential part of monolayer function, simple experimental methods to characterise monolayer mechanical properties are lacking. The major challenge is to characterise monolayer mechanics in isolation from their substrate.
Mechanical properties of suspended epithelial monolayers
We have developed a novel system in the lab for tensile testing of freely suspended cultured monolayers that enables the examination of their mechanical behaviour at multi-, uni-, and sub-cellular level [1]. We have used this system to measure monolayer mechanics [2]. We found that monolayers could withstand more than a doubling in length before failing through rupture of intercellular junctions. Measurement of stress at fracture enabled a first estimation of the average force needed to separate cells within truly mature monolayers. As in single cells, monolayer mechanical properties were strongly dependent on the integrity of the actin cytoskeleton, myosin, intercellular adhesions interfacing adjacent cells and keratin filaments. Currently, we are studying how monolayers can dissipate mechanical stresses to avoid fracture and how fracture occurs. We have found that, at time-scales of about one minute, monolayers composed of tens of thousands of cells behave mechanically like a single cell [4]. This is because intercellular junctions turn over slowly providing stable connections between cells whose actomyosin cytoskeleton turns over in about one minute.
Mechanical properties of cell monolayers. (A) Typical stress-extension curves of cell monolayers. (B) Representative force relaxation curve for an MDCK monolayer. Monolayers relax to equilibrium in ~25 s. (C) We are currently investigating how fractures occur within monolayers.
Cell divisions in suspended epithelial monolayers
We are also interested in characterising cell divisions in monolayers under mehcanical stress. We have recently found that cell divisions in monolayers align better with the long, interphase cell axis than with the monolayer stress axis [3]. Since the application of stretch induces a global realignment of interphase long axes along the direction of extension, this is sufficient to bias the orientation of divisions in the direction of stretch. Each division redistributes the mother cell mass along the axis of division. Thus, the global bias in division orientation enables cells to act collectively to redistribute mass along the axis of stretch, helping to return the monolayer to its resting state.
(A) Cell division in stretched and non-stretched monolayer. (B) Most of the cells orient their division axis along the stretch axis. (C) Here, the interphase shape is misaligned with the direction of monolayer stretch, and the division follows the interphase shape rather than the stretch direction.
Relevant publications:
[1] Harris, A.R., Bellis, J., Khalilgharibi, N., Wyatt, T., Baum, B., Kabla, A.J. and Charras, G.T., 2013. Generating suspended cell monolayers for mechanobiological studies. Nature protocols, 8(12), pp.2516-2530.
[2] Harris, A.R., Peter, L., Bellis, J., Baum, B., Kabla, A.J. and Charras, G.T., 2012. Characterizing the mechanics of cultured cell monolayers. Proceedings of the National Academy of Sciences, 109(41), pp.16449-16454.
[3] Wyatt, T.P., Harris, A.R., Lam, M., Cheng, Q., Bellis, J., Dimitracopoulos, A., Kabla, A.J., Charras, G.T. and Baum, B., 2015. Emergence of homeostatic epithelial packing and stress dissipation through divisions oriented along the long cell axis. Proceedings of the National Academy of Sciences, 112(18), pp.5726-5731.
[4] Khalilgharibi N., Fouchard J., Asadipour N., Barrientos R., Duda M., Bonfanti A., Yonis A., Harris A.R., Mosaffa P., Fujita Y., Kabla A., Mao Y., Baum B., Muñoz J.J., Miodownik M., Charras G.. Stress relaxation in epithelial monolayers is controlled by the actomyosin cortex. Nature Physics. 2019. link
Preprints:
[1] TActomyosin controls planarity and folding of epithelia in response to compression. Biorxiv. 2018. link
Collaborators on this project:
Buzz Baum (LMCB, UCL, London, UK)
Alexandre Kabla (Department of Engineering, Cambridge, UK)
Mark Miodownik (Department of Mechanical Engineering, UCL)
Jose Muñoz (Universitat Polytecnica de Catalunya, Spain).
Funding on this project:
ERC consolidator grant (MolCellTissMech) link
Biotechnology and Biological Sciences Research Council (BBSRC)
Rosetrees Trust
Charras Lab 