The mechanics of epithelial tissues

Lead Research Organisation: University College London
Department Name: London Centre for Nanotechnology

Abstract

Many of the cavities and free surfaces of the human body (e.g. gut, lungs, blood vessels) are lined by tissues just a few cells thick. These epithelial tissues separate the body's internal environment from the external environment. As part of their normal function, epithelial tissues are continuously exposed to large mechanical deformations: lung alveoli deform during respiration, intestinal epithelia resist peristaltic movements in the gut, and endothelia are exposed to pulsatile fluid shear stresses in blood flow. The mechanical function of epithelia is particularly apparent in disease when mutations or pathogens affecting the cell skeleton (cytoskeleton) or junctions linking cells to one another result in fragile tissues that tear during routine function (e.g. epidermis bullosa, staphylococcus blistering). Cells within epithelial tissues are tightly connected to one another by intercellular junctions: some junctions form a barrier restricting the passage of solutes across the tissue whilst others integrate the cytoskeletons of neighbouring cells to form a strong multicellular tissue that can withstand mechanical stresses. Despite their clear mechanical role, little is currently known about the mechanics of epithelial tissues and how this derives from the mechanical properties of the cells that make up the tissue and the proteins that make up the cells. This is primarily due to the lack of specific experimental techniques to measure the intrinsic mechanical properties of tissues while monitoring cellular and subcellular traits.

We have developed a novel tool to quantify the mechanics of epithelial tissues by stretching cultured epithelia. During tissue deformation, the applied mechanical tension can be measured and the tissues can be simultaneously imaged at subcellular, cellular and tissue length scales, such that the architecture of the sub-cellular components, the shape of the cells and their eventual reorganisation can be accurately monitored as a function of the imposed force. To complement this experimental tool, we have developed a novel computational model of epithelial tissues that can serve as a means to interpret and refine our experiments.

We now propose to use our new techniques to understand what proteins play a role in setting the mechanical properties of epithelial tissues. To do this, we will focus on three aims:

1) Develop a systematic methodology for characterizing the mechanics of tissues
2) Discover what proteins set tissue mechanical properties
3) Incorporate our findings into a computational model of tissues.

Aim1 is geared at creating a systematic methodology for collecting all of the necessary information to fully characterise the mechanics of normal tissues.

In aim 2, we will ask how the absence of a given protein affects the mechanics of a tissue. To answer this question, we will reduce the level of expression of a chosen gene in the cells that make up the tissue and measure how this affects the mechanics of the tissue. We will also examine how gene depletion changes the organization of the tissue and the cells that compose it. We will pay particular attention to proteins identified in clinical studies of fragile epithelia, as they have direct relevance to patients and potential palliative therapies.
In aim 3, we will use computational and statistical approaches to identify what cellular structures are the most important for setting tissue mechanics using the results of aim 2. Moreover, this analysis and the experiments from aim 2 will directly support the development of a model specifically tailored to study tissue mechanics at large deformation and used to refine our understanding of how changes in protein expression within cells can lead to failure of epithelial sheets, as in clinical cases.

In summary, the proposed investigations will greatly enhance our understanding of the mechanics of epithelial tissues and how pathologies can affect tissue strength.

Technical Summary

Exposure to mechanical stresses is a normal part of physiology for epithelial tissues. This mechanical function is particularly apparent in disease when mutations or pathogens affecting the cytoskeleton, adherens junctions, or desmosomes result in increased fragility of tissues. Despite clear physiological relevance, little is known about the mechanics of epithelia and how these relate to the mechanical properties of the tissue's constituent cells.

We have developed a new experimental tool for measuring the mechanical properties of cell monolayers which can be coupled to high magnification optical microscopy to image cellular phenotype and subcellular organization during mechanical testing. To complement this, we have developed a versatile new numerical modeling platform to serve as a means of interpreting and refining our experiments.

We propose to couple mechanical testing with chemical and genetic perturbations to understand what subcellular structures govern tissue mechanics. To do this, we will carry out a limited siRNA screen focusing on proteins that form or regulate subcellular structures thought to be important for tissue mechanics: adherens junctions, desmosomes, the apical actin cortex, intermediate filaments, contractile proteins and proteins identified in pathologies causing fragile epithelia. Statistical methods will be then used to cluster proteins in groups according to the changes their depletion induces on tissue mechanics. We expect that depletion of proteins participating to the assembly of the same substructures should result in similar mechanical phenotypes. These results will then be implemented into our computational model to tailor it for the study of tissues at large deformations. An iterative cycle of mechanical testing and simulations will be used to refine our understanding of how subcellular structures, cellular structures, and multicellular behaviours control the mechanics of epithelia in normal and pathological conditions.

Planned Impact

The proposed research will seek to bridge the gap between molecular, cellular, and tissue-scales to understand the molecular and cellular determinants of epithelial tissue mechanics and investigate how tissues adapt to their mechanical environment. This will primarily benefit academics in the fields of tissue and developmental biology but, in the longer term, through comprehension of the determinants of tissue strength, we envisage that it will benefit tissue engineering startups in the UK and clinical medicine.

Academic impact

Academic advancement and innovation:
We expect our research to attract interest from many fields in the global scientific community such as developmental biology, cell biology, biophysics, bioengineering, and clinical medicine.
To ensure our findings have the highest possible impact, we will present our technological developments and preliminary results generated at high profile conferences that cover relevant topics including tissue engineering, biophysics, developmental biology and cell biology throughout the duration of the grant. Where possible we will disseminate our findings in general audience journals.

Training and professional development:
Both GC and AK are actively involved in interdisciplinary training activities at UCL and Cambridge University. GC participates in teaching in the CoMPLEX DTP and is a member of the new interdisciplinary BBSRC DTP. AK is an important contributor to the development of the Bioengineering curriculum in Cambridge, and teaches a number of relevant subjects ranging from material sciences to physiology. The project described here will be used to introduce students from different backgrounds to interdisciplinary research in the life sciences. Elements of the work will be used as exemplar projects for students in the CoMPLEX and BBSRC DTPs. The mechanical aspects of the project will also form the basis of a couple of 4th year engineering projects in Cambridge and are likely to attract students with a Mechanical/Bio Engineering background.

Throughout the course of the project, the post-docs involved will receive cross-disciplinary mentoring and benefit from regular interactions both in Cambridge and London. In addition, they will be involved in mentoring students and develop their own mentoring and leadership skills. This will aid their progression towards an independent group leader position.

Societal and economic impact

Commercialisation and exploitation:
We envisage that, in the longer term, our integrated mechanical testing and computational modeling approach will be of interest to clinical medicine, bioengineering startups, and the pharmaceutical industry. Indeed, we anticipate that our approach could be utilized to study the effect of pathologic genetic mutations on tissue mechanical properties and test the efficacy of palliative treatments in restoring the mechanical properties of diseased tissues. Should there be industrial interest, we will study the possibility of designing a new prototype in a format suited to high throughput screening.

Both the UCL and Cambridge University have efficient mechanisms to assist academics in the development of commercial applications of their research outputs and in the management of intellectual property rights (see for instance Cambridge Enterprise or UCL Business).

Increasing public engagement and understanding:
Previously members of the team have been involved in interactions with the wider community through public discussions and school visits. Through this type of outreach we expect this work to reach a wide audience, giving the public a better understanding of multidisciplinary research and an appreciation of the remarkable natural world in which we live. The co-Is each expect to participate in one public engagement event per year during the course of this project. We will use these opportunities to stress the important role played by basic research in driving societal advances.

Publications

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Bonfanti A (2020) A unified rheological model for cells and cellularised materials in Royal Society Open Science

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Khalilgharibi N (2016) The dynamic mechanical properties of cellularised aggregates. in Current opinion in cell biology

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Wyatt T (2016) A question of time: tissue adaptation to mechanical forces. in Current opinion in cell biology

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Wyatt TP (2015) Emergence of homeostatic epithelial packing and stress dissipation through divisions oriented along the long cell axis. in Proceedings of the National Academy of Sciences of the United States of America

 
Description Epithelial monolayers are one-cell thick tissue sheets that separate internal and external environments. As part of their function, they withstand extrinsic mechanical stresses applied at high strain rate. However, little is known about how monolayers respond to mechanical deformations. In stress relaxation tests, monolayers respond in a biphasic manner and stress dissipation is accompanied by an increase in monolayer resting length, pointing to active remodelling of cell architecture during relaxation. Consistent with this, actomyosin remodels at a rate commensurate with mechanical relaxation and governs the rate of monolayer stress relaxation - as in single cells. By contrast, junctional complexes and intermediate filaments form stable connections between cells, enabling monolayers to behave rheologically as single cells. Together, these data show actomyosin cytoskeletal dynamics govern the rheological properties of monolayers by enabling active, ATP-dependent changes in the resting length. These findings have far-reaching consequences for our understanding of developmental morphogenesis and tissue response to mechanical stress. In another study, we examined the response of epithelial tissues to shortening in length. Epithelial tissues are subject to mechanical perturbations that vary greatly in magnitude and timescale during development, normal physiological function and regeneration. At timescales of hours to days, their response to perturbations involves cell proliferation and oriented cell division following a stretch and cell extrusion in response to tissue compression (Marinari and Baum, 2012; Eisenhoffer and Rosenblatt, 2012). While the response of epithelia to stretch has been widely studied, little is known about their response to compressive strain and the molecular mechanisms enabling it despite clear physiological relevance. Here, using monolayers devoid of a substrate, we probe the response of epithelia to compressive strains. When subjected to rapid application of significant (~50%) uniaxial compressive strains, monolayers first buckle as expected from their viscoelastic properties but remarkably, in the following seconds, autonomously driven cell-shape changes return the tissue to a flat configuration. When subjected to slower rate compressive strain, monolayers can retain their flat planar configuration without buckling by accommodating the reduction in surface area through an increase in apico-basal height. A combination of experiment and modelling reveals that these behaviours are driven by intrinsic tension generated by the actomyosin cytoskeleton. In physiological conditions, the generation of a resting tension enables epithelia to withstand both compressive and tensile strains while maintaining a planar shape.
Exploitation Route Better fundamental understanding of embryonic development and diseases linked to epithelial fragility.

Developmental of palliative treatments for diseases with symptoms of epithelial fragility.

General methodology for describing the mechanics of tissues and cells.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description EMBO Long term fellowship
Amount £75,000 (GBP)
Organisation European Molecular Biology Organisation 
Sector Charity/Non Profit
Country Germany
Start 09/2016 
End 09/2018
 
Description EMBO short term fellowship
Amount £10,000 (GBP)
Organisation European Molecular Biology Organisation 
Sector Charity/Non Profit
Country Germany
Start 03/2018 
End 05/2018
 
Description Multi disciplinary award
Amount £440,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2017 
End 09/2020
 
Description Royal Society international network grant
Amount £12,000 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2018 
End 02/2020
 
Description Uehara foundation post doctoral fellowship
Amount £40,000 (GBP)
Organisation Uehara Memorial Foundation 
Sector Charity/Non Profit
Country Japan
Start 04/2018 
End 03/2019
 
Description Deciphering the role of signalling in cell sheet morphogenesis 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution The goal of this collaboration is to understand how signalling controls cell mechanical properties and in turn the mechanical properties of cell sheets. When signalling is locally activated, this will create local differences in mechanics that will lead to morphogenesis. My team is generating the constructs and carrying out the experiments to control signalling using optogenetics. We are also carrying out experiments in which we measure mechanical properties of cell sheets before and after activation of signalling either locally or globally.
Collaborator Contribution Our collaborator is a group of theoretical physicists. They are using our experiments to model cell sheet morphogenesis based on changes that occur at the single cell level.
Impact This is a multidisciplinary collaboration involving Physics and Biology. This collaboration has given rise to a grant application to control signalling at the single cell level.
Start Year 2016
 
Description Optogenetics for investigating cell mechanics 
Organisation University of Grenoble
Department Laboratory for Interdisciplinary Physics
Country France 
Sector Academic/University 
PI Contribution My team is providing expertise in designing and actuating optogenetic probes for controlling cell signalling.
Collaborator Contribution My partners are providing expertise in measuring the forces exerted by cells on the substrate.
Impact N/A. Interdisciplinary collaboration.
Start Year 2018
 
Description Regulation of planar polarity by mechanical stresses 
Organisation University of Rennes 1
Country France 
Sector Academic/University 
PI Contribution I am providing expertise on applying mechanical forces to biological tissues. I am providing expertise on image analysis to examine the extent of recruitment of proteins in response to mechanical stresses.
Collaborator Contribution My partners are providing expertise in Developmental Biology.
Impact No impact yet. Multi-disciplinary collaboration.
Start Year 2018
 
Description Role of the Arp2/3 complex in monolayer resistance to fracture 
Organisation University of Helsinki
Department Viikki Biocentre
Country Finland 
Sector Academic/University 
PI Contribution My team is running mechanical tests on epithelial monolayers in which the function of Arp2/3 and WASF2 has been perturbed.
Collaborator Contribution Our partners have discovered the biological processes that use Arp2/3 and WASF2 at intercellular junctions. They have defined the basic observations.
Impact N.A
Start Year 2019
 
Description The impact of mechanical stress on endothelial cell cell signalling 
Organisation Semmelweiss University
Department Faculty of Medicine
Country Hungary 
Sector Academic/University 
PI Contribution We have helped our partner setup and design a new mechanical testing device for examining the response of endothelial cells to stress.
Collaborator Contribution Our partners have brought in expertise on human vascular endothelial cells as well as labour for bringing the new device to fruition.
Impact This is a multidisciplinary collaboration. Currently no impacts.
Start Year 2017
 
Description Understanding the mechanical control of YAP activation 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution The goal of this project is to understand how YAP signalling is controlled by mechanical cues. My team will carry out the experiments aiming to test under what mechanical conditions YAP relocalises to the nucleus.
Collaborator Contribution Our collaborators will generate the reagents necessary for visualising the different components of YAP in single cells. They will also engineer all of the mutants necessary to understand YAP signalling in response to mechanical cues.
Impact Multi disciplinary collaboration: Physics and Biology.
Start Year 2017
 
Title Rheos 
Description The software library provides a computational framework to model the rheological response of materials presenting a power law behaviour. Such behaviour has been observed across a broad range of biological materials. 
Type Of Technology Software 
Year Produced 2018 
Open Source License? Yes  
Impact It has provided us with a reliable mechanism to extract material parameters in monolayers and embryo tissues. This has allowed the prediction of complex behaviours in monolayers, as recently reported in https://www.biorxiv.org/content/10.1101/543330v1. 
URL https://github.com/JuliaRheology/RHEOS.jl
 
Description Interview with TV 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact I participated in an interview for NHK, a major TV network in Japan. I described how charities in the UK fund research.
Year(s) Of Engagement Activity 2017
 
Description Interview with newspaper 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact I provided opinions on the risk of Brexit to Science research in the UK.
Year(s) Of Engagement Activity 2018
 
Description Primary school visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Gave a presentation to 60 year 5 and 6 pupils at a local primary school.
Year(s) Of Engagement Activity 2015
 
Description Secondary school visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I gave a presentation of our research work to a group of 60 secondary school students.
Year(s) Of Engagement Activity 2015