Mechanotransduction at tight junctions and epithelial differentiation and dynamics

Lead Research Organisation: University College London
Department Name: Institute of Ophthalmology

Abstract

Epithelia are layers of cells that cover body surfaces and line internal organs. They form functional barriers that protect us from the environment and enable our organs to generate and maintain compartments of different compositions, such as the barrier that separates the retina from the blood at the back or the eye. For individual epithelial cells to interact and form epithelial tissues, they need to assemble adhesive complexes with neighbouring cells. One of these adhesive complexes is called tight junction and forms a barrier in between neighbouring cells; hence, tight junctions are essential for epithelia to form tissue barriers as they prevent random diffusion along the space in between neighbouring cells. Consequently, the integrity of tight junctions must be maintained in order to prevent epithelial barrier breakdown and tissue failure. However, epithelial cells are often under physical strain and undergo cell shape changes during cell division or during the development of our organs and tissues. Therefore, mechanisms are likely to exist that allow tight junctions to adapt to changing cell shapes and, possibly, help cells sense and adapt to external physical forces that act on tight junctions. Here, we focus on the questions of whether such mechanisms exist and how such molecular bridges are built.

Tight junctions are composed of many different proteins that form a molecular network that starts with cell-cell adhesion proteins at the cell surface by which cells interact with each other. These cell-cell adhesion proteins interact with a large range of proteins inside the cells that regulate the various junctional functions and that are thought to function as molecular scaffolds that support the structure of tight junctions. Some of these proteins can also interact with the cytoskeleton, a network of protein fibres that supports the cell's structure and shape. However, the functional relevance of these interactions is not well understood. We hypothesized that components that can interact with the cell-cell adhesion proteins at the cell surface and the internal cytoskeleton might work as force transducing linkers. Hence, we have constructed a sensor based on such a protein that allows us to determine whether the molecule is indeed under tension. Pilot experiments indicate that the sensor is functional and that tight junctions are indeed a force-bearing structure.

Our objectives now are to determine the junctional architectural principles that enable tight junctions to bear forces and transduce them between the cytoskeleton and the cell surface, and to make use of functional assays to determine the physiological function of these principles for epithelial tissue formation and development.

The expected results will help us to understand physiologically important processes relevant for organism development, and tissue function and regeneration. They will contribute to our understanding of common diseases that disrupt epithelial tissues such as cancer, viral and bacterial infections, and common chronic inflammatory and age-related conditions. We also expect that the results and principles to be discovered will support tissue engineering and regenerative medicine approaches.

Technical Summary

Tight junctions are essential for the formation of functional epithelial barriers and regulate epithelial proliferation, polarisation, and morphogenesis. Maintenance of epithelial barriers and junctional integrity requires tight junction to adapt to cell shape changes such as those occurring during cell division or migration. Tight junctions are formed by a protein network consisting of multiple transmembrane cell-cell adhesion proteins and cytoplasmic proteins. Several of its components are able to interact with the cytoskeleton, suggesting that the junctional architecture consists of a protein network that connects the membrane to the cytoskeleton; however, whether such interactions serve a scaffolding function or are part of a force-transducing link between the actin cytoskeleton and the junctional adhesion proteins is not known. We developed a force sensor based on a central component of tight junctions. Pilot experiments suggest that this molecule is indeed under actomyosin-generated tension and that tight junctions are a force-bearing adhesion complex. Our objectives are to determine the molecular architecture important for force transmission, to identify the relevant cell-cell adhesion proteins important for assembly of a junction able to bear tensile force, and to determine the functional relevance of this new molecular principle using recently developed in vitro and in vivo assays for the analysis of tight junctions in epithelial dynamics and morphogenesis. The expected results will establish the molecular architecture of a new force-transmitting linker between cell adhesion proteins and the cytoskeleton at tight junctions, and will be important for the understanding of how such mechanisms drive epithelial morphogenesis and early embryonic development. Such information will support our understanding of common diseases that involve epithelial tissue failure and support tissue engineering approaches.

Planned Impact

Who will benefit from this research?
The immediate beneficiaries will be scientists working in allied fields at Universities as well as in industry. Apart of the academic beneficiaries of allied fields, the research will impact on scientists working in areas such as infections and wound repair, as well as chronic inflammation and cancer biology. The research will thereby contribute to the BBSRC's research priority of healthy aging across the lifecourse. Approaches for tissue engineering and regenerative medicine will be important beneficiaries of our research. Hence, our results and reagents are likely to impact on translational and clinical scientists focusing on acute, chronic and age-related diseases affecting various organs including the eye, kidney and liver. Hence, the research will support BBSRC's research strategy of bioscience for health. In the long term, the research will thus benefit patients and, thereby, the NSH and the general public. The research will also help to support training of early career scientists in designing and using innovative and interdisciplinary methods, as well as enable them to participate in international collaborations (including training). Hence, the research will support BBSRC's enabling themes and the international partnership priority.

How will they benefit from this research?
The research will impact on other scientists as the expected new knowledge will help them to design new approaches to answer questions about tissue function and degeneration in disease, and the identified functional principles will facilitate the development of new approaches for tissue engineering and development of materials for such approaches. Translational and clinical scientists will then benefit from such research for the development of new therapies for their disease of interest. They will also benefit from experimental models and approaches that we have developed and will refine during the project (e.g., manipulation of matrix and cell-cell tension to analyse epithelial differentiation and morphogenesis). These scientists will also profit from tools that we develop (e.g., to monitor tension during tissue engineering approaches). Ultimately such research will lead to the development of new therapies and thereby profit patients by enhancing their quality of life and wellbeing, the NHS and the general public. We expect that at least part of that research will take place in industry and, thereby, profit the UK's and international economic performance. We will also train early career scientists in interdisciplinary methods and international collaborative research. Upon completion of the research, these trained scientists will move on to work in other academic, industrial or NHS laboratories and thereby benefit the economic performance and/or public services.

Timescale
Other basic and translational scientists will start to benefit from the research during the lifetime of the grant. Reagents and knowhow will be made available as soon as possible and certainly once published. However, translational approaches to reach the clinic is a long-term benefit. We expect that research staff that will be trained during the grant will move on and thereby benefit academic or industrial employers by the end of the funding period.
 
Description - a new molecular pathway of force transduction at tight junctions
- how interplay between the mechanical properties of the cell substrate and cell-cell junctions determines cell shape, function and genes
- how cell junctions generate mechanical force required for developmental processes
Exploitation Route - to engineer tissues for transplantation and regenerative medicine
- to repair tissues in patients that degenerate due to fibrotic processes
- to study the importance of the discovered mechanisms in other biological processes
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Project grant
Amount £40,596 (GBP)
Funding ID R180001A 
Organisation Moorfields Eye Charity 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2017 
End 07/2018
 
Description Seed funding
Amount £19,172 (GBP)
Funding ID M692 
Organisation Rosetrees Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2017 
End 07/2018
 
Title Cell lines overexpressing MRCK 
Description Epithelial cells to analyse the role of MRCK in cell polarization and function 
Type Of Material Cell line 
Year Produced 2017 
Provided To Others? Yes  
Impact Support of colleagues' research 
 
Title Transgenic and knockout zebrafish strains 
Description Transgenic and knockout zebrafish strains that express fluorescently tagged cytoskeletal/junctional proteins or junctional tension sensors, and knockout stains in which specific junctional proteins were deleted 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2018 
Provided To Others? No  
Impact The stains are currently used to complete a paper on the role of junctional cytoskeletal tension during early development and will then become available to others. 
 
Title tension sensors 
Description Probes to measure tension across tight junction proteins 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact The reagents are currently used to complete a first research paper describing their use and will then become available to others. 
 
Description Functional analysis of proteins encoded by retinal disease genes and analysis of patient derived induced pluripotent stem cells 
Organisation Andalusian Center for Molecular Biology and Regenerative Medicine
Country Spain 
Sector Private 
PI Contribution Design of the project
Collaborator Contribution Provision of human induced pluripotent stem cells from patients with inherited retinal degeneration
Impact grant application
Start Year 2016
 
Description MRCK signalling in epithelial polarity and function 
Organisation Beatson Institute for Cancer Research
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We are determining the functional importance of MRCK signalling in epithelia
Collaborator Contribution BICR provides small molecule inhibitors of MRCK
Impact A first paper has been published in 2017 describing part of this research
Start Year 2016
 
Description Mechanotransduction at tight junctions 
Organisation Austrian Institute of Technology
Country Austria 
Sector Private 
PI Contribution Organisation of the project and coordination wiht partners
Collaborator Contribution Application of specialized techniques, generation of reagents, academic discussion
Impact Multidisciplinary: Biophysics, developmental and cell biology
Start Year 2016
 
Description Mechanotransduction at tight junctions 
Organisation University College London
Department Sobell Department of Motor Neuroscience and Movement Disorders
Country United Kingdom 
Sector Hospitals 
PI Contribution Organisation of the project and coordination wiht partners
Collaborator Contribution Application of specialized techniques, generation of reagents, academic discussion
Impact Multidisciplinary: Biophysics, developmental and cell biology
Start Year 2016
 
Description Mechanotransduction at tight junctions 
Organisation University of Grenoble
Country France 
Sector Academic/University 
PI Contribution Organisation of the project and coordination wiht partners
Collaborator Contribution Application of specialized techniques, generation of reagents, academic discussion
Impact Multidisciplinary: Biophysics, developmental and cell biology
Start Year 2016
 
Description Cell polarity in cell and tissue function 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact General presentation of functional relevance of cell polarity and cell-cell adhesion in tissue function for an audience including graduate and postgraduate students as well as researchers from a wide spectrum of cell and developmental biology
Year(s) Of Engagement Activity 2017
 
Description Distinguished Lecture UCL Medicine 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Discussion of future work
Year(s) Of Engagement Activity 2017
 
Description Engagement with parliament 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Event with discussion with members of parliament and their staff organised by the Royal Society of Biology
Year(s) Of Engagement Activity 2016
 
Description Eye Research - an equal partner 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Vision Bridge is an organisation dedicated to informing the general public about contemporary eye research and to provide a platform to enable exchange between researchers, the general public and patients.
Year(s) Of Engagement Activity 2018,2019
URL http://visionbridge.org.uk/
 
Description Mechanobiology and epitelial differenation 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact International workshop on mechanical principles in biology.
120 people attended the workshop and most were postgraduate students
Year(s) Of Engagement Activity 2018
 
Description PhD students Berlin 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Lecture for students of a PhD programme in Germany and discussions about their own research projects
Year(s) Of Engagement Activity 2017