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.

Publications

10 25 50

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Balda MS (2023) Tight junctions. in Current biology : CB

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Balda MS (2016) Tight junctions as regulators of tissue remodelling. in Current opinion in cell biology

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Zihni C (2016) Tight junctions: from simple barriers to multifunctional molecular gates. in Nature reviews. Molecular cell biology

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Erasmus JC (2021) Rac1-PAK1 regulation of Rab11 cycling promotes junction destabilization. in The Journal of cell biology

 
Description The focus of the application was an adhesive structure between cells called tight junctions, as it is important to regulate permeability between cells. If it is defective, tissues like our skin are unable to form functional barriers; hence, water would leak out of the body.
The research led to significant new findings important for the understanding of how cells adhere to each other, and how tissues and organs form and adapt to different shapes and mechanical constraints.
- A new biophysical principle was discovered demonstrating that epithelial tight junctions in vivo assemble by phase transition. Phases are distinct physical states like ice or liquid water. This means that junction components exist in two physical states that differ before and after junction assembly. This is important as the required phase transition drives junction assembly and leads to a sorting process resulting in a strong enrichment of junctional proteins within a 'junctional' phase. Other complex biological structures may assemble along similar principles.
- We have established a new set of molecular tools enabling the analysis of mechanical tension on tight junctions. These tools will be valuable to us and others to investigate how morphogenetic processes and junctional tension influence each other.
- Using such tools, we have shown that interplay between cell-cell adhesion and the mechanical properties of the extracellular matrix (ECM) regulates monolayer tension and, thereby, regulates epithelial cell morphogenesis and gene expression. We have also discovered a new molecular mechanism that generates and transmits mechanical tension at tight junctions.
- We have investigated how tight junctions regulate tension and found that on physiologically relevant substrates as well as in vivo tight junctions function as a force generating molecular machine. Force generation at tight junctions is important for morphogenetic processes during fundamentally important processes during the early development of embryos (i.e., gastrulation).
- We have further discovered that current models of the molecular mechanisms underlying junction assembly and function need to be revised. Recently obtained data indicate that, unlike current assumptions, key junctional scaffolding proteins have specific functions and are not redundant in terms of their roles in junction assembly, transmission of mechanical tension within the monolayer, morphogenesis, as well as regulation of gene expression. Such data has also led to further insights into how such specific functions might be regulated by modifications of key regulatory domains within such scaffolding proteins, opening up new lines of research focusing on the cellular and molecular mechanisms that regulate tight junction assembly. To support such new lines of research we have generated new mutant expression constructs of junctional proteins, cell lines expressing such constructs, and generated antibodies recognising modified (phosphorylated) forms of such key junctional proteins. Future experiments will be based on such tools to decipher the structural and regulatory mechanisms that guide phase transition of junctional proteins and molecular assembly of functional tight junctions.
Exploitation Route Other academics might be interested in these findings who are investigating
- dynamic developmental processes that involve migration of cell sheets (e.g.., gastrulation, dorsal closure) of cell-cell fusion (placenta development or defects during human pregnancy);
- scientists studying inherited forms of tissue damage and permeability defects that impact on junctional membrane proteins (e.g., some forms of deafness)
- we have also discovered new mechanobiological mechanisms that are important for tissue function of specialised cells such as the retinal pigment epithelium, a tissue heavily affected by inherited and age-related diseases, that will believe are important for the treatment of retinal disease
- scientists focusing on chronic diseases in which epithelial and endothelial barriers play important roles in disease development, such as inflammatory diseases, infectious diseases and metabolic conditions such as diabetes.

The findings will also be important to scientists in industry and academia focusing to tissue engineering approaches as we identified novel principles of how substrate stiffness impacts on tissue integrity and function.
Sectors Healthcare

Manufacturing

including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology

 
Description The findings have contributed to the development of a new gene therapy approach for retinal degenerative diseases.
First Year Of Impact 2020
Sector Healthcare
Impact Types Economic

 
Description Apg-2: At the crossroads of tissue regeneration and degeneration
Amount £100,000 (GBP)
Funding ID R180018A 
Organisation Moorfields Eye Charity 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2018 
End 09/2021
 
Description Dbl3 signalling in RPE polarisation and function
Amount £124,999 (GBP)
Funding ID GR001497 
Organisation Moorfields Eye Charity 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2023 
End 07/2026
 
Description Epithelial apical membrane polarization, morphogenesis, and regulation of gene expression
Amount £692,791 (GBP)
Funding ID BB/X000575/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2023 
End 01/2026
 
Description Glaucoma - From genetic association studies to patient screening and disease mechanisms
Amount £127,278 (GBP)
Funding ID GR001476 
Organisation Moorfields Eye Charity 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2023 
End 09/2024
 
Description MarvelD3 in diabetic retinal disease
Amount £489,423 (GBP)
Funding ID 23/0006589 
Organisation Diabetes UK 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2024 
End 03/2028
 
Description MarvelD3 signalling and retinal tissue stress
Amount £122,242 (GBP)
Funding ID GR001000 
Organisation Moorfields Eye Charity 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2020 
End 12/2021
 
Description Project grant
Amount £40,596 (GBP)
Funding ID R180001A 
Organisation Moorfields Eye Charity 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2017 
End 07/2018
 
Description Seed funding
Amount £19,172 (GBP)
Funding ID M692 
Organisation Rosetrees Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2017 
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 Constitutive and conditional mouse strains deficient in ARHGEF18/p114RhoGEF 
Description Mouse strains to analyse the functions of the tight junction-associated RhoA GEF p114RhoGEF in development and disease 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2021 
Provided To Others? Yes  
Impact not yet 
URL https://www.ncbi.nlm.nih.gov/pubmed/33842485
 
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. 
URL https://www.ncbi.nlm.nih.gov/pubmed/32697990
 
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 Academic/University 
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 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 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 Structural and molecular analysis of tight junction 
Organisation Charité - University of Medicine Berlin
Country Germany 
Sector Academic/University 
PI Contribution We have generared and analysed epithelial cell lines mutant in particular tight junction proteins important for mechanotransduction in epithelial sheets.
Collaborator Contribution Freeze fracture of mutant epithelial cell lines followed by electron microscopy to assess tight junction structure.
Impact A manuscript describing the results is in progress. However, much fo the freeze fracture analysis is still in progress.
Start Year 2021
 
Title GENE THERAPY 
Description The invention relates to the use of vectors to improve vision by restoring RPE phagocytosis of photoreceptor outer segments in a patient suffering from retinal dysfunction and/or degeneration. 
IP Reference WO2021165685 
Protection Patent application published
Year Protection Granted 2021
Licensed Commercial In Confidence
Impact Funding for proof of concept study was received.
 
Title PEPTIDE INHIBITORS OF GUANINE NUCLEOTIDE EXCHANGE FACTOR-H1 
Description The present invention relates to peptide antagonists or inhibitors of GEF-H1, pharmaceutical compositions comprising said antagonists, polynucleotides encoding said antagonists, vectors encoding said polynucleotides, uses of said antagonists, pharmaceutical compositions and vectors in methods of medical treatment and kits comprising said antagonists, pharmaceutical compositions and vectors. The peptide antagonists of the present invention inhibit RhoA binding to the DH/PH module of GEF-H1 and, thereby, GEF-H1 function. 
IP Reference WO2021148763 
Protection Patent application published
Year Protection Granted 2021
Licensed Commercial In Confidence
Impact Funding for refinement of inhibitors has been obtained.
 
Title Gene therapy for age-related and inherited retinal degeneration 
Description The gene therapy is to rescue apical and junctional actomyosin activation in retinal pigment epithelial cells to restore functional epithelial cells in deficient patients. The therapy was successfully tested in two animal models with a proof-of-concept grant from UCL Technology Fund. 
Type Therapeutic Intervention - Cellular and gene therapies
Current Stage Of Development Refinement. Non-clinical
Year Development Stage Completed 2022
Development Status Actively seeking support
Impact None yet 
 
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
 
Description Plenary lecture - Cell and Experimental Biology meeting 2021 (USA) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Overiew of tight junctions and epithelial mechanobiology
Year(s) Of Engagement Activity 2021