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.
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.
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.
Organisations
- University College London (Lead Research Organisation)
- Charité - University of Medicine Berlin (Collaboration)
- University College London (Collaboration)
- University of Grenoble (Collaboration)
- Andalusian Center for Molecular Biology and Regenerative Medicine (Collaboration)
- Austrian Institute of Technology (Collaboration)
- Beatson Institute for Cancer Research (Collaboration)
- Institute of Science and Technology Austria (Project Partner)
- Grenoble Alpes University (Project Partner)
Publications
Zihni C
(2017)
An apical MRCK-driven morphogenetic pathway controls epithelial polarity.
in Nature cell biology
Zihni C
(2016)
Tight junctions: from simple barriers to multifunctional molecular gates.
in Nature reviews. Molecular 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 |