Mechanotransduction at tight junctions and epithelial differentiation and dynamics

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 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.

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.