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Dissecting the molecular organisation of Fat-Dachsous cadherin complexes to understand mechanisms of coordinated cell polarisation

Reference	BB/S001395/1
Principal Investigator / Supervisor	Professor David Strutt
Co-Investigators / Co-Supervisors	Professor Ashley Cadby, Dr Helen Strutt
Institution	University of Sheffield
Department	School of Biosciences
Funding type	Research
Value (£)	448,520
Status	Completed
Type	Research Grant
Start date	01/01/2019
End date	31/12/2022
Duration	48 months

Abstract

Cell signalling and cell polarisation are fundamental cellular properties that underlie many cellular functions. In epithelial cells these can be interlinked, giving coordinated cell polarisation in the tissue plane - referred to as 'planar polarity' and exemplified by hairs and cilia all pointing in the same direction on the tissue surface. Loss of planar polarity during embryogenesis results in congenital disease (e.g. cardiac valve defects and Van Maldergem/Hennekam syndromes) and its disregulation is associated with cancer tumourigenesis. Planar polarity is established by specialist signalling pathways that employ atypical cadherins. Cadherins are a diverse protein family, the best-studied being the classic 'adhesion' cadherins typified by E-cadherin (E-cad). Like E-cad, the planar polarity cadherins bind to each other at cell-cell contacts. However, unlike E-cad, they also mediate polarised cell-cell signalling and adopt planar polarised localisations within cells. We seek to understand these novel behaviours. This proposal investigates the properties of the Fat (Ft) and Dachsous (Ds) planar polarity cadherins. Ft and Ds bind heterophilically between cells - mediating polarised cell-cell signalling - and localise to opposite cell edges - providing subcellular planar polarity cues. We will use bulk and single molecule microscopy techniques to study Ft-Ds protein localisations, dynamics and clustering behaviours, together with structure-function analysis, in a reconstituted heterologous tissue culture system and in vivo in the Drosophila wing. We will map domains of Ft and Ds involved in their binding interactions, and determine how they cluster to form stable domains at cell-cell contacts and how they become 'sorted' to opposite cell edges. These studies will not only provide mechanistic insights into planar polarity, but also extend our knowledge of the functions of the atypical cadherins, a poorly studied group of proteins.

Summary

The cells that compose our bodies show great diversity, nevertheless some properties are shared by many cell types. These include the ability to communicate ('signal') to coordinate functions, and to adopt polarised shapes or behaviours. Thus, understanding cell signalling and cell polarisation are fundamental goals of cell biology. Notably, in some contexts, signalling and polarisation are coupled. An example is 'planar polarity', where cells in a sheet interact together so they all point in the same direction in the tissue plane. This results in hairs on the skin being able to point in one direction, and cilia lining the trachea and lungs to beat in the same direction. We will study two molecules that play important roles in establishing planar polarity in animal tissues, the cadherins Fat (Ft) and Dachsous (Ds). Cadherins mediate binding between cells, by sitting in the outer membrane and forming bonds with cadherins sitting on neighbouring cells. The best studied cadherin - Epithelial cadherin (E-cad) - binds between cells in epithelial sheets, and plays a structural role in holding tissues together. Ft and Ds cadherins also bind between cells, with Ft on one cell binding to Ds on the neighbouring cell. This 'heterophilic' binding between Ft and Ds mediates signalling between cells, as high Ds on one cell results in more Ft molecules being bound on the surface of a contacting cell. Furthermore, Ft-Ds heterodimers cluster together at cell-cell contacts, and within these clusters the molecules are 'sorted', such that clusters consist predominantly of Ft in one cell and Ds in the neighbouring cell. This local sorting appears to promote cell-level polarisation of Ft and Ds, such that most Ft goes to one cell end and most Ds goes to the other. These two properties of Ft and Ds - heterophilic binding between cells and segregation to opposite edges within cells - result in tissue-level planar polarisation of cell sheets, such that each cell has Ft at one edge and Dsat the other. We will investigate how Ft and Ds promote planar polarisation of tissues. We propose that their stable clustering at cell-cell contacts depends on a combination of cis- and trans-interactions between Ft and Ds, and we will test this by systematically mapping parts of Ft and Ds involved in binding to themselves and each other. Furthermore, we propose that local sorting into polarised clusters is due to a combination of stabilising interactions between Ft-Ds heterodimers of the same orientation, and destabilising interactions between heterodimers of the opposite orientation. We will seek to identify the molecular basis of such sorting interactions. To understand the behaviour of Ft and Ds, we will exploit advances in microscopy that allow imaging of individual molecules at high resolution. Moreover, as Ft and Ds are very large molecules, we will use leading-edge genetic engineering techniques to assist their fine-scale dissection. We will study the properties of Ft and Ds in a cultured cell system where they can be rapidly manipulated, and also in a simple animal model - the developing fruit fly wing - which is particularly amenable to genetic manipulation and imaging. This work will shed light on the molecular properties of a poorly studied group of 'atypical' cadherins which are required for human health. Disruption of Ft-Ds activity is associated with congenital diseases such as cardiac valve defects and Van Maldergem and Hennekam syndromes, as well as being implicated in cancer progression. Detailed knowledge of how Ft and Ds mediate cell signalling and cell polarisation will reveal mechanisms underlying fundamental aspects of cell function, and open the way to understanding how to therapeutically modulate their activities.

Impact Summary

Expected non-academic beneficiaries of the research are: - Members of society potentially afflicted with congenital disease or cancer resulting from disregulation of Fat-Dachsous pathway function - Pharmaceutical companies interested in developing diagnostic tools or drugs to modulate pathway function - Health services While the timescale for translation of basic research to clinical therapy is of the order of 20 years, we will nevertheless seek to accelerate this by actively seeking out collaborators to translate our findings made in Drosophila into vertebrate models (years 1-3). Furthermore, we will disseminate our findings at conferences and publish them in international peer-reviewed journals (years 2-3). - Organisations seeking to recruit scientifically trained staff, including business, industry and the public sector Research group members will improve their training, including transferable skills e.g. project management and leadership skills. Additionally, we will supervise undergraduate/postgraduate students, contributing to their training in scientific experimentation, experimental design, data analysis and transferable skills. Thus, this grant will contribute towards the health of UK science and higher education through developing expertise and training highly skilled researchers (years 1-3). - The public/wider society We will communicate the excitement, beauty and fundamental importance of scientific research to the public e.g. by presenting at our university-wide events such as Researchers' Night and Festival of the Mind. Examining molecular and cellular behaviour in living animals provides beautiful images that effectively capture and communicate the concepts of biomedicine, and encourages young people to consider careers in scientific research. Furthermore, discussing the genetic basis of disease and emphasising the importance of animal models (including the fruitfly) to the public is critical for maintaining their support for basic research (years1-3).

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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