Regulation of epithelial apical membrane differentiation and function

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

Abstract

Epithelia are continuous layers of cells that delineate our tissues and organs. Individual epithelial cells interact with each other via molecular complexes that mediate adhesion but also function as sensors that transmit information about the presence or absence of neighbouring cells to the cell interior. Integrity of epithelia is important for our organs to develop and function normally, and to protect us from our environment. For example, breaches in epithelial layers such as the skin or in the lining of the intestine can lead to serious infections and can occur due to chronic inflammations or acute infections by viruses and bacteria. Similarly, a characteristic of cancer cells is that they have lost the capability to sense the presence of neighbouring cells or how tightly they are packed, and hence continue to proliferate and migrate on top of their neighbours.

Epithelia are polarised, which means they have two cell surface domains that have different compositions and functions. The cell surface that faces the outside world or the internal lumen in our organs is called the apical cell surface. This apical domain often forms a highly specialised structure that mediates organ specific functions, such as digestion and nutrient absorption in the intestine or supportive functions for the cells that sense light in our eyes. Here, we propose to investigate a molecular mechanism that drives the formation of such specialised apical membranes in intestinal and retinal epithelial cells. This pathway is based on signalling proteins that we have recently identified and for which we have evidence that they form a mechanism that may also inhibit the function of proteins that have been linked to tumorigenesis and cancer by stimulating cells to differentiate and form tightly packed cellular sheets.

Knowledge of how epithelial cells differentiate to mediate organ specific functions and how such pathways inhibit mechanisms that can lead to cancer is important to understand how tissues behave in disease. Epithelial dysfunction has been linked to many diseases such as cancer, chronic inflammations in the intestine, and inherited and age-related diseases that lead to blindness. The expected results will help us to think of new ways how we can treat such diseases that lead to epithelial degeneration and cancer.

Technical Summary

Epithelial cells form sheets of cells that are connected by junctional complexes. Individual epithelial cells in simple epithelia are polarised and form apical and basolateral cell surface domains that are biochemically and functionally distinct. The apical domain often differentiates into a highly specialised structure such as the brush border membranes in the intestine or the phagocytic apical membrane in retinal pigment epithelial cells. The signalling pathways that regulate junction formation, positioning and apical membrane differentiation are linked and make use of overlapping sets of signalling proteins; however, we understand only poorly how these proteins are activated in process-specific manners and how development of epithelial cell morphology is linked to apical differentiation and junctional positioning. This proposal focuses on the signalling mechanisms activated by DOME, a Cdc42 exchange factor that we have recently discovered. DOME associates with the apical membrane and regulates columnar morphogenesis, apical differentiation and junctional positioning once initial junction assembly occurred, as well as morphogenesis in 3D cultures. Our preliminary data suggest that DOME activates two Cdc42 effector pathways, the Par6/aPKC pathway and actomyosin via MRCKs. Our aims are to identify the molecular mechanisms by which DOME activated MRCK signalling drives actomyosin dynamics, epithelial morphogenesis, and apical differentiation, and to establish the importance of this signalling mechanism for the differentiation and function of retinal pigment epithelial cells in vitro and in vivo. The expected results will be important for our understanding of the signalling networks and dynamic processes that guide and mediate cell polarization and epithelial differentiation, and how such pathways interact with mechanisms that guide epithelial proliferation and tumorigenesis.

Planned Impact

Who will benefit from this research?
The immediate beneficiaries will be basic and applied scientists working in related fields at Universities as well as in industry. This includes scientists working in areas such as epithelial biology, organ development, retinal function and physiology, tissue engineering, as well as chronic inflammation and cancer biology. The retinal pigment epithelium plays a crucial role in the retina and is the primary cell type affected in one of the major blinding diseases: age-related macula degeneration. Hence, insights into the biology and function of retinal pigment epithelial cells will benefit medical scientists, clinicians and, ultimately, patients as well as the NHS and the general public.

How will they benefit from this research?
The research will benefit allied scientists by providing them with the molecular details and functional principles of a new pathway that guides epithelial cell differentiation and function. This will benefit their research as such knowledge and tools generated can be applied to different epithelial cell types and organs. Translational and clinical scientists will benefit in a similar way, as, for example, scientists interested in cancer may be able to exploit the potential tumour suppressor properties of the DOME pathway. Similarly, scientists investigating retinal dysfunction can exploit our results to analyse inherited and age-related retinal diseases.

The project will also involve training of two postdoctoral fellows that can benefit the private sector as well as public services through the NHS as one of the fellows will be trained in techniques required for the development of treatments for retinal diseases involving gene therapy.

The expected results are likely to start to benefit other scientists within the lifetime of this grant.

Publications

10 25 50
 
Description We have discovered a new molecular mechanism that drives the formation of specialized apical membrane domains in various important epithelial cells such as those of the intestine, kidney and retinal pigment epithelium. The discoveries have now led to a project exploiting this mechanism to improve epithelial function in disease.
Exploitation Route Tissue engineering and therapeutic applications for epithelial degenerative diseases.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description The findings have led to further funding for a proof of concept study.
First Year Of Impact 2019
Sector Pharmaceuticals and Medical Biotechnology
 
Description Pathfinder
Amount £142,000 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Bloomsbury Centre
Sector Charity/Non Profit
Country United Kingdom
Start 02/2017 
End 08/2018
 
Description PhD fellowship
Amount £100,000 (GBP)
Organisation Fight for Sight 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2016 
End 09/2019
 
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 Dbl reagents 
Description Biological cell lines and reagents to study Dbl and apical membrane formation in epithelia 
Type Of Material Cell line 
Year Produced 2015 
Provided To Others? Yes  
Impact too early 
 
Title MDCK cell lines 
Description MDCK cells liens expressing inflammatory regulators to study effect interplay between inflammation and epithelial barrier properites 
Type Of Material Cell line 
Provided To Others? No  
Impact Used at talks and is being incorporated into a publication 
 
Description Evolutionary conservation of epithelial apical differentiation 
Organisation University College London
Department MRC Laboratory for Molecular Cell Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution The collaboration is to study the molecular pathways of epithelial apical differentiation in vertebrate and invertebrates. We are performing the experiments in vertebrate model systems.
Collaborator Contribution The collaborating laboratory performs analogous experiments in flies.
Impact fiirst outputs have been submitted
Start Year 2015
 
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 Role of tight junctions in infectious disease 
Organisation University of Zurich
Country Switzerland 
Sector Academic/University 
PI Contribution Analysis of junctional signalling mechanisms in relation of infectious agents
Collaborator Contribution Provision of reagents and information
Impact no outputs yet
Start Year 2015
 
Description ZONAB signalling in inflammation and cell survival, and apical differentiation in the retinal pigment epithelium 
Organisation University College London
Department Institute of Ophthalmology UCL
Country United Kingdom 
Sector Academic/University 
PI Contribution We are performing the analysis with primary cultures in vivo and the collaborators test our in vitro results with mouse experiments
Collaborator Contribution The collaborator performs the in vivo experiments with lentiviral vectors in mouse retinas
Impact A first publication has been published in 2010 (PMID: 21209887) Follow-up work has led to an an extension of this collaboration to apical differentiation in vivo in mouse RPE, which has been funded by the BBSRC
Start Year 2007
 
Description 4th China-UK Cancer (CUKC) Conference. Cardiff, Wales 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Discussions about junctions and cancer

I was asked whether I would be interested to participate in a follow-up meeting in China
Year(s) Of Engagement Activity 2015
 
Description Annual Student Lecture, IBMC, University of Porto, Portugal 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Interest of students for future laboratory visits

Discussions about collaborations
Year(s) Of Engagement Activity 2015
 
Description Cardiff-Peking Universities Cancer Institute, invited lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact lively discussions

exchange of information
Year(s) Of Engagement Activity 2013
 
Description Cell Press Tumour Microenvironment LabLinks Symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact diverse audience reached and interesting discussions

new collaboratoin
Year(s) Of Engagement Activity 2012
 
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 Epithelial Morphogenesis Symposium, Sapporo 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact interdisciplinary meeting

echange of reagents and protocols
Year(s) Of Engagement Activity 2012
 
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 Gordon Research Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Selected presentation by postdoctoral fellow paid from grant
Year(s) Of Engagement Activity 2015
 
Description Gordon Research Conference on Signalling by Adhesion Receptors 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Great discussions with peers

established collaborations
Year(s) Of Engagement Activity 2012
 
Description International Conference on the Molecular Structure and Function of Tight Junctions 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact interesting discussions with peers

exchange of research reagents
Year(s) Of Engagement Activity 2012
 
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 Regulation of Cdc42 in epithelial differentiation, Konstanz, Germany 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Invited lecture sparked interesting discussions

discussion of future collaborations
Year(s) Of Engagement Activity 2015
 
Description Regulation of Cdc42 in epithelial differentiation, Zurich, Switzerland 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Supporters
Results and Impact discussions about junctions and infectious disease

Start of a collaboration on tight junctions and infectious disease
Year(s) Of Engagement Activity 2015
 
Description Signal Transduction, Mexican Biochemical Society, Oaxaca, Mexico 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Talked led to discussions about future collaborations with Mexican laboratories

Possible interactions were discussed
Year(s) Of Engagement Activity 2015