Building epithelial cytoarchitecture:regulation of actin dynamics by cell-cell junctions

Lead Research Organisation: Imperial College London
Department Name: Life Sciences

Abstract

Cell-cell adhesion and migration are key events in the life of many different cell types, and are essential for the organization of cells into higher ordered structures such as blood vessels, lung, skin, muscle, etc. Epithelial cells differentiate and form sheets of tightly packed cells: these sheets form protective layers wrapping up the body (skin) and the inside of internal cavities (i.e. stomach, intestines, etc). The choice of whether to migrate or stick to their neighbours has implications for epithelial function in health and disease. Thus, it is important to understand how a cell is kept inside an epithelial sheet or detaches and become shapeless, homeless and aimless. Epithelial cells can not function properly in the absence of tight cell-cell adhesion and a cuboidal morphology. We are interested in understanding how cells glue to their neighbours in order to regulate their shape and differentiation. We aim at identifying the molecules that participate in these processes and investigate their role in epithelia. Insights into how epithelial cell shape is established and maintained will provide clues on how to ameliorate diseases that have compromised epithelial function.

Technical Summary

The acquisition of a cuboidal morphology (polarization) is a hallmark of epithelia and occurs after cell division, wound closure, and during morphogenesis. Extensive reorganization of epithelial actin cytoskeleton is triggered following assembly of cadherin-dependent cell-cell adhesion. This cytoskeleton remodelling is essential for the functionality of epithelial sheets, stability of adhesive receptors at junctions and for the appropriate localization of signalling complexes. Yet, in spite of the importance of epithelia in development, homeostasis and diseases, the mechanisms via which a cuboidal morphology is acquired are poorly understood. Understanding how actin structures are assembled following cell-cell adhesion will provide major insights into the relevant signalling pathways for polarization. We have found that when cell-cell contacts are induced in keratinocytes, an increase in actin polymerization and myosin function (contractility) is observed very quickly. These processes are coordinated spatially and temporally into two distinct actin populations: junctional-actin and thin bundles. Here we aim to identify specific cytoskeletal proteins that are modulated by cell-cell adhesion and participate in the formation and/or maintenance of junctional-actin and thin bundles. As strategy, we will deplete the protein levels of specific proteins using siRNA during induction of cell-cell contacts and perform a screening using a siRNA library of actin binding proteins. Once relevant proteins are identified, we will perform functional assays to determine in which step of epithelial polarization these actin binding proteins operate: stabilization of cell-cell contacts or cell height increase (which reflects the functions of junctional-actin and thin bundles). We will use a combination of dynamic studies (live imaging), biochemical, biophysical and cell biology experiments together with rescue analysis of the specific phenotypes induced by siRNA depletion.

Publications

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Schultz F (2019) Cardiomyocyte-myofibroblast contact dynamism is modulated by connexin-43. in FASEB journal : official publication of the Federation of American Societies for Experimental Biology

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Tzircotis G (2011) RhoG is required for both Fc?R- and CR3-mediated phagocytosis. in Journal of cell science

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McCormack J (2013) Cycling around cell-cell adhesion with Rho GTPase regulators in Journal of Cell Science

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Machesky L (2017) So far, yet so close: a-Catenin dimers help migrating cells get together. in The Journal of cell biology

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V. M.M. Braga (2017) Cell-cell junctions

 
Description We designed and optimized a methodology to identify regulators of cell-cell contacts and thus understand how cells stick to each other and how they regulate how strong their bond should be.
Exploitation Route The methodology developed in this grant may be of interest for screening of compounds that inhibit junction dissociation. It would be interesting for basic researchers, drug development and pharmaceutical companies. In addition, we developed an automated software to quantify distinct junction phenotypes.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description BHF Centre for Research Excellence
Amount £40,000 (GBP)
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2009 
End 10/2010
 
Description Cross-talk between Ajuba and Rac signalling in the stabilization of cadherin adhesion
Amount £435,943 (GBP)
Funding ID MR/J007668/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 03/2012 
End 12/2015
 
Description Multi-user equipment
Amount £784,699 (GBP)
Funding ID WT066143 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2015 
End 12/2020
 
Description Newton Fund (MRC-CONFAP)
Amount £84,730 (GBP)
Funding ID MR/M026310/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2016 
End 12/2016
 
Description Science without Borders
Amount £203,427 (GBP)
Funding ID 4803318142983356 
Organisation National Council for Scientific and Technological Development (CNPq) 
Sector Public
Country Brazil
Start 02/2014 
End 01/2016
 
Title Development of Machine Learning methods to quantify junction phenotypes driven by different stimuli (2017) 
Description A major road block in the analysis of junction phenotypes is the ability to recognize cell borders in a monolayer of epithelial cells, which is painstakinly done manually and with enorumous time invested in each image. Such detailed analysis is not feasible for high-throughput analysis in screens. Training datasets teach computer how to identify cell-cell contacts, which can then tbe deployed in large datasets with minimal input from user. Development of the program was done using a large dataset of images obtained under different conditions obtained via the distinct grants funding my research. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact Methodology is currenly being validated. It will be made available after thorough optimization, validation and adding a user-friendly interface. 
 
Title high-throughput image analysis of junctions 
Description A pipeline for automated quantification of phenotypes on cell-cell contacts as published in our paper: Erasmus, J, Bruche, S; Pizarro, L; Maimari, N; Pogglioli, T; Wheeler, A; Lees, J; Tomlinson, C; Zalivina, I; Alberts, A; Russo, A & Braga, VMM. Defining functional interactions during biogenesis of epithelial junctions. Nature Commun. 7, 13542, 2016 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact N/A methodology is available online. 
URL http://www.imperial.ac.uk/bioinformatics-data-science-group/resources/software/rnaiscreener/
 
Description Defining the role of MTSS1 in epithelial cell-cell contacts 
Organisation Beatson Institute for Cancer Research
Country United Kingdom 
Sector Academic/University 
PI Contribution This study identified the mechanism via which MTSS1 stabilizes cell-cell adhesion via modulation of actin filaments.
Start Year 2010
 
Description Sean Sun - Modelling of biophysical parameters that generate a mature cell-cell contact 
Organisation Johns Hopkins University
Country United States 
Sector Academic/University 
PI Contribution We provided the biological question and image datasets.
Collaborator Contribution Sean Sun - Hopkins Universitymodeller
Impact Manuscript submitted: Ranjan, K.; Alonso-Mardones, JS; Swiatlowska, P; Liu, S.;Rothery, S.; M. Stevens; Sun,S.; Gorelik, J & Braga, VMM. Cell confinement impacts on cortical elasticity, configuration and dynamics of junctions
Start Year 2015
 
Description 51. 3rd World Congress on Cancer Biology and Immunology-2019", Italy KEYNOTE SPEAKER 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Seminar at conference
Year(s) Of Engagement Activity 2019
 
Description Invited Speaker Valbonne France 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Invited to give a seminar at the Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne France

networking with academic staff from department
Year(s) Of Engagement Activity 2012
 
Description Keynote speaker Symposium Epithelial intercellular junctions as dynamic hubs to integrate forces, signals and cell behaviour". Aachen University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact conference Keynote speaker
Year(s) Of Engagement Activity 2016,2017
 
Description Lecture "Pioneers in Cell Dynamics and Imaging", Cells in Motion Clusters of Excellence, University of Muenster, Germany. 2018 
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 networking
Year(s) Of Engagement Activity 2018
 
Description Mechanical Forces in Biology, EMBL Symposia, Heidelberg - Germany. 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact networking, data dissemination
Year(s) Of Engagement Activity 2017
 
Description Physics and Biology confront cell-cell adhesion" Aussois, France October 14-19th 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Seminar presented at conference - networking and recognition
Year(s) Of Engagement Activity 2019
 
Description Summer Global School at Imperial College 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Schools
Results and Impact Together with Dr Anita Hall (Faculty of Natural Sciences) we were the leaders for the FoNS and FoM Stream Module. This stream aimed to provide students with insights and hands-on experience of cell biology experiments and their impact in medical research and drug development, cancer research in particular. All members of the Braga laboratory were involved in preparing cell samples to understand tumour metastasis and imaging techniques. The activities can be seen in the video https://spectrecom.wistia.com/medias/oipf0gc1je

Consolidate and develop current knowledge of science, engineering, technology and maths
Improve study skills whilst working in a range of stimulating environments
Develop practical skills as well as theoretical knowledge
Work in teams to develop interpersonal and organisational abilities
Find out about what university life is like
Year(s) Of Engagement Activity 2015
URL https://www.imperial.ac.uk/be-inspired/global-summer-school/
 
Description Talk at Brazilian Embassy Networking event 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Workshop "Innovation in Healthcare" - discussion on engagement and collaborations - how is it best fomented?
Year(s) Of Engagement Activity 2017
 
Description Workshop Women in Science 
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 Women Scientists discussing their career progression in Academia, what worked well, what could have been different
Year(s) Of Engagement Activity 2009
 
Description invited speaker Liverpool 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact invited to give a seminar at the Dept Cellular and Molecular Physiology, Liverpool, UK

networking with colleagues at the Dept.
Year(s) Of Engagement Activity 2012
 
Description talk conference Mechanobiology Institute Singapore, 10th Anniversary 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact networking, Science discussions, collaborations
Year(s) Of Engagement Activity 2018