Cross-talk between Ajuba and Rac signalling in the stabilization of cadherin adhesion
Lead Research Organisation:
Imperial College London
Department Name: National Heart and Lung Institute
Abstract
Cell-cell adhesion is a key event in the life of different cell types and is essential for their organization into higher ordered structures such as blood vessels, lung, skin, muscle, etc. Conversely, de-regulation of cell-cell contacts is found following bacterial infection, would healing or initiation of tumour invasion. We are interested in understanding how cells control their ability to glue to their neighbours as opposed to scatter into surrounding tissues. We identified a molecule, Ajuba, which plays a key role to strengthen cell-cell adhesion: in the absence of Ajuba, cell-cell aggregation is greatly reduced. Our unexpected finding is that Ajuba can manipulate the function of the small GTPase Rac, which is required to stabilize cell-cell contacts. In this proposal, we will address the mechanisms via which crosstalk between Ajuba and Rac leads to stronger cell-cell adhesion. Understanding the mechanisms via which Ajuba/Rac stabilize junctions will highlight potential strategies to interfere with tumour cell invasion and metastasis. Suitable strategies to stall metastatic growth will have important implications for the quality of life of cancer patients.
Technical Summary
We have evidence that Ajuba plays a role in Rac dynamics, and conversely, Rac/PAK1 signalling modulates Ajuba-dependent junction stabilization. We will address in three Work Packages:
1 - how Ajuba modulates Rac dynamic. We postulate that Ajuba interaction may prevent Rac.GTP hydrolysis or facilitate recruitment of an exchange factor to junctions to activate Rac. Rac GTP loading and intrinsic hydrolysis will be addressed in the presence/absence of wild-type Ajuba or its phosphomimetic (T172D) or non-phosphorylatable (T172A) mutants. Ajuba mutants unable to interact with Rac will be made and tested in vivo for junction stabilization and rescue of Rac activation. As depletion of DOCK180, a Rac GEF, phenocopies Ajuba RNAi, a potential role for DOCK180 in Ajuba-dependent Rac activation by cadherins will be addressed.
2 - how Ajuba reorganizes actin at cadherin complexes. We will address the relative contribution of Ajuba-dependent actin polymerization versus bundling for cadherin stabilization and how Rac/PAK1 signalling influences these two activities. Mutational analysis will identify the F-actin binding sites on Ajuba responsible for polymerization or bundling and the efficiency of these mutants to strengthen cadherin adhesion. The existence of ternary complexes Rac/Ajuba/Actin or Rac/Ajuba/alpha-catenin will be studied as a mechanism to integrate cadherin complexes with actin cytoskeleton.
3 - the relevance of Ajuba in Rac/PAK1 signalling during tumour progression will be tested. Up-regulation of Ajuba protein and phosphorylation levels will be investigated in tumour tissue microarrays and cell lines (primary and metastatic site) and correlated with malignancy and clinical outcome. We will test Ajuba sub-cellular localization in tumours/cell lines (nucleus, junction or focal adhesion) and which cellular process it regulates downstream of Ras/Rac hyper-activation during transformation (proliferation, invasion and/or junction disassembly).
1 - how Ajuba modulates Rac dynamic. We postulate that Ajuba interaction may prevent Rac.GTP hydrolysis or facilitate recruitment of an exchange factor to junctions to activate Rac. Rac GTP loading and intrinsic hydrolysis will be addressed in the presence/absence of wild-type Ajuba or its phosphomimetic (T172D) or non-phosphorylatable (T172A) mutants. Ajuba mutants unable to interact with Rac will be made and tested in vivo for junction stabilization and rescue of Rac activation. As depletion of DOCK180, a Rac GEF, phenocopies Ajuba RNAi, a potential role for DOCK180 in Ajuba-dependent Rac activation by cadherins will be addressed.
2 - how Ajuba reorganizes actin at cadherin complexes. We will address the relative contribution of Ajuba-dependent actin polymerization versus bundling for cadherin stabilization and how Rac/PAK1 signalling influences these two activities. Mutational analysis will identify the F-actin binding sites on Ajuba responsible for polymerization or bundling and the efficiency of these mutants to strengthen cadherin adhesion. The existence of ternary complexes Rac/Ajuba/Actin or Rac/Ajuba/alpha-catenin will be studied as a mechanism to integrate cadherin complexes with actin cytoskeleton.
3 - the relevance of Ajuba in Rac/PAK1 signalling during tumour progression will be tested. Up-regulation of Ajuba protein and phosphorylation levels will be investigated in tumour tissue microarrays and cell lines (primary and metastatic site) and correlated with malignancy and clinical outcome. We will test Ajuba sub-cellular localization in tumours/cell lines (nucleus, junction or focal adhesion) and which cellular process it regulates downstream of Ras/Rac hyper-activation during transformation (proliferation, invasion and/or junction disassembly).
Planned Impact
Our proposal has impact for public health, improving quality of life and UK economic competitiveness. Because epithelial cells are present in many body sites, understanding how their function is maintained has profound implications for the physiology of many different organs. Epithelial cells require constant signalling from junctions to maintain their polarized, differentiated phenotype. Improving knowledge on how cadherin-dependent junctions are maintained will provide potential mechanisms for therapeutic intervention in different clinical settings.
The biomedical aspect of the research may lead to applications for commercial exploitation, and hence increase economic competitiveness in the UK. Because of the dual aspect of the proposed work, including fundamental research as well as its biomedical applications, the project will support knowledge and technology translation and increase the economic competitiveness. The core principles investigated here (interplay between adhesion receptors and signalling) are frequently perturbed in different pathologies.
Cancer:
Over 80% of human tumours originate from epithelial cells and thus, specific knowledge on epithelial processes is relevant to identify processes amenable to intervention. Most deaths of cancer patients may be prevented by reducing dissemination/growth of metastasis or damage to distant organs by malignant lesions. Reduced cell-cell adhesion in tumours impairs epithelial differentiation and appropriate localization of signalling molecules and tumour suppressors. Understanding the mechanisms via which cell-cell adhesion is de-regulated during tumour progression will provide therapeutic strategies to ameliorate cell de-differentiation and detachment from solid tumours and metastasis. Such strategies have considerable clinical implications as adjuvant therapy, to sustain stable tumour mass of non-responsive tumours or tumours inaccessible to surgery.
Other diseases:
(a) A "leaky" epithelium underlies the causes of many chronic diseases such as asthma, atopic dermatitis, food intolerance/allergy, and chronic infections. When the tightness of epithelial sheets is compromised are defective (including cadherins and tight junctions), exogenous components such as airborn antigens (skin, lungs) or breakdown food products (intestines) are able to leak through the epithelium and stimulate the underlying immune system.
(b) Bacterial infections: different pathogens highjack adhesion receptor signalling to mediate their internalization. An example specifically involving E-cadherin is invasion of the pathogen Listeria monocytogenes, a serious infection for infants and immuno-compromised individuals. Another example is the intricate interplay between Helicobacter pylori and E-cadherin in stomach and gastric epithelia, a risk-factor in development of ulcers and gastric cancers.
To increase the impact of future therapies to specific cohorts of cancer patients, our work will define the type of tumour in which interfering with the cross-talk Rac>PAK1>Ajuba is most beneficial. Rac signalling is often up-regulated in different and, on their own right, Rac and PAK1 play an essential role in numerous diseases. Ajuba mRNA is increased in different tumours some of which are previously reported subsets that show Rac and PAK up-regulation. In addition, Ajuba gene is located in 14q11.2, a breakpoint site for leukemia mantel cell leukemia. Thus, identifying novel regulatory circuitry that controls Rac/PAK1 signalling will provide insights towards the design of small molecules to be used for translational purposes.
The biomedical aspect of the research may lead to applications for commercial exploitation, and hence increase economic competitiveness in the UK. Because of the dual aspect of the proposed work, including fundamental research as well as its biomedical applications, the project will support knowledge and technology translation and increase the economic competitiveness. The core principles investigated here (interplay between adhesion receptors and signalling) are frequently perturbed in different pathologies.
Cancer:
Over 80% of human tumours originate from epithelial cells and thus, specific knowledge on epithelial processes is relevant to identify processes amenable to intervention. Most deaths of cancer patients may be prevented by reducing dissemination/growth of metastasis or damage to distant organs by malignant lesions. Reduced cell-cell adhesion in tumours impairs epithelial differentiation and appropriate localization of signalling molecules and tumour suppressors. Understanding the mechanisms via which cell-cell adhesion is de-regulated during tumour progression will provide therapeutic strategies to ameliorate cell de-differentiation and detachment from solid tumours and metastasis. Such strategies have considerable clinical implications as adjuvant therapy, to sustain stable tumour mass of non-responsive tumours or tumours inaccessible to surgery.
Other diseases:
(a) A "leaky" epithelium underlies the causes of many chronic diseases such as asthma, atopic dermatitis, food intolerance/allergy, and chronic infections. When the tightness of epithelial sheets is compromised are defective (including cadherins and tight junctions), exogenous components such as airborn antigens (skin, lungs) or breakdown food products (intestines) are able to leak through the epithelium and stimulate the underlying immune system.
(b) Bacterial infections: different pathogens highjack adhesion receptor signalling to mediate their internalization. An example specifically involving E-cadherin is invasion of the pathogen Listeria monocytogenes, a serious infection for infants and immuno-compromised individuals. Another example is the intricate interplay between Helicobacter pylori and E-cadherin in stomach and gastric epithelia, a risk-factor in development of ulcers and gastric cancers.
To increase the impact of future therapies to specific cohorts of cancer patients, our work will define the type of tumour in which interfering with the cross-talk Rac>PAK1>Ajuba is most beneficial. Rac signalling is often up-regulated in different and, on their own right, Rac and PAK1 play an essential role in numerous diseases. Ajuba mRNA is increased in different tumours some of which are previously reported subsets that show Rac and PAK up-regulation. In addition, Ajuba gene is located in 14q11.2, a breakpoint site for leukemia mantel cell leukemia. Thus, identifying novel regulatory circuitry that controls Rac/PAK1 signalling will provide insights towards the design of small molecules to be used for translational purposes.
Publications
Kelly D
(2015)
Automated multiwell fluorescence lifetime imaging for Förster resonance energy transfer assays and high content analysis
in Analytical Methods
V. M.M. Braga
(2017)
Cell-cell junctions
McCormack J
(2013)
Cycling around cell-cell adhesion with Rho GTPase regulators.
in Journal of cell science
Erasmus JC
(2016)
Defining functional interactions during biogenesis of epithelial junctions.
in Nature communications
Braga VM
(2018)
Editorial Overview: Integration of dynamic processes in cell behaviour and tissue architecture.
in Current opinion in cell biology
Garcia-Cattaneo A
(2014)
Hold on tightly How to keep the local activation of small GTPases
in Cell Adhesion & Migration
Frasa MA
(2012)
Illuminating the functional and structural repertoire of human TBC/RABGAPs.
in Nature reviews. Molecular cell biology
Sri-Ranjan K
(2022)
Intrinsic cell rheology drives junction maturation.
in Nature communications
Machesky L
(2017)
So far, yet so close: a-Catenin dimers help migrating cells get together.
in The Journal of cell biology
McCormack JJ
(2017)
The scaffold protein Ajuba suppresses CdGAP activity in epithelia to maintain stable cell-cell contacts.
in Scientific reports
Fischer A
(2018)
Vascular Permeability: Flow-Mediated, Non-canonical Notch Signalling Promotes Barrier Integrity
in Current Biology
Jaber N
(2016)
Vps34 regulates Rab7 and late endocytic trafficking through recruitment of the GTPase-activating protein Armus.
in Journal of cell science
Description | Brunei PhD Studentship |
Amount | £151,000 (GBP) |
Organisation | Government of Brunei |
Sector | Public |
Country | Brunei Darussalam |
Start | 02/2011 |
End | 01/2015 |
Description | Cancer Research UK |
Amount | £196,665 (GBP) |
Funding ID | C1282/A11980 |
Organisation | Cancer Research UK |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 10/2010 |
End | 02/2015 |
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 | Wellcome Trust Project grant |
Amount | £263,000 (GBP) |
Funding ID | WT094371MA |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 07/2011 |
End | 12/2014 |
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 | Carlos Chavez |
Organisation | Federal University of Minas Gerais |
Department | Dept of Biochemistry and Immunology |
Country | Brazil |
Sector | Academic/University |
PI Contribution | We provided a cellular model to investigate different actions of snake venoms in cell structure and funcitonality. |
Collaborator Contribution | Prof Chavez provided expertise in the identification of binding domains for protein-protein interactions using peptides spotted onto membranes. We also collaborate closely in understanding the mechanisms of snake envenomation in cells. |
Impact | Manuscript submitted McCormack, JJ; Bruche,S; Ouadda, ABD; Ishii, H; Lu,H; Garcia-Cattaneo,A; Chávez-Olórtegui,C; Lamarche-Vane, N & Braga, VMM. The scaffold protein Ajuba suppresses CdGAP activity in epithelia to maintain stable cell-cell contacts. |
Start Year | 2012 |
Description | Lamarche - GAPs |
Organisation | McGill University |
Country | Canada |
Sector | Academic/University |
PI Contribution | My lab provide specific proteins |
Collaborator Contribution | Dr. Lamarche is an expert in GAP proteins and has contributed with reagents, expertise and technical advice |
Impact | We have published a paper together in 2000 and are currently preparing another for publication |
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 |
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Part Of Official Scheme? | No |
Geographic Reach | International |
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Year(s) Of Engagement Activity | 2019 |
Description | Invited Speaker Valbonne France |
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Geographic Reach | International |
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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 |
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Description | Mechanical Forces in Biology, EMBL Symposia, Heidelberg - Germany. 2017 |
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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 |
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Description | Talk Oncobiology Symposium, Brazil |
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Results and Impact | networking, share ideas |
Year(s) Of Engagement Activity | 2017 |
Description | Talk at Brazilian Embassy Networking event |
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Results and Impact | Workshop "Innovation in Healthcare" - discussion on engagement and collaborations - how is it best fomented? |
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Description | Workshop Women in Science |
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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 |
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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 Regulation and Function of Small GTPases, FASEB Conference, Miami - USA. 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 |
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Description | talk conference Mechanobiology Institute Singapore, 10th Anniversary |
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Geographic Reach | International |
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Results and Impact | networking, Science discussions, collaborations |
Year(s) Of Engagement Activity | 2018 |