E-cadherin subcomplexes: function and regulation by microtubules
Lead Research Organisation:
University of Cambridge
Department Name: Gurdon Institute
Abstract
The mechanism that attaches neighbouring units, or cells, in our body to each other is known as cell-cell adhesion. Recent work has demonstrated that cell-cell adhesion is also important for communication between the neighbouring cells to decide when to divide, migrate or die. Specific cell adhesion proteins ensure cell-cell adhesion: the proteins on the surface of one cell bind directly to similar proteins on the surface of adjacent cell. One of the major cell adhesion proteins is called E-cadherin. E-cadherin provides cell-cell adhesion between the epithelial cells: the cells that outline all cavities and surface structures of the body. In the cell, E-cadherin adhesion forms a thin belt, called zonula adherens that outlines the periphery of the cell and connects it to multiple neighbours. E-cadherin is vital for proper development of the body from very early stages. Furthermore, faulty E-cadherin adhesion contributes to cancer progression by increasing growth and metastasis.
For proper cell-cell adhesion it is vital to position the zonula adherens at a particular distance from the surface of the cell that faces the cavity. More unexpectedly, the cell also carefully controls how E-cadherin is distributed around the circumference of the cell, within the zonula adherens. While most cells have an even distribution, an increasing number of cases have been discovered where E-cadherin is distributed asymmetrically. The function of such asymmetric distribution in the developing animal has yet to be tested. We have chosen a simple animal to study this problem, the fruit fly Drosophila. Fruit flies use E-cadherin in the same way as we do. For example, if fruit fly embryos lack E-cadherin they die early in development because epithelial cells cannot maintain contacts to each other and tissues fall apart. Our recent findings demonstrate that there are two different populations of E-cadherin in epithelial cells in Drosophila embryo. One population is distributed uniformly around cell periphery. Another population is distributed asymmetrically. This second population of E-cadherin is specifically associated with protein called Bazooka/Par-3, and its asymmetry requires a subtype of cytoskeleton: long tubular structures called microtubules.
These findings raise several questions that are the focus of this proposal. First, do different E-cadherin populations have different functions? If they do then it may be possible to interfere with one without affecting the others, which could help control aberrant E-cadherin functions. Second, can we identify other proteins that work with the different E-cadherin populations to help us to understand what they do. We anticipate that proteins that are present in just one or other population may be used to regulate the levels, distribution or action of a particular E-cad population, and thus be targets for drug discovery, and in addition may prove to be mark out aberrant cells for diagnostic purposes. Third, we wish to discover how microtubules control E-cadherin asymmetry. Knowing this mechanism will allow us to manipulate E-cadherin asymmetry in the cells and specifically control this population.
We anticipate that we will discover basic mechanisms that are shared between all animals. In future, we will be able to apply this knowledge to treatment of medical conditions arising from defects in E-cadherin function such as epithelia-derived tumours. For example, if we find that only one population of E-cadherin prevents excessive cancer growth, and we identify the molecules that specifically bind this population of E-cadherin, it will be possible to search for drugs that attack this population of E-cadherin to reduce cancer growth, without disrupting E-cadherin adhesion in the surrounding non-tumour cells.
For proper cell-cell adhesion it is vital to position the zonula adherens at a particular distance from the surface of the cell that faces the cavity. More unexpectedly, the cell also carefully controls how E-cadherin is distributed around the circumference of the cell, within the zonula adherens. While most cells have an even distribution, an increasing number of cases have been discovered where E-cadherin is distributed asymmetrically. The function of such asymmetric distribution in the developing animal has yet to be tested. We have chosen a simple animal to study this problem, the fruit fly Drosophila. Fruit flies use E-cadherin in the same way as we do. For example, if fruit fly embryos lack E-cadherin they die early in development because epithelial cells cannot maintain contacts to each other and tissues fall apart. Our recent findings demonstrate that there are two different populations of E-cadherin in epithelial cells in Drosophila embryo. One population is distributed uniformly around cell periphery. Another population is distributed asymmetrically. This second population of E-cadherin is specifically associated with protein called Bazooka/Par-3, and its asymmetry requires a subtype of cytoskeleton: long tubular structures called microtubules.
These findings raise several questions that are the focus of this proposal. First, do different E-cadherin populations have different functions? If they do then it may be possible to interfere with one without affecting the others, which could help control aberrant E-cadherin functions. Second, can we identify other proteins that work with the different E-cadherin populations to help us to understand what they do. We anticipate that proteins that are present in just one or other population may be used to regulate the levels, distribution or action of a particular E-cad population, and thus be targets for drug discovery, and in addition may prove to be mark out aberrant cells for diagnostic purposes. Third, we wish to discover how microtubules control E-cadherin asymmetry. Knowing this mechanism will allow us to manipulate E-cadherin asymmetry in the cells and specifically control this population.
We anticipate that we will discover basic mechanisms that are shared between all animals. In future, we will be able to apply this knowledge to treatment of medical conditions arising from defects in E-cadherin function such as epithelia-derived tumours. For example, if we find that only one population of E-cadherin prevents excessive cancer growth, and we identify the molecules that specifically bind this population of E-cadherin, it will be possible to search for drugs that attack this population of E-cadherin to reduce cancer growth, without disrupting E-cadherin adhesion in the surrounding non-tumour cells.
Technical Summary
Cell adhesion is vital for attachment between adjacent cells and cell-cell communication. E-cadherin is one of the major transmembrane proteins to provide cell-cell adhesion in epithelial cells. Positioning of E-cadherin along the cell's apical-basal axis is important for adhesion, and it has recently become clear that the distribution of E-cadherin around the periphery of the cell, within the zonula adherens, is also tightly controlled. It is an active process to distribute E-cadherin evenly around the periphery, and there are increasing numbers of cases where an asymmetric distribution is generated in the plane of the cell layer. This proposal describes research aimed at discovering the mechanisms that generate planar asymmetry in E-cadherin and its function in development and disease.
This proposal follows on from our discovery that dynamic polarized microtubules generate the asymmetrical distribution of a newly defined sub-population of E-cadherin at cell-cell junctions in epidermal cells of Drosophila embryos. This E-cadherin pool is more dynamic (mobile) in comparison to the rest of E-cadherin (as defined by its rate of exchange), and is uniquely associated with the scaffolding protein Bazooka/Par-3 (Baz).
The goals of this research are to identify the function of this asymmetrically distributed mobile E-cadherin and how microtubules ensure its junctional asymmetry. By manipulating Baz, we can downregulate mobile E-cadherin to test its function in diverse E-cadherin-dependent processes. We will identify other proteins that are part of the complex of mobile E-cadherin and Baz. We will focus on interacting proteins with enzymatic activity, e.g. kinases, and will elucidate how they contribute to mobile E-cad function. In parallel, we will study how microtubules ensure the asymmetry of Baz-bound E-cadherin. We will study how microtubules become polarized, and which molecular mechanism links them with Baz-bound E-cadherin to produce E-cadherin asymmetry.
This proposal follows on from our discovery that dynamic polarized microtubules generate the asymmetrical distribution of a newly defined sub-population of E-cadherin at cell-cell junctions in epidermal cells of Drosophila embryos. This E-cadherin pool is more dynamic (mobile) in comparison to the rest of E-cadherin (as defined by its rate of exchange), and is uniquely associated with the scaffolding protein Bazooka/Par-3 (Baz).
The goals of this research are to identify the function of this asymmetrically distributed mobile E-cadherin and how microtubules ensure its junctional asymmetry. By manipulating Baz, we can downregulate mobile E-cadherin to test its function in diverse E-cadherin-dependent processes. We will identify other proteins that are part of the complex of mobile E-cadherin and Baz. We will focus on interacting proteins with enzymatic activity, e.g. kinases, and will elucidate how they contribute to mobile E-cad function. In parallel, we will study how microtubules ensure the asymmetry of Baz-bound E-cadherin. We will study how microtubules become polarized, and which molecular mechanism links them with Baz-bound E-cadherin to produce E-cadherin asymmetry.
Planned Impact
Beneficiaries:
-Future patients suffering from cancer, especially carcinomas
-Pharmaceutical and Biotech industries
-Medical diagnosis of cancers
-Those recruiting scientifically trained staff, including, business, industrial and public sector
-The general public, who we hope will be inspired by our images, movies and discoveries
Potential for cancer treatment: As E-cadherin is expressed in epithelia our studies are most relevant to carcinomas, which derive from epithelial cells. Carcinomas include breast, prostate, lung and colorectal cancers: the four most common cancers in UK (54% of all cancers; 165,876 diagnoses in 2008 and 73,803 deaths). Loss of or faulty E-cadherin is closely correlated with carcinoma progression, e.g. in breast cancer 90% of invasive lobular carcinomas had lost E-cadherin, and reciprocally, some aggressive breast cancers have elevated E-cadherin. This research could benefit patients in two ways:
1) by providing biomarkers that specifically label different E-cadherin populations with specific functions, thus permitting earlier diagnoses, e.g. if a specific E-cadherin complex inhibits cell proliferation, levels of proteins in this complex will reveal loss of anti-proliferative activity, before changes in overall E-cadherin levels can be detected. Early diagnosis is crucial for patient survival and well being, as it allows early treatment. Timescale 5 years.
2) by providing drug targets through the discovery of proteins specific to individual populations of E-cadherin. These proteins could be used to regulate the levels, distribution or function of a particular E-cadherin population. For example, if a population of E-cadherin promotes collective cell migration, inhibiting the proteins associated with it may inhibit breast cancer metastasis without destroying E-cadherin adhesion. Timescale for identification of targets and initial screening: 5 years.
As carcinomas are predominantly diagnosed in older adults with, for example, 81% of breast cancers occurring in women aged 50 years or over, better diagnostics and treatment could contribute to healthy aging of affected individuals and allow sufferers to live longer and remain active longer contributing to society as a whole. Overall, the quality of life of the sufferers and their families could increase contributing to nation's health. Timescale 5-10 years.
Biotech and Pharmaceutical Industries licensed to use and develop the above biomarkers and drug targets will benefit from their successful identification. Development of such products will be beneficial for the UK economy and attract investment from global business. Timescale 5 years.
Medical services would benefit as consumers of these new products and services increasing efficiency in diagnosis and treatment of E-cadherin-associated diseases. Furthermore, medical services could benefit from better understanding of functions of E-cadherin in adhesion and signalling in healthy and diseased cells, as this would highlight new ways to prevent, diagnose and treat associated diseases. Timescale 5 years.
Staff training: Not only will the two postdoctoral fellows supported by this grant improve their training but they will supervise A-level students doing work experience, and undergraduate projects, contributing to their rigorous training in scientific experimentation, good experimental design, and data analysis. Thus, this grant will contribute towards health of UK science and higher education through developing expertise in these scientific areas and training highly skilled researchers. Timescale 3 years.
Inspiration of the general public: Through our engagement with the public through talks, websites, and general audience publications we seek to communicate the excitement and beauty of scientific research. Examining molecular and cellular behaviour in living developing animals provides beautiful images and movies that effectively capture and communicate the concepts of biomedicine.
-Future patients suffering from cancer, especially carcinomas
-Pharmaceutical and Biotech industries
-Medical diagnosis of cancers
-Those recruiting scientifically trained staff, including, business, industrial and public sector
-The general public, who we hope will be inspired by our images, movies and discoveries
Potential for cancer treatment: As E-cadherin is expressed in epithelia our studies are most relevant to carcinomas, which derive from epithelial cells. Carcinomas include breast, prostate, lung and colorectal cancers: the four most common cancers in UK (54% of all cancers; 165,876 diagnoses in 2008 and 73,803 deaths). Loss of or faulty E-cadherin is closely correlated with carcinoma progression, e.g. in breast cancer 90% of invasive lobular carcinomas had lost E-cadherin, and reciprocally, some aggressive breast cancers have elevated E-cadherin. This research could benefit patients in two ways:
1) by providing biomarkers that specifically label different E-cadherin populations with specific functions, thus permitting earlier diagnoses, e.g. if a specific E-cadherin complex inhibits cell proliferation, levels of proteins in this complex will reveal loss of anti-proliferative activity, before changes in overall E-cadherin levels can be detected. Early diagnosis is crucial for patient survival and well being, as it allows early treatment. Timescale 5 years.
2) by providing drug targets through the discovery of proteins specific to individual populations of E-cadherin. These proteins could be used to regulate the levels, distribution or function of a particular E-cadherin population. For example, if a population of E-cadherin promotes collective cell migration, inhibiting the proteins associated with it may inhibit breast cancer metastasis without destroying E-cadherin adhesion. Timescale for identification of targets and initial screening: 5 years.
As carcinomas are predominantly diagnosed in older adults with, for example, 81% of breast cancers occurring in women aged 50 years or over, better diagnostics and treatment could contribute to healthy aging of affected individuals and allow sufferers to live longer and remain active longer contributing to society as a whole. Overall, the quality of life of the sufferers and their families could increase contributing to nation's health. Timescale 5-10 years.
Biotech and Pharmaceutical Industries licensed to use and develop the above biomarkers and drug targets will benefit from their successful identification. Development of such products will be beneficial for the UK economy and attract investment from global business. Timescale 5 years.
Medical services would benefit as consumers of these new products and services increasing efficiency in diagnosis and treatment of E-cadherin-associated diseases. Furthermore, medical services could benefit from better understanding of functions of E-cadherin in adhesion and signalling in healthy and diseased cells, as this would highlight new ways to prevent, diagnose and treat associated diseases. Timescale 5 years.
Staff training: Not only will the two postdoctoral fellows supported by this grant improve their training but they will supervise A-level students doing work experience, and undergraduate projects, contributing to their rigorous training in scientific experimentation, good experimental design, and data analysis. Thus, this grant will contribute towards health of UK science and higher education through developing expertise in these scientific areas and training highly skilled researchers. Timescale 3 years.
Inspiration of the general public: Through our engagement with the public through talks, websites, and general audience publications we seek to communicate the excitement and beauty of scientific research. Examining molecular and cellular behaviour in living developing animals provides beautiful images and movies that effectively capture and communicate the concepts of biomedicine.
Organisations
People |
ORCID iD |
| Nicholas Brown (Principal Investigator) |
Publications
Bulgakova NA
(2017)
Diverse integrin adhesion stoichiometries caused by varied actomyosin activity.
in Open biology
Bulgakova NA
(2013)
Dynamic microtubules produce an asymmetric E-cadherin-Bazooka complex to maintain segment boundaries.
in The Journal of cell biology
Bulgakova NA
(2012)
Cell adhesion in Drosophila: versatility of cadherin and integrin complexes during development.
in Current opinion in cell biology
Bulgakova NA
(2016)
Drosophila p120-catenin is crucial for endocytosis of the dynamic E-cadherin-Bazooka complex.
in Journal of cell science
Gomez JM
(2016)
Microtubule organization is determined by the shape of epithelial cells.
in Nature communications
Panamarova M
(2016)
The BAF chromatin remodelling complex is an epigenetic regulator of lineage specification in the early mouse embryo.
in Development (Cambridge, England)
| Description | We made substantial progress on the four main objectives of the proposal: 1. What are the processes in multicellular organism development that require asymmetric Bazooka/Par-3-bound E-cadherin We have identified three functions of the dynamic (mobile) fraction of the cell adhesion molecule E-cadherin in Drosophila development: (a) preventing the cells from crossing segment boundaries in the epidermis of Drosophila embryos, and therefore maintaining tissue pattern, (b) regulating proliferation of epithelial cells within the sacs of cells in the larva that give rise to the adult epidermis, (c) also regulating the shape and apical area of these cells. Using our discovery that p120catenin inhibits internalisation of mobile E-cadherin without changing its amount at the cell surface, we demonstrated that it is the mobility of E-cadherin and not its total amount that is required for these functions. 2. What is the difference in molecular composition between the E-cad complexes containing Baz (mobile) and lacking Baz (immobile) We created the tools for protein purification of mobile and immobile E-cadherin sub-complexes. Unfortunately, our attempts to purify sub-complexes using biotinylated variants of Bazooka protein have not been successful due to the low abundance of these complexes. By testing likely candidate molecules, we have discovered that p120catenin protein is a cue for internalization of mobile E-cadherin from plasma membrane, but does not affect the level or distribution of immobile E-cadherin. 3. How polarized microtubules generate asymmetry of Bazooka/Par-3-bound E-cadherin; We have discovered that the cytoskeletal components microtubules regulate the level of mobile E-cadherin sub-complex through the inhibition of Rho signalling. We developed an automated computational approach to quantify junctional levels of E-cadherin at individual cell-cell borders, to ensure reliable quantification of protein levels at the sites of cell-cell adhesion. This led to a discovery that there is a continuous relationship between E-cadherin level and the angle of the cell-cell border (relative to the dorso-ventral axis of the embryo). To help explain this relationship, we developed a computer model that revealed that if the density of microtubules was fixed then changing the angle was sufficient to increase the number of microtubules contacting the cell border, and elevating E-cadherin. 4. How microtubules get polarized in epithelial cells. We have developed an automated method to measure the degree of alignment of the microtubules within a cell from high resolution micrographs. Using this tool, we found a linear relationship between the rate of microtubule alignment and the elongation of the apical surface of the epithelial cells during development, suggesting a causal relationship. In order to elucidate if cell elongation controls microtubule alignment or vice versa we used genetic manipulations to disrupt either microtubules or interfere with cell elongation. This demonstrated that cell elongation is the main driver of microtubule alignment. To explore how cell shape drives microtubule alignment, we developed a computational model of dynamic microtubules within a cell. This model recapitulated the experimental data and predicted that a set of simple rules on microtubule dynamics (how they grow and shrink) and interaction between microtubules and cell boundaries (what happens to a microtubule when it reaches the cell boundary) are sufficient to explain the observed results. We then tested the model predictions and by examining microtubule behaviour when it reaches cell boundaries, and found that it is as predicted by the model. Finally, we examined several other epithelial tissue in flies and discovered that microtubule alignment in these tissue also display linear correlation with cell shape, suggesting that this is a general rule in epithelia. |
| Exploitation Route | 1. We have identified three functions of mobile E-cadherin in two distinct epithelia: cell mobility, cell shape and proliferation. Additionally, we have found several approaches to manipulate the level, distribution and dynamics of mobile E-cadherin in cells. In future these findings can be taken further to understand the cellular and molecular mechanisms of how E-cadherin functions in these processes, and the relevance of E-cadherin role in these processes for medical conditions, for example cancer progression or wound healing, which rely on cell mobility and proliferation. 2. We have identified p120catenin as critical component required for mobile E-cadherin internalisation. This finding can be taking further to understand how E-cadherin is internalised from the cell surface. In vertebrate cell p120catenin both promotes and inhibits E-cadherin internalisation, making it difficult to study only the former function without complications of the latter. Therefore, our finding that in fly cells p120catenin has only internalisation promoting function, which is likely to be ancestral, provides a new tool for studying p120catenin role in E-cadherin internalisation. 3. We have identified a pathway, in which cell shape regulates microtubule organization, which in turn regulates Rho signalling distribution and, consequently E-cadherin distribution. The discovery of this pathway can be taken forward to test its relevance in other Drosophila and vertebrate tissue. 4. We created a testable computer model of how microtubules become aligned within epithelial cells. This model can be further developed to model how microtubules modify signalling through protein sequestration by the plus ends. The model can then be used to predict outcomes following experimental manipulations and severity of clinical conditions. 5.We have developed a method to quantify the degree of alignment of microtubules in a cell-by-cell basis and found that cell shape is the main cue to align microtubules in epidermal cells. Furthermore, we found that correlation between cell shape and microtubule alignment is also observed across fly epithelia. Across different tissues and cell types, the alignment of microtubules is essential for proper cell function. Hence, the next step would involve validation of this dependency in other cell types that require aligned microtubules for proper cell function, as neurons, ciliated cells and vertebrate epithelial tissues. In addition, quantification of the degree of microtubule alignment could be used as a signature of normal cell development or conversely, to stage and identify different pathological conditions. Examples of this would include (but are not restricted to): normal and degenerative axonal microtubules alignment or cancer cell development (metastatic vs. non-metastatic). 6. We have developed a computational approach to quantify levels and distribution of cell-cell adhesion proteins from micrographs. The approach was published and the Matlab script is available upon request. This approach can be applied in research to study other adhesion proteins in various conditions and tissue, and in clinics, for example to detect a minimum initial changes in E-cadherin levels in cells that are likely to become cancerous. |
| Sectors | Education,Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | Brown Lab Twitter |
| Form Of Engagement Activity | Engagement focused website, blog or social media channel |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Other audiences |
| Results and Impact | We set up the BrownLab Twitter account to disseminate our own research and exciting developments related to our research. We currently have sent 228 tweets and have 489 followers |
| Year(s) Of Engagement Activity | 2015,2016,2017 |
| URL | https://twitter.com/nickbrownlab |
| Description | BrownLab Twitter |
| Form Of Engagement Activity | Engagement focused website, blog or social media channel |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Public/other audiences |
| Results and Impact | We set up a lab twitter account to promote our publications and network with other researchers. We currently have 270 followers. |
| Year(s) Of Engagement Activity | 2015,2016 |
| URL | https://twitter.com/nickbrownlab |
| Description | Invited Seminar (University of Toronto) |
| 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 | Talk sparked questions and promoted a current collaboration Useful discussion with colleagues at the University of Toronto |
| Year(s) Of Engagement Activity | 2014 |
| Description | Science Uncovered at the Natural History Museum |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Public/other audiences |
| Results and Impact | The purpose was to highlight the research at the Gurdon Institute to the general public as part of the Science Uncovered event organised at the Natural History Museum on 25th September, 2015. The audience was particularly excited by the Drosophila part of the presentation, and asked many questions about this model organism. We were also able to encourage A-level students towards a scientific career. |
| Year(s) Of Engagement Activity | 2015 |
| URL | http://www.nhm.ac.uk/visit/exhibitions/science-uncovered-2015.html |
| Description | Seminar Yale |
| 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 | Good discussion with colleagues following seminar, sparked potential future collaborations Involved in organising future research meetings in my field |
| Year(s) Of Engagement Activity | 2014 |
| Description | Talk at PDN Postdoc symposium |
| 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 | Increased knowledge of colleagues in our department about our research Have shared expertise on matlab programming and FRAP analysis |
| Year(s) Of Engagement Activity | 2014 |
| Description | Talk to Core Staff, Gurdon Institute |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Public/other audiences |
| Results and Impact | These talks are aimed at the administrative and technical staff at our Institute, to inform them of the research activities in our labs that they so admirably support. They were excited to discover more about our research programme. Improved interactions between my research group and the core staff |
| Year(s) Of Engagement Activity | 2014 |