Spatial and temporal regulation of cell adhesion and intracellular trafficking by Armus

Epithelial cells form an important component of many different tissues and organs in the body, where they wrap up any cavity and external surfaces as separate compartments, protect against water loss, pathogen infection and facilitate the exchange of fluids, air and nutrients. To do so, epithelial cells must be tightly attached to each other. Many studies investigate how these cells glue strongly among themselves and how their attachment can be manipulated when cells divide, during wound healing migration, invasion by pathogens and other chronic diseases. Disruption of cell-cell contacts is thus a key feature of epithelial pathologies and must be tightly regulated to maintain healthy tissues and organs.

A key event to keep epithelial sheets intact is the amount of adhesive receptors at cell-cell contacts. By impairing the levels and localization of adhesion receptors at junctions, epithelial integrity is compromised and easier to disrupt. Our proposal will dissect mechanisms leading to removal and the destruction of a cell-cell adhesion molecule named E-cadherin following stimulation with the growth factor EGF. Although EGF is important for epithelial development and maintenance, aberrant activation of its receptor in cells is observed in different pathologies, including cancer. Yet, the precise mechanisms via which EGF over-stimulation leads to E-cadherin degradation are not well understood.

Armus is a protein identified in our lab that controls the degradation of E-cadherin in a cell compartment called lysosome, where degradation of unwanted material is processed. Here we aim to dissect how Armus is regulated when cells are stimulated to move away from their peers by EGF treatment, what controls its localization at junctions or lysosomes, and the binding proteins that are important for the precise activation of Armus in space and time. Our studies will provide insights into fundamental biological questions as to what controls the distribution and function of the same protein into different compartments inside the cell at steady-state and upon stimulation.

By investigating the mechanisms of Armus regulation, we will identify potential ways to block Armus function to impair degradation of E-cadherin. Importantly, such knowledge will be beneficial in therapeutic strategies beyond our model system: (i) specific inhibitors of lysosomal function are not available and (ii) there are a number of pathologies where degradation of intracellular material is mal-functioning, such as in neurodegenerative diseases, killing internalised bacteria, etc.

This proposal aims to dissect mechanisms via which cell-cell-adhesion receptors are specifically routed for degradation. Our model studies the regulation of surface receptor turnover by EGFR signalling leading to degradation of E-cadherin receptors in lysosomes, cell detachment from epithelial colonies and scattering. Using this model, a fundamental question will be addressed on how a single protein is differentially regulated at distinct intracellular compartments. We focus on Armus, a regulator of the small GTPase Rab7 activity and lysosomal fusion. Armus localises at junctions and at vesicular compartments and is required for efficient E-cadherin down-regulation downstream of EGF stimulation. We identified novel partners that are likely to localise Armus at cell-cell contacts (alpha-catenin and p120CTN) or at distinct intracellular compartments (Rabs at endosomes or lysosomes). Following EGFR stimulation and at steady state, we address whether these partners are necessary to regulate (a) Armus spatial distribution, (b) Armus activation status at different intracellular compartments and (c) Rab cascades controlling E-cadherin route from junctions to lysosomes. Using mass spectometry and RNAi screen, we will compile a network of regulators as novel Rab proteins that participate in cadherin degradation. Molecular mechanisms of Armus activation are dissected using different approaches: binding to the small GTPase Rac1, potential phosphorylation by EGFR signalling and interaction with selected Rab/catenin partners. We will map phosphorylation and partners binding sites with motifs necessary for Armus conformation switch leading to activation and put into context of polymorphism hot-spots and truncation mutants available. We will use state-of-art microscopy to perform dynamic studies in combination with functional, biochemical and GTPase activity assays in vivo and in vitro.

Who will benefit from the research?
Our work will benefit researchers with a broad range of interests on intracellular trafficking and specificity of signalling, tumour cell biology and pathologies with impaired lysosomal function, such as neuropathies and retinal degeneration, destruction of intracellular pathogens or bone remodelling. From the clinical point of view, the core signalling and cellular events investigated here are likely to be applicable to a number of diseases and types of tumours. Future translational aspects of our research will be particularly beneficial to companies driving drug screening and compound validation, as there are currently no specific inhibitors of lysosomal fusion. Patients will also benefit from potential drug targets that may originate from our research. Systematic analysis of the pathways altered in distinct diseases will highlight the subset of patients amenable to therapeutic intervention of Armus or lysosomal function.

How they will benefit from the research?
The fundamental and biomedical aspects of the research will support knowledge and technology translation, lead to applications for commercial exploitation and increase economic competitiveness in the UK. The novel screening methods and biosensors developed herein will have considerable impact for industry interested in small GTPase regulation, target discovery and validation.

From the translational point of view, failure of specific compounds in clinical trial is substantially increased by lack of knowledge of the cellular functions of the drug target. This proposal will address precisely this key issue, which will be beneficial to drug discovery companies and academic groups. We will provide considerable functional insights on the identified targets, from regulatory circuitry to specificity of localization and activation during E-cadherin degradation. Understanding the coordination of these signalling events is essential for (i) prediction of off-target effects and (ii) evaluation of the impact of target inhibition in additional cellular events.

From the societal point of view, this proposal has the potential to improve the nation health. Our work will generate in-depth understanding of different diseases in which the functions of cadherins or lysosomes are compromised. For example, different pathogens use adhesion receptors to invade cells and subvert targeting to lysosomes for degradation to ensure their survival in the intracellular milieu. Among pathogens that rely on E-cadherin are Listeria monocytogenes, a serious infection for infants and immuno-compromised individuals, and Helicobacter pylori a risk-factor in development of ulcers and gastric cancers. Another aspect is to understand how adhesive systems between epithelial cells become defective and compromise the impermeability of epithelial sheets. A "leaky" epithelium underlies the causes of many chronic diseases such as asthma, atopic dermatitis, food intolerance/allergy, and chronic infections. 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 inappropriately. Finally, lysosomes play and important role in the dynamic remodelling of bones (formation and reabsorption), which can result in serious diseases such as osteoporosis, osteopetrosis, osteopenia, etc.

Skills Training and Scientific Advancement is an important component of our proposal. In addition to specific technical training (see Pathways to Impact), appointed staff will acquire skills in project and time management, supervision training, leadership and career progression which are essential for the next steps of their careers. Imperial College Staff Development Unit provides excellent courses and support for Research staff with respect to career progression and personal development. A well-developed mentoring scheme for post-docs is also available.