Live imaging of 3D multicellular tumour models to elucidate the dynamics of the cell adhesion complex during cell migration and invasion

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Cell migration is essential during embryonic development, immune response, inflammation, and is also involved in metastasis, a process where cancer cells colonise distant organs. The aim of the project is to elucidate the cell invasion and migration mechanisms in physiological conditions, where cells are appropriately interacting with their matrix environment. The main molecular driver for cell migration is the focal adhesion protein complex, which coordinates interactions between the cell and extracellular matrix. Our current knowledge of this complex is primarily based on 2D cell cultures on rigid substrates which are poorly representative of adhesion in a physiological context. Migration in tissues has different control mechanisms, requiring studies of adhesion proteins in 3D systems in the presence of biologically relevant matrices. A better understanding of the adhesion regulation in space and time is a prerequisite to the future development of therapies that promote or prevent migration. The development of new 3D cell culture models and novel imaging technologies enable us to now address this challenge. To achieve this aim, we have assembled an interdisciplinary team with a structural biologist in the NMR Centre for Structural Biology (I. Barsukov), a cell biologist (V. See) and a physicist (R. Levy) from the Liverpool Centre for Cell Imaging and a cell biologist at the University of Sheffield (S. Winder). Specifically, the project involves the use of key proteins involved in cell adhesion/migration as fluorescent fusion proteins that will then be studied with advanced imaging technologies. Small GTPases of the Rho family are part of the focal adhesion complex and regulate cell migration. The Rho-GAP DLC1 is essential for the optimal cell migration at all stages of organism development. The role of DLC1 is associated with its direct connection to the adhesion complexes through the interaction with the structural adhesion proteins talin and tensin. Furthermore the extracellular matrix receptor dystroglycan not only links the actin cytoskeleton to the extracellular matrix, but also recruits the Rho-GEF Dbl to the plasma membrane where it can further modulate actin dynamics in migrating and invading cells. To evaluate the importance of specific interactions, the effects of DLC-1 and dystroglycan, mutations (design guided by structural biology studies ongoing at the University of Liverpool NMR Centre) will be investigated. Imaging will include quantitative measurement of protein-protein interaction using fluorescence life time (FLIM) imaging as well as time-lapse measurement of cell invasion in 3D spheroids. The work will be performed in the Centre for Cell imaging in Liverpool, a world-class imaging facility for live cells and one of the few facilities in the UK to possess a lightsheet fluorescent microscope and a fluorescence life time imaging system. The student will spend 6 months in Sheffield in the Department of Biomedical Science screening facility, using high throughput high content analysis of 3D spheroids to investigate the effects of RNAi knockdown of relevant proteins.