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

细胞迁移在胚胎发育、免疫反应、炎症过程中是必不可少的，而且还涉及癌细胞迁移到远处器官的转移过程。该项目的目的是阐明在生理条件下细胞的入侵和迁移机制，其中细胞适当地与它们的基质环境相互作用。细胞迁移的主要分子驱动因子是黏附蛋白复合物，它协调细胞和细胞外基质之间的相互作用。我们目前对这种复合物的了解主要是基于刚性底物上的二维细胞培养，这在生理背景下很难代表粘附性。组织中的迁移具有不同的控制机制，这需要在存在生物学相关基质的3D系统中研究粘附蛋白。在空间和时间上更好地理解粘附调节是未来发展促进或防止迁移的治疗的先决条件。新的3D细胞培养模型和新型成像技术的发展使我们现在能够应对这一挑战。为了实现这一目标，我们组建了一个跨学科的团队，其中包括核磁共振结构生物学中心的结构生物学家（I. Barsukov），利物浦细胞成像中心的细胞生物学家（V. See）和物理学家（R. Levy），以及谢菲尔德大学的细胞生物学家（S. Winder）。具体来说，该项目涉及使用参与细胞粘附/迁移的关键蛋白质作为荧光融合蛋白，然后将使用先进的成像技术进行研究。Rho家族的小gtpase是局灶黏附复合体的一部分，并调节细胞迁移。Rho-GAP dcl1在生物发育的各个阶段对最佳细胞迁移至关重要。dcl1的作用与其通过与结构粘附蛋白talin和tensin的相互作用与粘附复合物的直接联系有关。此外，细胞外基质受体糖代谢障碍不仅可以将肌动蛋白骨架与细胞外基质连接起来，还可以将Rho-GEF Dbl募集到质膜上，在那里它可以进一步调节肌动蛋白迁移和入侵细胞的动力学。为了评估特定相互作用的重要性，将研究DLC-1和糖醛酸异常聚糖的作用，突变（由利物浦大学核磁共振中心正在进行的结构生物学研究指导设计）。成像将包括使用荧光寿命（FLIM）成像的蛋白质-蛋白质相互作用的定量测量，以及三维球体中细胞入侵的延时测量。这项工作将在利物浦的细胞成像中心进行，这是一个世界级的活细胞成像设施，也是英国少数拥有光片荧光显微镜和荧光寿命成像系统的设施之一。该学生将在谢菲尔德生物医学科学系的筛选设施中度过6个月，使用高通量高含量的3D球体分析来研究RNAi敲除相关蛋白质的影响。

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.