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，也参与了癌细胞在远处器官定植的转移过程。该项目的目的是阐明细胞在生理条件下的入侵和迁移机制，在生理条件下，细胞与其基质环境适当地相互作用。细胞迁移的主要分子驱动力是粘着斑蛋白复合体，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体的了解主要是基于刚性底物上的2D细胞培养，这在生理环境中不能很好地代表粘连。组织中的迁移有不同的控制机制，需要在生物相关基质存在的情况下研究3D系统中的黏附蛋白。更好地了解黏附在空间和时间上的调节是未来促进或防止迁移的治疗方法发展的先决条件。新的3D细胞培养模型和新的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团队，成员包括核磁共振结构生物学中心的一名结构生物学家(I.Barsukov)、利物浦细胞成像中心的一名细胞生物学家(V.See)和一名物理学家(R.Levy)以及谢菲尔德大学(S.Winder)的一名细胞生物学家。具体地说，该项目涉及使用参与细胞黏附/迁移的关键蛋白作为荧光融合蛋白，然后将利用先进的成像技术进行研究。Rho家族的小GTP酶是焦点黏附复合体的一部分，调节细胞迁移。Rho-GAP DLC1对于生物体发育的所有阶段的最佳细胞迁移都是必不可少的。DLC1的作用与其通过与结构黏附蛋白Talin和tensin相互作用而直接连接到黏附复合体有关。此外，细胞外基质受体营养不良不仅可以将肌动蛋白细胞骨架连接到细胞外基质上，还可以将Rho-Global DBL募集到质膜上，在质膜上进一步调节肌动蛋白在迁移和入侵细胞中的动态变化。为了评估特定相互作用的重要性，DLC-1和营养不良葡聚糖的影响，突变(设计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)将被调查。成像将包括使用荧光寿命成像(FLIM)定量测量蛋白质-蛋白质相互作用，以及在3D球体中进行细胞侵袭的延时测量。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，也是英国为数不多的拥有光片荧光显微镜和荧光寿命成像系统的设施之一。这名学生将在谢菲尔德的生物医学科学系筛查设施中呆上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.