Quantitative in vivo analysis of ligand-receptor interactions in the Wnt signalling network

细胞间的通讯对于调节所有多细胞生物体的发育和内稳态是必不可少的。像Wnt信号一样的分泌信号调节细胞的增殖和分化、细胞的极性和迁移等基本过程。这种信号网络的失调会导致严重的疾病，如阿尔茨海默病等神经退行性疾病。Wnt配体与细胞表面的七通跨膜FrizzledFZD受体结合是激活Wnt信号网络的主要途径。19个不同的Wnt配体可以与10个不同的FZD受体结合，以指定下游信号事件的性质。准确地量化活细胞中的这些相互作用对于了解信号的特异性至关重要，然而，由于技术障碍，这直到今天才成为可能。我们将与徕卡微系统公司合作，在细胞生物学家和生物物理学家的监督下，首次使用关联光谱来定量三种Wnt配体和三种不同的Fzd蛋白在体内的有效结合亲和力-Kd(有效)。学生将应用分子生物学技术，如mRNA过表达和基于CRISPR/Cas9的神经元敲除方法来可视化配体和受体。然后，我们将使用基于成像的定量测量细胞中的配体-受体相互作用，例如神经元和斑马鱼的荧光波动技术。学生将与徕卡的研发部门合作，开发和完善徕卡的单分子检测单元Falcon，以用于活体标本。被选为该项目的学生将在实验遗传学、活体技术、生物物理方法(如光谱和显微镜)方面发展宝贵的技能，同时也对了解WNT信号网络做出重大贡献。这种综合技能组合，包括与行业合作伙伴的互动，将使应聘者成为未来学术界和行业雇主非常理想的招聘前景。生命系统研究所(LSI)在生物科学和物理学方面拥有互补的专业知识，将是进行这些博士培训研究的最佳环境。LSI为博士生提供了独特的培训机会，因为它允许学生在跨学科环境中使用最先进的设备解决生命科学中的关键问题。该项目包括与加的夫大学、伦敦大学(KCL)和布里斯托尔大学密切合作，以补充所需的技能。

Cell-cell communication is essential for the regulation of the development and homeostasis of all multicellular organisms. Secreted signals like Wnt signals regulate fundamental processes including cell proliferation and differentiation, cell polarity and migration. Misregulation of this signalling network causes severe diseases like neurodegenerative diseases like Alzheimer's disease. Binding of a Wnt ligand with a seven-pass transmembrane Frizzled (Fzd) receptor on the surface of cells is the principal way to activate the Wnt signalling network. 19 different Wnt ligands may bind to 10 different Fzd receptors to specify the nature of the downstream signalling event. Accurate quantification of these interactions in living cells is crucial to understand signalling specificity, however, due to technical obstacles, this was not possible until today.In collaboration with Leica Microsystems and under the supervision of cell biologists and biophysicists, we will employ correlation spectroscopy to quantify for the first time the effective binding affinities - Kd (effective) - between three Wnt ligands and three different Fzd proteins in vivo. The student will apply molecular biological techniques such as mRNA overexpression and CRISPR/Cas9- based knock-out methods in neurons to visualize ligands and receptors. Then we will use imaging-based, quantitative measurements of ligand-receptor interaction in cells e.g. neurons and in zebrafish by fluorescence fluctuation techniques. In a collaboration with the R&D unit of Leica, the student will develop and refine Leica's single-molecule detection unit FALCON for its usage in the living specimen.The student selected for this project will develop invaluable skill sets in experimental genetics, in vivo techniques, biophysical methods such as spectroscopy and microscopy, whilst also making a significant contribution to the understanding of the Wnt signalling network. This combined skill set including the interactions with the industrial partner will make the candidate a highly desirable recruitment prospect for future academic and industrialemployers. The Living Systems Institute (LSI), with complementary expertise in biosciences and physics, will be an optimal environment to conduct these doctoral training studies. The LSI offers unique training opportunities for the PhD student as it allows the student to address key problems in life sciences with state-of-the-art equipment in an interdisciplinary environment. The project includes close collaboration with the universities of Cardiff, London (KCL) and Bristol to compliment the required skill sets.