A fully integrated FLIM-FRET system for imaging dynamic protein - protein interactions and protein turnover in single live cells and model organisms

细胞中的大多数生物过程都是由蛋白质完成的。所有蛋白质都有严格的周转调节，即蛋白质合成和蛋白质降解之间的平衡。此外，大多数蛋白质参与特定的蛋白质-蛋白质相互作用。蛋白质周转和蛋白质相互作用的能力在疾病中经常改变，包括癌症、心血管疾病和神经退行性疾病，以及在衰老过程中。因此，了解蛋白质周转和蛋白质相互作用的分子机制对于详细了解生理和病理中的生物过程以及开发新的治疗方法至关重要。为了可视化活细胞中蛋白质-蛋白质的相互作用，有必要以非常高的(亚微米)分辨率测量蛋白质的接近程度。使用常规显微镜是不可能的，因为光学显微镜的分辨率是有限的。Förster共振能量转移(FRET)技术可以克服可见光分辨率造成的这种限制。FRET是激发态能量从一个分子(称为给体)向附近的另一个分子(称为受体)的转移，可以通过荧光寿命成像(FLIM)来测量。该项目计划购买和使用一种独特的系统--徕卡SP8X猎鹰FLCS显微镜，这是唯一可以进行此类测量的商用全集成系统。它将能够测量活细胞动态过程、蛋白质周转和蛋白质-蛋白质相互作用，这些都是邓迪大学现有设备无法实现的。新系统的实际位置将设在邓迪成像设施，这将为用户培训以及图像处理和分析所需的通用成像和计算基础设施作出重大贡献。该系统对用户非常友好，使这项技术可以向广泛的研究人员开放。与英国BioImagingUK和欧洲生物成像公司建立的联系将提供机制，支持社区在当地、国家和国际上获取这一最先进的成像资源。申请者是来自邓迪大学生命科学学院、医学院和科学与工程学院的一组首席研究人员。所有申请者都对使用活细胞成像技术解决生命科学中的关键生物学问题和提供解决方案有着浓厚的兴趣和过往记录。邓迪大学由100多个研究小组组成的多个学院的许多研究人员的研究将随着这一多用户多项目使用显微镜的获得而大大加强。这些发现将带来关于一系列基本生物学过程的新知识，包括细胞极性、细胞分裂、胚胎发育、基因组完整性、神经元通讯和应激反应，这些过程在衰老和疾病中往往是失调的。

The majority of biological processes in the cell are performed by proteins. All proteins have strictly regulated turnover, the balance between protein synthesis and protein degradation. In addition, most proteins engage in specific protein - protein interactions. Both protein turnover and the ability of proteins to interact with one another are frequently altered in disease, including cancer, cardiovascular, and neurodegenerative diseases, as well as during ageing. Thus, knowledge of the molecular mechanisms underlying protein turnover and protein - protein interactions are crucial for detailed understanding of biological processes in physiology and pathology, and for development of new therapies. To visualize protein - protein interaction in live cells, it is necessary to measure the proximity of proteins with a very high (sub-micrometre) resolution. This is not possible by using conventional microscopy, as the resolution of the light microscope is limited. This limitation imposed by the visible light resolution can be overcome by the Förster resonance energy transfer (FRET) technique. FRET is the transfer of excited-state energy from one molecule (termed the donor) to another nearby molecule (termed the acceptor), and can be measured by Fluorescence Lifetime Imaging (FLIM). This project proposes the acquisition and use of a unique system, the Leica SP8X Falcon FLCS microscope, which is the only commercial fully-integrated system on which such measurements can be made. It would enable measurements on live cell dynamic processes, protein turnover and protein - protein interactions that are not possible on the existing equipment at the University of Dundee. The new system will be physically located at the Dundee Imaging Facility, which will provide substantial contributions to user training and the general imaging and computing infrastructure necessary for image processing and analysis. The system is very user friendly, giving the benefit of opening the technology to a wide range of researchers. Established links with BioImagingUK and Euro-BioImaging will provide mechanisms to support community access to this state-of-the-art imaging resource locally, nationally and internationally.The applicants are a group of Principal Investigators from the School of Life Sciences, the School of Medicine and the School of Science and Engineering of the University of Dundee. All applicants have strong interest and track record in the use of live cell imaging techniques to address critical biological questions and provide solutions in life sciences. The research of many investigators across multiple Schools of the University of Dundee, comprising more than 100 research groups, will be greatly enhanced by the acquisition of this multi-user multi-project use microscope. The findings will bring new knowledge of a wide range of fundamental biological processes, including cell polarity, cell division, embryonic development, genome integrity, neuronal communication and stress responses, which are frequently dysregulated in ageing and disease.