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

Reference	BB/T017546/1
Principal Investigator / Supervisor	Professor Albena Dinkova-Kostova
Co-Investigators / Co-Supervisors	Dr Paul Appleton, Professor Kevin Hiom, Dr Jens Januschke, Professor Michael MacDonald, Dr Adrian Saurin, Professor Jason Swedlow, Professor Tomoyuki Tanaka, Professor Kees Weijer
Institution	University of Dundee
Department	Cellular Medicine
Funding type	Research
Value (£)	595,000
Status	Completed
Type	Research Grant
Start date	01/11/2020
End date	31/03/2021
Duration	5 months

Abstract

This application is for a state-of-the-art Leica SP8X Falcon FLCS confocal microscope with capabilities for imaging of protein - protein interactions, protein turnover and dynamics by Förster Resonance Energy Transfer (FRET) using fluorescence lifetime imaging microscopy (FLIM) in single live cells. The applicants are Principal Investigators from the Schools of Life Sciences, Medicine and Science and Engineering of the University of Dundee. Their research projects illustrate the potential of this unique fully integrated FLIM-FRET system to empower research across multiple Schools. In the Dinkova-Kostova lab, a new FLIM-FRET methodology will be developed for visualization and quantification of changes in protein turnover in live cells at cellular and subcellular resolution, providing insights into the regulation of proteins of fundamental biological importance: the anaphase promoting complex/cyclosome (APC/C) co-activator Cdc20, which is rapidly synthesized and degraded during mitosis as part of the spindle assembly checkpoint response; UNC-13, a protein involved in synaptic transmission, and Nrf2, the master regulator of the cellular redox homeostasis. In the Tanaka lab, FLIM-FRET will inform how chromosomes are dynamically regulated during mitosis. The Saurin lab will use FLIM-FRET to develop single cell assays to quantify spindle assembly checkpoint complexes; the Hiom lab, to characterize the assembly of multimeric protein complexes in the DNA damage response; the Weijer lab, to quantify the in-vivo dynamics of cAMP signaling; the Januschke lab, to investigate the establishment of cell polarity in neural stem cells of Drosophila. The MacDonald and the Krstajic groups will use the SP8X Falcon system to further the laser and sensing technologies into life sciences research and industry. The OME team led by Jason Swedlow will provide support for storage and management of all data produced in this project using the Dundee Life Sciences OMERO installation.

Summary

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.

Impact Summary

Numerous internal and external academic researchers will benefit from the establishment of the SP8X Falcon FLCS system through access to this state-of-the-art microscope and its advanced imaging capabilities. The researchers will be able to do experiments that are uniquely possible on this system, gaining new knowledge into the function and the regulatory mechanisms underlying a wide array of fundamental biological processes, and moving forward their research in new directions. Young investigators, such as PhD students and postdoctoral fellows will have unique opportunity to receive training in advanced live imaging and data processing and apply this expertise to their ongoing and future projects. The wider biology, biomedical and biotechnology research community will also benefit from the novel findings in key areas of fundamental life science research. Thus, increased understanding of the control of asymmetric divisions of stem cells and cell fate specifications will be critical for the development of the rational use of stem cells in regenerative medicine. Novel insights in cell migration will be critical to understanding diseases such as immune system diseases and cancer. Understanding the regulation of cellular adaptation to stress will be essential for combating chronic diseases, all of which have oxidative stress and inflammation as key factors underlying disease pathogenesis. The findings made using these imaging techniques in live cells and model organisms will contribute to the identification and validation of novel biomarkers and drug targets, which are particularly relevant to the biotechnology and pharmaceutical industries. The project will bring together biologists, physicists, engineers, and data analysts, driving further interdisciplinary collaborations both in the academia and with industry. These interactions will be mutually beneficial and synergistically impactful.

Committee	Not funded via Committee
Research Topics	Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	Advanced Life Sciences Research Technology Initiative (ALERT) [2013-2014]
Funding Scheme	X – not Funded via a specific Funding Scheme

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