Development of live cell imaging from single cells to single molecules

A major challenge in biology is to understand how cells recognize external signals and give appropriate responses. Now that the sequence of the human genome is complete it is important to assign functions to each gene and to identify the corresponding proteins that control key cellular functions. We pioneered the development of microscopy-based methods for the visualization and timelapse measurement of biological processes in single living cells. We have used natural light-emitting proteins from fireflies, jelly fish and fluorescent corals. Synthesis (expression) of these proteins causes mammalian cells to become luminescent (light emitting in the dark) or fluorescent (change the colour of light). By placing the gene that codes for a luminescent protein next to a promoter that controls a gene of interest, we can use luminescence from living cells as a way of measuring when the gene of interest is normally switched on and off. Fluorescent proteins have also been used to genetically label proteins of interest, so that the movement of the protein can be visualized in a living cell. White, See and Spiller have previously used timelapse fluorescence and luminescence microscopy to investigate cell decision making. We discovered that a set of important signalling proteins, called NF-kappaB, move repeatedly into and out of the nucleus of the cell, suggesting that cells may use proteins as timers to encode complex messages (like Morse Code). Only timelapse measurements in single living cells were able to see this. We now propose to develop simultaneous imaging of luminescence from two different colour firefly (green) and click beetle (red) proteins in single cells to track the expression of two different genes at the same time. We will also develop and apply new fluorescent tools for more accurate measurement of protein movement rates and protein stability in single cells. One approach will explore new uses for fluorescent proteins that have been engineered so that they can be optically switched between light-emitting and non-light emitting forms (highlighters). This will include improved measurement of whether and when two proteins of interest bind to each other in cells. Until now, it has been a major challenge to track when such protein interactions occur. Levy, Fernig and Brust have considerable experience in synthesis of very small metal (gold and silver) nanoparticles, which have been coated to make them more stable in water and hence in the interior of cells. We will develop generic methods to precisely couple these nanoparticles to any protein (or sequence of DNA) of interest. We will build a novel photothermal microscope to allow the visualization and tracking of single nanoparticles in single cells. We will target the nanoparticles to particular DNA sequences in the nucleus of living cells to allow the localization of genes of interest within the nucleus. We will also target nanoparticles to bind to the artificially tagged RNA of genes of interest. This will allow the immediate visualization of when RNA is synthesized (transcription) from the gene of interest with no delay. We will use this technology to measure the movement of proteins that bind to DNA, such as NF-kappaB proteins. These approaches will for the first time allow us to study how single proteins regulate transcription at single genes. The NF-kappaB signalling system will be used as a model, since we have a great deal of experience and interest in its regulation and excellent computer models that predict how it may work. This technology will have widespread applications in cell biology and systems biology. The Centre for Cell Imaging is an ideal site for this project, since it is a resource used by a large number of scientists in the North West and is well placed to transfer technology to other labs through training and conferences. We also have close links with pharmaceutical and instrumentation industries who will benefit from these developments.

Non-invasive maging of biological processes in living cells has become an important tool in molecular and cell biology research. There is an increasing need for new multiparameter imaging technologies that can provide more quantitative measurement of a greater range of dynamic biological processes in single cells. The development of such improved technologies can provide a major contribution to systems biology, analysis of cell signalling, transcription and cell fate. The aims of this project are firstly to develop new technology based on our current expertise in luminescence and fluorescence imaging. Secondly, we will develop new bionanotechnology tools for the imaging and tracking of single molecules in cells. Specifically we will develop: 1) Dual imaging of transcription in single living cells; 2) Improved assays for quantitative measurement of protein translocation rates, protein half-life and the kinetics of protein interactions; 3) Single molecule imaging in living cells based on photothermal microscopy of biomimetic nanoparticles and 4) The application of multiparameter imaging to study single gene transcription in single cells. This work will be carried out by a multidisciplinary team from Molecular Cell Biology, Chemistry and Physics backgrounds. The nanotechnology part of the project will involve the building of the world's second photothermal microscope for single metal nanoparticle imaging. This will be the first instrument to be specifically designed for imaging of nanoparticles in living cells. The work is based on our existing expertise in cell imaging (White, See and Spiller) and nanoparticle development (Levy, Brust and Fernig). The work will project will contribute to our ongoing systems biology analysis of the important NF-kappaB system, which will be used as a model for application of the technology. In addition, the new technology will have generic applications in systems and cell biology.