Development of FLIM analysis tools for the in vivo study of cellular distribution of regions of protein stability within microbial systems.

The development and use of fluorescence microscopy technologies has allowed significant breakthroughs to be made in our understanding of the fundamental processes within a cell. This includes the ability to observe differences in the behaviour of molecules depending upon their cellular location. One break-through which has facilitated this has been the development of Fluorescence Lifetime Fluorescence Imaging Microscopy (FLIM), where specific lifetime determination of any fluorophore tagged molecule within a cell can be determined. Research within the Mulvihill and Warren labs at the University of Kent have highlighted the importance of mechanisms for spatially regulating protein stability within the bacterial and fungal cell. Compartmentalization of metabolic activities represents an important tool by which defined microenvironments can be created for specific metabolic functions. This provides challenges for bacteria, which lack membrane bound organelles, however some overcome this by making specialized proteinaceous metabolic compartments called bacterial micro-compartments (BMCs) or metabolosomes. The Warren and Mulvihill labs have recently reported (J. Biol, Chem. 283: 14366-75; Mol. Cell 38: 305-15) that using synthetic biology techniques not only could the shell of an empty BMC can be produced within E. coli cells but proteins of interest can be targeted to the empty BMC, thus providing a controlled microenvironment within the cell to optimize the stability of recombinant proteins. Although this finding is likely to have a significant impact for both biopharmaceutical and biotechnology applications, its full potential in terms of controlling the environment within the BMC and subsequent stabilisation of target proteins within them have yet to be explored. Coincident work in the Mulvihill lab using the fission yeast has recently uncovered a novel mechanism in which a class V myosin modulates the spatial coordination of proteolysis of the S. pombe CLIP-170 homologue (J.Cell Sci. 122: 3862-72.). The myosin works in concert with a ubiquitin receptor to enhance Clip170 removal from the plus end of growing microtubules at the cell tips and target it for degradation, and thus regulate microtubule dynamics. However the sites of Clip170 degradation, stability and high turn over remain unresolved, as do the turnovers of other cytoskeletal regulators. This project sets out to develop an imaging system to facilitate Fluorescence Lifetime Fluorescence Imaging Microscopy (FLIM) to determine differences in the exponential decay rate of fluorescence of targeted molecules, depending upon their cellular location in both bacterial and fission yeast systems, and will allow the PhD student to determine (i) the protection synthetic bacterial micro-compartments allow molecules that have been targeted to their interior; and (ii) spatial differences in the turnover of regulators of eukaryote cytoskeleton dynamics, which provides a means to control cell polarity and growth. These research questions coincide exactly with current development projects within Cairn Research, a leading developer of LED illumination technology for biological applications, who have recently developed a proprietary FLIM system to be used in conjunction with their world leading LED light sources. These state-of-the art LEDs provide a stable light source in which intensity and intensity modulation can be exquisitely controlled. The facility to 'gate' the light source directly from the camera and thus only expose the specimen for extremely short (msec) controlled periods is crucial for FLIM analyses and make it an optimum light source for this application. This will have the potential to provide significant cost savings over conventional laser based systems. Therefore this synergy of research interests and close proximity in geographical locations provide an excellent opportunity for both researchers at Cairn and UKC for developing and optimising this FLIM system.