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

荧光显微镜技术的发展和使用使我们对细胞内基本过程的理解取得了重大突破。这包括根据分子的细胞位置观察分子行为差异的能力。促进这一点的一个突破是荧光寿命荧光成像显微镜（FLIM）的发展，其中可以确定细胞内任何荧光团标记分子的特定寿命测定。肯特大学Mulvihill和Warren实验室的研究强调了细菌和真菌细胞内空间调节蛋白质稳定性机制的重要性。代谢活动的区室化是一种重要的工具，通过它可以为特定的代谢功能创建定义的微环境。这为缺乏膜结合细胞器的细菌提供了挑战，然而一些细菌通过制造称为细菌微区室（BMC）或代谢体的专门的蛋白质代谢区室来克服这一挑战。Warren和Mulvihill实验室最近报道了（J. Biol. Chem. 283：14366-75; Mol. Cell 38：305-15），使用合成生物学技术不仅可以在大肠杆菌中产生空BMC的壳。大肠杆菌细胞，但感兴趣的蛋白质可以靶向空BMC，从而在细胞内提供受控的微环境以优化重组蛋白质的稳定性。虽然这一发现可能对生物制药和生物技术应用产生重大影响，但其在控制BMC内环境和随后稳定其中靶蛋白方面的全部潜力尚未被探索。在Mulvihill实验室使用裂殖酵母的巧合工作最近揭示了一种新的机制，其中V类肌球蛋白调节S.粟酒裂殖酵母CLIP-170同源物（J.Cell Sci. 122：3862-72.）。肌球蛋白与泛素受体协同工作，以增强Clip 170从细胞尖端生长的微管的正端去除，并靶向其降解，从而调节微管动力学。然而，Clip 170的降解位点、稳定性和高转化率仍然没有得到解决，其他细胞骨架调节因子的转化率也没有得到解决。该项目旨在开发一种成像系统，以促进荧光寿命荧光成像显微镜（FLIM），以确定靶分子荧光指数衰减率的差异，这取决于它们在细菌和裂变酵母系统中的细胞位置，并将允许博士生确定（i）保护合成细菌微区室允许分子被靶向其内部;和（ii）真核生物细胞骨架动力学调节因子周转的空间差异，其提供了控制细胞极性和生长的手段。这些研究问题与Cairn Research目前的开发项目完全一致，Cairn Research是生物应用LED照明技术的领先开发商，最近开发了一种专有的FLIM系统，可与世界领先的LED光源结合使用。这些最先进的LED提供了一个稳定的光源，其中强度和强度调制可以精确控制。直接从相机“选通”光源的设施，因此仅在极短（毫秒）的受控时间内曝光样品，这对于FLIM分析至关重要，并使其成为该应用的最佳光源。这将有可能比传统的基于激光的系统提供显著的成本节约。因此，这种研究兴趣的协同作用和地理位置的紧密联系为Cairn和UKC的研究人员提供了开发和优化FLIM系统的绝佳机会。

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.