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。肯特大学的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。代谢活动的划分是一个重要的工具，通过它可以为特定的代谢功能创建定义的微环境。这给缺乏膜结合细胞器的细菌带来了挑战，然而一些细菌通过制造专门的蛋白质代谢室（称为细菌微室（BMCs）或代谢体）来克服这一问题。Warren和Mulvihill实验室最近报道（J. Biol, Chem. 283: 14366-75; Mol. Cell 38: 305-15），利用合成生物学技术不仅可以在大肠杆菌细胞内产生空BMC的外壳，而且可以将感兴趣的蛋白质靶向到空BMC上，从而在细胞内提供受控的微环境，以优化重组蛋白的稳定性。尽管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内环境和随后稳定靶蛋白方面的全部潜力尚未得到探索。最近，在Mulvihill实验室使用裂变酵母的同时，发现了一种新的机制，其中V类肌球蛋白调节S. pombe CLIP-170同源物的蛋白质水解的空间协调（j .细胞科学，122:3862-72）。肌凝蛋白与泛素受体协同作用，增强细胞尖端生长微管正端的Clip170去除，并将其作为降解目标，从而调节微管动力学。然而，Clip170的降解位点、稳定性和高周转率仍未得到解决，其他细胞骨架调节因子的周转率也未得到解决。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）来确定目标分子的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)合成细菌微室允许靶向分子进入其内部的保护；（ii）真核细胞骨架动力学调节因子周转的空间差异，这为控制细胞极性和生长提供了一种手段。这些研究问题与凯恩研究公司目前的开发项目完全吻合，凯恩研究公司是生物应用LED照明技术的领先开发商，他们最近开发了一种专有的FLIM系统，用于与世界领先的LED光源结合使用。这些最先进的led提供了一个稳定的光源，其中的强度和强度调制可以精确地控制。直接从相机“门控”光源的设备，从而仅在极短（毫秒）的控制周期内暴露样品，这对于FLIM分析至关重要，并使其成为该应用的最佳光源。与传统的激光系统相比，这将有可能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接近为凯恩大学和英国大学的研究人员提供了开发和优化该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.