Non-perturbative use of fluorescent proteins for super-resolution microscopy

荧光蛋白在超分辨率显微镜中的非扰动使用

• 批准号: 2276379
• 金额: --
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2276379
• 关键词: Non; perturbative; use; fluorescent; proteins

Fluorescence microscopy allows the dynamic observation of phenomena in living cells. Until recently, however, the wavelike nature of light and its associated diffraction, limited the resolution of optical microscopy to ~200 nm. New 'super-resolution' fluorescence microscopy methods, have been developed to overcome the diffraction limit of 'traditional' microscopy. The enormous impact of super resolution microscopy methods was recognized by its inventors being awarded the Nobel Prize in Chemistry (2014). Super resolution methods can increase the resolution of fluorescence microscopy down to ~5 nm, thus enabling the observation of molecular processes in great detail. Despite these advances in optical methods, labelling the biomolecule of interest remains a challenge, especially within live cells. Direct fusion of the protein of interest to a fluorescent protein is common strategy, because it is genetically encodable, can be used for live cell imaging, and is amenable to super-resolution microscopy (by judicious choice of fluorescent protein). Unfortunately, fluorescent proteins are quite large (>27 kDa) and a direct fusion can perturb the function, assembly, and/or location of the protein of interest.To address these issues, the student will develop a novel super-resolution imaging method based on a labelling strategy that is fully genetically encodable, does not require the use of exogenous substrates, and adds a minimally disruptive tag to the biomolecule of interest. Rather than fusing the fluorescent protein itself to the protein of interest, a short peptide tag will be genetically encoded onto it. This tag can then be linked either covalently or non-covalently to a partner protein that is also expressed, in a controlled fashion, within the cell. Such a method has been demonstrated to work well to label proteins within bacteria and yeast1,2. The student will build on this work, adapting the strategy for use in mammalian cells. Most importantly, they will develop this approach using the photoswitchable fluorescent protein, mEoS, which will enable super-resolution images to be generated using photo-activated localization microscopy3. We are particularly interested in applying this novel strategy to visualize amyloid protein formed within human-derived neurons. Training: the supervisors of this project will combine their expertise in protein structure and design (Regan), neuroscience (Horrocks) and super-resolution microscopy techniques (Horrocks). The training will be achieved primarily through practical application of the research techniques within the supervisors' and collaborators' laboratories. Skills that will be developed include cell and molecular biology techniques, cell culture (including differentiated induced pluripotent stem cells), gene editing, advanced microscopy (single-molecule and super-resolution), data analysis and coding. The student will be encouraged to participate in training workshops, to present in various multi-group meetings at the University, and to participate in public engagement activities. Science is a global endeavour, and the supervisors will ensure that the student has the opportunity to attend and present at international conferences.

荧光显微镜允许动态观察活细胞中的现象。然而，直到最近，光的波动性质及其相关的衍射将光学显微镜的分辨率限制在~200 nm。新的“超分辨率”荧光显微镜方法，已被开发，以克服“传统”显微镜的衍射极限。超分辨率显微镜方法的巨大影响得到了其发明者的认可，他们被授予诺贝尔化学奖（2014年）。超分辨率方法可以将荧光显微镜的分辨率提高到~5 nm，从而能够非常详细地观察分子过程。尽管光学方法取得了这些进展，但标记感兴趣的生物分子仍然是一个挑战，特别是在活细胞内。目标蛋白与荧光蛋白的直接融合是常见的策略，因为它是遗传编码的，可用于活细胞成像，并且适合于超分辨率显微镜（通过明智地选择荧光蛋白）。不幸的是，荧光蛋白非常大（>27 kDa），直接融合可能会干扰目标蛋白的功能，组装和/或位置。为了解决这些问题，学生将开发一种基于标记策略的新型超分辨率成像方法，该方法完全可遗传编码，不需要使用外源底物，并在目标生物分子中添加最小破坏性标签。不是将荧光蛋白本身融合到感兴趣的蛋白质上，而是将一个短的肽标签遗传编码到它上面，然后该标签可以共价或非共价地连接到一个伴侣蛋白上，该伴侣蛋白也以受控的方式在细胞内表达。这种方法已被证明可以很好地标记细菌和酵母中的蛋白质1，2。学生将在这项工作的基础上，调整该策略以用于哺乳动物细胞。最重要的是，他们将使用光开关荧光蛋白mEoS开发这种方法，这将使超分辨率图像能够使用光激活定位显微镜生成。我们特别感兴趣的是应用这种新的策略来可视化在人类来源的神经元内形成的淀粉样蛋白。培训内容：该项目的主管将联合收割机结合他们在蛋白质结构和设计（里根），神经科学（霍罗克斯）和超分辨率显微镜技术（霍罗克斯）方面的专业知识。培训将主要通过在主管和合作者的实验室内实际应用研究技术来实现。将开发的技能包括细胞和分子生物学技术，细胞培养（包括分化的诱导多能干细胞），基因编辑，高级显微镜（单分子和超分辨率），数据分析和编码。学生将被鼓励参加培训研讨会，在大学的各种多组会议上发言，并参加公众参与活动。科学是一项全球性的努力，导师将确保学生有机会参加并出席国际会议。