A correlative, ultra-stable, optical tweezers-confocal microscope for high-resolution molecular and cellular mechanobiology

用于高分辨率分子和细胞力学生物学的关联、超稳定光镊共聚焦显微镜

• 批准号: BB/X019047/1
• 负责人: Carlos Penedo
• 金额: $ 81.84万
• 依托单位: University of St Andrews
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX019047%2F1
• 关键词: correlative; ultra; stable; optical; tweezers

The roles played by mechanical forces manifest across of biological scales, from the nanometre-size structural changes observed in proteins to the large-scale relaxation and contraction phenomena happening in muscle tissue that allows body movement. There is mounting evidence that these forces have a fundamental role in a myriad of molecular and cellular processes. Unfortunately, biological forces cannot be investigated using tools such as NMR, circular dichroism, fluorescence spectroscopy, X-ray crystallography or cryo-EM, simply because these methods cannot measure the magnitude and location of the forces and cannot apply mechanical stress to replicate molecular or cellular mechanical conditions. However, recent technical advances using optical tweezers (OT) are giving unprecedented access to what these biological forces do and how they do it. This proposal relates to the purchasing of a LUMICKS C-trap correlative imaging microscope that combines OT for mechanical manipulation with simultaneous fluorescence imaging using confocal microscopy. Combining both elements with a multi-channel microfluidic device to alter conditions in real time provides a highly versatile and unique tool for nano-mechanics. OT use a tightly focused beam of light to trap a micrometre-size spherical object (a bead), a cell organelle or an entire cell. Once trapped, the object can be held in place or moved by changing the beam position. To apply or sense forces at cellular level, the trapped bead is coated with 'glue-like' molecules such receptor or antibodies that will interact with the cell membrane thus providing a hook to apply forces at specific locations. At molecular level, proteins, nucleic acids, and multi-subunit complexes can be tethered between two trapped beads. By altering the distance between them is possible to apply stretching forces or sense changes in bead(s) position from which to extract information about structure, folding, dynamics, interactions, and biological function. At molecular level, the C-trap capabilities offer an endless range of applications, and the team will use them to investigate the mechanobiology of DNA replication, recombination, repair, packaging, RNA folding and regulation, CRISPR-based editing and to understand the effect of mechanical stress in the function of proteins involved in bacterial, viral and parasite infection. At cellular level, applying local forces to distort membrane curvature will enable to determine the impact of mechanical stress in receptor signalling, microtubule dynamics, polarized trafficking, invadopodia formation in cancer cells and the function of mechanosensitive protein channels. OT is also revolutionizing plant cells biology and the team will use it to evaluate how force remodels membrane-contacts, membrane-constricting sites, protein-protein interactions and the conformation of receptors involved in the plant defence system.Although the technology is being quickly and widely adopted worldwide, there is no confocal C-trap microscope in Scotland (and North England). In this proposal, we have put together a broad range of research projects to showcase the transformative impact that this instrument will have across the participating groups, giving access to information otherwise inaccessible. We have designed a program of training and support to integrate the C-trap with existing capabilities across the partner institutions and ensure its long-term sustainability. To facilitate access to the wider bioscience community, promote new science and collaborations, we will operate in a 'donated time' format during a three-year period. In summary, the LUMICKS C-trap system is a state-of-the-art 'turn-key' multi-user, multi-project equipment that will increase imaging capability in Scotland and will enable a deeper understanding of fundamental biological processes related to cancer, ageing, and infection pathways that are part of the BBSRC Forward Look for Bioscience strategy

机械力的作用在生物尺度之间表现出来，从蛋白质中观察到的纳米大小的结构变化到在肌肉组织中发生的大规模松弛和收缩现象，从而使人体运动。有越来越多的证据表明，这些力在无数分子和细胞过程中具有基本作用。不幸的是，不能使用NMR，圆形二色性，荧光光谱，X射线晶体学或冷冻EM等工具研究生物力，仅仅是因为这些方法无法测量力的幅度和位置，并且无法在复制分子或细胞机械条件下应用机械应力。但是，使用光学镊子（OT）的最新技术进步使人们可以访问这些生物力量的所作所为以及如何做。该建议涉及购买Lumicks C-Trap相关成像显微镜，该成像显微镜将OT与使用共聚焦显微镜同时进行机械操作与同时荧光成像相结合。将这两个元素与多通道微流体设备相结合以实时更改条件，为纳米机械学提供了高度通用和独特的工具。 OT使用紧密聚焦的光束捕获微米大小的球形物体（珠子），细胞器细胞器或整个细胞。一旦被困，可以通过更改梁位置将物体固定或移动。为了在细胞水平上施加或赋予力量，被捕获的珠子涂有“胶状”分子，例如与细胞膜相互作用的受体或抗体，从而提供了钩子在特定位置施加力。在分子水平上，蛋白质，核酸和多亚基络合物可以在两个被困的珠子之间束缚。通过改变它们之间的距离，可以应用拉伸力或珠子位置的感觉变化，从中提取有关结构，折叠，动力学，相互作用和生物学功能的信息。在分子水平上，C陷阱功能提供了无限范围的应用，该团队将使用它们来研究DNA复制，重组，修复，包装，RNA折叠和调节，基于CRISPR的编辑，基于CRISPR的编辑以及理解机械应力在涉及细菌，病毒，病毒，病毒，病毒和par虫感染功能的作用的机械生物学。在细胞水平上，将局部力应用于扭曲膜曲率，将使机械应力在受体信号传导，微管动力学，极化运输，癌细胞中的侵袭性疾病形成以及机械敏感蛋白通道的功能中的影响。 OT还彻底改变了植物细胞的生物学，该团队将使用它来评估力如何重塑膜 - 接触，膜收缩的位点，蛋白质 - 蛋白质相互作用以及植物防御系统所涉及的受体的构象。在此提案中，我们整理了广泛的研究项目，以展示该工具在参与小组中会产生的变革性影响，从而使信息访问其他方式无法访问。我们设计了一项培训和支持计划，以将C-TRAP与合作伙伴机构的现有能力相结合，并确保其长期可持续性。为了促进进入更广泛的生物科学社区，促进新科学和合作，我们将在三年期间以“捐赠的时间”格式运作。总而言之，Lumicks C-Trap系统是一种最先进的“交钥匙”多用户，多项目型设备，它将提高苏格兰的成像能力，并能够更深入地了解与BBSRC前进策略的一部分有关的与癌症，衰老和感染途径相关的基本生物学过程