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

机械力所起的作用体现在生物尺度上，从蛋白质中观察到的纳米级结构变化，到肌肉组织中发生的允许身体运动的大规模放松和收缩现象。越来越多的证据表明，这些力量在无数的分子和细胞过程中发挥着重要作用。不幸的是，无法使用核磁共振、圆二色谱、荧光光谱学、X射线结晶学或低温电子显微镜等工具来研究生物力，原因很简单，因为这些方法无法测量力的大小和位置，也无法施加机械应力来复制分子或细胞的力学条件。然而，使用光学镊子(OT)的最新技术进步使人们能够前所未有地了解这些生物力量做什么以及它们是如何做到的。这项建议涉及购买LUMICKS C-TRAP相关成像显微镜，该显微镜将用于机械操作的OT与使用共焦显微镜的同步荧光成像相结合。将这两种元素与多通道微流控装置相结合，以实时改变条件，为纳米力学提供了一种高度通用和独特的工具。不要使用紧密聚焦的光束来捕捉微米大小的球形物体(珠子)、细胞器或整个细胞。一旦被困住，物体就可以固定在原地或通过改变光束位置来移动。为了在细胞水平施加或感觉作用力，捕获的珠子上覆盖着类似胶水的分子，如受体或抗体，它们将与细胞膜相互作用，从而提供一个钩子，在特定位置施加力。在分子水平上，蛋白质、核酸和多亚单位复合体可以被拴在两个捕获的珠子之间。通过改变它们之间的距离，可以施加拉伸力或感觉珠子(S)位置的变化，从而提取关于结构、折叠、动力学、相互作用和生物功能的信息。在分子水平上，C-TRAP功能提供了无穷无尽的应用，该团队将利用它们来研究DNA复制、重组、修复、包装、RNA折叠和调节、基于CRISPR的编辑等机械生物学，并了解机械应力对参与细菌、病毒和寄生虫感染的蛋白质功能的影响。在细胞水平上，施加局部力来扭曲细胞膜的曲率，将能够确定机械应力在受体信号转导、微管动力学、极化运输、癌细胞内足的形成以及机械敏感蛋白通道的功能中的影响。OT也正在给植物细胞生物学带来革命性的变化，该团队将利用它来评估FORCE如何重塑植物防御系统中涉及的膜接触、膜收缩部位、蛋白质-蛋白质相互作用和受体的构象。尽管这项技术正在世界范围内迅速和广泛地采用，但在苏格兰(和英格兰北部)还没有共焦C-TRAP显微镜。在这项提案中，我们汇集了广泛的研究项目，以展示这一工具将在参与群体中产生的变革性影响，使人们能够获得否则无法获取的信息。我们设计了一项培训和支持计划，将C-TRAP与合作机构的现有能力相结合，并确保其长期可持续性。为了促进更广泛的生物科学界的接触，促进新的科学和合作，我们将在三年内以“捐赠时间”的形式运作。总而言之，LUMICKS C-TRAP系统是一种最先进的“交钥匙”多用户、多项目设备，它将提高苏格兰的成像能力，并将使人们能够更深入地了解与癌症、衰老和感染途径相关的基本生物过程，这些过程是BBSRC前瞻性生物科学战略的一部分

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