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

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

Molecules and cells are exposed to a range of mechanical stimuli, and they must sense the frequency, magnitude, and duration of these signals to generate responses that allow them to adapt to the environment. The field of mechanobiology relates to the understanding of these mechano-adaptation processes and this proposal aims to install the first C-trap confocal microscope in Scotland for mechanobiology research. A crucial advantage of the C-trap is its correlative imaging capability, so that in addition to measuring endogenously generated or externally applied forces, a simultaneous fluorescence readout of the system using confocal fluorescence imaging can be performed in real time. The ultra-high stability dual-trap configuration of the C-trap will allow us to measure the smallest (0.1 pN), transient, changes in force with high spatial resolution over long periods of time. In addition, the C-trap includes a multi-channel laminar flow microfluidics device for rapid changing of conditions, and it can be combined with multi-color colocalization or with Förster Resonance Energy Transfer (FRET) to report nanometre-size changes in local structure. Thus, the instrument will allow researchers to directly measure the effects of mechanical forces using a multidimensional approach including force, distance, fluorescence, position and their changes over time. The team will cover a broad range of topics that can be classified in four major groups: i) the understanding of nucleic acids processing complexes involved in DNA repair, recombination, editing, remodelling, transcription, protein-protein interactions and enzymatic function, ii) cellular membrane interactions involved in receptor signalling, polarized trafficking, invadopodia and cytoskeleton dynamics, iii) mechanics of host-pathogen interactions in both animal and plant cells and iv) the development of novel technologies for nucleic-acid imaging and protein-targeted degradation.