A multi-user access laser tweezers, fluorescence and interference microscopy facility for understanding force at the molecular level

点击翻译按钮获取中文摘要

Biological systems are affected by numerous external factors such as temperature, osmotic stress, and force. The latter occurs on multiple size scales, DNA repair proteins are subject to collisions with other proteins bound to DNA, immune cells migrating to a site of infection must push past obstacles and muscles contract against load. These are all examples of the impact of force on biological systems. At present, we lack a clear understanding of these processes because of insufficient access to instruments capable of studying the effects of force. In this proposal, we aim to fill this gap by installing an optical trapping system at the University of Kent known as the Lumicks C-trap. Optical trapping (also known as laser tweezers), the discoverers of which were awarded the Nobel prize last year, is a technique that allows beads or other objects, including vesicles, cells or organelles to be captured by a focused beam of light. This 'tractor beam'-like technology then enables investigators to physically manipulate the biological system of interest. The architecture of such assays could include suspending a single molecule of DNA between two beads and then assembling protein complexes on the DNA by dipping the DNA into different solutions made possible by the Lumicks C-trap microfluidic chamber. Using fluorescence to check for assembly the force-dependence of the system can then be investigated. By applying these forces in vitro we learn the properties of the system and how it would respond in its native environment in the cell, where it is not presently possible to perform such measurements.Alternatively, we can capture pathogenic yeast cells and measure their adhesion to materials on the flow chamber surface, and their response to drugs affecting adhesion can be directly measured. The uses of this system are vast and in this proposal we present seven projects with a diverse spectrum of applications.The system we are proposing to install possesses multiple combined functionalities, microfluidics, optical trapping, TIRF, widefield fluorescence and interference reflection microscopy (IRM). This latter technology uses the interference of light to detect objects without any label. This means that in some experiments where labelling affects activity, using IRM overcomes this limitation. Indeed, one project in this proposal seeks to use IRM to measure the formation of protein complexes in a membrane coated surface. By combining these capabilities the resulting system is very powerful, and also unique. In the UK there are no systems with this capability, and across the world there are only two. We want to ensure wide access to this technology and therefore we are reserving 25% of instrument time for external use. We will bring investigators to Kent to train on the system and to try out force experiments on their biological systems. The environment at Kent is ideal for the C-trap, the PI is an expert in the use and development of optical trapping technologies and this project also includes a second expert in optical trapping from the University of Nottingham. Also, at the University of Kent we have a diverse range of investigators that will be exposed to the capabilities of this system and therefore we will achieve more rapid diversification of application, which in turn will bring more investigators to Kent to use the C-trap. Finally, this system is not an add-on to an existing system, nor is it an incremental advance in our capabilities. The C-trap offers a genuine step change in the capabilities of researchers across the UK, and this is the right time and right group of investigators to support such an instrument.