A toolkit of customised tension sensors for interrogating mechanical forces in the cell

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Proteins are the workhorses of the cell, but despite significant advances in our understanding of the physical and chemical principles underlying their structures and functions, one fundamental property - protein mechanics - remains poorly understood. Mechanical forces are involved in varied biological processes such as force-bearing proteins in the muscle and tension upon chromosomes separation during cell division, and disruption of the cell's ability to sense the mechanical properties of its surroundings represents a hallmark of many diseases, including muscular dystrophy, arteriosclerosis, cardiomyopathies, and cancer. It is clear, therefore, that specific proteins must be able to sense mechanical signals and convert them into biological responses, but determining how they do so presents some major challenges: First, measurements on single molecules are required, as force cannot be applied to a bulk solution of protein molecules but rather to individual protein molecules, the response being highly dependent on how and where the force is applied. Second, the tiny (piconewton) force scales involved require extremely sensitive instrumentation that is difficult to build and use of which necessitates the introduction of complex modifications into the proteins under study. Third, for a true understanding of how mechanics translates into function we will need to characterise these forces within the complex physiological environment of the cell, adding a further layer of difficulty. At the heart of our proposal are so-called "repeat proteins" - a striking class of proteins that appear to behave as nano-sized biological springs. These ubiquitous proteins have unique properties. Unlike "typical" proteins, which are globular in shape, repeat proteins form linear, horseshoe-shaped structures from the stacking of small structural units repeated multiple times in tandem, like steps in a spiral staircase or a column of Lego blocks. This simple, modular architecture makes it straightforward to design novel repeat proteins from scratch and with exquisite precision, and we are only now starting to realise the range of potential applications for which this "design-ability" can be exploited. We will use a class of artificial repeat proteins that we have found to possess very special, exploitable physical characteristics. Applying our knowledge of protein engineering we will alter the spring-like properties of these proteins. Foremost, this will enable us to decipher the code that makes our springs work in the way they do. Once we have this code, we can then program the spring to adopt any stiffness we like. Our primary aim is to translate our findings into the development of a toolkit of spring-like tension sensors able to resolve forces in living cells well beyond the current technologies and which can be customised to the individual researcher's needs. The sensors will be coupled to fluorescent molecules that light up and thereby enable us to "see" the mechanics in action inside living cells.A builder is limited by the quality and versatility of their tools. Moreover, the more specialised the tools, the fewer people can use them. Thus, although existing force sensors highlight the potential for such tools to provide us with remarkable new insights into the inner workings of the cell, they will be of limited value unless we design them in such a way that they can be readily customised. This is our goal. We will create a new type of sensor - one that researchers can program with whatever specifications they require. We believe that our design strategies will prove to be a versatile resource for any researcher wanting to investigate mechanosensory process in living systems. Just like a screwdriver set, our toolkit will have a range of different "bits" that can be selected according to the biological question being tackled.