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

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.

A fundamental question in biology is how proteins mediate mechanical force transduction. Our understanding is very limited, due to the lack of methods that are able to visualize cell-generated forces at the molecular scale. Novel techniques with molecular resolution, pN sensitivity, and live-cell applicability are required. To date researchers have developed FRET-based force sensors, composed of a pair of fluorescent proteins connected by a small spring-like protein or peptide. These sensors are limited in terms of both sensitivity and the range of forces they can detect. Equally importantly, it is difficult to design such sensors de novo, and current tools must instead rely on the fortuitous identification of suitably force-responsive proteins rather than rational, customised design. Subsequently determining their mechanical characteristics is a labour- and time-intensive process. It would be much better if we had at our fingertips a sensor 'system' that was based on proteins for which these properties are intrinsic and can be reliably predicted from the sequence. This is the goal of our research. We will create a novel toolbox of sensors, based on tandem-repeat proteins, which will be highly tuneable, sensitive and able to resolve very low forces as well as responding to high forces - well beyond what is possible currently.

We will complement our expertise in repeat-protein engineering, folding and stability with single-molecule force spectroscopy expertise (collaborator Rief) and in-cell visualisation of protein mechanotransduction processes (collaborator Grashoff). Our approach is distinctive in its use of first-principles knowledge of chemical and force-induced protein unfolding to design and fine-tune force-sensing switches that can be used in the cell. We anticipate that these tools will be widely exploited by scientists working in diverse branches of mechano-transduction research and will enable us to illuminate the physics-biology interface.

Disruption of the ability of cells to sense mechanical forces represents a hallmark of many diseases, including muscular dystrophy, arteriosclerosis, cardiomyopathies, and cancer. Yet we are a long way from being able to understand these processes, as we lack the tools required. Addressing this lacuna is the goal of our proposal. Our research will therefore have beneficiaries within academia and the commercial (biotechnology) private sector and will benefit the general public on various timescales. Academia will benefit through our acquisition and dissemination of new knowledge, through training of young scientists and future scientists in a world-class environment and through the advances that we make and new tools and methodologies that we generate, which will be shared with fellow academics upon request.

My recent move to the Department of Pharmacology has strengthened my research interests in the area of molecular therapeutics, both targeting repeat proteins that are deregulated in disease and also exploiting the extraordinary design-ability of repeat proteins to develop nanomaterials and new biologics against intracellular targets. In respect to this proposal, the novel approaches to modifying protein mechanics and to interrogating the processes by which forces are sensed and propagated in physiological and disease settings in live cells will help to advance several different fields, from protein chemistry and biophysics to molecular therapeutics.

The general public will benefit on different timescales: In the short term, they will benefit from our dissemination and outreach activities aimed at engaging a wider audience with our research, in the medium term from the training we provide that will contribute to the global future of science, and in the long term, from the discovery and exploitation of mechansims underlying pathways that are deregulated in diseases such as cancer and neurodegenerative disorders. The development of new medicines relies on our ability to understand the biological details of a disease, and one fundamental biological mechanism is the process by which proteins sense and respond to force.

We are committed to presenting our research to lay audiences as part of our public engagement. We contribute to the annual Cambridge Science Festival and, since moving to the Pharmacology Department, our activities have additionally been focused on the "Young Pharmas" programme, which I helped to set up and run with two other members of the Department. The programme is for Year 12 students from three local sixth-form colleges, and it comprises a year-long series of lab rotations, student presentations and summer placements. The PDRA and I will develop a new rotation within the programme, built around the proposed research, on the theme of proteins as cellular workhorses and as targets for therapeutic intervention.