Harnessing protein unfolding and aggregation in mechanotransduction

Mechanical forces shape how our bodies develop and function. For instance as our muscles enlarge and contract with greater force, a mechanism senses these forces and strengthens the attachment of muscle ends to tendons so they are strong enough to withstand the increased force. This process is called mechanotransduction and it is central to many of our body's functions. The proposed research focuses on the molecular machinery involved in how cells sense these mechanical forces.

All cells in the human body are held in the correct place via adhesion to neighbouring cells, and to a dense meshwork of proteins that surround cells, the extracellular matrix. Cells adhere to this matrix via cell surface proteins called integrins. Talin is the main linker protein coupling integrins to the cell's force generating machinery, engaging integrin at one end and coupling it to the cytoskeleton. As the cytoskeleton pulls on the integrin anchors, talin stretches like a spring and unfolding of talin recruits the protein vinculin, which reinforces the adhesion in a force-dependent manner. While this paradigm provides a feasible mechanism for force to induce a chemical change, namely the redistribution of vinculin within the cell, it also raises many questions, which are the focus of this research.

In this proposal we build on our recent discovery of two new and unexpected pieces of the puzzle of how mechanotransduction works. We have discovered that talin unfolding can lead to self-assembly of talin molecules by a process called aggregation. This is an unexpected discovery, as protein aggregates are best known for their role in disease, for instance dementia and Alzheimer's disease are both caused by accumulation of protein aggregates. Our cells protect themselves from such aggregates using "chaperone" proteins that dissolve and refold misfolded proteins.

Our central hypothesis is that these two harmful processes, protein unfolding and protein aggregation, have been harnessed by the cell to serve as elegant force sensing mechanisms that enable the cell to sense forces and convert them into biological signals. The hypothesis that we would like to test is that a normal feature of anchor sites is the formation of a meshwork of stretched talin molecules, which provide a solid platform for the assembly of many additional components required for integrin adhesion. Our pilot data suggest that the formation and rearrangement of this meshwork involves specific chaperones to control this process and to ensure it does not go wrong.

We will test this hypothesis by combining the expertise of our two labs. The Goult lab will use biochemical, biophysical and structural methods to characterize how the components work together, and to identify specific changes that can be made to the molecules to alter their activity. The Brown lab will exploit the powerful genetics and imaging approaches that can be used in the fruit fly Drosophila to test the importance of the formation and remodelling of the talin meshwork in different processes that require the integrin machinery within the organism, such as attachment of muscles and anchoring of stem cells.

This research is important at several levels. Our discoveries will improve our understanding of how forces strengthen cell adhesion, and how pathological protein aggregation is avoided, with potential benefits to the understanding of human disease. Diseases caused by weakening of cell adhesion may be improved by interventions that mimic the force signal and strengthen adhesion. Similarly, movement of cancer cells, or metastasis, renders cancers much more difficult to treat, and strengthening adhesion will anchor cancer cells and restrain cell movement. The experimental advantages of Drosophila will allow us to investigate the role of specific protein-protein interactions within an organism throughout its life cycle, and this knowledge will then be applied to humans.

The proposed research addresses the mechanisms underlying integrin-mediated adhesion to the extracellular matrix and mechanotransduction. These mechanisms are critical for movement of cells and their assembly into tissues, during development and throughout life. We focus on the key mechanotransducing protein talin, which by linking integrins to the actin cytoskeleton can transduce mechanical force into chemical signals. It achieves this by having protein domains that act like a series of mechanochemical switches, unfolding at intracellular forces, dislodging already bound proteins and revealing new binding sites for mechanoeffector proteins. Both biochemical and molecular genetic preliminary data from the two labs points to a novel hypothesis, that self-interactions between mechanically stretched talin molecules form an 'intracellular meshwork' (ICM), which provides a stable protein super-complex that recruits additional components of integrin adhesions. Furthermore, we have evidence to suggest that both integrin and vinculin can induce talin to form the ICM, and that vinculin and specific components of the unfolded protein response machinery have an important chaperone role in regulating talin self-interactions. We propose a wide range of experiments to test our hypotheses, from structure determination, single molecule biophysics and biochemistry, to molecular genetics and advanced imaging within intact, living Drosophila. Our findings will enhance our understanding of how protein unfolding and controlled aggregation contribute to mechanotransduction and cell adhesion. This will provide important insights into how functionally important protein aggregation can become misbalanced and lead to devastating diseases such as dementia.

The beneficiaries of this study will be:
1. Pharmaceutical and Biotech industries. The increasing recognition of the importance of mechanotransduction in fitness, obesity and disease, means that our work will be relevant for these industries. They will potentially be interested in using our findings to develop new drugs and technologies for the patients described below (2-4). The improved understanding of mechanosensing that will arise from this project could lead to the design of nanoparticles that sense and respond to mechanical force. We will contact the Cambridge Nanoscience Centre to develop such technologies, benefiting the economy of the UK and strengthen our position as a world leader in drug development. (5-10 years)
2. Those wishing to develop high levels of fitness and longevity. The mechanisms of mechanotransduction are an essential part of how the body ensures that tissues can withstand the forces produced by everyday and strenuous activity. For example, increasing levels of the mechanoeffector vinculin in the heart of the model organism Drosophila extends its lifespan 150%. The improved understanding of the beneficial activities of vinculin, and its harmful activity when over active, will aid in developing fitness regimes and promoting healthy ageing. (10-15 years)
3. Patients suffering from obesity. Mechanotransduction mechanisms play an important role in sensing the amount of food in the gut. Discovering whether hereditary impairment of mechanotransduction contributes to obesity may provide new routes for treatment. (10-15 years)
4. Patients suffering from genetic disorders that affect cell adhesion, such as muscle dystrophies or Kindler syndrome. We aim to understand how talin, vinculin and specific components of the unfolded protein response work together in tissue development and function. This could for example lead to the identification of small molecules that activate vinculin and therefore strengthen cell adhesions. New drugs would be developed and applied to strengthen cell adhesions of patients with such disorders. (10-15 years)
5. Patients suffering from neurodegenerative diseases, such as Alzheimer's or Parkinson's disease, and muscle diseases such as myofibrillar myopathies, all of which are associated with the presence of protein aggregates in the affected tissues. Our proposed project aims to elucidate a process where aggregation is harnessed by the cell to mediate cell adhesion. This understanding will help improve the selectivity of treatments aimed at preventing disease-causing protein aggregation. (10-15 years)
6. Patients suffering from cancer. A vast majority of cancer deaths are caused by metastasis, the process by which cancer cells spread within the body. The invasive behaviour of cancer cells is critically regulated by mechanotransduction. Our improved understanding of mechanotransduction mechanisms will be exploited to design ways of inhibiting the metastatic capability of cancer cells. (10-15 years)
7. Organisations and Companies recruiting scientifically trained staff, including both public and private sectors. The two postdoctoral researchers funded by this work will develop their training and expertise, as well as supervising A-level, undergraduate and postgraduate students. Thus, the work will benefit a new generation of scientists. After the completion of the work, the researchers will be able to contribute to the scientific economy of the UK by applying the skills gained in the project, whether in public or private sectors.
8. The general public. Through engagement with the public through talks, websites, social media and general audience publications we seek to communicate the excitement and beauty of scientific research. Our work will involve a substantial amount of compelling images that serve as an important starting point for public engagement with biomedicine. We will submit such images to competitions (e.g. Nikon/Wellcome Trust) to reach the widest audience.