Orchestration of adhesion signalling by the mechanosensors talin and vinculin.

Cells continuously sense and produce their surrounding environment, which consists of fibrillar material the cells can attach to and is called extracellular matrix (ECM). Cell-ECM communication is particularly important during development or regeneration processes that require specific cellular responses to changing environments. Cellular responses comprise changes in motile behaviour (e.g. closing of wounds), contractility (e.g. functioning of the cardiovascular system) but also active remodelling of their ECM for the purpose of formation of new functional tissue. Many studies have focused on how cells sense their environment, but we are still far from understanding the mechanisms how cells perceive environmental signals and how they are translated into signals within cells that promote specific cellular responses.

The environment of cells alters enormously during development, normal ageing, injury and certain diseases. For example, the mechanical properties of the ECM is thought to influence tumour progression and increased breast matrix stiffness is associated with poor survival. Stiffening of ECM also causes cardiovascular malfunctioning. Intriguingly cells contribute to the production of specific matrix on one hand but also respond to this produced environment on the other hand. Therefore, understanding how cells sense and produce their ECM environment is critically important if we want to get a step closer to treating the roots of diseases and promote regeneration.

Cells can feel or sense their environment by exerting forces on it and probing its deformation. To transmit forces, they 'grab' neighbouring structures using surface proteins, which are called integrins. These integrins not only bind to the environment of the cells but also connect to a skeleton inside the cells. This link is not direct but is regulated by components that couple or uncouple the two. We published a number of manuscripts showing that two of these coupling proteins, called talin and vinculin, are central to sensing of environmental changes. They are particularly important to measure the stiffness of their environment, they control cell migration, as well as cell growth and differentiation. In this proposal we also present important pilot data demonstrating that vinculin is critical for ECM remodelling. However how they do this is still unclear. In order to investigate how these proteins regulate the response to their environment, and to what extent they are involved in telling cells how to behave, the two laboratories in the prestigious Cell-Matrix Centre at the University of Manchester will team up and combine their long-standing expertise with the field of integrins signalling and cell-matrix interactions.

The proposed research aims to to (i) understand the role of mechanical signals in the activation of talin and vinculin, (ii) how this activation helps vinculin and talin to associate with a large number other proteins that serve to exert specific signals (e.g. cell migration or cell growth) (iii) how vinculin with the newly found association of another protein called tensin is contributing to the formation and remodelling of ECM environment.

To reach our goals, we will not only use cutting edge microscopy, biochemisty and molecular biology techniques but also a newly generated intracellular system whereby we can target proteins to specific compartments (mitochondria) in the cells which enable us to visualise and probe molecular interactions and behaviour under defined conditions. Our results will be combined into a model that outlines and potentially predicts how cells interpret and remodel their environment. Ultimately, the knowledge gained may lead to important changes in how we currently envisage environmental changes and their contribution to diseases. This may also lead to changes in treatment of patients, and it might thus, for example, contribute to improvements in disease prevention and in regeneration processes.

Cells interact with the extracellular matrix (ECM) through transmembrane adhesion receptors (integrins) that are linked to the actin cytoskeleton via proteins that dynamically regulate this link. Our published data suggest that two of these linker proteins, talin and vinculin, are involved in assembly of a large network of proteins which influence how cells interpret and remodel their ECM. As ECM sensing and remodelling affect cell motility and differentiation, our overarching aim is to understand the role of the talin-vinculin axis in transmission and transduction of signals to and from the ECM.

Using structure-based mutations, in combination with a novel mitochondrial targeting system (MTS) that allows visualisation of complexes assembled under defined conditions, we will first determine the contribution of force to talin and vinculin activation. Using the same system, we will then investigate how the two adapter proteins and their individual domains participate in the recruitment of other regulatory proteins. Based on our pilot data, we will also explore the hypothesis that association of vinculin with the adapter protein tensin, regulates fibronectin fibrillogenesis/ECM remodelling. The use of cutting edge imaging will enable us to gain specific knowledge about interaction mechanisms and strength, and the use of mass spectrometry will enable us to gain insight in the wider signalling network associated with talin and vinculin under defined conditions.

Insights from these studies may ultimately lead to novel strategies both to prevent diverse diseases and to promote tissue regeneration.

The proposed project combines biological, physical, and medical aspects, is concerned with the design of novel techniques and investigates molecular mechanisms potentially relevant to development, pathology and medical treatment. Thus, there is a wide range of direct and indirect beneficiaries from the research:

(1) Biotechnology. Understanding how cells respond to and remodel their mechanical biochemical environment and establishing methodologies that enable cellular responses to be directed will be of benefit for biotechnology research and industry, particularly for tissue engineering. Cell lines stably expressing GFP-tagged proteins may become valuable for the screening of materials and drugs affecting cellular behaviour. We expect a high potential impact in the biotechnology area and will actively search for relevant systems/companies to share our knowledge. The impact will be direct and mid-term.

(2) Pharmaceutical industry. Unravelling how talin and vinculin-mediated signalling is involved in mechanosensitivity will provide a starting point for the development of pharmaceutical products influencing cellular responses to mechanical stimuli. Modulating cell responses to changing matrix properties may promote regeneration. Thus, there is the potential to commercialise products used to modulate force sensing. It will be direct and mid- to long-term.

(3) General public. Images generated from this project are colourful, intuitive, attractive and make the science more accessible. They are useful for educating the public, and particularly children through school lectures, about science. We will further set up a website about "The Cell's Sense of Touch" which will contain sections accessible to the lay person. This will focus on how disciplines can be integrated to deliver tangible benefits for society, in terms of finding new ways to understand and treat disease. Moreover, contributing to the successful treatment of tissue injuries has an enormous impact on general health. Promoting regeneration processes will improve life quality of thousands of people in the UK and beyond the borders. Furthermore, it will drastically reduce treatment costs, thus directly and indirectly impacting the healthcare system. The impact is indirect and mid- to long-term.

(4) Researchers of various backgrounds. Understanding cellular responses to mechanical cues is highly relevant to biology and biophysics. It is known that mechanosensitivity is involved in many physiological and pathological processes ranging from embryo formation to liver cirrhosis, adding an impact on medical research. The development of novel methods is particularly relevant to engineers. Accordingly, scientists working in any of those areas might be highly interested in the outcome of the project. The impact will be direct and immediate.

(5) Staff working on the project. Researchers will work interdisciplinary, interact with many scientists of different backgrounds and companies and creatively solve problems. They will further develop communication, problem solving and entrepreneurial skills and acquire new technical and IT skills, which will be useful in any later profession.