The role of talin and vinculin in neuronal mechanosensing.

During development, billions of neurons form long cellular extensions called axons, which find their way to specific target sites such as other neurons. Through these connections neurons form a highly organised network and transmit information in form of electrical and chemical signals that govern our organ functions. Neurons need to find specific targets not only during the development of the nervous system but also after injury, when they have to regrow through damaged tissue. Failure to reconnect often leads to severe health problems - for example, to date there is no treatment for recovering function after spinal cord injuries. Neuroscience has focused on chemical signals regulating growth and regrowth of neurons for decades, but we are still far from understanding why neurons in human brains and spinal cords do not regenerate.

The environment in the brain alters enormously during development, normal ageing, injury and certain diseases. Brain tissue is extremely soft. However, it becomes stiffer during ageing (in men more than in women), and under pathological conditions it can change dramatically in structure and stiffness. Prominent examples are scarring after injury or stroke, and the formation of rigid plaques or tangles in diseases such as Alzheimer's. Neurons sense such mechanical changes in their environment and respond with drastic changes in their behaviour, as best illustrated by the failure of axons to regrow after spinal cord injury due to the presence of scars. Therefore, understanding how neurons respond to their mechanical environment is important if we want to get a step closer to treating, for example, spinal cord injuries.

Neurons can feel the stiffness of their environment by exerting forces on it and probing its deformation. In order to transmit forces, they 'grab' neighbouring structures using special 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 did some first experiments that suggest that two of these coupling proteins (which are called talin and vinculin) allow neurons to measure the stiffness of their environment, but how they do this is still unclear. In order to investigate how these proteins regulate the neuronal response to their mechanical environment, and to what extent they are involved in telling neurons where to grow, two laboratories in Manchester and Cambridge team up and combine their long-standing expertise with integrins, neurons and forces.

The proposed research aims to (i) determine how talin and vinculin transmit mechanical information between the outside world and the inside skeleton, (ii) investigate their role in sensing stiffness differences in the environment of neurons and how this affects neuronal outgrowth and guidance, and (iii) understand how talin and vinculin interact with each other and with another protein named RIAM, which likely explains how mechanical signals control axon outgrowth and pathfinding.

To reach our goals, we will not only use cutting edge microscopy, biophysics and molecular biology techniques but also develop new tools to mimic the mechanically altered environmental conditions that neurons encounter. Our results will be combined into a model that outlines and predicts how environmental signals and intracellular processes contribute to neuronal outgrowth and guidance during development, ageing and disease. Ultimately, the knowledge gained may lead to important changes in how we currently treat patients with different neuronal disorders, and it might thus, for example, contribute to successful treatment approaches to spinal cord injuries.

Cells interact with the extracellular matrix through transmembrane adhesion receptors (integrins) that are linked to the actin cytoskeleton through proteins that dynamically regulate this link. Our pilot data suggest that two of these linker proteins, talin and vinculin, are involved in sensing mechanical signals, which influences axonal outgrowth and guidance. As neuropathies affect brain mechanics, our overarching aim is to understand how talin and vinculin transmit and transduce signals of matrix stiffness.
Using traction force microscopy, we will first determine how talin and vinculin regulate the connectivity between integrins and the force generating actomyosin machinery. We will measure forces growth cones exert on their substrate and compare control cells with cells that are depleted of talin and vinculin or express mutants that are inhibited for actin binding. Using the same cell systems, we will then investigate how different mechanical properties of the environment influence axon growth and pathfinding and how talin and vinculin are involved in sensing these differences. In order to enable these studies, we will develop substrates with incorporated stiffness patterns. Furthermore, we will test a model in which axon growth and guidance are regulated through a balance between talin/vinculin and talin/RIAM interactions (RIAM is another linker protein). While talin binding to RIAM favours growth cone protrusion, talin/vinculin stabilises adhesion complexes and suppresses axon outgrowth. This hypothesis will be tested in traction force and mechanosensing experiments, combined with advanced fluorescence imaging and mutagenesis to specifically perturb protein-protein interactions.
By combining cell biological with biophysical techniques, we will unravel a currently unknown mechanotransduction mechanism, which may ultimately lead to novel strategies promoting neuronal regeneration in the mammalian CNS.

The proposed project is highly interdisciplinary in nature. It combines biological, physical, engineering 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 their mechanical environment and establishing methodologies/materials that enable directing cellular responses will be of enormous 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 neuronal 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) Companies commercialising cell culture equipment. Currently, most commercial cell culture substrates consist of plastics or glass, both orders of magnitude stiffer than the physiological cell environment. Most tissue cells, however, respond to mechanical cues. Securing and marketing novel cell culture substrates incorporating appropriate mechanical cues will be highly interesting for these companies. The commercialisation of such substrates and the implementation of the know-how in the portfolio of UK companies will impact their national and international competitiveness. This direct impact will be short- to mid-term.

(3) Pharmaceutical industry. Unravelling how talin and vinculin-mediated signalling is involved in neuronal mechanosensitivity will provide a starting point for the development of pharmaceutical products influencing cellular responses to mechanical stimuli. Preventing neurons from being repelled by stiff obstacles might help overcoming their failing regeneration in the vicinity of potentially stiff glial scars. Thus, there is a great potential of commercialising products used to treat neuronal damage. It will be direct and mid- to long-term.

(4) 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 neural tissue injuries has an enormous impact on general health. Treating spinal cord injuries 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.

(5) 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.

(6) 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.