The role of talin and vinculin in neuronal mechanosensing.

Lead Research Organisation: University of Manchester
Department Name: School of Biological Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Title Arts exhibition "Immortality II" 
Description Participating with images of cells which were screen printed in collaboration with Sally Gilford at an arts gallery exhibition called "Immortality II" 
Type Of Art Artistic/Creative Exhibition 
Year Produced 2016 
Impact knowledge to the general public audience; stimulating thoughts about health and disease; artistic value; 
 
Title Arts exhibition "Immortality" 
Description Artistic prints or scientific images. 
Type Of Art Artistic/Creative Exhibition 
Year Produced 2015 
Impact Engaging the public; see movie on provided link with participation of C.Ballestrem. 
 
Description We have made progress on two lines: (1) we were able to improve methodology of how we test sensing of specific extracellular matrix proteins on substrates of different stiffnesses. (2) We have also progressed on aspects of Aim2 where with experiments determining the molecular aspects of ECM & stiffness sensing. In contrast to the adhesion machinery of normal neurons, the one of neurons that do not have the adapter protein vinculin do not sense matrix of different stiffness. Time-lapse recordings also suggest show that the protein controls outgrowth speeds by influencing the length of resting phases of axon outgrowth. Some of the knock down data of successful vinculin depletion have to be verified biochemically before making it suitable for publication but we are currently preparing 2 manuscripts.
Exploitation Route We are currently preparing three manuscripts for publication. One method paper about axon outgrowth and pathfinding assays using microprinting will be submitted within the month of March to JoVE, a second methods paper will be submitted within the next 2 month to a methods journal publishing a new and improved protocol for efficient the generation substrates of different rigidities; a third manuscript will be submitted in an important cell biological journal to publish the findings about how the finding that the adhesion regulatory adapter protein vinculin is critical for sensing of mechanical changes in the neuronal environment.
Sectors Education,Healthcare,Manufacturing, including Industrial Biotechology

 
Description Material (videos & images) were used in lectures at school visits for educational teaching purposes.
First Year Of Impact 2016
Sector Education
 
Description A novel health promoting device and its potential in accelerating wound healing
Amount £20,000 (GBP)
Organisation University of Manchester 
Sector Academic/University
Country United Kingdom
Start 12/2018 
End 03/2019
 
Description Center for doctoral training (CDT) in regenerative medicine (University of Manchester)
Amount £120,000 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 04/2016 
End 09/2019
 
Description Deep Learning for the Analysis of Label-Free Ptychographic Imaging
Amount £120,000 (GBP)
Organisation University of Manchester 
Sector Academic/University
Country United Kingdom
Start 10/2017 
End 09/2020
 
Description Wellcome Trust multiuser equipment grant
Amount £255,000 (GBP)
Funding ID 202923/Z/16/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2016 
End 06/2020
 
Title Micro-patterning of polyacrylamide gels 
Description Micro-patterning of polyacrylamide gels will enable us and others to perform higher quality of pathfinding assays for neurons along stiffness gradients. The postdoc has established that very recently and knowledge has been only transferred yet to neighbouring labs that will help refinement. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact Help defining cellular responses to changing environments which is relevant for many physiological aspects in the body (e.g. ageing processes) 
 
Description The Role of the Cysteine and Glycine-Rich Protein-3 (CRP3) in Mechanosensing of Cardiovascular Smooth Muscle Cells 
Organisation University of Sao Paulo
Department Medical School
Country Brazil 
Sector Academic/University 
PI Contribution - Successful application to FAPESP for funding of mutual visits. - access granted to facilities, data and equipment - our lab trained Ayumi in advanced fluorescence microscopy
Collaborator Contribution - mutual intellectual input through first visit of Ayumi in the summer 2018 to the lab. - reagents (CRP plasmids) and knock out cells contributed by Ayumi to perform follow up studies
Impact FAPESP funding: Sprint grant for collaboration with Ayumi Aurea Miyakawa, (Sao Paulo, Brazil); The Role of the Cysteine and Glycine-Rich Protein-3 (CRP3) in Mechanosensing of Cardiovascular Smooth Muscle Cells. Oct 2017 - Sept 2020. £28,000.
Start Year 2017
 
Description The role of tensin in normal and cancerous cells 
Organisation Agency for Science, Technology and Research (A*STAR)
Department Institute of Molecular and Cell Biology,
Country Singapore 
Sector Academic/University 
PI Contribution - Collaborative student through funding of a studentship in the Manchester-A*STAR PhD program - training and intellectual input - home laboratory for the student
Collaborator Contribution will host to the student for 2 years.
Impact First data about potential interaction partners for this protein.
Start Year 2018
 
Description The role of the extracellular matrix in neuroregeneration 
Organisation University of Manchester
Department Manchester Medical School
Country United Kingdom 
Sector Academic/University 
PI Contribution The collaboration lead to attract a PHD student who works under the supervision of myself with the collaborator to explore the role of the extracellular matrix and adhesion receptor signalling in neuroregeneration.
Collaborator Contribution The contribution is the isolation of peripheral nerve cells that are tested on our models to explore axon outgrowth on micro patterned substrates. There is also a large intellectual input as co-supervisor in this project.
Impact - funding or a PhD studentship; this is multi-disciplinary and involves micro-engineering, cell biology and microscopy, clinical aspects in term of isolation of neuronal cells with the aim to establish a regeneration model that will then be tested also in vivo (long term).
Start Year 2016
 
Description Lab shadowing sessions 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Two pupils were visiting our lab shadowing the lab members' project work. This gave them insight the lab environment, how scientists work and in the topics that are investigated in our laboratory.
Year(s) Of Engagement Activity 2016
 
Description Placement visit of High School pupils (year10) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 2 week long placement of pupil
Year(s) Of Engagement Activity 2018
 
Description Primary School science-art masterclass - "what makes us human" 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Alongside scientists, children were looking at biology through an artistic lens. Pupils investigated how to make beautiful patterns from the unexpected. They looked through microscopes to identify patterns in the "nature" (e.g. cells), compare it with patterns in the museum and produce their own pattern design to print on cloth. At the end patterns of life was discussed with the children; how humans and how cells are affected by their environment in health and disease.
Year(s) Of Engagement Activity 2016
URL http://www.whitworth.manchester.ac.uk/learn/schoolscollegesanduniversities/primary/masterclass/
 
Description School Visit (Withington High School for Girls), Manchester UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Lesson about "How cells sense their environment and what happens if this goes wrong".

Sparked questions and discussions and the school increased interest in related subject areas and regular visits.
Year(s) Of Engagement Activity 2015,2016
 
Description School visit St Bedes (Manchester) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Info evening about potential careers in Science.
Year(s) Of Engagement Activity 2018
 
Description School visits abroad (Singapore, Indonesia and Malaysia) and participation and recruitment fairs for international undergraduate students. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact Several high school classes were visited with a selection of talks around the topic of "How cells sense their environment and the role in health and disease". Around 20-50 students visited per school visit (some with parents); 4 school visits in 2015 (Singapore only); 10 in 2016 (Singapore, Indonesia, Malaysia). Besides engagement, the talks about my labs research aimed to attract international undergraduate students to the University of Manchester.
Year(s) Of Engagement Activity 2015,2016