The mechanism of stretch activation in muscle: a multidisciplinary approach

Lead Research Organisation: University of York
Department Name: Biology

Abstract

Understanding how muscle works is important, both for our in depth comprehension of physiology, and to allow us to design new therapies to cure muscle diseases. In particular, understanding how cardiac muscle, works is essential for the prevention and treatment of all cardiomyopathies. Regulation of muscle contraction is now known to involve two different mechanisms, one triggered by calcium, the second initiated by mechanical stretch. While the first is relatively well studied, very little is known about the second.
The goal of this application is to understand the mechanism of stretch activation in all muscles. We plan to use insect flight muscle, because in these muscles, stretch activation plays a predominant role, and can thus be studied in the absence of the masking effects of the otherwise predominant calcium-activation mechanism. We hope to find out what aspects of regulation are common to all striated muscles. We will use a giant Asiatic waterbug, since its flight muscles are particularly suitable for physiological studies, thanks to their appreciable size. There have been ground breaking studies on the flight muscle of the giant waterbug, some of which were carried out by members of this team. The flight muscle has the most ordered structure of any muscle, which makes it ideal for following structural changes when it contracts.

We will exploit the expertise of two teams, one in London and the other in York, both of which have previous long experience of working on muscle proteins using different but complementary analytical approaches to tackle this challenging and fascinating problem. Both teams have state-of-the-art facilities in their own discipline, and both have many years experience in developing analytical methods for studying biomolecules, and biomolecular interactions and pathways. The two teams have also been working in collaboration before, producing published results that have increased our understanding of muscle functioning.

The project holds enormous promise, not only for understanding the mechanism of stretch activation, but also for providing new tools for the future development of compounds able to intervene in muscle activation. Both cardiac muscle and insect flight muscle contract rhythmically and are activated by stretch. Thus, our research will help us to understand how the regulation of contraction has evolved; we will gain more insight into the way cardiac and other muscles work. This has ultimately the potential to increase our understanding of the causes of human cardiomyopathies.

Technical Summary

We aim to advance the field of muscle research by gaining an understanding of the molecular basis of stretch activation, one of the two mechanisms that allow muscles to contract.

We intend to combine two teams with complementary expertise in developing different analytical, structural and functional methods, to understand the regulation of muscle contraction through protein-protein interactions. We propose to map in atomic detail the changes in troponin structure that are involved in stretch activation, and to suggest how these could lead to muscle contraction. Despite being debated for several decades, and the increasing interest in the effects of mechanical stress on biological systems, stretch activation is poorly understood. The results will add to our knowledge of the physiology of all muscles, and the way in which regulatory mechanisms have evolved; importantly, the proteins responsible for regulating contraction will be described at atomic resolution.

The project is challenging from a structural viewpoint, in that it involves proteins of >70 kDa in size that are filamentous, and in many cases, intrinsically unstructured, which assemble into large complexes. It is also challenging because some components are prone to denaturation and aggregation. We plan to use a combination of state-of-the-art NMR methods (to provide atomic resolution information about protein binding sites) and small angle scattering (to obtain the overall shape), supported by other biophysical approaches (ITC, SEC-MALS, MST). We also plan to substitute mutated proteins into muscle fibres and to make mechanical measurements.

Our results will significantly advance our understanding of stretch activation and increase our knowledge of the mechanisms that allow muscle to contract.

Planned Impact

Scientific impact: Insects are the most diverse of any group of living organism: there are more than a million documented species, which make up 80% of all species. Knowledge of how insects fly will benefit our species, both for the potential of controlling insect-borne disease and for ensuring the survival of beneficial insects. Stretch activation of flight muscle is the mechanism that enables insects to fly.

The mechanical basis of stretch activation has been studied for more than 50 years but little is known about the structural changes that occur in the contractile proteins. Our study of stretch activation has both biochemical and medical applications, since well functioning muscle is important for health and wellbeing, especially in the elderly. The results will interest scientists, pharmacists and medical researchers in both academia and industry. Muscles researchers will benefit, as will clinicians involved in myopathies. The results may be exploited by scientific centres associated with our institutions: KCl has a strong muscle community who will be particularly interested in the output of this project.

Supporting knowledge: Pastore and Bullard regularly present their work at major national and international scientific conferences in their disciplines. When appropriate, press releases will be issued by the University of York, King's College and BBSRC, which may disseminate the results to the general public. While at NIMR, AP organized several press releases to spread information on her research and she intends to continue to do it at KCL. We will ensure that the research staff involved will participate in the promotion and dissemination of the research results by attending scientific meetings and publishing the results in high profile, peer-reviewed journals.

Industrial impact and technology translation: It is envisaged that the long term findings of the proposed research could be exploited in translational research for developing strategies for structure-based design of new drugs and hence, may result in social and economic benefit to the UK, and the community dedicated to improving human health. Intellectual properties at KCl and University of York are protected by their central offices. One of the teams has produced a panel of monoclonal antibodies to flight muscle proteins that are sold by the BBSRC Babraham Institute (Babraham Bioscience Technologies) and Abcam. Other antibodies could potentially be marketed.

Delivering highly skilled people: The proposed project will train two PDRFs in new technologies; they will develop skills in advanced methods of structural molecular biology, nano-science and biochemistry. This will benefit their future careers, whether in academia or industry. Over the past few years, skilled post-graduate and post-doctoral fellows from the laboratories of both applicants have gone on to post-doctoral fellowships and lectureships in other universities, and positions in industry.

Public engagement: It is important that the public is involved in understanding and valuing scientific research, as this is the basis for the support and development of research. It will be important to find the most effective ways to communicate and disseminate the results of the proposed research to the non-scientific community. AP and BB have been committed to giving seminars to students at different levels, and to a general audience. The Wohl Institute of KCl is also committed to creating a specific interface with the public, and to involving it in their research. The Biology Department at York has Open Days for school children, and encourages their participation in research projects.
 
Title Drosophila JMRCM 
Description Cover image for journal of Muscle Research and Cell Motility. The image shows Drosophila in flight and mechanical recordings from the flight muscle. 
Type Of Art Image 
Year Produced 2014 
Impact The journal has a wide circulation among people working on muscle and motility. The paper has been accessed by a large number of people attracted by the cover image, many of whom have corresponded with the authors. This has added to the reputation of Renssalear Polytechnic NY and the University of York. 
 
Description Insect flight muscle can contract at high frequency because it is activated when it is stretched repetitively. We have isolated a complex of the proteins that regulate contraction and determined the shape of the complex in collaboration with the PI (Annalisa Pastore at King's college, London) and Dr Svergun at Hamburg (Germany), using X-ray methods. We have also measured the shape of the complex with truncated subunits and shown that the atomic structure of the individual subunits can be fit to the overall shape of the complex. Knowing the size and shape will help us to understand how the complex is oriented in the lattice of filaments in the muscle fibre.
Contraction is produced by the interaction of thick and thin filaments. We have isolated thick filaments from flight muscle and shown that the regulatory complex binds to these power-producing filaments, as well as to the thin filaments. We have found there are two types of the rod-like protein tropomyosin, which is part of the regulatory complex. One tropomyosin binds to thick filaments, as well as thin filaments. Cross-linking between the filaments will result in transmission of force between them, which is essential for the way in which flight muscle is activated by stretching. Our findings are consistent with a model in which the heads of some myosin molecules in thick filaments extend to tropomyosin on thin filaments. On stretching, the tropomyosin would be pulled away from a blocking position on thin filaments, and contraction would follow. This mechanism would allow the high frequency activation needed for rapid wing beats.
Our collaborators in Shanghai have obtained a draft version of the waterbug genome. This year we have obtained the complete sequence of the genome, using a new technique in which DNA is sequenced as it passes through a small pore (nanopore). When the genome has been annotated, it can be used to identify any protein sequence. This will be used to identify additional components of thick filaments involved in regulating contraction.
The unusual stiffness of insect flight muscle is needed for a rapid response to stretching. Kettin is a large protein that contributes to the stiffness by anchoring the ends of thick and thin filaments. We have determined that four molecules of kettin bind to each thin filament. The long kettin molecules bind along the two grooves of the thin filament. This result was achieved using kettin tagged with a fluorescent molecule, in collaboration with the physics department at York.
We now have a model for how flight muscle is activated by stretching and how the stiffness of fibres is maintained. This contributes towards understanding the control of contraction in all muscles.
Two postdoctoral workers have been trained in the techniques of protein chemistry and electron microscopy.
Exploitation Route Our collaborators at Rensalier Polytechnic have followed up our finding that one isoform of troponin C in the regulatory complex is needed for activation of flight muscle by stretching. They have used transgenic techniques with Drosophila to swap isoforms of troponin C in flight and non-flight muscles. They found that activation by stretch only occurs in flight muscle, and that replacing troponin C is not enough to make non-flight sensitive to stretching.
Our collaborators at Duke University have used our nucleic acid (cDNA) preparation to determine the sequence of waterbug myosin in thick filaments. We have also supplied them with a plasmid for a myosin light chain, which will be used in assembling the myosin molecule with heavy and light chains. This will contribute to understanding the structure of the thick filament.
We have supplied waterbug muscle and RNA extracts to our collaborators in Shanghai, which was used in partial sequencing the Lethocerus genome. Our finding that the tropomyosin-troponin complex forms part of crosslinks between thick of thin filaments is important in a comparison of activation in flight muscle and cardiac muscle. In cardiac muscle, myosin binding protein C forms similar links, which can activate contraction. Finding out how flight muscle is activated will help in understanding a similar mechanism in cardiac and skeletal muscle.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Crystal structure of obscurin kinase 
Organisation University of Konstanz
Department Department of Biology
Country Germany 
Sector Academic/University 
PI Contribution We made a construct of protein kinase 1 of Drosophila obscurin in flight muscle and cloned it in to a pET vector for expression. There are two kinase domains in Drosophila obscurin. Both are inactive pseudo-kinases and we showed that the two domains bind to each other. We tested binding between kinase 2 expressed in vivo and recombinant kinase 1. This interaction may affect the activity of protein kinase 2.
Collaborator Contribution Olga Mayans and her postdoc, Tom Zacharencho, expressed protein kinase 1 of Drosophila obscurin in E. coli. They purified the protein, which we used for binding studies. They crystallised the kinase and determined the structure. The structure confirmed the observed inactivity of the kinase. This is the first determination of a structure for the muscle obscurin kinase domain.
Impact The association of 2 kinase domains in Drosophila obscurin has been observed and this is expected to affect the activity of kinase2. The crystal structure of kinase 1 has been determined. This is the first structure of a muscle obscurin kinase domain. The result will be useful for predicting the function of both Drosophila and vertebrate obscurin kinases. The collaboration is multidisciplinary, involving molecular biology, protein chemistry and crystallography.
Start Year 2015
 
Description Drosophila kettin 
Organisation Eastern Virginia Medical School
Country United States 
Sector Academic/University 
PI Contribution I have obtained Drosophila expressing fluorescently labelled kettin and I have isolated flight muscle for microscopy. I have trained 3 students in dissection and microscopy. The students used the samples for high resolution microscopy with Mark Leake (Physics, York). My lab has made recombinant kettin fragments. These have been combined with actin and the structure of the filaments has been determined by tomography of electron microscopy images by Vitold Galkin (East Virginia Medical School) .
Collaborator Contribution Chris Elliott (Biology) crossed flies to produce heterozygotes and checked for fluorescence of the thorax. He trained 3 students in fly culture. Mark Leake (Physics) used his bespoke high resolution microscope to examine the muscle. The students obtained the results. Vitold Galkin analysed Kettin-actin complexes and determined the structure.
Impact Kettin was observed in the middle of the sarcomere, which was unexpected, and on the Z-disc, which was expected. The Z-disc label had blinking fluorescence, possibly indicating rapid turnover of kettin. We have determined that stoichiometry of kettin relative to actin is one Ig domain of kettin to one monomer of actin. We have run Western blots of gels suitable for identifying high molecular weight proteins like kettin and its isoforms. This has shown that the relative amounts of kettin tagged with GFP to untagged kettin in the flight muscle of heterozygous flies is 30:70. This measurement is needed in order to estimate the stoichiomentry of kettin domains and actin in the sarcomere. The collaboration is multidisciplinary, involving biology, molecular biology and physics.
Start Year 2016
 
Description Drosophila kettin 
Organisation University of York
Department York Environmental Sustainability Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution I have obtained Drosophila expressing fluorescently labelled kettin and I have isolated flight muscle for microscopy. I have trained 3 students in dissection and microscopy. The students used the samples for high resolution microscopy with Mark Leake (Physics, York). My lab has made recombinant kettin fragments. These have been combined with actin and the structure of the filaments has been determined by tomography of electron microscopy images by Vitold Galkin (East Virginia Medical School) .
Collaborator Contribution Chris Elliott (Biology) crossed flies to produce heterozygotes and checked for fluorescence of the thorax. He trained 3 students in fly culture. Mark Leake (Physics) used his bespoke high resolution microscope to examine the muscle. The students obtained the results. Vitold Galkin analysed Kettin-actin complexes and determined the structure.
Impact Kettin was observed in the middle of the sarcomere, which was unexpected, and on the Z-disc, which was expected. The Z-disc label had blinking fluorescence, possibly indicating rapid turnover of kettin. We have determined that stoichiometry of kettin relative to actin is one Ig domain of kettin to one monomer of actin. We have run Western blots of gels suitable for identifying high molecular weight proteins like kettin and its isoforms. This has shown that the relative amounts of kettin tagged with GFP to untagged kettin in the flight muscle of heterozygous flies is 30:70. This measurement is needed in order to estimate the stoichiomentry of kettin domains and actin in the sarcomere. The collaboration is multidisciplinary, involving biology, molecular biology and physics.
Start Year 2016
 
Description Mutation of titin Ig domains Liverpool 
Organisation University of Liverpool
Country United Kingdom 
Sector Academic/University 
PI Contribution I measured binding between actin and recombinant protein from Mayans' lab. The peptides consisted of wild type and mutant Ig domains from the elastic muscle protein, titin. I showed the peptides did not bind to actin.
Collaborator Contribution Mayans' lab showed that a mutation in a domain of titin in the I-band region destabilises the domain, which affects the elasticity of titin and the compliance of the muscle. The effects of the mutation on the heart muscle can be explained by these changes.
Impact The work on the effects of a mutation in cardiac titin was published. This will lead to a better understanding of the cause of cardiac disease at the molecular level. PMID 27683155 The work is multidisciplinary. Collaboration is between a molecular biologist and a protein chemist and a physiologist.
Start Year 2015
 
Description Sequence of the Lethocerus genome 
Organisation Duke University Medical Centre
Country United States 
Sector Academic/University 
PI Contribution We provided muscle tissue to Shanghai, which was used in an initial attempt at sequencing the genome. This was unsuccessful. RNA prepared from fresh muscle provided by us, was made by our collaborators at Duke University and taken to Hong Kong. A team from Duke University visited my lab at the University of York. We imported Lethocerus from Thailand and dissected 200 water bugs. Fresh tissue was prepared for isolating DNA for sequencing the Lethocerus genome. Our Bioinformatics collaborators at York have prepared long stretches of genomic DNA for sequencing. By using the nanopore technique, we have now determined the complete genome sequence of Lethocerus indicus. This was possible once we obtained MinION and PromethION cells from the Oxford Nanopore company.
Collaborator Contribution Our collaborators in Shanghai prepared a draft version of the Lethocerus genome, obtained by the Illumina sequencing method. They provided the personal needed for the task. They obtained a scaffold sequence. The sequences of particular genes have been provided when needed. For example, the sequence of the myosin gene has been used for identifying isoforms in the flight and non-flight muscles by our collaborators at Duke University. Sequences of 2 tropomyosin genes have enabled us to identify the isoforms in flight muscle. More recently, colleagues in Bioinformatics in York, have isolated genomic DNA from frozen flight muscle. This had been used for sequencing with a MinION and PromethION nanopore cells. Our Shanghai collaborators are assembling and annotating the 450 Mb genome for publication.
Impact We now have the sequence of the Lethocerus genome. 8 myosin isoforms in flight and non-flight muscle have been identified and sequenced. This work led to a publication PMID: 28707142 Two tropomyosin isoforms have been identified in flight muscle and sequenced. One of these is likely to be required for stretch-activation of the muscle. The collaboration is multi-disciplinary involving Genomics, Biology and Bioinformatics.
Start Year 2015
 
Description Sequence of the Lethocerus genome 
Organisation Shanghai Center for Bioinformation Technology
Country China 
Sector Academic/University 
PI Contribution We provided muscle tissue to Shanghai, which was used in an initial attempt at sequencing the genome. This was unsuccessful. RNA prepared from fresh muscle provided by us, was made by our collaborators at Duke University and taken to Hong Kong. A team from Duke University visited my lab at the University of York. We imported Lethocerus from Thailand and dissected 200 water bugs. Fresh tissue was prepared for isolating DNA for sequencing the Lethocerus genome. Our Bioinformatics collaborators at York have prepared long stretches of genomic DNA for sequencing. By using the nanopore technique, we have now determined the complete genome sequence of Lethocerus indicus. This was possible once we obtained MinION and PromethION cells from the Oxford Nanopore company.
Collaborator Contribution Our collaborators in Shanghai prepared a draft version of the Lethocerus genome, obtained by the Illumina sequencing method. They provided the personal needed for the task. They obtained a scaffold sequence. The sequences of particular genes have been provided when needed. For example, the sequence of the myosin gene has been used for identifying isoforms in the flight and non-flight muscles by our collaborators at Duke University. Sequences of 2 tropomyosin genes have enabled us to identify the isoforms in flight muscle. More recently, colleagues in Bioinformatics in York, have isolated genomic DNA from frozen flight muscle. This had been used for sequencing with a MinION and PromethION nanopore cells. Our Shanghai collaborators are assembling and annotating the 450 Mb genome for publication.
Impact We now have the sequence of the Lethocerus genome. 8 myosin isoforms in flight and non-flight muscle have been identified and sequenced. This work led to a publication PMID: 28707142 Two tropomyosin isoforms have been identified in flight muscle and sequenced. One of these is likely to be required for stretch-activation of the muscle. The collaboration is multi-disciplinary involving Genomics, Biology and Bioinformatics.
Start Year 2015
 
Description Sequence of the Lethocerus genome 
Organisation University of York
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided muscle tissue to Shanghai, which was used in an initial attempt at sequencing the genome. This was unsuccessful. RNA prepared from fresh muscle provided by us, was made by our collaborators at Duke University and taken to Hong Kong. A team from Duke University visited my lab at the University of York. We imported Lethocerus from Thailand and dissected 200 water bugs. Fresh tissue was prepared for isolating DNA for sequencing the Lethocerus genome. Our Bioinformatics collaborators at York have prepared long stretches of genomic DNA for sequencing. By using the nanopore technique, we have now determined the complete genome sequence of Lethocerus indicus. This was possible once we obtained MinION and PromethION cells from the Oxford Nanopore company.
Collaborator Contribution Our collaborators in Shanghai prepared a draft version of the Lethocerus genome, obtained by the Illumina sequencing method. They provided the personal needed for the task. They obtained a scaffold sequence. The sequences of particular genes have been provided when needed. For example, the sequence of the myosin gene has been used for identifying isoforms in the flight and non-flight muscles by our collaborators at Duke University. Sequences of 2 tropomyosin genes have enabled us to identify the isoforms in flight muscle. More recently, colleagues in Bioinformatics in York, have isolated genomic DNA from frozen flight muscle. This had been used for sequencing with a MinION and PromethION nanopore cells. Our Shanghai collaborators are assembling and annotating the 450 Mb genome for publication.
Impact We now have the sequence of the Lethocerus genome. 8 myosin isoforms in flight and non-flight muscle have been identified and sequenced. This work led to a publication PMID: 28707142 Two tropomyosin isoforms have been identified in flight muscle and sequenced. One of these is likely to be required for stretch-activation of the muscle. The collaboration is multi-disciplinary involving Genomics, Biology and Bioinformatics.
Start Year 2015
 
Description Structural dynamics of stretch activation in muscle and Myofibrillar structure of striated muscle 
Organisation Duke University
Department Department of Cell Biology
Country United States 
Sector Academic/University 
PI Contribution I have made and supplied muscle proteins and antibodies for Duke. I have also given my collaborators glycerinated insect muscle. My collegues at Duke University are long-standing collaborators. I am subcontracted to two NIH grants awarded to Dr M. K. Reedy. I have measured mechanical properties of flight muscle and discussed optimal conditions with Duke. A team from Duke visited my lab to dissect muscles from Lethocerus that we imported from Thailand. Cryo-preserved muscle was sent to Durham, North Carolina. I have given Dr Reedy's group cDNA from Lethocrus flight muscle to enable them to sequence myosin isoforms.
Collaborator Contribution Duke has provided York with washed glycerinated muscle fibres. They have also collected water bugs from Thailand, which we both use in our research. We share our results and are in constant communication, which has helped our work enormously. They have also provided enzymes and proteins needed for isolating myosin filaments from muscle. They have critically evaluated the mechanical results we obtained from fibres, and they designed many of the methods for the mechanics.
Impact We have detemined the position of regulatory proteins in insect flight muscle. We have made the regulatory complex from myofibrils provided by Duke University. We have also found optimal conditions for exchanging troponin components in flight muscle for mechanical measurements. Duke has sequenced a muscle protein (myosin) using cDNA provided by us. We have investigated the regulation of flight muscle contraction by calcium, using mechanical techniques suggested by Duke. The collaboration is multidisciplinary: between biochemists (York) and electron microscopists (Duke) and experts in X-ray diffraction (Duke). A recent publication arising from the work is PMID: 28707142.
Start Year 2015
 
Description The mechanism of stretch activation in muscle: a multidiciplinary approach-2 
Organisation King's College London
Department Maurice Wohl Clinical Neuroscience Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution We have prepared muscle specimens for spectroscopic work. The King's group, and the PI of this investigation at the Wohl Institute (also King's College) are collaborators. We have prepared a new batch of Lethocerus thoraces for isolation of muscle fibres. These were used for mechanics and simultaneous measurement of changes in the angle of troponin C. We have tested different methods of exchanging troponin C in fibres in order to incorporate the protein labelled with a divalent probe.
Collaborator Contribution The King's group are studying the changes in the regulatory protein, troponin C, in insect flight muscle. They contribute essential spectroscopic measurements. The Wohl group have made proteins that are labelled with fluorescent probes for replacing endogenous troponin C in the muscle fibres. King's have made preliminary measurements of changes in troponin C when the muscle is activated. The angle of troponin C changes when calcium is added to a fibre. After stretching, there is a further change in troponin C and force increases more slowly.
Impact The Wohl Institute has made divalent probes on troponin C. King's have preliminary spectroscoic results with a single probe on troponin C. Preliminary results show that the orientation of troponin C changes very little when calcium is added to fibres and no force is developed. A larger change occurs when fibres are stretched and force increases after a delay. The collaboration is between biochemists (York), who provide insect muscles. spectroscopists (Wohl) and physicists (King's), who perform spectroscopy.
Start Year 2015
 
Description The mechanism of stretch activation in muscle: a multidiciplinary approach-2 
Organisation King's College London
Department Randall Division of Cell & Molecular Biophysics
Country United Kingdom 
Sector Academic/University 
PI Contribution We have prepared muscle specimens for spectroscopic work. The King's group, and the PI of this investigation at the Wohl Institute (also King's College) are collaborators. We have prepared a new batch of Lethocerus thoraces for isolation of muscle fibres. These were used for mechanics and simultaneous measurement of changes in the angle of troponin C. We have tested different methods of exchanging troponin C in fibres in order to incorporate the protein labelled with a divalent probe.
Collaborator Contribution The King's group are studying the changes in the regulatory protein, troponin C, in insect flight muscle. They contribute essential spectroscopic measurements. The Wohl group have made proteins that are labelled with fluorescent probes for replacing endogenous troponin C in the muscle fibres. King's have made preliminary measurements of changes in troponin C when the muscle is activated. The angle of troponin C changes when calcium is added to a fibre. After stretching, there is a further change in troponin C and force increases more slowly.
Impact The Wohl Institute has made divalent probes on troponin C. King's have preliminary spectroscoic results with a single probe on troponin C. Preliminary results show that the orientation of troponin C changes very little when calcium is added to fibres and no force is developed. A larger change occurs when fibres are stretched and force increases after a delay. The collaboration is between biochemists (York), who provide insect muscles. spectroscopists (Wohl) and physicists (King's), who perform spectroscopy.
Start Year 2015
 
Description The mechanism of stretch activation in muscle:a multidiciplinary approach-1 
Organisation European Molecular Biology Laboratory
Country Germany 
Sector Academic/University 
PI Contribution We have provided DNA constructs of the regulatory proteins in insect muscle, which are used for expressing proteins. In some cases, we have provided bacterial pellets of expressed proteins. We have helped with the expression and assembly of the troponin complex from engineered subunits. We have checked some complexes by electron microscopy. We have isolated the tropomyosin-troponin complex from Lethocerus flight muscle and demonstrated specific binding between the complex and myosin filaments. We have identified two tropomyosin isofoms in flight muscle and shown that one binds with high affinity to myosin. The shape of a complete tropomyosin-troponin complex and tropomyosin alone has been determined by small angle X-ray diffraction (SAXS) in collaboration with the Wohl Institute and EMBL Hamburg.
Collaborator Contribution Collaborators at the Wohl Institute have expressed proteins and made NMR measurements on the subunits and complexes of troponin. They have reconstituted the regulatory complex containing truncated subunits and determined the structure of the complexes by NMR. This showed similarities and differences in comparison with the complex assembled with vertebrate proteins. Collaborators at EMBL Hamburg (Dmitri Svergun) have determined the shape of the tropomyosin-troponin complex and tropomyosin isolated from muscle by SEC-MALS and SAXS. They have also determined the shape of the complex with truncated subunits.
Impact Collaborators at the Wohl Institute have determined the NMR structure of the flight muscle troponin complex assembled from truncated subunits. Collaborators at EMBL Hamburg have determined the shape of the whole tropomyosin-troponin complex by SAXS. We have shown that a link between tropomyosin and myosin is likely to be a stretch-sensor in flight muscle. Isolation of the native tropomyosin-troponin complex and the demonstration that it binds to thick filaments in flight muscle will pave the way for further measurements on the structure and mechanical properties of the complex. There is a publication from this work PMID: 27226601 The collaboration is multidisciplinary between biochemists (York) and spectroscopists (Wohl) and X-ray diffraction experts (EMBL Hamburg)
Start Year 2015
 
Description The mechanism of stretch activation in muscle:a multidiciplinary approach-1 
Organisation King's College London
Department Maurice Wohl Clinical Neuroscience Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided DNA constructs of the regulatory proteins in insect muscle, which are used for expressing proteins. In some cases, we have provided bacterial pellets of expressed proteins. We have helped with the expression and assembly of the troponin complex from engineered subunits. We have checked some complexes by electron microscopy. We have isolated the tropomyosin-troponin complex from Lethocerus flight muscle and demonstrated specific binding between the complex and myosin filaments. We have identified two tropomyosin isofoms in flight muscle and shown that one binds with high affinity to myosin. The shape of a complete tropomyosin-troponin complex and tropomyosin alone has been determined by small angle X-ray diffraction (SAXS) in collaboration with the Wohl Institute and EMBL Hamburg.
Collaborator Contribution Collaborators at the Wohl Institute have expressed proteins and made NMR measurements on the subunits and complexes of troponin. They have reconstituted the regulatory complex containing truncated subunits and determined the structure of the complexes by NMR. This showed similarities and differences in comparison with the complex assembled with vertebrate proteins. Collaborators at EMBL Hamburg (Dmitri Svergun) have determined the shape of the tropomyosin-troponin complex and tropomyosin isolated from muscle by SEC-MALS and SAXS. They have also determined the shape of the complex with truncated subunits.
Impact Collaborators at the Wohl Institute have determined the NMR structure of the flight muscle troponin complex assembled from truncated subunits. Collaborators at EMBL Hamburg have determined the shape of the whole tropomyosin-troponin complex by SAXS. We have shown that a link between tropomyosin and myosin is likely to be a stretch-sensor in flight muscle. Isolation of the native tropomyosin-troponin complex and the demonstration that it binds to thick filaments in flight muscle will pave the way for further measurements on the structure and mechanical properties of the complex. There is a publication from this work PMID: 27226601 The collaboration is multidisciplinary between biochemists (York) and spectroscopists (Wohl) and X-ray diffraction experts (EMBL Hamburg)
Start Year 2015
 
Description The roles of troponin C isoforms in the mechanical function of Drosophila indirect flight muscle 
Organisation Rensselaer Polytechnic Institute
Department Department of Biological Sciences
Country United States 
Sector Academic/University 
PI Contribution We analysed proteins in Drosophila mutants, including RNAi lines in which expression of troponin C isoforms was eliminated or reduced. We ran flight tests and determined the structure of mutant muscle by fluorescent and electron microscopy. Reduction of TnC4 resulted in abnormal structure of the flight muscle.
Collaborator Contribution Collaborators bred mutant flies and performed mechanics on the muscle. Reduction of troponin C isoform, TnC1, in flight muscle had no effect of the mechanics. Reduction of TnC4 resulted in the elimination of tension development in the muscle. TnC4 is essential for the function of flight muscle.
Impact We have a joint publication, with cover picture (PMID 25134799). 2 graduate students and an Erasmus student were trained in fluorescent microscopy and RNAi techniques with Drosophila regulatory proteins. Multi-disciplinary project: Rensselaer Polytechnic performed mechanical experiments. York did spectroscopy and Biochemistry. The work has been continued by our Rensselaer collaborator, using Drosophila mutants in which troponin C isoforms were exchanged using CRISPR-Cas9 gene editing.
Start Year 2015