The structure and assembly of collagen VI using cryo-EM and SAXS

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

The extracellular matrix is all the material that is found outside of the cell and it makes up most of the tissues in our bodies. The matrix is composed of an intricate network of proteins which provides support for cells and the framework for tissues. The structure and organisation of most matrix proteins is poorly understood as they are very complicated, and it has proved a major challenge to understand their organisation. However, new approaches are now beginning to reveal important information on their assembly and structural organisation. Collagen VI microfibrils are important components of the extracellular matrix. These microfibrils have widespread distributions throughout the body where they form many linkages between cells and proteins. Mutations in collagen VI are linked to inherited muscular dystrophies, highlighting how important it is to study this molecule. The aims of this project are to investigate the structure and function of collagen VI microfibrils using the technique of cryo-Electron Microscopy. Many low contrast images of frozen hydrated microfibrils will be averaged to produce a 3D structure. Further details will be added by looking at the shape of small fragments of the collagen VI protein and mapping these onto the 3D structure of the microfibril. We will also look at larger assemblies of collagen VI in lattice-like structures, similar to those found in human tissues. The structure produced from these data will increase our understanding of the organisation of these complicated microfibrils.

Technical Summary

Collagen VI is expressed in virtually all connective tissues, where it forms a complex and extensive microfibrillar network. The cell adhesive and extracellular matrix protein binding capabilities of collagen VI suggests it plays an important role in the interconnection between the cell and the structural scaffolding of the extracellular matrix. One critical tissue-specific role has been demonstrated by the detection of mutations in the collagen VI genes which give rise to heritable muscular dystrophies, highlighting its vital role in muscle anchorage. This study will determine the 3D organisation of hydrated collagen VI microfibrils using cryo-EM and single particle image analysis, which will generate the first model of these unique biopolymers in physiological hydrated state. Recombinant microfibrils will be generated to define the change in microfibril structure in different splice forms. Model building into the EM envelope will be aided using structures of contiguous blocks of A-domains determined by small angle x-ray solution scattering. The locations of these domains within the microfibril will be found using antibody epitope mapping. Contiguous blocks of A-domains, which equate to naturally occurring splice forms will be expressed, in order to determine the arrangement and configuration of domains upon splicing. Suprafibrillar assemblies will be reconstituted in vitro, in order to study higher order collagen VI assemblies. Electron tomography and image analysis will be utilised to study the 3D structure and organisation of higher order assemblies, to increase our understanding of collagen VI network formation in both healthy and diseased tissues. Refined alignment models produced from these data will increase our understanding of collagen VI molecular architecture. This fundamental biology of collagen VI is required for understanding its biological roles, tissue engineering applications, and defining the abnormal structures found in pathological tissues.

Publications

10 25 50
 
Description The extracellular matrix is all the material that is found outside of the cell and it makes up most of the tissues in our bodies. The matrix is composed of an intricate network of proteins which provides support for cells and the framework for tissues. The structure and organisation of most matrix proteins is poorly understood as they are very complicated, and it has proved a major challenge to understand their organisation. However, new approaches are now beginning to reveal important information on their assembly and structural organisation.

Collagen VI microfibrils are important components of the extracellular matrix. These microfibrils have widespread distributions throughout the body where they form many linkages between cells and proteins. Mutations in collagen VI are linked to inherited muscular dystrophies, highlighting the importance of this molecule in tethering the extracellular matrix to cells.

The aims of this project were to investigate the structure and function of collagen VI microfibrils using the technique of Electron Microscopy with image analysis. We collected many thousand images of microfibrils extracted from tissue or cell cultures. These were imaged either using negative staining or frozen in a thin layer of ice. The microfibril images were computationally processed to calculate a 3D structure. The 3D structure highlighted flexibility in one region of collagen VI which we had previously suggested could be responsible for interacting with extracellular matrix molecules. In order to gain further details on this region we produced it in cells and looked at its shape and the interactions of this region of collagen VI with other extracellular matrix molecules found in human tissues. These data highlighted competition for binding between molecules and a role for metal-ions in changing the conformation of this region and its assembly. We mapped this region into the 3D structure of the microfibril, filling in a piece of this jigsaw. We also analysed the structure of collagen VI microfibrils isolated from cells taken from patients with Ullrich's congenital muscular dystrophy (UCMD). Although these microfibrils appeared to have a normal structure they were only able to form short microfibrils compared to normal individuals. The structures and information produced from these data has increase our understanding of the organisation of these complicated microfibrils, the effects of UCMD-causing mutations on microfibril assembly and has highlighted a novel role for metal-ions in stabilising collagen VI structure and assembly.
Exploitation Route An understanding of the structure and assembly of collagen VI and the defects seen in microfibrils from UCMD patients may lead to the development of therapeutics for this disease. The use of collagen VI as a scaffold protein for regenerative medicine is also of interest to this field.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description We have developed improved methods for purifying collagen VI microfibrils from tissue (cornea) which has led to two new collaborations where we have provided material for stem cell and vascular endothelial cell studies. We have also generated a stably transfected mammalian cell line expressing the N9-N1 region of the alpha 3 chain of collagen VI. The protein is expressed to high amounts and is stable. UG and MSc student projects have used this material for teaching purposes. Collagen VI microfibrils have proved a suitable model for Dr Alan Roseman, The University of Manchester, to use as a test sample in software development for heterogeneous samples. We have subsequently obtained a BBSRC DTP Studentship to further this collaboration.
First Year Of Impact 2010
Sector Education,Healthcare
Impact Types Societal

 
Description Shireen Lamande, Melbourne 
Organisation University of Melbourne
Department Murdoch Children's Research Centre
Country Australia 
Sector Academic/University 
PI Contribution Electron microscopy of collagen VI microfibrils from Ullrich Congenital Muscular Dystrophy patients.
Collaborator Contribution Provided skin fibroblasts isolated from patients. Provided SaoS-2 cell line transfected with a truncated alpha3(VI) chain
Impact Collagen VI microfibril formation is abolished by an {alpha}2(VI) von Willebrand factor type A domain mutation in a patient with Ullrich congenital muscular dystrophy. Tooley LD, Zamurs LK, Beecher N, Baker NL, Peat RA, Adams NE, Bateman JF, North KN, Baldock C, Lamandé SR. J Biol Chem. (2010) 285:33567-76.
Start Year 2008
 
Description Elastin and Microfibrils Gordon Research Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other audiences
Results and Impact Biannual invited talk at the Elastin GRC for PI Clair Baldock since 2003. Talk facilitated discussion of the use of SAXS for other elastic fibre proteins.

Elected vice-chair of GRC for 2015 (chair in 2017).
Collaboration on SAXS of tropoelastin has led to 2 PNAS, 1 JBC and 1 Matrix Biology papers to date.
Talks led to invitation to visit two Institutions in Canada (Sick Kids Hospital, Toronto and Simon Fraser University, Vancouver) and to Shriners Hospital in Portland in 2011.
Year(s) Of Engagement Activity Pre-2006,2006,2007,2009,2011,2013,2015,2017,2019
 
Description European Elastin Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact PI Clair Baldock and group members attend biannual European Elastin Meetings to network with European community and raise profile of research.

Invitation to participate in a Marie Curie Sklodowska ITN network and elected vice-chair of the Elastin Gordon conference for 2015 (Chair in 2017).
Year(s) Of Engagement Activity 2008,2010,2012,2014,2016,2018
 
Description Matrix Biology Europe 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talks and posters stimulated discussion.

Mukti Singh (BBSRC DTP PhD student) was awarded a poster prize.

After talks I was asked to talk at National Societies including the German Connective Tissues Society Meeting in Cologne in 2011 and the French Connective Tissue Society Meeting in 2012.
Led to participation in an application for a Marie Curie Sklodowska ITN network.
Year(s) Of Engagement Activity 2010,2012,2018
 
Description Nuffield Bursary Scheme 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact A-level students hosted for up to four weeks in the lab. This involved designing a project, supervising the student in the lab and helping the student write a scientific report.

More students to get involved in science A-levels.
Year(s) Of Engagement Activity 2008,2010,2012,2014
 
Description S4SAS Workshop and Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact In 2010, PI Clair Baldock gave an invited seminar at the inaugral S4SAS meeting.
In 2012, PDRA Dr Chris Bayley attended a training workshop in small angle X-ray scattering to gain skills in this technique. He also presented a poster to the user community for discussion and advice.
In 2014, PhD student then PDRA Dr Helen Troilo gave a talk and poster and was awarded the poster prize for the meeting. Helen subsequently was award the runner up proze for the Diamond PhD Investigator of the year Award 2014.

CB invited on the steering committees for Diamond's new protein scattering beamline B21 and software.
New methods were suggested in discussion and use of the OPPF facility which we followed up.
Year(s) Of Engagement Activity 2010,2012,2014
 
Description Wellcome to the Matrix 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Annual event to provide the opportunity for Key Stage 3 and 4 students from local schools in Manchester to gain insight into biological research. Each 'Wellcome to the Matrix' day encourages students to work in teams to design, create and present models of particular areas of research in which we are involved.

When surveyed after the visit more students expressed an interest in taking science GCSEs.
Year(s) Of Engagement Activity 2008,2009,2012,2013