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

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.

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.