A multilevel approach to define the hierarchical organisation of extracellular matrix microfibrils A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy (PhD) in the School of Biological Sciences in the the Faculty of Biology, Medicine and Health 2016 Alan Robert Francis Godwin List of contents List of contents................................................................................................................ 2 List of figures .................................................................................................................. 6 List of tables ................................................................................................................... 9 List of abbreviations ........................................................................................................ 9 Abstract ........................................................................................................................ 13 Declaration ................................................................................................................... 14 Copyright statement ...................................................................................................... 14 Acknowledgements ....................................................................................................... 15 1 Introduction ............................................................................................................ 16 1.1 Fibrillin microfibrils .................................................................................................. 16 1.1.1 Fibrillin family ................................................................................................... 16 1.1.2 Fibrillin domain organisation ............................................................................ 17 1.1.2.1 Fibrillin unique N-terminal domain ............................................................ 18 1.1.2.2 EGF domains ........................................................................................... 19 1.1.2.3 TB domains .............................................................................................. 19 1.1.2.4 Hybrid domain .......................................................................................... 19 1.1.3 Fibrillin microfibril assembly ............................................................................. 20 1.1.4 Fibrillin microfibril structure .............................................................................. 21 1.1.5 Fibrillin packing models ................................................................................... 23 1.1.6 Fibrillin tissue organisation............................................................................... 26 1.1.7 The fibrillin microenvironment .......................................................................... 27 1.1.7.1 Elastic fibre associated proteins ............................................................... 27 1.1.7.2 Elastin ...................................................................................................... 27 1.1.7.3 Fibulins ..................................................................................................... 28 1.1.7.4 LOX .......................................................................................................... 28 2 1.1.7.5 Elastic fibre assembly ............................................................................... 29 1.1.7.6 Integrins ................................................................................................... 29 1.1.7.7 Proteoglycans ........................................................................................... 30 1.1.7.8 ADAMTS and ADAMTSL .......................................................................... 31 1.1.7.9 MAGPs ..................................................................................................... 32 1.1.7.10 Regulation of growth factor bioavailability ................................................. 33 1.1.7.11 LTBPs ...................................................................................................... 33 1.1.7.12 BMPs ........................................................................................................ 34 1.1.8 Fibrillinopathies ................................................................................................ 34 1.2 Fibrillin summary..................................................................................................... 38 1.3 Collagen VI microfibrils ........................................................................................... 39 1.3.1 Collagen VI α chains ........................................................................................ 39 1.3.2 Collagen VI α chain domain organisation ......................................................... 39 1.3.2.1 Collagen VI helical region ......................................................................... 41 1.3.2.2 Globular domains ..................................................................................... 42 1.3.3 Collagen VI microfibril assembly ...................................................................... 43 1.3.4 Collagen VI microfibril structure ....................................................................... 44 1.3.5 Collagen VI function......................................................................................... 46 1.3.5.1 Collagen VI and the cell surface ............................................................... 47 1.3.5.2 Collagen VI and PCM structure ................................................................ 48 1.3.6 Collagen VI tissue organisation ....................................................................... 48 1.3.7 Collagen VI diseases ....................................................................................... 49 1.3.8 Knockout mouse models of Bethlem myopathy ............................................... 50 1.4 Collagen VI summary ............................................................................................. 50 1.5 Project aims ............................................................................................................ 51 2 Materials and methods .......................................................................................... 53 2.1 Tissue sources ....................................................................................................... 53 2.2 Tissue culture ......................................................................................................... 53 2.3 Microfibril extractions .............................................................................................. 53 2.3.1 Fibrillin tissue microfibrils ................................................................................. 53 2.3.2 Collagen VI tissue microfibrils .......................................................................... 54 3 2.3.3 N6-C5 collagen VI microfibrils .......................................................................... 54 2.4 Sodium dodecyl sulphate polyacrylamide gel electrophoresis ................................. 55 2.5 Western blotting ...................................................................................................... 55 2.6 Transmission electron microscopy .......................................................................... 55 2.6.1 The transmission electron microscope ............................................................. 55 2.6.2 Image formation and contrast .......................................................................... 57 2.6.3 Sample preparation ......................................................................................... 58 2.6.4 Single particle 3D reconstruction ..................................................................... 59 2.6.4.1 CTF correction .......................................................................................... 59 2.6.4.2 Alignment ................................................................................................. 59 2.6.4.3 Classification ............................................................................................ 60 2.6.4.4 Determination of particle orientation ......................................................... 60 2.6.4.5 3D reconstruction ..................................................................................... 61 2.7 Negative stain TEM ................................................................................................ 62 2.8 Cryo-TEM ............................................................................................................... 63 2.8.1 Fibrillin microfibrils ........................................................................................... 63 2.8.2 Collagen VI microfibrils .................................................................................... 63 2.9 Single particle averaging and 3D model reconstruction of matrix microfibrils .......... 63 2.9.1 Fibrillin ............................................................................................................. 63 2.9.1.1 Fibrillin sub-models ................................................................................... 66 2.9.2 Collagen VI ...................................................................................................... 66 2.9.2.1 Single bead model .................................................................................... 66 2.9.3 Docking of fibrillin-1