A Multilevel Approach to Define the Hierarchical Organisation of Extracellular Matrix Microfibrils

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

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

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

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 fragment molecular models ...... 66

2.10 SBF-SEM and electron tomography sample preparation ...... 66 2.10.1 Bovine ciliary zonule ...... 66 2.10.2 Murine ciliary zonule ...... 67 2.10.3 Murine cartilage ...... 67

2.11 SBF-SEM ...... 67 2.11.1 Bovine ciliary zonule ...... 67 2.11.2 Murine ciliary zonule ...... 68 2.11.3 Murine articular cartilage ...... 68

2.12 Electron tomography ...... 68 2.12.1 Bovine ciliary zonule ...... 68 2.12.2 Mouse articular cartilage ...... 68 2.12.3 Particle analysis in ImageJ ...... 69

2.13 Bovine ciliary zonule microfibril averaging ...... 69

2.14 AFM ...... 69

3 Results chapter 1: Nano-scale structure of fibrillin microfibrils...... 71

3.1 Microfibril single particle averaging and model reconstruction...... 71 3.1.1 Individual microfibril region reconstructions ...... 77 3.1.1.1 Optimising symmetry restrained sub-model reconstruction ...... 78 3.1.1.2 Bead region model ...... 78 3.1.1.3 Arm region model ...... 81 3.1.1.4 Interbead region model ...... 84 3.1.1.5 Shoulder region model...... 86 3.1.2 Docking of molecular models ...... 89 3.1.3 Cryo-TEM ...... 95

3.2 Discussion and conclusions ...... 98

4 Results chapter 2: Fibrillin microfibril tissue micro-structure ...... 103

4.1 Murine ciliary zonule structure ...... 104

4.2 Bovine ciliary zonule structure ...... 110 4.2.1 Correlative bovine ciliary zonule tomography ...... 111 4.2.2 Ciliary zonule fibres contact the basement membrane of non-pigmented ciliary epithelial cells ...... 116

4.3 Discussion ...... 118

5 Results chapter 3: Nano and micro-scale organisation of collagen VI microfibrils 121

5.1 Collagen VI microfibril single particle averaging and 3D model reconstruction ...... 121 5.1.1 AFM of extracted bovine collagen VI microfibrils ...... 130

5.2 N6-C5 α3 chain collagen VI microfibril ...... 131

5.3 3D reconstructions of murine chondrocyte PCM ...... 134

5.3.1 Murine articular cartilage PCM structure ...... 134

5.4 Discussion and conclusions ...... 139

6 Final discussion and conclusions ...... 142

6.1 Fibrillin microfibril nanoscale structure ...... 142

6.2 Fibrillin microfibril microscale structure ...... 144

6.3 Collagen VI nanoscale structure ...... 146

6.4 Collagen VI microscale structure ...... 147

7 Future work...... 149

7.1 Fibrillin microfibril organisation ...... 149

7.2 Collagen VI microfibril organisation ...... 150

7.3 Summary ...... 152

8 References ...... 153

Appendix 1: Initial fibrillin microfibril processing ...... 182

Word Count: 48,246

List of figures

Figure 1.1 Domain organisation of fibrillin-1 ...... 18

Figure 1.2 Diagram of fibrillin microfibril assembly...... 21

Figure 1.3 Diagram of a fibrillin microfibril ...... 23

Figure 1.4 Microfibril organisation models...... 25

Figure 1.5 Elastic fibre assembly ...... 29

Figure 1.6 Domain organisation of Collagen VI α chain 1-6...... 41

Figure 1.7 Collagen VI microfibril assembly...... 44

Figure 1.8 Collagen VI microfibril structure ...... 46

Figure 2.1 A schematic diagram of an electron microscope...... 57

Figure 2.2 Single particle averaging and 3D model reconstruction method ...... 65

Figure 3.1 Size exclusion chromatography of ciliary zonule extracts...... 72

Figure 3.2 Extracted bovine ciliary zonule microfibrils...... 73

Figure 3.3 3D EM model of extracted bovine fibrillin...... 75

Figure 3.4 Microfibril model resolution estimation ...... 76

Figure 3.5 Microfibril sub-region masks...... 78

Figure 3.6 3D reconstruction of the fibrillin microfibril bead region...... 80

Figure 3.7 Symmetry analysis and resolution estimation of the bead region sub-model ...... 81

Figure 3.8 3D reconstruction of fibrillin microfibril arm region...... 82

Figure 3.9 Symmetry analysis and resolution estimation of the arm region sub-model ...... 83

Figure 3.10 3D reconstruction of the fibrillin microfibril interbead region...... 85

Figure 3.11 Symmetry analysis and resolution estimation of the interbead region sub-model ...... 86

Figure 3.12 3D reconstruction of the fibrillin microfibril shoulder region...... 87

Figure 3.13 Symmetry analysis and resolution estimation of the shoulder region sub-model 88

Figure 3.14 SAXS structures and domain schematic of fibrillin fragments...... 90

Figure 3.15 Docking of fibrillin molecular models according to the molecular pleated model. 92

Figure 3.16 Docking of fibrillin molecular models according to the half-staggered model ...... 94

Figure 3.17 Cryo-TEM micrographs of extracted bovine ciliary zonule microfibrils ...... 96

Figure 3.18 Schematic diagram of the fibrillin microfibril folding models ...... 102

Figure 4.1 Schematic diagram of a cross section of a human eye...... 104

Figure 4.2 Murine ciliary zonule tissue imaged with SBF-SEM...... 106

Figure 4.3 3D reconstructions of Murine ciliary zonule...... 108

Figure 4.4 Murine ciliary zonule fibre diameter ...... 109

Figure 4.5 SBF-SEM of bovine ciliary zonule tissue...... 111

Figure 4.6 Correlative bovine ciliary zonule tomography ...... 113

Figure 4.7 3D reconstructions of a ciliary zonule microfibrils ...... 115

Figure 4.8 Tomography of ciliary body non-pigmented epithelium...... 116

Figure 4.9 Ciliary zonule microfibrils associate with the basement membrane of the NPCE...... 117

Figure 5.1 Size exclusion chromatography of bovine corneal extracts ...... 122

Figure 5.2 A cryo-TEM micrograph of extracted bovine collagen VI microfibrils...... 123

Figure 5.3 3D reconstruction of the bead region from collagen VI microfibrils ...... 125

Figure 5.4 3D model of the bead region of bovine corneal extracted collagen VI microfibrils ...... 126

Figure 5.5 Collagen VI half-bead 3D reconstruction ...... 128

Figure 5.6 3D reconstruction of the collagen VI half-bead...... 129

Figure 5.7 AFM analysis of collagen VI...... 130

Figure 5.8 Collagen VI N6-C5 microfibril purification ...... 132

Figure 5.9 Cell culture extracted N6-C5 collagen VI microfibrils ...... 133

Figure 5.10 Murine articular cartilage imaged with SBF-SEM ...... 135

Figure 5.11 Murine articular cartilage electron tomography ...... 137

Figure 5.12 A Histogram of chondrocyte PCM globular-density diameters ...... 138

Figure 5.13 A schematic model of the collagen VI bead region...... 140

List of tables

Table 2.1 Summary of sample imaging techniques used ...... 70

Table 3.1 Dimensions of fibrillin 3D model ...... 77

Table 3.2 Predicted volumes of microfibril sub-models ...... 89

Table 3.3 Docking of molecular models ...... 95

Table 3.4 Dimensions of fibrillin microfibril cryo-average ...... 97

Table 5.1 Dimensions of the half-bead of bovine corneal and N6-C5 collagen VI microfibrils ...... 134

List of abbreviations

AA Amino acid

AD Acromicric dysplysia

A disintegrin and metalloproteinase (reprolysin type) with ADAMTS thrombospondin type i motifs

A disintegrin and metalloproteinase (reprolysin type) with ADAMTSL thrombospondin type i motifs like

AU Absorbance units

BM Basement membrane

BMP Bone morphogenetic protein

CB Ciliary body

cbEGF Calcium binding epidermal growth factor like domain

CCA Contractural arachnodactyly

CS Chondroitin sulphate

CSPG Chondroitin sulphate proteoglycans

CZ Ciliary zonule

Da Dalton

DDD Direct detection devices

DMEM Dulbecco‟s modified medium

DNA Deoxyribonucleic acid

DS Dermatan sulphate

ECM Extracellular matrix

EDTA Ethylenediaminetetraacetic acid

EGF Epidermal growth factor like domain

EM Electron microscopy

ESEM Environmental scanning electron microscopy

FSC Fourier shell correlation

FUN Fibrillin unique N-terminal domain

GAG Glycosaminoglycan

GD Gelophysic dysplysia

GDF Growth differentiation factor

HS Heparan sulphate

HSPG Heparan sulphate proteoglycans kDa Kilodalton

LAP Latency associated peptide

LC Lens capsule

LLC Large latent complex

LOX Lysyl oxidase

LOXL Lysyl oxidase like

LTBP Latent TGFβ binding proteins

M Molar mA Milliamp

MAGP-1 Microfibril-associated glycoprotein-1 mAU Milli absorbance units

MFS Marfan syndrome

MIDAS Metal ion-dependent adhesion site

MMPs Matrix metalloproteinases

NEM N-ethylmaleimide nm Nanometre

NMR Nuclear magnetic resonance

NPCE Non-pigmented ciliary epithelium

OA Osteoarthritis

PBS Phosphate buffered saline

PCM Pericellular matrix

PF Protein fragment

PM Plasma membrane

PMSF Phenylmethane sulfonyl fluoride

RGD Arg-gly-asp

SAXS Small angle x-ray scattering

SBF-SEM Serial block face scanning electron microscopy

SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis

SEM Scanning electron microscopy

SEM Standard error of the mean

SLC Small latent complex

SLRP Small leucine rich proteoglycans

SNR Signal to noise ratio

SSS Stiff skin syndrome

STEM Scanning transmission electron microscopy

TB TGFβ binding protein like domain

TBS Tris buffered saline

TBST Tris buffered saline containing 0.05% tween20

TEM Transmission electron microscopy

TGFβ Transforming growth factor-β

UBM Ultra-sound biomicroscopy

UCMD Ullrich‟s congenital muscular dystrophy

UV Ultraviolet

V Volts

VWA Von Willibrand factor a domain

WMS Weill Marchesani syndrome

Abstract

The University of Manchester Alan Robert Francis Godwin Degree title: Doctor of Philosophy (PhD) A multilevel approach to define the hierarchical organisation of extracellular matrix microfibrils June 2016 Extracellular matrix (ECM) microfibrils are critical components of connective tissues with a wide range of mechanical and cellular signalling functions. The focus of this PhD thesis is the study of two microfibrillar assemblies, fibrillin-1 and collagen VI. Fibrillin is a large ECM glycoprotein which facilitates the deposition of elastin in elastic tissues such aorta, skin and lung and sequesters growth factors in the matrix. Collagen VI is a heteromeric network-forming collagen which is expressed in tissues such as skin, lung, blood vessels and articular cartilage where it anchors cells into the ECM allowing for the transduction of biochemical and mechanical signals. The structures of some individual domains and short fragments of both fibrillin and collagen VI have been solved, but it is not fully understood how they are arranged into microfibrils and how these microfibrils are arranged into tissues. Therefore the aim of this project has been to determine the hierarchical organisation of fibrillin and collagen VI across multiple length scales. The nanoscale structure of the fibrillin microfibril was determined using negative stain TEM and single particle reconstruction. Microfibrils had a hollow tube-like structure with well-defined bead, arm, interbead and shoulder regions. To overcome flexibility observed in the microfibril, separate sub-models of the different fibrillin regions were modelled. The bead region had a complex double layered structure with an interwoven core and ring structures. The arm region had four separate densities which are potentially formed from dimers of fibrillin molecules. Serial block face scanning electron microscopy (SBF-SEM) and electron tomography allowed for the in situ 3D imaging of individual fibrillin microfibrils in ciliary zonule tissue. Microfibrils in ciliary zonule fibres were held together by cross bridges between microfibrils. These ciliary zonule fibres were then organised into larger fascicle-like structures which were stabilised by circumferentially arranged ciliary zonule fibres. The frozen hydrated structure of the collagen VI half-bead was reconstructed using cryo-TEM. The half- bead region had a compact hollow head, and flexible tail regions, the tail regions were linked together by the collagenous interbead region. SBF-SEM and electron tomography of the pericellular matrix (PCM) of murine articular cartilage revealed that the PCM had a meshwork-like organisation formed from globular densities ~30 nm in diameter. Together a combinatorial approach to image ECM microfibrils from the sub- molecular level to intact tissue structures spanning nanometre to millimetre length scales is presented. This provides a better understanding of how fibrillin and collagen VI microfibrils are organised in tissues.

Declaration

No portion of the work referred to in this thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning.

I. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and s/he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. II. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. III. The ownership of certain Copyright, patents, designs, trade marks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. IV. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property University IP Policy (see http://documents.manchester.ac.uk/display.aspx?DocID=24420), in any relevant Thesis restriction declarations deposited in the University Library, The University Library‟s regulations (see http://www.library.manchester.ac.uk/about/regulations/) and in The University‟s policy on Presentation of Theses.

Acknowledgements

Firstly I would like to thank my supervisor Professor Clair Baldock and my co-supervisors Dr Alan Roseman and Dr Michael Sherratt for all the help and guidance they have given me during this degree.

I would also like to thank all the past and present members of the Baldock group for all their practical advice and help.

Thanks to Professor Anthony Day for being my advisor throughout this project.

I would also like to thank Dr Tobias Starborg and Dr Aleksandr Mironov and the rest of the EM facility for their help and advice duing this project.

Lastly I would like to thank my family and especially my wife Kayley for the years of love and support which she has given me and without whom I wouldn‟t be where I am today.

The work described here was funded by the BBSRC and was part of the Wellcome Trust centre for matrix research.

1 Introduction

Extracellular matrix (ECM) microfibrils are critical components of connective tissues with a wide range of mechanical and cellular signalling functions. To carry out these functions microfibrils have specific hierarchical organisations which are highly dependent on the tissue context. The focus of this PhD thesis is the study of two microfibrillar assemblies, fibrillin-1 and collagen VI. This introduction will outline the current knowledge of how fibrillin-1 and collagen VI are assembled and organised in the ECM.

1.1 Fibrillin microfibrils

Fibrillins are large ~350 kDa glycoproteins and are the main component of a class of connective tissue microfibrils which are expressed in many mammalian tissues such as the skin, lung and vasculature (Sakai et al., 1986). Fibrillins 1, 2, and 3 form microfibrils with a beads-on-a-string appearance which are a major component of elastic fibres conferring long range extensibility and contributing to the elastic deformation of tissues. Microfibrils also play a key role in tissue homeostasis through their interaction with growth factors such as transforming growth factor-β (TGFβ) and bone morphogenetic proteins (BMPs) and through interaction with cell surface receptors such as the integrins and syndecans. The importance of fibrillin-1 in the function of tissues is further highlighted as mutations in fibrillin-1 cause a number of heritable connective tissue disorders termed fibrillinopathies, such as Marfan syndrome (MFS) and Weill Marchesani syndrome (WMS). Disruption of fibrillin has also been implicated in stiffening of elastic tissues with age (reviewed in (Sherratt, 2013)).

1.1.1 Fibrillin family

The fibrillin family has three members fibrillin-1, fibrillin-2 and fibrillin-3 which are all products of the separate genes FBN1, FBN2 and FBN3 (Lee et al., 1991, Corson et al., 1993, Pereira et al., 1993, Zhang et al., 1994, Nagase et al., 2001, Corson et al., 2004). Mice do not have fibrillin-3 which is thought to be due to a chromosomal reorganisation (Corson et al., 2004).

Fibrillins 1 and 2 have been shown to have overlapping functions in development as mice with the double knockouts of fibrillin-1 and fibrillin-2 die at mid-gestation, much earlier than knockout of either gene (Carta et al., 2006). In humans fibrillin-2 and fibrillin-3 are primarily expressed in foetal tissues where fibrillin-2 is highly expressed in elastic fibre rich tissues such as the medial layer of the aorta (Zhang et al., 1994, Zhang et al., 1995) and fibrillin-3 in

16 the brain (Corson et al., 2004). The expression of fibrillin-1 increases after morphogenesis and is the dominant form of fibrillin in adult tissues (Zhang et al., 1995). Fibrillin-1 was identified by immunolabelling with monoclonal antibodies in connective tissue matrices of skin, lung, kidney, vasculature, cartilage, tendon, muscle, cornea, and ciliary zonule (Sakai et al., 1986).

1.1.2 Fibrillin domain organisation

The domain structure of fibrillin-1 is shown in Figure 1.1. Fibrillin-1 is mainly comprised of arrays of tandem pairs of epidermal growth factor like (EGF) domains, 43 of which contain a calcium binding site (cbEGF), interspersed by globular TGFβ binding protein like domains (TB) and hybrid domains. Fibrillin-1 has a proline rich sequence which in fibrillin-2 is glycine rich and in fibrillin-3 is proline and glycine rich (Zhang et al., 1994, Corson et al., 2004). Fibrillin undergoes processing by members of the furin/PACE family of proprotein convertases at two sites, one at the consensus sequence RAKR/R at residues 41-45 in the N-terminus (Pereira et al., 1993, Reinhardt et al., 1996, Ritty et al., 1999) and at the motif RKR/R at residues 2731-2732 in the C-terminus (Ritty et al., 1999). Cleavage at these sites is thought to be required for microfibril assembly (Milewicz et al., 1995, Raghunath et al., 1999).

Figure 1.1 Domain organisation of fibrillin-1

The top panel shows cartoons of a representative nuclear magnetic resonance (NMR) model of the domains FUN-EGF1-3 (Yadin et al., 2013), the X-ray crystal structure of domains cbEGF9-hyb2- cbEGF10 (Jensen et al., 2009) and cbEGF22-TB4-cbEGF23 (Lee et al., 2004). The domain cartoons are rainbow coloured with blue representing the N-terminus and red the C-terminus. The middle panel is a cartoon illustrating the domain organisation of fibrillin-1, theamino acid (AA) residue numbers are labelled below. The bottom panel shows small angle X-ray scattering (SAXS) structures of recombinant fibrillin fragments (Baldock et al., 2006, Cain et al., 2012) (left to right) Protein fragment 2 (PF2) is composed of TB1 a proline rich region EGF6-10-TB2 domains, PF5 contains EGF11-15-hyb2-TB3 domains, PF7 is composed of TB3-EGF15-20, PF8 contains EGF20-26-TB4 PF12 is composed of domains TB6-EGF36-41-TB7 and PF13 contains domains TB7-EGF 41-47.

1.1.2.1 Fibrillin unique N-terminal domain

Fibrillin-1 has a unique N-terminal fold and this domain has been termed the fibrillin unique N- terminal (FUN) domain. The FUN domain adopts an overall compact conformation apart from an unstructured N-terminus between residues R45–A52 (Figure 1.1). The domain comprises two loops connected by two disulphide bonds, between C59-C68 and C67-C80 (C1-C3 and C2-C4), with an N-terminal segment which packs in between (Yadin et al., 2013).

1.1.2.2 EGF domains

EGF domains are found in a wide range of proteins, such as the elastic fibre associated latent TGFβ binding proteins (LTBP) and fibulins, cell surface receptors such as notch, and are also involved in blood coagulation (Jensen et al., 2012). The structures of several fibrillin EGF domains have been solved by NMR or X-ray crystallography (Downing et al., 1996, Jensen et al., 2009) see Figure 1.1. EGF domains consist of a major and minor double stranded beta sheet which are stabilised by intermolecular disulphide bridges (Yuan et al., 1997). EGF domains contain six cysteines which form three disulphide bonds in the 1-3, 2-4, 5-6 configurations. The EGF domains have a conserved core structure but can vary in the length of loops. The majority of EGF domains in fibrillin are cbEGF domains and contain the consensus sequence (D/N)X(D/N)(E/Q)Xm(D/N)Xn(Y/F) (Pereira et al., 1993).

1.1.2.3 TB domains

The TB domains, also referred to as eight cysteine domains, are only found in the fibrillin- LTBP family of ECM proteins. The TB domain has a globular structure and consists of six β- strands and two α-helices which are stabilised by four disulphide bridges between the eight cysteine residues in a 1–3, 2–6, 4–7, 5–8 arrangement (Yuan et al., 1997, Lee et al., 2004)(Figure 1.1). The β-strands are arranged to form a four-stranded β-sheet which packs closely with α-helix one and a two stranded β-sheet which packs closely to α-helix two. At the end of the first α-helix there is a cysteine triplet motif. TB4 contains an Arg-Gly-Asp (RGD) motif which has been shown to bind to integrin hetero-dimers α5β1, αvβ3 and αvβ6 in an RGD dependant manner (Bax et al., 2003, Jovanovic et al., 2007) see section 1.1.7.6.

1.1.2.4 Hybrid domain

The hybrid domains have a similar structure to TB domains with a C-terminal region which resembles EGF domains (Jensen et al., 2009). The hybrid domain has eight cysteine residues which form disulphide bridges in a 1-3, 2-5, 4-6, 7-8 pattern (Figure 1.1). The hybrid domain has a similar arrangement of beta sheet as the TB domains with one β-sheet formed

19 from three β-strands (four in TB domains) in the N-terminal half of the domain and two β- strands in the C-terminal half. The Hybrid domain only has one α-helix in comparison to the TB domain which has two. The first hybrid domain in fibrillin contains an unpaired cysteine residue which is thought could be important in forming intermolecular disulphide bridges in the formation of fibrillin microfibrils (Reinhardt et al., 2000). However the first hybrid domain is not essential for microfibril assembly or function as mice missing the first hybrid domain can form microfibrils and have normal tissue architecture (Charbonneau et al., 2010).

1.1.3 Fibrillin microfibril assembly

Fibrillin is initially expressed as a pro-protein which undergoes N and C-terminal cleavage by enzymes of the furin/PACE family. Processing of the C-terminal pro-peptide occurs extracellularly as it is required for fibrillin secretion (Wallis et al., 2003, Jensen et al., 2014). Fibrillin monomers then polymerise linearly, though head-to-tail association of the N and C- termini (Reinhardt et al., 1996, Lin et al., 2002) see Figure 1.2. Lateral assembly of microfibrils is also a critical assembly step. Scanning transmission electron microscopy (STEM) mass mapping and electron microscopy (EM) analysis of microfibrils suggests that there are up to eight fibrillin monomers in a microfibril (Baldock et al., 2001, Wang et al., 2009). This lateral assembly of microfibrils is thought to be stabilised through homotypic associations between N and C-termini (Trask et al., 1999, Marson et al., 2005), this hypothesis is supported by the ability of C-terminal fibrillin-1 fragments to form beaded structures similar in appearance to the bead region of extracted microfibrils (Hubmacher et al., 2008). The microfibril structure is further stabilised through formation of crosslinks which are catalysed by transglutaminase (Qian and Glanville, 1997).

Microfibril assembly is thought to occur at the cell surface facilitated by association with heparan sulphate proteoglycans (HSPG) and/or integrins. N-terminal fibrillin fragments can promote the formation of focal adhesions and facilitate cell spreading through interaction with HSPGs (Cain et al., 2008) also addition of exogenous heparin (a highly sulphated heparan sulphate (HS) analogue) and removal of HS chains from HSPG core proteins ablates microfibril assembly, which further highlights the importance of HS interactions (Tiedemann et al., 2001, Ritty et al., 2003a, Sabatier et al., 2014). HSPG will be discussed in greater detail in section 1.1.7.7.

Fibronectin has also been shown to have a key role in fibrillin assembly in a cell type specific context. Depletion of fibronectin in fibroblast cell cultures using siRNA prevented fibrillin

20 microfibril assembly and could be rescued by the addition of exogenous fibronectin (Kinsey et al., 2008, Sabatier et al., 2013). In contrast epithelial cells can assemble microfibrils in the absence of fibronectin, but instead require activity of syndecan-4 (Baldwin et al., 2014) Epithelial cells which are undergoing an epithelial to mesenchymal transition, require fibronectin and syndecan-4 for microfibril assembly.

Figure 1.2 Diagram of fibrillin microfibril assembly.

Fibrillin molecules are cleaved at their N and C-termini before or soon after secretion by furin/PACE pro-protein convertases. They then undergo linear head to tail assembly and lateral assembly to form microfibrils. Microfibril assembly is thought to occur at the cell surface and may involve interactions with fibronectin, integrin receptors and/or HSPGs such as syndecans. After assembly microfibrils form arrays in the ECM (adapted from (Baldwin et al., 2013)).

1.1.4 Fibrillin microfibril structure

Fibrillin microfibrils imaged in tissues by transmission electron microscopy (TEM) have a diameter of ~10-12 nm with a beads-on-a-string appearance, when imaged in cross-section the microfibrils appear hollow (Sakai et al., 1986, Davis et al., 2002, Wang et al., 2009). X-ray

21 fibre diffraction studies of ciliary zonules show microfibrils have an un-tensioned periodicity of ~56 nm and diameter of ~20 nm (Wess et al., 1997), and EM studies have shown microfibrils can be irreversibly stretched to a periodicity of ~160 nm (Baldock et al., 2001) see Figure 1.3. Fibrillin microfibrils have a mass per repeat which ranges from ~1400 kDa in some early foetal tissues and cell culture to ~2500 kDa in adult tissues (Sherratt et al., 1997, Wess et al., 1998b, Baldock et al., 2001, Sherratt et al., 2010) this mass is consistent with up to eight fibrillin monomers per repeat.

The different regions of fibrillin microfibrils have been named the bead, arm, interbead and shoulder regions (Baldock et al., 2001, Baldock et al., 2002) see Figure 1.3. The bead region is heart shaped and is highly compact, containing roughly half of the mass of the microfibril repeat (Baldock et al., 2001). Epitope labelling studies have localised the N and C-termini to opposite sides of the bead regions (Reinhardt et al., 1996, Baldock et al., 2001). Emerging from the bead region are two arm like structures which bow out and surround a stain accessible cavity, the arms join back together at the interbead region. The interbead and shoulder regions have a more compact structure (Baldock et al., 2001). As microfibrils are extended from ~56-100 nm microfibril regions undergo conformational change, there is a reduction in mass in the arm, interbead and shoulder regions potentially due to unfolding in these regions. The structure of the bead region remains constant as microfibril extend up to a periodicity of ~90 nm after which it undergoes a conformational change resulting in a loss of mass (Sherratt et al., 2001, Wang et al., 2009).

Microfibrils are stabilised by salt bridges, lowering the ionic concentration of buffers leads to conformational change in the structure of the microfibril, allowing the periodicity of microfibrils to extend to up to 145 nm (Wang et al., 2009). The structure of microfibrils is also dependant on calcium ion binding which affects interbead periodicity, mass distribution and flexibility of the microfibril (Reinhardt et al., 1997a, Wess et al., 1998b). Addition of calcium ions causes fibrillin to become more rigid, this is thought to be due to calcium binding stabilising the interfaces between pairs of cbEGF domains and cbEGF and TB domains (Werner et al., 2000). The changes in conformation on calcium binding have also been shown to protect the microfibril from proteolysis by matrix metalloproteinases (MMPs) (Reinhardt et al., 1997b).

Figure 1.3 Diagram of a fibrillin microfibril

A schematic diagram of a fibrillin microfibril repeating unit. The bead, arm, interbead and, shoulder regions are highlighted, (adapted from (Baldock et al., 2001))

1.1.5 Fibrillin packing models

Although there is a general consensus that fibrillin arranges into microfibrils in a head to tail fashion how each fibrillin molecule packs into a microfibril is controversial. There are three models which have been proposed to explain how fibrillin monomers pack together to form a microfibril (reviewed in (Kielty et al., 2005, Jensen et al., 2012)), these are; the molecular pleated model (Baldock et al., 2006, Lu et al., 2006) or the half (Kuo et al., 2007) or one third linear staggered models (Lee et al., 2004)(see Figure 1.4).

In the molecular pleated model a single fibrillin-1 molecule spans one repeat between beads (Figure 1.4A). The theoretical length of a fibrillin-1 molecule when fully extended is ~180 nm, in this model the molecule folds back on its self to be consistent with the observed 56 nm repeat (Baldock et al., 2001, Baldock et al., 2006, Lu et al., 2006). This model is based on epitope mapping, EM stain exclusion patterns and STEM mass mapping. This model best explains the extensibility of microfibrils as the unpleating of the fibrillin-1 molecules supports the observed periodicities ranging from 56 nm to ~160 nm (Baldock et al., 2001, Wang et al., 2009). The pleating of arrays of fibrillin EGF domains is supported by SAXS structures of fibrillin-1 fragments which have a non-linear arrangement of domains (Baldock et al., 2006) see Figure 1.1.

In contrast to the molecular pleated model, in the linear staggered models the fibrillin monomer spans either two (in the half-staggered model (Figure 1.4B)) or three bead periods of 56 nm (in the one third staggered model (Figure 1.4C)). These linear staggered models are

23 based on epitope mapping and predictions from NMR and X-ray crystallography data of EGF domain pairs which suggest a rod like organisation of these domains (Lee et al., 2004). Extensibility in the staggered microfibril comes from interdomain flexibility and unfolding of domains.

X-ray Crystallography, NMR and SAXS have revealed the high resolution domain structure and the structure of short fibrillin fragments, but it is still unclear how individual fibrillin monomers are organised in the microfibril. This will be essential in understanding how microfibrils form and how disease causing mutations in fibrillin-1 might affect the overall structure of the microfibril and impact on their hierarchical organisations in tissues.

Figure 1.4 Microfibril organisation models.

There are two main categories of microfibril organisation models, the molecular pleated model (A) and the linear staggered models (B and C). (A) In the molecular pleated model a single fibrillin-1 molecule spans one repeat between beads, in this model the molecule folds back on its self to be consistent with the observed 56 nm repeat. In the linear staggered models the fibrillin monomer spans either two (in the half-staggered model) (B) or three bead periods of 56 nm (in the one third staggered model) (C). Shown in (C) are positions of transglutaminase cross-links (red circles) and MAGP-1 molecules (green diamonds). (Figure adapted from (Jensen et al., 2012)).

1.1.6 Fibrillin tissue organisation

Fibrillin microfibrils form a number of higher order structures within tissues which are tissue specific and depend on the function of the tissue (reviewed in (Kielty et al., 2002, Sherratt, 2009)). In the medial layer of the aorta and elastic arteries, elastic fibres form concentric fenestrated lamella, surrounded by circumferentially oriented smooth muscle cells, which imparts elasticity and resilience to the blood vessels (Wagenseil and Mecham, 2012). In the lung, elastic fibres are present in the blood vessel walls and through the respiratory tree where they form branched networks which support alveolar expansion and recoil during breathing (Kielty et al., 2005). In skin, the reticular dermis contains a horizontal arrangement of thick elastic fibres which are connected to thinner perpendicular arranged microfibrils called elaunin fibres. These elaunin fibres then merge with bundles of microfibrils devoid of elastin called oxytalan fibres and intercalate in the dermal-epidermal junction (Braverman and Fonferko, 1982). Elastic fibres in auricular cartilage contribute to the deformability of the tissue by forming thin networks which intersperse with collagen fibrils in the interterritorial zone (Kielty et al., 2002).

Another tissue where fibrillin forms bundles of microfibrils devoid of elastin is the ciliary zonule of the eye, which holds the lens in dynamic suspension and transmits force from the ciliary muscles to deform the lens in accommodation. The ciliary zonule is made up of laterally aligned ciliary zonule fibres which are mainly comprised of bundles of fibrillin microfibrils, these bundles of microfibrils are also organised into larger fascicle-like structures which are held together by circumferentially organised zonule fibres (Hiraoka et al., 2010). The main component of ciliary zonule fibres is fibrillin-1 (Cain et al., 2006), in murine ciliary zonule microfibrils also contain fibrillin-2 (Beene et al., 2013). These microfibrils associate with the basement membrane of the non-pigmented epithelium of the ciliary body on one side, and are anchored by insertion into the lens epithelium on the other. Fibrillin microfibrils are thought to attach to the basement membrane by interaction with perlecan (Tiedemann et al., 2005). A recent study of a LTBP-2 knock out mouse model of ectopia lentis has suggested that the ciliary zonule microfibrils are stabilised by LTBP-2 (Inoue et al., 2014). The a disintegrin-like and metalloproteinase domain with thrombospondin-type 1 motifs (ADAMTS) and ADAMTS like (ADAMTSL) family of proteins are also thought to be important in assembly and maintenance of ciliary zonule structure. ADAMTS-10 is found throughout the zonule and ADAMTSL-4 is found in the ciliary body and at the lens epithelium and is thought

26 to be involved in anchorage of zonules (Collin et al., 2015). One aspect of this PhD study will be on the organisation of fibrillin microfibrils in the ciliary zonule.

1.1.7 The fibrillin microenvironment

The structure and function of fibrillin microfibrils in tissues is dependent on a complex assortment of scaffold associated proteins, growth factors, and cellular receptors which constitute the fibrillin microenvironment. This niche is involved in the regulation of tissue homeostasis as well as mechanical resistance and elasticity of tissues (reviewed in (Baldwin et al., 2013, Hubmacher and Apte, 2015, Mecham and Gibson, 2015, Sengle and Sakai, 2015)).The following sections will cover the role of molecules which interact with fibrillin on microfibril function and assembly of elastic fibres as well as how fibrillin assemblies are involved in the regulation of growth factor signalling.

1.1.7.1 Elastic fibre associated proteins

Fibrillin microfibrils are an essential component of elastic fibres where they form a template for the deposition of tropoelastin to form a core of cross linked elastin. In mature elastic fibres this elastic core is surrounded by a sheath of microfibrils, some microfibrils also appear to be embedded in the elastin core. As well as tropoelastin a number of other proteins have been localised to elastic fibres and are important in the process of elastogenesis such as the fibulins, lysyl oxidase (LOX) and lysyl oxidase like (LOXL) crosslinking enzymes (reviewed in (Yanagisawa and Davis, 2010, Baldwin et al., 2013)) see Figure 1.5.

1.1.7.2 Elastin

Elastin is expressed from cells such as fibroblasts, auricular chondrocytes and vascular smooth muscle cells as the soluble precursor tropoelastin (Figure 1.5) (reviewed in (Wagenseil and Mecham, 2007, Baldwin et al., 2013)). Tropoelastin is ~60-70 kDa and has a multi-domain structure made up of alternating hydrophobic and lysine (lys-ala or lys-pro) containing crosslinking domains which can participate in forming desmosine crosslinks, a process catalysed by the enzyme LOX or LOXL-1. Tropoelastin has three sub regions, an N- terminal elastic coil, a C-terminal cell interactive module which contains the GRKRK motif which binds αvβ3 integrin (Bax et al., 2009) and a bridge region which is important for elastin coacervation (Yeo et al., 2011, Yeo et al., 2012). Tropoelastin can bind to fibrillin through interaction with the N-terminus of fibrillin (Rock et al., 2004). Elastin can also bind to the

27 fibulin family of glycoproteins which are important for its coacervation and deposition on microfibrils.

1.1.7.3 Fibulins

The fibulins are a family ECM glycoproteins, there are seven members of the fibulin family which can be sub categorised into classes I or II based on their length and domain structure (Yanagisawa and Davis, 2010). The fibulins have a fibulin-type C-terminal domain and are mainly comprised of tandem cbEGF domains. Fibulin-1 associates with the elastic fibre core and fibulins 2, 4 and 5 bind to bind to the N-terminus of fibrillin-1 (El-Hallous et al., 2007). Fibulins-1 -2 4 5 have been show to bind tropoelastin in vitro (Kobayashi et al., 2007). Studies of knock out mouse models have determined that fibulin-4 and fibulin-5 have a critical role in the formation of elastic fibres. Fibulin-5 homozygous knockout mice survive to adulthood but have loose skin, emphysema, tortuous aortas and disorganised elastic fibres (Nakamura et al., 2002, Yanagisawa et al., 2002). Fibulin-5 is thought to act as an adaptor complex regulating the formation of elastin microaggregates and facilitating their deposition onto microfibrils (Figure 1.5), as addition of recombinant fibulin-5 to skin fibroblasts promotes coacervation (Hirai et al., 2007). Fibulin-5 may also be involved in elastin cross-linking as it can bind to the LOXL enzymes (Hirai et al., 2007).

Loss of fibulin-4 has a more severe phenotype as homozygous fibulin-4 knockout mice die perinataly with severe vascular and lung defects such as emphysema, artery tortuosity, aneurysms and disorganised elastic fibres (McLaughlin et al., 2006). These mice also have a drastic reduction in desmosine cross links. Fibulin-4 is thought to have an essential role in elastin crosslinking by mediating the formation of complexes between elastin and LOX (Horiguchi et al., 2009) (Figure 1.5).

1.1.7.4 LOX

LOX and the homologous LOXL1-4 enzymes are involved in crosslinking of several ECM components such elastin and collagen I (Lucero and Kagan, 2006). LOX catalyses the oxidative deamination of peptidyl lysine residues to alpha-aminooadipic-delta-semialdehde this then spontaneously condenses to form desmosine and isodesmosine crosslinks. LOX and LOXL1-4 are secreted as zymogens and are activated by removal of their N-terminal propeptide by the tolloid protease BMP-1 (Lucero and Kagan, 2006). Fibronectin plays a critical

28 role in the activation of LOX by regulating cleavage of the propeptide (Fogelgren et al., 2005) see Figure 1.5.

1.1.7.5 Elastic fibre assembly

The formation of elastic fibres is a complex multi-step process which is not fully understood. Initially secreted tropoelastin self-associates into aggregates in a process termed coacervation. The size and rate of formation of elastin droplets is regulated by temperature, pH and the presence of regulatory proteins fibulin-4 and fibulin-5. Elastin aggregates are then deposited onto microfibrils where they form larger aggregates which are stabilised by the formation of desmosine and isodesmosine crosslinks by LOX and LOXL-1 see Figure 1.5.

Figure 1.5 Elastic fibre assembly

Tropoelastin is secreted from cells and coacervates into microarregates regulated by fibulin 5. This may occur at the cell surface as elastin has been shown to bind to integrin receptors. Elastin aggregates are deposited onto the fibrillin microfibril scaffold where they are cross-linked by LOX in complex with fibulin 4. LOX is activated by the removal of is propeptide by BMP-1 this process is enhanced by binding to fibronectin. (Figure adapted from (Baldwin et al., 2013)).

1.1.7.6 Integrins

Fibrillin microfibrils provide a crucial link between the cell surface and the ECM. In several elastic tissues, such as arteries and lungs, microfibrils can be seen to attach to the surface of cells clustering at focal adhesion sites (Davis, 1993). These cell matrix contact sites are thought to be critical in allowing cells to sense their surrounding environment. Fibrillin can mediate this connection to the cell surface through interaction with the integrin family of cell surface receptors. Fibrillin has been shown to bind to α5β1 αvβ3and αvβ6 through interaction with an RGD motif which is located in TB4 (Bax et al., 2003, Jovanovic et al., 2007). The affinities vary between the integrin receptors which may result in differences in downstream signalling (Jovanovic et al., 2007). Binding of fibrillin to integrins has been shown to affect cell motility, cell spreading, cytoskeletal organisation, and enhanced the extracellular deposition of fibrillin-1 (Bax et al., 2003). Mutations in the RGD motif disrupt integrin binding and cause the disease stiff skin syndrome (SSS) (Loeys et al., 2010) see section 1.1.8 for further discussion. More recently it has been demonstrated that binding of fibrillin to integrins can modulate cell signalling through changes in microRNA expression (reviewed in (Zeyer and Reinhardt, 2015)). The ability of fibrillin fragments to influence cell spreading and focal adhesions was further enhanced by a downstream HS binding site in TB5 (Bax et al., 2007). This led to the hypothesis that fibrillin interaction with integrin could be supported by binding to HSPGs

1.1.7.7 Proteoglycans

Proteoglycans are important in fibrillin assembly and incorporation into the ECM. Proteoglycans comprise of one or more glycosaminoglycan (GAG) chains, such as chondroitin sulphate (CS), HS, and dermatan sulphate (DS) attached to a protein core (Gandhi and Mancera, 2008).

Fibrillin has been shown to have up to six high affinity HS binding sites (Tiedemann et al., 2001, Ritty et al., 2003a, Cain et al., 2005, Cain et al., 2008). Mutations in fibrillin-1 which disrupt HS binding have been implicated in a number of fibrillinopathies such as WMS, acromicric dysplasia (AD) and gelophysic dysplasia (GD) (Cain et al., 2012). Sulphation patterns are thought to be important in regulation of HS binding affinity, fibrillin binding to heparin is most strongly inhibited by highly sulphated and iduronated HS (Tiedemann et al., 2001). HS chains are found on large number of proteoglycans such as the cell surface receptors syndecans or glypicans and the basement membrane HSPG perlecan. Perlecan has been shown to bind to fibrillin through interaction of its type I and II domains and a central

30 region on fibrillin (Tiedemann et al., 2005). Perlecan is a large ~440 kDa proteoglycan and is thought to be involved in anchoring fibrillin to basement membranes. Fibrillin and perlecan have been shown to colocalise at the epidermal-dermal junction in skin and the basement membrane surrounding non-pigmented ciliary body epithelial and may be important in ciliary zonule structure (Tiedemann et al., 2005).

Other proteoglycans which fibrillin has also been shown to interact with are the chondroitin sulphate proteoglycans (CSPG) decorin, biglycan and versican. Versican is a large CSPG of the lectican family which has been shown to co-localise with microfibrils in the skin (Zimmerman 1994). Versican interacts with microfibrils through its C-terminal lectin domain binding to the N-terminus of fibrillin (Isogai et al., 2002). Versican is thought to play a role in integrating microfibrils into the ECM.

Fibrillin also binds to the small leucine rich CSPGs decorin and biglycan. The small leucine rich proteoglycans (SLRPs) family consists of 9 different proteins which contain the leucine rich motif. Decorin and biglycan are closely related proteins sharing 55% amino acid identity (Hocking et al., 1998). They have a core protein which contains 10 leucine-rich repeats each of 25 amino acids. The core proteins are substituted with glycosaminoglycan (GAG) chains, in a tissue specific manner; CS in bone, DS in cartilage and CS or DS in cornea {Axelsson, 1980 #648}. Decorin and biglycan have one or two CS/DS chains, respectively. Decorin and biglycan can both bind to fibrillin-1 microfibrils (Trask et al., 2000, Reinboth et al., 2002). The decorin binding site is localised to the N-terminus of fibrillin. Decorin can bind fibrillin on its own or in a ternary complex with MAGP-1 (Trask et al., 2000). Biglycan can also bind to MAGP-1 and tropoelastin suggesting a potential role in elastogenesis (Reinboth et al., 2002). Overexpression of CS has been shown to disrupt microfibrillar assembly in keloid scarring (Ikeda et al., 2009), which have disorganised elastic fibres. Keloid tissue was shown to express higher levels of CS, also addition of exogenous CS to keloid fibroblast cultures disrupted assembly of fibrillin microfibrils.

1.1.7.8 ADAMTS and ADAMTSL

Another family of proteins which play an important role in regulation of microfibrils are ADAMTS and ADAMTSL proteins, which are involved in a number of microfibrillar related functions such as, microfibrillar assembly, stability and anchorage (reviewed in (Hubmacher and Apte, 2015)). There are 19 ADAMTS and 7 ADAMTSL members of the superfamily, which are present in the ECM or associated to the cell surface. The ADAMTS N-terminus

31 comprises a highly homologous N-terminal protease domain a disintegrin module, a thrombospondin type 1 repeat (TSR) and a cysteine rich module. The ADAMTSL proteins lack the protease domain and are not proteolytically active, but have similar C-termini. ADAMTS-10, ADAMTSL-2, -3, -4 and -6 have been shown to bind directly to recombinant fibrillin and can accelerate microfibril biogenesis when added to cell cultures (Tsutsui et al., 2010, Kutz et al., 2011, Sengle et al., 2012). The further importance of these proteins in microfibrillar function is highlighted by the fact that they phenocopy a number of fibrillinopathies. Mutations in ADAMTS-10 can cause WMS (Dagoneau et al., 2004, Kutz et al., 2008) and mutations in ADAMTS-17 causes a WMS-like syndrome (Morales et al., 2009). While ADAMTSL-2 mutations can lead to GD (Le Goff et al., 2008) and ADAMTSL-4 mutations result in isolated ectopia lentis (Aragon-Martin et al., 2010) and ectopia lentis et pupillae (Christensen et al., 2010). As mutations in ADAMTS-10 ADAMTS-17 and ADAMTSL- 4 can cause ectopia lentis this shows they are critical in maintaining the structure of the ocular zonule. ADAMTS-10 has been localised across the whole ciliary zonule (Kutz et al., 2011) and ADAMTSL-4 is found in the ciliary body and is highly expressed in the equatorial lens epithelium (Gabriel et al., 2012). The role of ADAMTSL-4 in ciliary zonule structure was studied by the creation of the tvrm267 mouse which has a nonsense mutation in the ADAMTSL-4 gene (Collin et al., 2015). Homozygous tvrm267 mice suffer from isolated ectopia lentis. Initially ciliary zonules form in these mice but by the age of three months the zonule structure is disrupted and does not connect to the lens epithelium. This study suggests that ADAMTSL-4 plays a critical role in the maintainance of the ciliary zonule structure and is most likely involved in anchoring microfibrils to the lens epithelium.

1.1.7.9 MAGPs

The microfibril-associated glycoproteins (MAGP) are a family of two small glycoproteins MAGP-1 and MAGP-2 which associate with fibrillin microfibrils. The MAGPs share a homologous 60 residue matrix binding region. MAGP-1 has an acidic N-terminus with a high proportion of proline and glutamine residues, and a cysteine rich C-terminus. MAGP-2 is rich in serine and threonine and has a central region which is cysteine rich. MAGP-2 also contains an RGD motif (Mecham and Gibson, 2015). Both MAGPs bind to the N-terminus and an internal site near TB3 of recombinant fibrillin fragments (Jensen et al., 2001, Rock et al., 2004) and have been shown to localise to the bead region of microfibrils (Hanssen et al., 2004). Knockout mouse models have shown that MAGPs are not essential for the assembly of microfibrils or elastic fibres (Weinbaum et al., 2008, Combs et al., 2013).

MAGP-1 and MAGP-2 are often grouped together with proteins MFAP1,3 and 4 which were grouped with the MAGP family of proteins due to their small size and localisation to microfibrils (Mecham and Gibson, 2015). MFAP-1 participates in RNA splicing and MFAP-3 is a nuclear kinase. MFAP-4 however has been shown to bind to fibrillin-1, fibrillin-2 and tropoelastin (Pilecki et al., 2016) and has been shown to be promote tropoelastin self- association.

MAGPs can bind to active TGFβ and may be involved in regulating TGFβ and BMP-7 signalling by sequestering the growth factors into the ECM by forming a complex with fibrillin microfibrils (Weinbaum et al., 2008). Knockout MAGP-1 mice have altered metabolism and longer bones with reduced mineralisation which is thought to be due to perturbed TGFβ signalling (Walji et al., 2016). The role of fibrillin in regulating growth factor signalling will be discussed in the following section.

1.1.7.10 Regulation of growth factor bioavailability

One of the key roles of fibrillin is to regulate the bioavailability of members of the TGFβ and BMP superfamily of growth factors by sequestering them in the ECM. The N-terminus of fibrillin-1 binds to these growth factors in their latent form through interaction with latent TGFβ binding proteins (LTBPs) (Isogai et al., 2003, Hirani et al., 2007, Ono et al., 2009) or the propeptide of BMP-2, BMP-4, BMP-5, BMP-7, BMP-10 and growth differentiation factor 5 (GDF- 5) (Gregory et al., 2005, Sengle et al., 2008a, Sengle et al., 2008b, Sengle et al., 2011). Binding of these BMPs in complex with thier pro-domains to fibrillin has been hypothesised as a mechanism for regulating their activity {Wohl, 2016 #629}. These BMPs have also been shown to interact with fibrillin-2 (Sengle et al., 2008a). The TGFβ superfamily are involved in regulating cellular processes such as proliferation, differentiation, cytoskeletal organisation, adhesion, and migration and also are essential in developmental processes including body axis formation and patterning (reviewed in (Weiss and Attisano, 2013)).

1.1.7.11 LTBPs

The LTBPs are part of the fibrillin-1/LTBP family of ECM proteins and are structurally very similar to the fibrillins. These multidomain glycoproteins contain arrays of cbEGF domains interspersed with TB domain and have a cysteine rich N-termini. There are four members of the LTBP family 1-4. Furin processing of the TGFβ precursor generates the small latent complex (SLC) which is comprised of mature disulphide linked TGFβ dimer bound to its pro-

33 peptide, latency associated peptide (LAP). LTBP1, 3 and 4 bind to the SLC to form the large latent complex (LLC) before it is secreted from cells (Saharinen and Keski-Oja, 2000). The LLC is then deposited onto fibrillin microfibrils in the ECM. The C-termini of LTBP-1,-2,-4 have been shown to interact with the first hybrid domain of fibrillin-1 (Isogai et al., 2003, Hirani et al., 2007, Ono et al., 2009). Depletion of fibrillin microfibrils disrupts the ECM deposition of TGFβ leading to disorganised TGFβ signalling which has been shown to be important in fibrillinopathies such as MFS (Neptune et al., 2003) and WMS (Le Goff et al., 2011). The LTBPs can also have functions which do not involve TGFβ. For example, LTBP-2 does not interact with TGFβ but has recently been shown to be important in maintaining the structure of the ciliary zonule (Inoue et al., 2014) and LTBP-4 has been implicated in elastic fibre assembly (Noda et al., 2013).

1.1.7.12 BMPs

The N-terminus of fibrillin binds to the pro-domains of BMP-2, BMP-4, BMP-5, BMP-7, BMP- 10, GDF-5 (Gregory et al., 2005, Sengle et al., 2008a, Sengle et al., 2011). Fibrillin does not interact with the growth factor in the absence of the pro-domain (Gregory et al., 2005). Unlike TGFβ the pro-domain does not prevent BMP signalling but binding of the growth factor to fibrillin recombinant fragments causes a conformation change which inactivates the growth factor (Wohl et al., 2016). Disruption of fibrillin microfibrils has been shown to lead to an increase in BMP signalling in fibrillin null osteoblasts (Nistala et al., 2010) and is involved in causing congenital contractural arachnodactyly (CCA) in fibrillin 2 null mice (Nistala et al., 2010, Sengle et al., 2015). CCA is characterised by arachnodactyly (long slender fingers), dolichosetenomelia (unusually long limbs), scoliosis and multiple congenital contractures as well as abnormalities of the external ears {BEALS, 1971 #649}.

As described in this section the TGFβ family of growth factors are involved in several fibrillinopathies such as MFS and WMS; the following section will cover this group of heritable connective tissue disorders in more detail.

1.1.8 Fibrillinopathies

Mutations in fibrillin can lead to several heritable connective tissue disorders termed fibrillinopathies (reviewed in (Baldwin et al., 2013)) such as MFS, SSS (Loeys et al., 2010), WMS (Faivre et al., 2003), AD and GD (Le Goff et al., 2011). Mutations in several microfibril associated proteins such as LTBP-2 and ADAMTS-10, ADAMTS-17, ADAMTS-2 and

ADAMSTSL-4 have been linked to WMS, AD, GD and isolated ectopia lentis. Mutations in fibrillin-2 cause a disease phenotypically related to MFS known as CCA (Putnam et al., 1995).

MFS is the most common fibrillinopathy; it is an autosomal dominant disease with prominent manifestations in the cardiovascular, skeletal and ocular tissues. Patients are often tall with long slender limbs, have arachnodactyly, scoliosis and pectus excavatum or carinatum. Approximately 80% of patients have bilateral ectopia lentis. MFS can be life threatening due to dilation of the ascending aorta which can then lead to rupture. Mutations in fibrillin can cause a wide range of MFS associated phenotypes from isolated skeletal phenotypes to the most severe neonatal form of MFS which can cause death of affected children within one year from congestive heart failure.

There are over 1000 MFS disease causing mutations, point mutations are the most common with nonsense and missense mutations comprising about 10% and 60% of all reported mutations. Mutations often disrupt calcium binding and disulphide bond formation (Robinson et al., 2006). Nonsense mutations, splicing errors, insertions, and duplications, as well as in- frame or out-of-frame deletions often result in nonsense-mediated decay resulting in reduction in the level of the mutant allele. A cluster of mutations between exons 24–32 cause a more severe form of MFS and this region is often referred to as the „neonatal‟ region as mutations in this region generally result in death within the first year of life (Kainulainen et al., 1994, Booms et al., 1999). Mutations in fibrillin-1 are thought to cause a reduction in the amount of fibrillin which is deposited in the ECM, this leads to less functional fibrillin microfibrils and disruption of TGFβ signalling which results in MFS. This hypothesis was supported by Jensen et al., 2015 who demonstrated that three MFS disease causing mutation C1564Y, C1719Y and C1720Y were not secreted by cells leading to a reduction of fibrillin microfibrils in the ECM.

Several mouse models have been generated to study the mechanism of MFS disease progression these include; mg∆, which has a deletion of exons 19–24 (Pereira et al., 1997), mgN which expresses a fibrillin-1 null allele (Carta et al., 2006), mgR which has a reduced expression of FBN1 (Pereira et al., 1999), C1039G a knocked-in MFS causing mutation (Judge et al., 2004), GT8 which expresses a truncated construct with an N-terminal eGFP tag (Charbonneau et al., 2010) and H∆ in which the first hybrid domain has been deleted (Charbonneau et al., 2010).

The mg∆/+ is indistinguishable from the wild type mouse; homozygous mg∆/ mg∆ mice die within 3 weeks after birth and have cardiovascular problems such as haemothorax, haemopericardium or pulmonary haemorrhage and some mice have thinning of the proximal ascending aorta which suggests potential for aortic dilatation (Pereira et al., 1997). These mice do not have skeletal problems and their elastic fibre architecture appears normal.

The mgR/mgR homozygous mice have a reduced expression and produce ~15% of normal fibrillin-1 and survive to early adulthood (Pereira et al., 1999). Homozygous mgR mice have a similar skeletal and aortic phenotype as MFS patients. Mice gradually develop severe kyphosis and exhibit over growth of the ribs. More severe problems occur in the vascular system such as medial calcification and infiltration of macrophages into lesions in the elastic lamella potentially causing collapse of the aortic wall by elastolysis mediated by expression of MMP-12. Heterozygous mgR/+ develop normally.

The complete knock out of fibrillin-1 was investigated by the creation of the mgN/mgN mouse which does not express fibrillin-1 (Carta et al., 2006). These mice die soon after birth from ruptured aortic aneurysm, impaired pulmonary function, and/or diaphragmatic collapse. The medial layer of the aorta of mgN/mgN mice has been shown to be disorganised and poorly developed but elastin cross-linking is not affected. Mice also have malformed elongated ribs and fragile skin and internal organs.

The C1039G/+ heterozygote mouse has classical MFS with aortic dilatation and fragmentation of elastic lamellae, as well as skeletal muscle and mitral valve abnormalities (Judge et al., 2004). The mutant phenotype could be rescued by the introduction of a trans- gene expressing the WT gene. Mice homozygous for the mutant C1039G allele die perinataly similarly to mgN homozygous mice.

Heterozygous GT8 mice suffer from fragmentation of aortic elastic lamellae and disruption of microfibril networks in the skin (Charbonneau et al., 2010). Microfibrils in the GT8 mice could be imaged by fluorescence suggesting the mutant fibrillin can be incorporated into microfibrils. Mice which are homozygous for the GT8 construct die postnatally.

The H∆ mouse which is missing exon 7 which encodes the first hybrid domain, have a normal life span and fibrillin microfibrils form in both the heterozygous and homozygous animals (Charbonneau et al., 2010). The H∆ mice show that the first hybrid domain is not essential for microfibril formation and that its removal does not cause MFS. The hypomorphic

36 mgR/mgR mice which produce only around 15% of fibrillin and the ability of trans-genes to rescue mutant phenotypes of the C1039G/+ heterozygote mice suggest that reduction in the expression of fibrillin-1 beneath a certain threshold level could be sufficient to cause MFS.

Newborn mgR homozygous mice were shown to exhibit developmental emphysema similar to a number of MFS patients. Mice with impaired lung development were shown to have enhanced Smad2/3 signalling and heightened apoptosis which could be reversed by treatment with a TGFβ blocking antibody (Neptune et al., 2003). Several other studies have also shown a causal link between disregulation of TGFβ signalling and MFS (Ng et al., 2004, Habashi et al., 2006, Cohn et al., 2007). This has led to the development of a model for MFS pathogenesis where reduced expression of fibrillin leads to an increase in active TGFβ, as there is a reduced ability for the ECM to sequester the growth factor. This increase in TGFβ signalling then leads to disregulated tissue remodelling which causes MFS. Blocking TGFβ signalling using the angiotensin II type 1 receptor (AT1) blocker losartan has been tested as a potential therapy for MFS. Treatment of mice with losartan can prevent aortic aneurism (Habashi et al., 2006) and clinical trials have shown losartan may be an effective treatment for MFS (Lacro et al., 2014). Controversially a recent study of a compound mutant mouse model with the C1039G/+ mutation and knock out of TGF-β type II receptor, which was thought would rescue the MFS phenotype, actually accelerated aortic aneurism formation (Li et al., 2014). This has led to a contrasting hypothesis where upregulation of TGFβ signalling might be an attempt to repair tissues in response to damage. Also studies showing that non canonical Smad2/3 signalling have been shown in MFS mouse models (Carta et al., 2009) reveal that TGFβ involvement in MFS disease progression is more complex than originally thought.

Abnormal TGFβ signalling is also thought to be a contributing factor in a number of other fibrillinopathies such as SSS and the acromelic dysplasias, autosomal dominant WMS, AD and GD (Loeys et al., 2010, Le Goff et al., 2011). SSS and the acromelic dysplasias are often considered to have the opposite phenotype to MFS.

SSS is a congenital form of scleroderma which is characterised by hard, thick skin found usually all over the body. Patients can also suffer limited joint mobility and flexion contractures. The disease is caused by mutations in TB4 of fibrillin-1 which contains the integrin binding RGD motif (Loeys et al., 2010). SSS causing mutations increase microfibrillar deposition, impaired elastogenesis and have an increase in active TGFβ concentration. It was

37 proposed that phenotypes could be caused by alteration in integrin-ECM interactions. Using a cell co-culture model it has been demonstrated that fibrillin with one of two SSS causing missense mutantions, C1564S and W1570C could be secreted from cells and was deposited into the ECM (Jensen et al., 2015).

The acromelic dysplasias are three disorders: WMS, GD and AD which have overlapping characteristics. Patients with acromelic dysplasias present with short stature and short stubby hands and feet, in some cases patients suffer joint stiffness, with skin thickening.

WMS is characterised by proportionate shortstature, brachydactyly (short stubby hands), joint stiffness, and abnormalities of the ocular lens, which includes microspherophakia, ectopia lentis, and glaucoma. WMS patients also suffer from cardiac anomalies such as mitral valve prolapse, aortic and pulmonary valve stenosis and ventricular septal defects {Faivre, 2003 #374}.The autosomal dominant form of WMS has been shown to be caused by several mutations in fibrillin, which include mutations which disrupt TB5 (Faivre et al., 2003, De Backer et al., 2007, Cecchi et al., 2013), a mutation in the first hybrid domain (Stheneur et al., 2009) or loss of TB1 to EGF4 (De Backer et al., 2007).

AD and GD can be differentiated from WMS due to differnces in symptoms, such as mild facial dysmorphism, premature death due to cardiac valve thickening, or tracheal narrowing leading to difficult or laboured breathing or pulmonary infection in patients with GD {Faivre, 2001 #656}. AD and GD are caused by mutations which so far only affect TB5. GD differs to AD in that it is inherited in an autosomal recessive mechanism. These mutations were shown to disrupt HS binding (Cain et al., 2012). Recent studies of AD, GD and WMS mutations using a cell culture co-expression system have suggested that mutant fibrillin can still assemble into microfibrils (Jensen et al., 2015). The acromelic dysplasias are potentially caused by altered interaction between the cell surface and the ECM through disruption of integrin and HS binding which causes secondary effects such TGFβ activation, fibrosis and microfibril aggregation.

1.2 Fibrillin summary

Fibrillin microfibrils are an essential component of the ECM and play a key role in the homeostasis of tissues through their ability to sequester growth factors and organise interactions with several other ECM proteins. Fibrillin microfibrils are also essential for conferring mechanical resistance and elastic recoil to tissues. To fully understand how fibrillin

38 fulfils these functions it is key to understand how fibrillin is organised into microfibrils and how microfibrils are organised into larger networks in tissues.

1.3 Collagen VI microfibrils

Collagen VI is a large heteromeric network-forming collagen, which forms microfibrils with an appearance similar to beads on a string (Engvall et al., 1986). Microfibrils are formed by the end to end assembly of tetramers of collagen VI. Collagen VI monomers are composed of 3 α-chains which form two globular regions separated by a short collagenous region. Collagen VI anchors cells into the ECM and allows the transduction of biochemical and mechanical signals. Collagen VI is critical for the function of the muscular skeletal system as mutations in collagen VI lead to diseases such as Bethlem myopathy, Ullrich‟s congenital muscular dystrophy (UCMD) and osteoarthritis (OA) (reviewed in Bönnemann 2011; Lampe & Bushby 2005).

1.3.1 Collagen VI α chains

There are six collagen VI α-chains which have been identified α1-6 (VI) which are products of the separate genes COL6A1-6 (Chu et al., 1987, Fitzgerald et al., 2008, Gara et al., 2008, Sabatelli et al., 2011). The expression of collagen VI chains α1, 2, 3 and 6 have been localised to a wide range of tissues, such as skin, lung, blood vessels and articular cartilage (Keene et al., 1988, Fitzgerald et al., 2008). The α5 chain of collagen VI has a more restricted pattern of expression and is found in skin, lung, testis and colon (Soderhall et al., 2007, Fitzgerald et al., 2008, Gara et al., 2008, Sabatelli et al., 2011). In humans and chimpanzees the α4 chain is not functional due to a chromosome inversion which leads to the COL6A4 gene being separated into two segments, which are not thought to be translated (Fitzgerald et al., 2008, Gara et al., 2008) but have been implicated in increasing susceptibility to OA (Wagener et al., 2009). Collagen VI is predominantly found at the epithelial surface of basement membranes. In tissues which do not have a basement membrane, such as tendon fibroblasts and chondrocytes in articular cartilage, collagen VI is localised to the pericellular matrix (PCM) (Keene et al., 1988, Ritty et al., 2003b).

1.3.2 Collagen VI α chain domain organisation

Chains α1 and α2 are similar in size (~120 kDa) and domain structure (see Figure 1.6). The globular regions of the collagen VI α-chains are comprised of domains which share (18-25%) amino acid identity with the A domains of Von Willibrand factor (VWA) (Chu et al., 1987, Chu

39 et al., 1989). The helical regions in the α1 and α2 chains are flanked by one N-terminal domain (N1) and two C-terminal VWA domains (C1-C2) (Chu et al., 1987). The α2 chain undergoes alternative splicing to form three separate mRNA transcripts α2C2, α2C2a and α2c2a` but only the α2C2 variant has been identified at the protein level (Saitta et al., 1990).

The α3 chain is much larger, with a predicted size of ~340 kDa (Chu et al., 1990), containing ten N-terminal domains and five C-terminal domains. Domains N1-10 and C1 and C2 are VWA domains, domain C3 has a proline rich repeat region, C4 domain is homologous to type III domains in fibronectin and domain C5 shares 40-50% amino acid identity with Kunitz type protease inhibitors (Chu et al., 1990). The α3 N-terminal VWA domains are encoded by single exons from the N10-N3 and domains N10, N9, N7, N5 and N3 can be alternately spliced (Dziadek et al., 2002). The most common splice variants identified were N1-N8, N1-N6+N8 and N1-N6. Collagen VI also undergoes proteolytic cleavage. In a study of articular cartilage the C5 domain was shown to be present after synthesis and initial incorporation into microfibrils and has been shown is required for assembly (Lamande et al., 2006) but was not present in mature microfibrils (Aigner et al., 2002). A more recent mass-spectroscopy analysis of extracted collagen VI from bovine cornea by Beecher at al (2011), did not detect peptide fragments after the C1 domain. The mass estimated by mobility on SDS-PAGE suggested an α3 chain which consisted of N8-N6-C1 domains (Beecher et al., 2011).

The α4, 5 and 6 are closely related to the α3 chain, each of these chains has a 336 residue collagenous region and contains 7 N-terminal VWA domains (Fitzgerald et al., 2008). The α4 and α6 chains have two C terminal VWA domains whereas the α5 chain has 3 C-terminal VWA domains. The α4 chain also contains a C-terminal Kunitz domain (Gara et al., 2008).

Figure 1.6 Domain organisation of Collagen VI α chain 1-6.

The top panel shows cartoons of the crystal structure of the α3 chain N5 VWA domain (Becker et al., 2014) and an NMR structure of the α3 chain Kunitz domain (Zweckstetter et al., 1996). The domain cartoons are rainbow coloured with blue representing the N-terminus and red the C-terminus. The centre panel is a cartoon illustrating the domain organisation of Collagen VI α chain 1-6. The AA residue numbers are labelled below.

1.3.2.1 Collagen VI helical region

Collagen VI contains a relatively short collagenous region which separates the N and C- terminal globular domains. The collagenous domains of the α1-3 are 335-336 residues in length and 98% of the amino acid sequence is comprised of Gly-X-Y repeats (Chu et al., 1988). The collagenous regions contain a cysteine residue at residue 50 in the α3, α4, α5 and α6 chains and the α1 and α2 chains have a cysteine residue at position 89 (Chu et al., 1988, Gara et al., 2008). The cysteine residues at position 89 in the α1 and α2 chains are involved in forming an anti-parallel dimer. The cysteine residue at position 50 is involved stabilising the

41 formation of tetramers (Furthmayr et al., 1983). More cysteine residues are found in cysteine rich regions which flank the collagenous region and could be involved in the formation of intramolecular disulphide bridges. These disulphide bonds explain why collagen VI has a high thermal stability and is resistant to proteases (Chu et al., 1990).The triple helical region of collagen VI is also involved in cell adhesion. Binding studies using extracted collagen VI have shown that it can interact with integrins α1β1and α2β1 through interaction with its helical domains. Collagen VI binding to α1β1 and α2β1 can be inhibited by the addition of synthetic RGD peptides (Pfaff et al., 1993) and extracted collagen VI can also promote cell spreading (Aumailley et al., 1989, Doane et al., 1992).

1.3.2.2 Globular domains

The globular regions of the collagen VI α-chains are mainly comprised of VWA domains. VWA domains are found in several ECM proteins such as integrins (Emsley et al., 1997), and matrilins (Klatt et al., 2011). VWA domains adopt a Rossmann fold which comprises a central hydrophobic β-sheet with amphipathic α-helices on opposing sides (reviewed in Hassan et al. 2012). Recently, the mouse α3N5 VWA domain was solved by X-ray crystallography (Becker et al., 2014). The domain has a central six stranded hydrophobic β-sheet flanked on either side by 3 amphipathic α-helices. The N and C-termini are on opposite ends of the domain allowing multiple VWA domains to form a beads-on-a-string arrangement.

VWA domains often contain metal ion-dependent adhesion site (MIDAS) motifs which can also be found in the VWA domains of integrins. In collagen VI, only the C1 and C2 domains of the α1 and α2 chains and the N4 domain of the α3 chain contain conserved MIDAS motifs (Ball et al., 2001). MIDAS motifs are involved metal ion dependant ligand binding in integrins, binding of Mg2+ and Mn2+ activates integrins allowing ligands to bind to the MIDAS site (Zhang and Chen, 2012). Recombinant VWA domains α3 N9-N1 weakly interacts with Mg2+ and Mn2+ but the presence of Zn2+ causes the VWA domains to adopt a more compact conformation and also promotes dimerization (Beecher et al., 2011). Binding of Zn2+ ions is not thought to involve the MIDAS site as Zn2+ ions are more likely to interact with cysteine residues.

The C3 domain in the α3 chain is characterised by 22 repeats of a Lys-Pro sequences which is separated by 2-5 other amino acid residues. This 120 residue domain is also unusual as it contains a high concentration of the residues Lys, Pro, Thr, Ala and Val (90%). The domain also shares a high homology, 37% identity, with the salivary protein sgs-3 found in drosophila

42 and is also similar to other proline-rich salivary proteins found in humans (Chu et al., 1990). The C4 domain shows typical features of type III domains of fibronectin. The domain has similar features such as size and conserved residues but has a low sequence identity (Chu et al., 1990). The final domain of the C-terminal globular domains of the α3 chain is a 70 residue segment which has high similarity to Kunitz-type serine protease inhibitors such as the trypsin inhibitor aprotinin and human urinary inhibitor (Chu et al., 1990). The structure of the α3 C5 domain has been solved by X-ray crystallography and NMR (Arnoux et al., 1995, Zweckstetter et al., 1996) and has a hydrophobic core comprised of two twisted antiparallel β- sheets bordered by two α-helices. The last 56 residues have 6 highly conserved cysteine residues which form 3 disulphide bridges in a 1-6, 2-4, 3-5 configuration. Although the C5 domain is similar in structure to serine protease inhibitors it does not inhibit trypsin or other serine proteases so far tested (Arnoux et al., 1995). The final domain of the α4 chain is also a Kunitz-like domain (Gara et al., 2008).

1.3.3 Collagen VI microfibril assembly

Three collagen VI α-chains associate to form heterotrimeric monomers (Furthmayr et al., 1983, Engvall et al., 1986) (Figure 1.7). The collagen VI microfibrils do not form in the absence of the α1 chain (Bonaldo et al., 1998) so it is likely that monomers are formed from one α1 and one α2 chain and that the α3, α4, α5 or α6 chains can be interchangeable based on their similarity (Gara et al., 2011). The C-terminal domains of each chain are thought to be involved in chain association and selection (Ball et al., 2001). Inter-chain disulphide bonds stabilise the formation of the monomer (Furthmayr et al., 1983, Chu et al., 1988). Two collagen VI monomers then associate to form a disulphide bonded anti-parallel homo-dimer. The two monomers are staggered by 30nm to form a dimer with an overlap ~75nm. In turn the dimers form a homo-tetramer which is then secreted into the extracellular space. Dimer and tetramers do not contain mixtures of different α3, α4, α5, or α6 chains (Maass et al., 2016). Microfibrils are formed extracellularly by the end-to-end assembly of tetramers (Furthmayr et al., 1983). Heteromeric microfibrils can form from different α3, α4, α5 or α6 chain homo-tetramers (Maass et al., 2016).

Non collagenous domains have been shown to be crucial for the formation of microfibrils, as deletion of the C-terminal VWA domains prevents the assembly of microfibrils (Ball et al., 2003, Tooley et al., 2010). The N-terminal domains N5-N1 of the α3 chain are also required for the assembly of microfibrils. Constructs missing the N10-N5 domains were unable to form

43 microfibrils (Lamande et al., 1998, Fitzgerald et al., 2001), however if the N10-N6 domains were absent microfibrils still formed.

Figure 1.7 Collagen VI microfibril assembly.

Collagen VI heteromeric monomers form from an α1 α2 and αX chain where X can be α chains 3- 6. Triple-helical monomers then form disulphide linked dimers and then tetramers before being secreted into the extracellular space where microfibrils are formed. C-terminal globular regions are shown in red, N-terminal domains are shown in blue.

1.3.4 Collagen VI microfibril structure

Collagen VI microfibrils have a beads on a string appearance with a periodicity of ~112 nm (Baldock et al., 2003) see Figure 1.8. The globular bead regions are ~53 nm in length, ~20 nm in width and are separated by a collagenous region which is 60 nm in length and has a width of ~4 nm. The bead regions can be sub-divided into two distinct half-beads which have a length of 26 nm and a width of ~20 nm. A three dimensional model of a half-bead of collagen VI using negative stain TEM was previously published by Beecher et al, (2011) (Figure 1.8). This three dimensional model of the half-bead has three distinct regions which have been termed the head, intermediate, and tail regions. The head region of the model is likely formed from the C1 domains, where six VWA domains fit in an oval shape, which is

44 consistent with two triple helical monomers. The intermediate region is less dense and more poorly defined, but the rectangular structure was proposed to contain four C2 domains. It was suggested that N- and C-terminal domains interact in this region. The tail regions are thought to contain the N-terminal domains. These regions show a higher degree of heterogeneity than the homogenous head region suggesting a degree of flexibility. EM and SAXS studies of recombinant α3 N9-N1 region and α4, α5, α6 N1-N7 region show that the VWA domains form a very similar compact C-shape (Beecher et al., 2011, Maass et al., 2016).

The collagenous region in the microfibril forms a segmented twisted supercoil (Knupp and Squire, 2001). The collagenous regions of the anti-parallel dimer can be seen twisting round each other in TEM images, and bifurcation of the strands can be seen at the bead regions (Baldock et al., 2003). The collagenous region is segmented and this is predicted to be due to the imperfections in the repeating Gly-X-Y motif (Knupp and Squire, 2001).

Figure 1.8 Collagen VI microfibril structure

(A) A schematic diagram of a collagen VI microfibril. The bead region can be subdivided into two half-beads as shown. (B) The 3D structure of the collagen VI half-bead (Beecher et al., 2011). C- terminal globular regions are shown in red, N-terminal domains are shown in blue.

1.3.5 Collagen VI function

Collagen VI has been shown to interact with a large number of extracellular matrix (ECM) components such as; collagen II (Bidanset et al., 1992), collagen IV, aggrecan, fibronectin (Tillet et al., 1994, Kuo et al., 1997), HS, perlecan and hyaluronan (Specks et al., 1992). These interactions can occur through direct binding but may also be organised by adaptor complexes through interaction with matrilins and the small leucine rich proteoglycans decorin and biglycan (Wiberg et al., 2001, Wiberg et al., 2002, Wiberg et al., 2003). The two main hypothesised functions of collagen VI are to anchor cells into the ECM and to organise the structure of the PCM surrounding chondrocytes in cartilage.

1.3.5.1 Collagen VI and the cell surface

In the ECM, collagen VI is hypothesised to act as an anchor between cell surface receptors, such as integrins (Aumailley et al., 1989, Pfaff et al., 1993) and the NG2 receptor, and the ECM (Stallcup et al. 1990; Burg et al. 1996). This allows the ECM to transduce biomechanical signals from the surrounding ECM to cells.

The NG2 receptor chondroitin sulphate proteoglycan is a membrane–associated receptor which has been shown to bind to the α2 chain of collagen VI (Burg et al., 1996; Stallcup et al., 1990). Collagen VI binds to the core protein of NG2, and competes with decorin suggesting a similar binding site (Burg et al., 1996). The NG2 receptor is thought to be crucial for mediating collagen VI interactions with the cell surface. The importance of the NG2 receptor binding is also highlighted by its involvement in collagen VI related diseases. It has been shown to be down regulated at the protein level in muscle tissue from patients suffering from UCMD and NG2 receptor is also down regulated in cartilage tissue from OA patients (Nugent et al., 2009; Petrini et al., 2005).

This link between the cell surface and the ECM has been shown to be cytoprotective as disruption of the collagen VI in the ECM has been shown to cause an increase in apoptosis through perturbation of autophagy (Irwin et al., 2003, Grumati et al., 2010). Collagen VI has also been shown to be a key component of the stem cell niche and is involved in maintaining the adult muscle stem cells ability to self-renew (Urciuolo et al., 2013).

1.3.5.2 Collagen VI and PCM structure

Another hypothesised role of collagen VI is to organise the structure of the PCM through its interaction with major structural components of the PCM and ECM such as collagen II, aggrecan, fibronectin, hyaluronan and perlecan (Poole, 1997, Vincent et al., 2007). These interactions occur through direct binding but may also be organised by adaptor complexes of members of the matrilins and the SLRPs decorin and biglycan.

Binding of decorin and biglycan has been studied using in vitro binding studies and TEM (Wiberg et al., 2001). Both SLRPs are show to compete for a binding site in a region between the N-terminus of the helical domain and the adjacent VWA N1 globular domain. Solid phase binding assays suggested that the α2 chain may play a role in this binding (Wiberg et al., 2001). Biglycan is thought to play an important role in the organisation of collagen VI assemblies in the PCM. In vitro studies have shown that purified collagen VI assembles into hexagonal networks in the presence of biglycan. In these structures biglycan is localised to intra-network junctions. Although only the core protein of biglycan is necessary for collagen VI binding (Wiberg et al., 2001), DS chains were required for network formation (Wiberg et al., 2002).

Matrilins are ECM proteins which act as adaptor molecules which mediate interactions between collagen fibres and other matrix constituents. The matrilins are a family of 4 multi- subunit proteins which comprise of VWA domains interconnected by arrays of epidermal growth factor like (EGF) domains. Subunits form trimers and tetramers through interactions in their C-terminal α-helical coiled coil domains. Matrilins-1 and 3 are predominantly expressed in cartilage tissues whereas matrilins-2 and 4 have a wider pattern of expression. The SLRPs decorin and biglycan have been shown to form assemblies with matrilin-1 to facilitate the matrix interactions of collagen VI with proteins such as collagen II and aggrecan (Wiberg et al., 2003).

1.3.6 Collagen VI tissue organisation

In skin, collagen VI forms an irregular web-like network of fibrils which associate with collagen II and III fibrils. The collagen VI fibres can be seen running parallel and in-between banded collagen fibrils (Keene et al., 1988). Collagen VI microfibrils are found throughout the corneal stroma where they associate with thin diameter collagen fibrils {Chen, 2015 #653}. In tissue culture, collagen VI can form bundles of aligned filaments which have a banding pattern with

48 a periodicity of ~100 nm (Bruns, 1984). These large aggregate like structures can also be seen in diseased tissue. Collagen VI tetramers were identified as the most likely main components of large banded aggregates found in the Bruch‟s membrane of the eye from patients with adult macular degeneration (AMD) and Sorsby‟s Fundus Dystrophy (Knupp et al., 2000, Knupp et al., 2002, Knupp et al., 2006). Large aggregate structures identified as collagen VI have also been identified in the trabecular meshwork (Koudouna et al., 2014). In vitro assays using purified collagen VI tetramers have demonstrated that collagen VI can assemble to form large hexagonal networks when incubated with biglycan (Wiberg et al., 2002). Hexagonal arrangements of collagen VI networks can also be observed in tissue culture (Engvall et al., 1986) suggesting that this is a potential structure collagen VI forms in tissues.

Collagen VI is also highley expressed and widely distributed in the PCM surrounding chondrocytes in the articular cartilage (Kuo et al., 1997, Wilusz et al., 2014) where it plays a key role in the PCM structure. The correct organisation of the PCM is essential for maintaining the mechanical properties of cartilage and for transducing biomechanical signals from the surrounding ECM to the chondrocytes (Zelenski et al., 2015). Immuno-fluorescence (Choi et al., 2007), and helium ion microscopy (Vanden Berg-Foels et al., 2012) shows that the PCM is formed from a basket-like meshwork of ECM surrounding the chondrocyte. One aspect of this PhD study will be on the organisation of collagen VI microfibrils in the PCM surrounding chondrocytes in murine articular cartilage.

1.3.7 Collagen VI diseases

Although collagen VI is widely expressed, mutations in the COL6A1 COL6A2 and COL6A3 genes have been mainly linked to diseases of the muscular skeletal system, causing a spectrum of myopathies which range from the mild diseases such as Bethlem myopathy to the much more severe UCMD and the degeneration of cartilage in OA. In addition, mutations in COL6A3 and COL6A6 have been linked to neuromuscular diseases (NMD) (Hunter et al., 2015), mutations in COL6A1, COL6A2 or COL6A3 have been linked to a number of skin disorders including dry skin, follicular keratosis, keloids and striae rubrae (stretch marks) (Lettmann et al., 2014) and variants of COL6A5 have been associated with atopic dermatitis (Soderhall et al., 2007).

The main symptoms of UCMD (reviewed in Bönnemann 2011; Lampe & Bushby 2005) are early onset joint weakness and laxity, as well as severe and progressive proximal joint

49 contractures. Bethlem myopathy exhibits many of the same symptoms but in a milder form than seen in the more severe disease UCMD

A large number of mutations which have been reported in UCMD patients arise from premature stop codons which lead to nonsense mediated mRNA decay. These mutations are caused by frame shifts, which arise from splice site mutations and intragenic deletions and insertions (Lampe & Bushby 2005). Another common form of mutations found in patients are splice site mutations which lead to in frame exonic deletions (Lampe & Bushby 2005). One of the most common type of Bethlem myopathy causing mutations are splice site mutations which cause skipping of exon 14 in the α1 chain. The loss of this exon prevents the formation of dimers as a key cysteine residue is lost, which in turn reduces the amount of collagen VI in the ECM (Lampe & Bushby 2005).

1.3.8 Knockout mouse models of Bethlem myopathy

To study the role of collagen VI in disease, collagen VI genes COL6A1 and COL6A3 have been disrupted in mice. Homozygous mutant mice lacking a functional COL6A1 gene had no collagen VI in the ECM of tissues showing that microfibrils were unable to assemble in the absence of the alpha1 chain (Bonaldo et al. 1998). These mice had signs of myopathy such as muscle necrosis, differences in muscle fibre diameter and signs of muscle fibre degeneration. Heterozygous null mice had a similar but milder myopathy phenotype than that of the homozygous null mice. Loss of collagen VI also led to mitochondrial dysfunction due to disruption of autophagy and changes in metabolism (Irwin et al., 2003, Grumati et al., 2010, De Palma et al., 2013). Absence of collagen VI microfibrils also led to the development of OA, delayed ossification and reduced mineralisation of bones and the appearance of osteophytes (Alexopoulos et al. 2010; Christensen et al. 2012). COL6A3 knockout mice have a myopathic phenotype similar to that of the COL6A1 knockout mouse (Pan et al. 2013).

1.4 Collagen VI summary

Collagen VI microfibrils are an essential component of the ECM and play a key role in the homeostasis of tissues through interactions with several other ECM proteins allowing for the anchoring of cells into the ECM. Collagen also plays a key role in organisation of the PCM surrounding chondrocytes in articular cartilage. To fully understand how collagen VI fulfils these functions it important to understand how collagen VI microfibrils are organised across multiple length scales from individual microfibrils to larger networks in tissues.

1.5 Project aims

The overall aim of this PhD project is to determine how fibrillin and collagen VI microfibrils are organised in their different levels of hierarchy, from individual molecules in microfibrils, to larger networks of microfibrils in tissues.

The high resolution structure of some individual fibrillin domains and short fibrillin fragments have been solved using X-ray crystallography, NMR and SAXS, but it is still not understood how individual fibrillin monomers are arranged in the microfibril. To address this, a high resolution 3D model of a fibrillin microfibril repeating unit will be constructed. Tissue extracted microfibrils will be imaged using negative stain TEM and will be reconstructed using single particle averaging approaches. This model will then be used to fit fibrillin domain models to determine how fibrillin may be arranged into mature microfibrils.

Studies of fibrillin have shown that it has a complicated tissue specific organisation but little is known about the 3D organisation of individual microfibrils into larger structures. To investigate how fibrillin microfibrils are organised into large scale networks in tissues, thick section electron tomography using the FEI Tecnai G2 Polara and serial block face scanning electron microscopy (SBF-SEM) imaging using the Gatan 3-view system will be used to create 3D reconstructions of the bovine and murine ciliary zonule. Previous studies of the 3D orgainsation of ciliary zonules using SEM, ultra-sound biomicroscopy (UBM)(Lutjen-Drecoll et al., 2010), environmental SEM (ESEM) (Sherratt et al., 2001, Nankivil et al., 2009) and immunofluorecense microscopy (Shi et al., 2013a) have provided a detailed understanding of the gross structure of the ciliary zonule but do not have the resolution to image individual microfibrils in situ. SBF-SEM and electron tomography can image tissues at a much higher resolution then these techniques and will allow the modelling of individual microfibrils in tissues up to bundles of microfibrils across nanometre to millimetre length scales.

Collagen VI microfibrils also have a critical role in tissues. Structures of the N and C-terminal fragments of α chains have been modelled using SAXS (Maass et al., 2016) and crystal structures of several domains have been solved. Previously a 3D reconstruction of collagen VI using negative stain-TEM has revealed the organisation of C- and N-terminal VWA domains in the bead region, where C-terminal VWA domains form a compact head structure with N-terminal VWA domains forming the more flexible tail regions (Beecher et al., 2011). However it is still not fully understood how collagen VI molecules are arranged in a microfibril. To understand this extracted corneal collagen VI microfibrils will be imaged in a frozen

51 hydrated state in the absence of stain using cryo-TEM. Single particle averaging will be used to construct a high resolution 3D model of collagen VI.

To determine how collagen VI is organised into higher order structures in tissues the PCM surrounding chondrocytes in murine articular cartilage will be imaged. Collagen VI is expressed highly in the PCM surrounding chondrocytes where it plays a role in signalling and mechanical properties. Studies of the structure of the PCM have used immuno-fluorescence (Choi et al., 2007), and helium ion microscopy (Vanden Berg-Foels et al., 2012) showing that PCM forms a basket-like meshwork surrounding the chondrocyte. These techniques however cannot determine the high resolution 3D organisation of the PCM. SBF-SEM and electron tomography will be used to create nanoscale 3D reconstructions of the PCM surrounding chondrocytes in situ in murine cartilage to determine how collagen VI microfibrils are organised into large networks.

2 Materials and methods

2.1 Tissue sources

Bovine eyes were sourced from a local abattoir. Murine articular cartilage was extracted from 6 month old C57BLKS-Leprdb/+ heterozygote diabetic mice (Jackson Labs). Murine eyes were taken from 27 day old C57BL/6 mice (Jackson Labs). Mice were sacrificed by asphyxiation using CO2 gas following home office guidance.

2.2 Tissue culture

Tissue culture reagents were obtained from Sigma-Aldrich and tissue culture plastics from Corning. SaOS-2 cells transfected with a N6-C5 collagen VI α3 chain (Fitzgerald et al., 2001) were a gift from Dr Shireen Lamande. SaOS-2 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% foetal calf serum, 100 U/ml penicillin/streptomycin and 400 µg/ml of G418 at 37°C in 5% CO2. When cells reached confluency they were sub-cultured. Media was removed and cells were washed with 1 x phosphate buffered saline (PBS). Cells were detached from flasks by incubating cell layers with 1 ml/25 cm2 of 1 x porcine trypsin-EDTA for 2 mins before neutralising the trypsin with an equal volume of cell culture medium. The cell suspension was then centrifuged in a desktop centrifuge at 450 g for 4 mins and the supernatant was removed. The resulting cell pellet was then suspended in fresh media before being split into fresh flasks at a ratio of 1:3.

2.3 Microfibril extractions

2.3.1 Fibrillin tissue microfibrils

Fibrillin microfibrils were extracted from bovine ciliary zonule tissue using collagenase as described previously (Lu et al., 2005). Approximately 0.2g (wet weight) of bovine ciliary zonule was broken up with a razor blade before being suspended in 2 ml of digestion buffer (400mM NaCl, 20 mM Tris-HCl, 2.5 mM CaCl (pH 7.4)) containing 0.1 mg/ml of chromatographically purified bacterial collagenase type VII (Sigma-Aldrich), and protease inhibitors (3 mM N-ethylmaleimide (NEM), 5 mM phenylmethane sulfonyl fluoride (PMSF)). Collagenase type VII is a purified collagenase from Clostridium histolyticum free of both caseinase and clostripain activity which cleaves various collagen types.Digestions were incubated overnight at 4 °C whilst undergoing gentle stirring. Digested tissue was centrifuged

53 for 3 mins at 800 g using a bench top centrifuge (Sigma-Aldrich) and the supernatant was size fractionated on a Sepharose CL-2B column (GE Healthcare). The column was either equilibrated in a high salt buffer (400 mM NaCl, 20 mM Tris-HCl, 2.5 mM CaCl (pH 7.4)) for subsequent imaging using negative stain TEM or a lower salt buffer (150 mM NaCl, 20 mM Tris-HCl, 2.5 mM CaCl (pH 7.4)) for imaging under cryo-conditions. The sample was separated on the column at a flow rate of 0.2 ml/min, fractions of 0.5 ml across the void peak of the column were collected and saved as microfibrils elute in the column void volume due to their large size. Protein concentration was monitored throughout by measuring UV absorbance at 280 nm.

2.3.2 Collagen VI tissue microfibrils

Collagen VI microfibrils were extracted from bovine cornea using collagenase as described previously (Beecher et al. 2011). Approximately 0.2 g (wet weight) of bovine cornea was diced with a razor blade before being suspended in 2ml of digestion buffer (400 mM NaCl, 20 mM Tris-HCl (pH 7.4)) which contained 0.1 mg/ml of chromatographically purified bacterial collagenase type VII (Sigma-Aldrich), and protease inhibitors (3 mM NEM, 5 mM PMSF). Digestions were performed as previously described in section 2.3.1. Cornea digestions were size fractionated on a Sepharose CL-2B column equilibrated in a high salt buffer (400mM NaCl, 20 mM Tris-HCl (pH 7.4)) as previously described in 2.3.1.

2.3.3 N6-C5 collagen VI microfibrils

N6-C5 collagen VI was purified from SaOS-2 N6-C5 cell layers using collagenase. SaOS-2 N6-C5 cells were cultured in 100 mm cell culture dishes in DMEM supplemented with 10% foetal calf serum, 100 U/ml penicillin/streptomycin and 25 nM sodium ascorbate for two weeks. Media on cell cultures was replaced with fresh media every two days. To extract collagen VI, cell layers were washed in PBS and scraped off into a 15 ml tube (Corning). The cell layers were pelleted by centrifugation for 5 mins at 450 g and the supernatant was removed. The cell pellet was then resuspended in digestion buffer (400 mM NaCl, 20 mM Tris-HCl (pH 7.4)) which contained 0.1 mg/ml of chromatographically purified bacterial collagenase type VII (Sigma-Aldrich), and protease inhibitors (3 mM NEM, 5 mM PMSF) and was incubated over night at 4 °C whilst undergoing gentle stirring. The digested cell layer was then centrifuged for 3 mins at 800 g and the supernatant was size fractionated using a CL-2B column equilibrated in high salt buffer as previously described in section 2.3.1.

2.4 Sodium dodecyl sulphate polyacrylamide gel electrophoresis

Collagen VI samples were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Samples were diluted in LDS loading buffer (Life technologies) with 5 % (v/v) β-Mercaptoethanol and were loaded onto 4- 20% Mini-PROTEAN TGX precast gels (Biorad). Precision plus protein marker ranging from 10 kDa to 250 kDa (Biorad) were used for molecular weight comparison. Electrophoresis was performed at 180 V for 45 minutes with Tris-glycine SDS (TGS) (25 mM Tris-HCl 192 mM glycine 0.1 % (v/v) SDS) running buffer using a Mini-PROTEAN Tetra Cell (Biorad). Protein bands were visualised by staining gels using Instant Blue (Expedion) according to manufacturer‟s instructions.

2.5 Western blotting

Proteins were separated by SDS-PAGE as described previously in section 2.4 and were transferred to nitrocellulose membranes (Whatman). Transfers were performed at 35 V for 90 minutes using the NuPAGE gel blotting system (Life technologies) using a Tris-glycine transfer buffer (96 mM Tris-HCl, 780 mM glycine and 0.075% (v/v) SDS) with 20% methanol (v/v). Following protein transfer, membranes were blocked in 5 % milk (w/v) in Tris buffered saline and Tween 20 (TBST) (50 mM Tris-HCl, 150 mM NaCl pH 7.4, 0.05% (v/v) Tween 20) for 30 mins. Membranes were incubated with the polyclonal rabbit anti collagen VI primary antibody (NB120-6588 (Novus Biologicals) diluted at a ratio of 1:1000 in 5 % milk (w/v) in TBST) overnight. After incubation with primary antibody membranes were washed three times in TBST for 10 mins. To detect bound primary antibody, membranes were incubated with a secondary goat anti rabbit antibody conjugated with horse radish peroxidase (Dako) (diluted at a ratio of 1:3000 in 5 % milk (w/v) in TBST), for 2 hours. Unbound secondary antibody was removed by washing membranes three times for 10 mins using TBST before bands were visualised using enhanced chemiluminescence (Interchim) and imaged using a ChemiDoc imaging system (Biorad).

2.6 Transmission electron microscopy

2.6.1 The transmission electron microscope

In TEM a beam of electrons is used to image samples; as electrons have a shorter wavelength than visible light they can therefore be used to resolve smaller structures than

55 light microscopy. The electron microscope has an electron source, a series of electromagnetic lenses, which focus the electron beam, and a detector, see Figure 2.1 for a schematic diagram of a transmission electron microscope. For reviews on single particle averaging and 3D reconstruction using TEM see (van Heel et al., 2000, Frank, 2002, Orlova and Saibil, 2011).

Electrons are emitted from the electron source and are accelerated through an electric field up to 300 kV. Electron sources commonly used are, tungsten filaments, or lanthanum hexaboride (LaB6) crystals where electrons are emitted through thermal emission. The most advanced electron source is the field emission gun (FEG), which is comprised of a sharpened tungsten crystal coated with zirconium oxide; electrons are emitted from the tip via a process called field emission. FEGs can create a beam which has a higher intensity with greater coherence than either the LaB6 or tungsten filament sources. The emitted electron beam is focused and magnified using several electromagnetic lenses. The lens system consists of, condenser, objective, intermediate and projector lenses. The condenser lens converts the diverging electron beam from the electron source into a parallel beam which interacts with the sample. The objective and intermediate lenses focus and magnify the image onto the detector. Apertures are placed in the path of the electron beam to remove electrons which have scattered to high angles from reaching the detector. Originally micrographs were taken using photographic film but this has largely been replaced by the use of charged couple device (CCD) detectors which are more convenient for computer based image processing. Recently a new type of detector has been developed, the direct detection devices (DDD). These detectors have a much higher sensitivity with the ability to detect individual electrons.

Figure 2.1 A schematic diagram of an electron microscope.

(A) A schematic diagram of an electron microscope adapted from (Orlova and Saibil, 2011). (B) Shown is a diagram of the different forms of scattering when the incident electron beam interacts with an atom adapted from (Orlova and Saibil, 2011). Electrons are shown as blue circles and the atomic nucleus is shown in purple. Incident electrons which collide with electrons or the nucleus resulting in energy loss and are inelastically scattered. Deflection of electrons results in elastic scattering where no energy is lost. Elastically scattered electrons are focussed by the microscope lens system onto the detector creating an image. The majority of inelastically scattered electrons are either absorbed or scattered to high angle and are removed by apertures so do not contribute to the image.

2.6.2 Image formation and contrast

Images are generated by detecting electrons which have passed through the sample. Contrast in the image (the difference between brightest and darkest points in the image) is made up of amplitude contrast, which is contrast resulting from absorption of the electron beam (inelastic scattering) and phase contrast resulting from interference between the incident electron beam with elastically scattered electrons (Figure 2.1). The image is also affected by the operating conditions of the microscope. Aberrations in the lens system such as chromatic aberration and astigmatic aberration as well as defocus, sample damage, fluctuations in magnetic fields, beam coherence and vibration limit the signal transfer for high resolution information. This fall off caused by imperfections in the microscope leads to blurring of fine details and in real space is referred to as the point spread function (PSF). The

PSF can be described in Fourier space as the contrast transfer function (CTF) multiplied by an envelope function, which describes the instabilities in the microscope and specimen. As biological samples consist of mainly light atoms such as hydrogen, oxygen, nitrogen and carbon they do not absorb electrons and scatter electrons weakly so the contribution of amplitude contrast in an image is small. To increase contrast in the image samples can be stained with heavy metal salts in techniques such as negative stain TEM or phase contrast can be enhanced by using lens aberration and defocusing the electron beam to increase the phase shift of scattered electrons (Erickson and Klug, 1970). Energy filters can also be used to improve the contrast of images. A small fraction of inelastically scattered electrons can reach the objective lens. Due to their loss of energy from interaction with the sample, they have a lower energy than elastically scattered electrons and therefore have a longer wavelength. This causes inelastically scattered electrons to be focused in a different plane than elastically scattered electrons. The result of this is chromatic aberration which leads to blurring and degradation of the image. Energy filters can deflect electrons of different wavelengths along different paths preventing inelastically scattered electron from contributing to the image. The filter can be either in the column like a Ω filter or post column such as a Gatan imaging filter (GIF).

2.6.3 Sample preparation

As electrons interact with matter strongly, the electron microscope column must be kept under a high vacuum to prevent the electron beam being scattered by gas molecules. This means however that samples need to be in a solid state. Sample preparation techniques such as drying or freezing allow samples to be imaged.

In negative stain TEM a sample of a protein solution is absorbed onto a carbon support film and is embedded in a layer of a heavy metal salt such as uranyl acetate (UA). Grids are then dried and the structure of the protein is visualised as the exclusion of stain, hence the name negative stain. This technique gives good contrast to samples due to the heavy metal salts high electron potential which can absorb the electron beam. The technique also allows imaging of small protein assemblies as small as 60 kDa. Negative staining however has a few draw backs, as samples are dried this can cause structures to collapse which leads to flattening in 3D reconstructions. Also if the stain does not cover the whole protein some parts of the sample will not be imaged.

Another sample preparation technique allows for the imaging of proteins in their hydrated state by freezing proteins in a layer of vitreous ice, known as cryo-TEM. A thin layer of protein solution is created on a grid by blotting excess sample away before being plunge frozen in liquid ethane, cooled by liquid nitrogen. Samples are imaged at -170 °C which prevents the formation of ice crystals and helps prevent damage from the electron beam. Thickness of the ice layer is critical to giving good contrast; ice needs to be as thin as possible without causing damage to the sample. One of the drawbacks of cryo-TEM is that for sub-nanometer reconstructions of proteins with no symmetry there is currently a practical limit to the sample mass of ~200-300 kDa, as smaller proteins require higher defocus to generate enough contrast to be detected {Cheng, 2015 #651}. Increasing the defocus however limits the higher spatial resolution information in an image.

2.6.4 Single particle 3D reconstruction

Single particles in electron micrographs represent 2D projections of a 3D volume but also contain noise from a variety of sources; from variation in staining or ice thickness, damage from sample preparation or detector noise. This noise prevents accurate reconstruction of the 3D structure of interest. To increase the signal to noise ratio (SNR) in the data, many different particles are superimposed and averaged together. Once particles have been averaged to increase the SNR the 2D projections can be used to calculate a 3D reconstruction.

2.6.4.1 CTF correction

As previously mentioned in section 2.6.2 an electron micrograph in Fourier space is a convolution of the CTF and the imperfections of the microscope. To recreate a true representation of the sample the image must be corrected for the CTF. This deconvolution however is complicated by the fact the CTF function oscillates around zero and therefore has points where information is lost. To compensate for the loss of information images are collected at several different defocuses and are combined. The most common method of CTF correction is phase flipping, where negative regions of the CTF spectra are flipped to positive.

2.6.4.2 Alignment

Before particles can be averaged to increase the SNR they need to be aligned. Particles in a data set are aligned by comparing images to a reference image using cross correlation. This determines rotations and translational shifts required to align the target to reference images. Initial reference images are often a rotationally averaged sum of all images in the dataset.

Noisy images are difficult to cross-correlate so this is often an iterative process with the references improving with each round of alignment. As well as the different orientations of particles in the plane of the image, the particles also have different out-of-plane orientations which result in different projections. Several statistical tools have been developed to group images into classes of particles with similar orientations. Classified images can then be averaged and used as improved references for further alignments.

2.6.4.3 Classification

There are several statistical methods which can be used to determine variation between images, such as principal component, multivariate, or covariance analysis. After determining variance between images, images can then be sorted into classes using hierarchical or K- means clustering (van Heel et al., 2000, Orlova and Saibil, 2011).

To determine the variation between images, images are represented as vectors in an N- dimensional hyper-space with coordinates which are determined by the density of the pixels in an image. This allows images to be represented as clouds of points in hyper-space. Images which are similar will be close to each other in the cloud. Multivariate statistical analysis and principal component analysis methods can then be used to simplify the variables between images and determine what are the components which do not correlate between images which are termed principle components. Images can then be sorted into clusters based on the principal components. There are two main methods to do this either using hierarchical or K-means clustering.

There are two different approaches to hierarchical clustering, in oligomeric hierarchical clustering clusters of points are considered as a class and the most similar classes are merged until a predefined number of classes are reached (van Heel, 1984). The divisive method starts with one class which is then split into further classes based on dissimilarity between classes. In each method algorithms try to minimise the intraclass variance and maximise the interclass variance.

2.6.4.4 Determination of particle orientation

To reconstruct a 3D object from its 2D projections the orientations of the projections must be determined. There are two main strategies to determine particle orientations either experimentally by collecting tilt pairs and using methods such as random conical tilt and

60 orthogonal tilt reconstruction or computationally using angular reconstitution methods or projection matching.

In random conical tilt methods, tilt pairs of images are taken at ~50 ° and at 0 °. The particles at 0 ° are grouped into classes of particles with the same in plane orientation (Radermacher, 1988). This is then used to determine the orientation of the tilted particles. This method suffers from a missing cone of data which is not represented due to samples not being imaged at high tilt angles. In orthogonal tilt reconstructions tilted image pairs are collected at 45 ° and -45 ° this method does not suffer from missing cone of data as long as particles represent a large number of orientations on the grid (Leschziner and Nogales, 2006).

The main methods to computationally determine orientations of particles are projection matching or angular reconstitution. Angular reconstitution is based on finding common lines between pairs of 2D projections. Any set of 2D projections of a 3D object will have common features in all projections. Any pairs of projections will have a 1D (line) projection in common (Van Heel, 1987). These common lines can be determined using real space or Fourier space methods. Common lines can be used to determine the orientation between images. Two images only have one common line so at least three images are needed to determine the angles between projections (Van Heel, 1987).

Projection matching requires an initial model which is used to generate a set of reprojections with known orientations. This set of reprojections are used as a set of references which can be compared against each image in a set using cross correlation (Harauz and Ottensmeyer, 1984). Each image is assigned the orientation of the reference it most highly correlates to. This projection matching method can be done in real space using IMAGIC (van Heel and Keegstra, 1981) and SPIDER (Frank et al., 1996), or reciprocal space using FREALIGN (Grigorieff, 2007) or FindEM (Roseman, 2004).

2.6.4.5 3D reconstruction

Once the orientation of the 2D projections is known they can be used to create a 3D reconstruction. Several approaches have been developed for the reconstruction of 3D objects from the 2D projections. These methods are either performed in real space such as back projection or in reciprocal space such as Fourier inversion.

Back projection works by stretching each pixel of a projection over the volume which is being reconstructed in the direction of the projection. These pixel projections are called ray sums.

The density of the reconstructed volume is estimated from the addition of all the rays sums which pass through it. This method however creates artefacts in the reconstruction where details smear out in a halo which surrounds the reconstruction. These distortions can be removed by using filtering methods (Harauz and Ottensmeyer, 1984). More complicated methods of back projection use algebraic methods such as algebraic reconstruction technique (ART) (Gordon et al., 1970), simultaneous iterative reconstruction technique (SIRT) (Herman and Rowland, 1973), and simultaneous algebraic reconstruction technique (SART) (Andersen and Kak, 1984). These algebraic methods estimate which pixels ray-sums pass through to determine the density of an object. To reduce the number of calculations, these methods approximate points where rays sums pass through and these approximations are refined iteratively.

Fourier inversion works on the central section theorem where projections of an object corresponds to the central section of the Fourier transform of the object. Particles represent central slices through the Fourier transform in their corresponding orientation, when particles sufficiently fill in the volume in Fourier space, an inverse Fourier transform can be performed to reconstruct the object (De Rosier and Klug, 1968).

2.7 Negative stain TEM

Purified fibrillin or collagen VI microfibrils were adsorbed onto glow discharged carbon coated copper grids. Commercial carbon coated copper 400 mesh grids (Agar Scientific) or home- made grids were used. To make “home-made” grids, carbon films were created by sputter coating cleaved mica (Agar Scientific) with carbon using a Cressington 308R vacuum sputter coater. Carbon films were then transferred to grids by slowly submerging the mica sheet in distilled water allowing the carbon film to float on the surface. Water was then pumped away to allow the carbon film to adhere onto submerged copper 400 mesh grids (Agar Scientific). Grids were dried for 24 hours before use. Approximately 3 µl of 200 µg/ml of sample protein was incubated on glow discharged (25 s at 25 mA) grids for 1 min before grids were washed using 60 µl of 2 % (w/v) UA stain. Excess UA was then wicked off using filter paper (Whatman) before being left to dry. Grids were imaged under low dose conditions using a FEI

Technai 12 twin TEM with a LaB6 tip operating at an accelerating voltage of 120 kV. Microfibrils were imaged at a magnification of 21000X to give a sampling of 4 Å/pixel with an exposure time of 1 second. The defocus was varied across a range from -0.5 to -1 µm.

2.8 Cryo-TEM

2.8.1 Fibrillin microfibrils

Fibrillin microfibril samples were adsorbed onto glow discharged 0.2 μm holey carbon Quantifoil 2/2 grids for 1 minute before grids were blotted and plunge frozen in liquid ethane using a FEI Vitrobot plunge freezer. Blot times ranged between 2.5 and 5 seconds and the Vitrobot was maintained at 4 °C and at 95% humidity.

Samples were imaged under low dose conditions on a FEI Tecnai G2 Polara TEM operating at an accelerating voltage of 200 KV at a magnification of 39000X. Images were collected using a Gatan Ultrascan 4000 CCD camera with a defocus range of -2 to -5μm. Images were exposed to a dose of approximately 20 e-/Å2

2.8.2 Collagen VI microfibrils

Collagen VI samples were adsorbed onto glow discharged 0.2 μm holey carbon Quantifoil grids, which had been coated with a thin layer (~2 nm) of carbon. Samples were pipetted onto the surface of grids and were left for 1 minute before being washed 3 times with 20 μl of water. Grids were frozen and imaged as previously described in section 2.8.1.

2.9 Single particle averaging and 3D model reconstruction of matrix microfibrils

2.9.1 Fibrillin

Single particle averaging and model reconstruction was used to create a 3D model of a fibrillin microfibril repeat; a flow chart outlining the main steps of the method is shown in Figure 2.2. Micrographs were evaluated and 2797 microfibril particles were manually boxed using the EMAN-2 suite of programs (Tang et al., 2007) with a box size of 512 X 512 pixels. Particles were CTF corrected by phase-flipping before being edge-mean normalised and band-pass filtered, with a high pass filter of 600 Å and lowpass filter of 20 Å, using a top-hat filter to remove noise from the images using SPIDER (Frank et al., 1996).

Particle stacks were iteratively rotationally and translationally aligned to an initial template. Rotational and translational shifts were determined using the local projection matching program FindEM (Roseman, 2004). FindEM uses a fast local correlation function which uses Fourier space cross correlation between a reference image and a target image, the local area

63 which is correlated is defined using a binary mask. The initial template was an average image of the unaligned stack of particles. A binary mask which covered the microfibril particle was drawn using WEB (Frank et al., 1996).

An initial 3D model was then constructed by creating a cylindrical average model from the sum average of the aligned particle set using SPIDER. The sum-average was back projected using a SIRT based method and with C-100 symmetry imposed. The initial model was then iteratively refined using a projection matching procedure. The initial model was projected at an angular increment of 5° to give an angular sampling sufficient for 20 Å resolution, for an object of this size, around the fibre axis. The model projections were used as references for multi-reference based rotational and translational alignment using FindEM as previously described. After the orientation of particles was assigned based on cross correlation to the model projections, particles with the same orientation were summed into class averages and were back projected using SIRT in SPIDER to construct a new model. This procedure was repeated for 44 iterations until a stable model was generated. During the iterative refinement 2-fold symmetry was imposed along the fibre axis of the model.

To evaluate the models, projections were compared non-quantitatively to classum images created with a non-reference based classification method using IMAGIC 5 (van Heel and Keegstra, 1981). The resolution of the final models was calculated using Fourier shell correlation (FSC) of two models reconstructed from two halves of the data set using SPIDER. The resolution of the model was estimated by taking the resolution at the 0.5 FSC threshold.

. Figure 2.2 Single particle averaging and 3D model reconstruction method

A flow chart outlining the main steps used in creating 3D models of fibrillin and collagen VI microfibrils.

2.9.1.1 Fibrillin sub-models

Sub-models of the final fibrillin microfibril model were generated to reduce heterogeneity in the model and to refine the model without imposing symmetry. Sub-models were generated by alignment of the bead, arm, interbead and shoulder regions of the microfibril using binary masks specific for these regions using the same methods as previously described 2.9.1.

2.9.2 Collagen VI

Collagen bead regions were boxed using a 256 X 256 pixel box using EMAN-2 (Tang et al., 2007) resulting in a dataset of 1060 particles. Particles were CTF corrected by phase-flipping before being edge-mean normalised and low pass filtered to 20Å using SPIDER. Particle stacks were iteratively aligned, and a 3D model was reconstructed as previously described in section 2.9.1.

2.9.2.1 Single bead model

To improve the resolution of the collagen VI globular regions and avoid flexibility between beads, a half-bead model was created. Each half-bead of the collagen VI bead region was shifted to the centre of the image using SPIDER to create a half-bead particle stack. The half- bead particle stack had 2120 images. A single half-bead of the bead model was used as the initial model for a model based refinement using the half-bead data set as previously described in section 2.9.1.

2.9.3 Docking of fibrillin-1 fragment molecular models

Molecular models published by Baldock et al., 2006 were initially docked by hand based on the molecular pleated (Baldock et al., 2006) or the half staggered models of fibrillin packing (Kuo et al., 2007) using UCSF Chimera (Pettersen et al., 2004). The fit of the models was refined using UCSF Chimera fit in map. Fit in map uses cross correlation to iteratively maximise the fit between the maps. Molecular models were fitted with a simulated resolution corresponding to the estimated resolution of the target model.

2.10 SBF-SEM and electron tomography sample preparation

2.10.1 Bovine ciliary zonule

Samples were prepared for SBF-SEM and electron tomography as previously published (Starborg et al., 2013). Bovine ciliary zonule tissue was fixed in 2.5% (v/v) glutaraldehyde and

4% paraformaldehyde (w/v) in 0.1 M cacodylate buffer for 24 hours, samples were then washed two times in 0.1 M cacodylate buffer before being further fixed and stained for 1 hour in 1% OsO4 and 1.5% C6N6FeK4 (w/v) in 0.1 M cacodylate buffer. Samples were then treated with 1% (w/v) tannic acid in 0.1 M cacodylate buffer for 1 hour, after which samples were washed two times in distilled water before further staining in 1% (w/v) OsO4 in distilled water. After osmium staining samples were washed two times in distilled water before being incubated with 1% (w/v) uranyl acetate in distilled water for 1 hour. Samples were then dehydrated in a series of ethanol dilutions (50-100%) followed by incubation in acetone before being embedded in TAAB 100 hard resin (Agar Scientific). Resin was polymerised at 60 ºC.

2.10.2 Murine ciliary zonule

Mouse eyes were fixed in fixed in 2.5% (v/v) glutaraldehyde and 4% paraformaldehyde (w/v) in 0.1 M cacodylate buffer for 24 hours. The posterior half of the fixed eye was removed before being stained and embedded as previously described in section 2.10.1.

2.10.3 Murine cartilage

Cartilage samples were decalcified to remove bone, which may cause damage to the knife during sectioning, by incubation in 14% EDTA at 4ºC for 7 days. Samples were lightly perturbed and the EDTA solution was changed every day as previously described (Jiang et al., 2005). Samples were then stained and embedded as previously described in section 2.10.1.

2.11 SBF-SEM

2.11.1 Bovine ciliary zonule

Bovine ciliary zonule samples were oriented so the ciliary zonule was imaged in transverse cross-section by SBF-SEM using the Gatan 3view system (Quanta FEG 250 (FEI) equipped with a Gatan3View ultramicrotome) using a 3.8 kV accelerating voltage. Primary electron back-scatter was used to image the block face before a section of 100 nm was removed from the surface. A dataset of 1500 images was collected at a magnification which gave a sampling of 7 nm/pixel. A depth of 150 µm of tissue was imaged.

2.11.2 Murine ciliary zonule

Murine ciliary zonule samples were orientated so that samples were sectioned through sagittal or coronal planes of the eye. Samples were imaged at a magnification which resulted in a sampling of 26 nm/pixel pixels using the Gatan 3view system, a section of 100 nm in thickness was removed after each image. The dataset of the sample imaged through the coronal plane of the eye is composed of 500 images and the data set of the sample imaged through the sagittal plane of the eye contains 917 images. A depth of 50 and 91.7 µm of tissue was imaged in the data sets respectively.

2.11.3 Murine articular cartilage

Murine articular cartilage samples were imaged at a magnification which resulted in a sampling of 10 nm/pixel using the Gatan 3view system. The data set is composed of 314 images; a section thickness of 100 nm was removed after each image. A total depth of 31.4 µm of tissue was imaged.

2.12 Electron tomography

2.12.1 Bovine ciliary zonule

Thick sections (~250 nm) were cut from samples embedded in resin, as previously described in section 2.10, using a Diatome diamond knife and Leica ultramicrotome. Sample sections were mounted on formvar carbon coated copper slot grids (Agar Scientific) and 10 nm colloidal gold solution was applied to both sides. Single axis tilt series were taken from -65 º to +65 º in 1 ° steps using a FEI Tecnai G2 Polara TEM operating at an accelerating voltage of 300 KV, at a magnification of 23000X. Images were collected using a Gatan Ultrascan 4000 CCD camera and using the software SerialEM (Mastronarde, 2005). The tilt series of images were aligned and tomograms were generated by back projection in IMOD using the Etomo workflow (Kremer et al., 1996).

2.12.2 Mouse articular cartilage

Mouse articular cartilage samples were prepared as described in 2.10.3. Tilt series were collected of the PCM surrounding chondrocytes and tomograms were generated as described previously 2.12.1.

2.12.3 Particle analysis in ImageJ

Analysis of microfibril diameter and spacing was carried out using ImageJ (Abràmoff et al., 2004). Representative virtual z slices from tomograms were extracted using trimvol in IMOD and were converted into the Tiff image format. Tomogram slices were converted to 8 bit files and were converted to binary format using the threshold tool in ImageJ. Particles were analysed using the particle analysis tool. Individual particles were selected and fitted to an ellipse. The minor diameter of the ellipse was used as a measure of particle diameter. The nearest neighbour plugin for image J was used to calculate the shortest distance between the centres of adjacent particles to determine an average particle packing distance.

2.13 Bovine ciliary zonule microfibril averaging

Ciliary zonule tomograms were segmented in UCSF Chimera using the segger tool (Pintilie et al., 2010). Individual microfibrils regions were extracted from the tomogram and were aligned using Chimera fit in map. Aligned microfibrils were exported in the same coordinate system using the “vop resample” command and were saved as individual densities. Exported microfibrils were converted to the SPIDER file format (.spi) before being averaged using the AS R command in SPIDER.

2.14 AFM

Bovine collagenase extracted collagen VI microfibrils were adsorbed onto 1.5 mm ethanol washed glass coverslips. A sample volume of 25 µl was pipetted onto the coverslip for 1 min before coverslips were washed in ddH2O. Cover slips were imaged using a Multimode 8, with a scan assist tip, whilst operating in ScanAssyst air mode (Brucker). Images were processed using Nanoscope v8.15 (Brucker). The volume of collagen VI beads was calculated with Image J. Individual beads were selected with a box with dimensions 78 nm x 78 nm and were analysed using the ImageJ measure tool. The median intensity value for the whole image was subtracted from the volume measured for each bead to give a final background subtracted bead volume.

Table 2.1 Summary of sample imaging techniques used

The table highlights which methods were used to image each tissue or collagenase extracted microfibril sample.

3 Results chapter 1: Nano-scale structure of fibrillin microfibrils

Fibrillin microfibrils are large glycoprotein polymers rich in disulphide bonds, which require post translation processing and association with both a cell surface and other ECM proteins to assemble into microfibrils; this makes them virtually impossible to make recombinantly. Microfibrils are also difficult to extract from tissues as they are heavily cross-linked and insoluble. Microfibril extraction methods rely either on enzymatic digestion of tissues (Lu et al., 2006) or by using chaotropic agents to denature tissues, releasing microfibrils (Keene et al., 1991). Although a lot is known about the high resolution structure of individual fibrillin domains and short fibrillin fragments which have been solved using X-ray crystallography, NMR and SAXS, it is still not understood how individual fibrillin monomers are arranged in the microfibril. To address this, a 3D model of a fibrillin microfibril repeating unit will be constructed. Microfibrils extracted from bovine ciliary zonule will be imaged using negative stain TEM and will be reconstructed using single particle averaging approaches. This model will then be used to determine how fibrillin molecules are arranged in microfibrils by fitting individual domain models.

3.1 Microfibril single particle averaging and model reconstruction

To determine the 3D structure of a fibrillin microfibril repeat, microfibrils were extracted from dissected bovine ciliary zonule tissue and imaged using negative stain TEM. Ciliary zonule tissue was used as it is a rich source of microfibrils devoid of elastin and is easy and cheap to source {Cain, 2006 #312}. Microfibrils were extracted by enzymatic digestion using type VII chromatographically purified collagenase. Type VII collagenase was used to reduce the potential of degradation by non-specific protease activity. Microfibrils were purified from the zonule extract using size exclusion chromatography; due to their large size microfibrils eluted in the void volume of the size exclusion column see Figure 3.1.

Figure 3.1 Size exclusion chromatography of ciliary zonule extracts.

Shown is an example of a chromatogram obtained from the size exclusion of collagenase extracted ciliary zonule tissue using a Cl-2B column. The absorbance (mAu) at 280 nm is plotted against the elution volume (ml). The first peak represents the void volume of the column; the second peak represents the included volume.

Extracted microfibrils were absorbed onto a carbon film supported on a copper grid and were imaged using negative stain TEM. A representative microfibril micrograph is shown in Figure 3.2. Imaged microfibrils had the characteristic periodic beads-on-a-string appearance. Micrographs were analysed using EMAN2 and approximately ~3000 microfibril repeating units as seen in Figure 3.2A were extracted from the micrographs. Initially extracted particles were centred and aligned to the bead region of the microfibril as shown previously by Wang et al., 2009. However this made it difficult to define a full microfibril repeat (see Appendix 1). In the reconstruction presented here particles were centred on the interbead region of each microfibril which included two bead regions. Particle stacks were iteratively rotationally and translationally aligned to an average of the unaligned particle stack using FindEM. The final aligned particle set was averaged and the average image was used to create an initial cylindrically average model using SPIDER. The initial model was iteratively refined against the data by projection matching using FindEM to create a final 3D microfibril model. During refinement 2-fold symmetry was applied along the fibre axis to help with model reconstruction as models would iteratively degrade during model refinement when symmetry was removed (data not shown). A representative set of aligned particles is shown in Figure 3.2B these aligned particles were classified using reference free classification using IMAGIC5 shown in

Figure 3.2C. Classum images reveal the distinct asymmetrical banding pattern with clearly defined bead, arm, interbead and shoulder regions. Some heterogeneity is apparent in the classum images of the microfibril. Some microfibrils appeared to be flexible along the fibre axis, the largest movement appeared to occur in the arm and shoulder region of the microfibril (highlighted by white arrows in Figure 3.2C).This flexibility observed along the length of the microfibril could be responsible for the degradation of the microfibril model when imposed symmetry was relaxed during model refinement.

Figure 3.2 Extracted bovine ciliary zonule microfibrils.

A) An image of a negatively stained purified bovine fibrillin microfibril, arrows highlight the bead regions of the microfibril. The boxed region shows a magnified single microfibril repeat. B) Aligned particles extracted from TEM micrographs, using EMAN2 and aligned using FindEM. C) Reference free classum images of the aligned particles. Aligned particle stacks were classified using reference free classification using IMAGIC. White arrows highlight regions where the microfibril appears flexible. The box sizes shown in B and C are 102 x 102 nm.

The final microfibril 3D model is shown in Figure 3.3. The particles, classum images and model projections shown in Figure 3.3A are very similar, with the distinctive fibrillin banding

73 pattern. Model projections resemble the classum images and individual particles suggesting that the final model is representative of the data. The reconstructed model has a bead-to- bead periodicity of 59.2 nm and has diameter of 20.8 nm at its widest point, see Table 3.1, which is similar to previously published measurements of microfibril dimensions (Baldock et al., 2001). The different regions of the model are labelled the bead, arms, interbead and shoulder. The 3D model has a hollow tube like structure.

The bead regions have a double layered structure and have a comparatively large volume when compared to other microfibril regions as can be seen in the volume map in Figure 3.3B. The arm regions emerge from the bead region and bow out and surround a stain including cavity and meet in the interbead region. The arm regions are made up of thin strands which have a globular region close to the bead and a double globular structure which is often referred to as a the interbead striation at the bottom (highlighted in Figure 3.3). The interbead region has a solid double layered structure. The shoulder region is less well defined in the model, in some of the reference free classums shown in Figure 3.2C individual strands can be seen which appear to have been averaged out in the model classes and projections in Figure 3.3A.

Figure 3.3 3D EM model of extracted bovine fibrillin.

A) The top panel shows classum images of the aligned particles and the bottom panel are 2D projections of the final reconstructed 3D model shown in B. The right panel is a slice through the centre of the rotational average of the 3D model shown in B. The region often refered to as the interbead striation is highlighted. The box size is 102 x 102 nm. B) A 3D model of the fibrillin repeat. The different regions of the model are labelled. The model has 2 fold symmetry imposed along the fibre axis. The right panel shows a volume map of the fibrillin model. Red represents areas with the highest density.

The resolution of the microfibril model was estimated by splitting the data set in two and calculating a new model from each half set. The resulting two models were low pass filtered to 20 Å and then compared using Fourier shell correlation (FSC) in SPIDER. The FSC curve for the microfibril model is shown in Figure 3.4. Taking the FSC value at the 0.5 threshold gives an estimated resolution for the model as 43 Å.

Figure 3.4 Microfibril model resolution estimation

The FSC for the microfibril reconstruction is plotted against spatial resolution. The green line shows the 0.5 threshold for resolution estimation. The estimated resolution of the reconstruction is 43 Å.

The volume of a single repeat was calculated in UCSF Chimera (Pettersen et al., 2004). The model was cropped to remove the volume of the second bead and set at a threshold value where all the densities were connected. The reconstruction had an enclosed volume of 10.1x106 Å3 which corresponds to a protein mass of 8.4x106 Da. This volume was converted to a protein mass using the volume factor of 1.2 Å3/Da (Harpaz et al., 1994). The calculated mass of the model was much larger than predicted from mass measurement from previously published STEM data which was 2,5 x106 Da (Baldock et al., 2001). The difference between the calculated mass of the reconstruction and the mass of microfibrils measured by stem was 5.9 x106 Da.This discrepancy between the measured mass by STEM and the estimated volume of the model may potentially be due to flattening of the microfibril during sample preparation which would increase the diameter of the microfibril.

Table 3.1 Dimensions of fibrillin 3D model

Shown are measured dimensions of the central slice of a radial average of the fibrillin microfibril model and the volume and calculated molecular weight of the microfibril 3D model.

3.1.1 Individual microfibril region reconstructions

To overcome potential heterogeneity caused by the flexibility of microfibrils, separate sub- models of different microfibril regions were created. Sub-models were created by aligning particles to specific regions using binary masks for the bead, arm, interbead or shoulder regions, see Figure 3.5. Sub-models were constructed and iteratively refined from an initial rotational average of the full model, during the initial model refinement no symmetry was applied along the fibre axis.

Figure 3.5 Microfibril sub-region masks

Shown are the regions of interest which were used to align particles against to create sub-region reconstructions of the microfibril repeat.

3.1.1.1 Optimising symmetry restrained sub-model reconstruction

To determine if there was any apparent symmetry in the different regions of the fibrillin microfibril, separate sub-models, refined with no symmetry constraints, were projected at angles ranging from 0-355 ° at 5 ° increments and were cross correlated against the data set using FindEM. The particles which correlated most highly to the projection at 0 ° were selected and their cross-correlation scores to projections ranging from 0-355 ° for each model were plotted. All the microfibril regions showed a second strong correlation at 180 ° suggesting that fibrillin microfibrils have a two-fold symmetry along the fibre axis (cross correlation analysis will be presented in greater detail in subsequent sections). Two-fold symmetry was then applied to subsequent rounds of refinement and all sub-models presented here were refined with two-fold symmetry imposed along the fibre axis of the models. Determination of the rotational symmetry of each of the sub-models may also give more information about how fibrillin microfibrils are organised in the different regions of the molecule.

3.1.1.2 Bead region model

Alignment of particles to the bead region led to an apparent sharpening of detail (Figure 3.6A). The bead can be seen to have two distinct layers. The 3D model shown in Figure 3.6B was set to a surface threshold which corresponds to the mass measured by STEM mass

78 mapping (Baldock et al., 2001).The bottom layer of the bead is comprised of a ring structure in the 3D model, this can be observed as two bright spots in the radial average image highlighted in Figure 3.6. The three dimensional model of the fibrillin-1 bead region shows a complicated arrangement of fibrillin molecules. If the model is rendered at a threshold of 40% of the estimated volume the core structure can be seen. The core of the bead forms a complex interweaving structure which can be seen to connect to the arm regions. Using segmentation in UCSF Chimera the bead can be separated into the bead core region and the ring region. The core structure has a volume of 8.1x105 Å³ which corresponds to a mass of 675 kDa see Table 3.2. The ring structure has a volume and a mass of 5.2x105 Å³ and 436 kDa, respectively.

Rotational cross correlation analysis of the microfibril bead region when no symmetry has been applied shows that there is a second strong correlation at 180 ° (Figure 3.7). This suggests that the bead region is 2-fold symmetric. The smaller peaks at angles approximately 30 °, 45 ° and 90 ° intervals suggests potential features with 4, 8 and 12 fold pseudo- symmetries. This potentially gives information about the microfibril composition and structure of the bead region. The presence of 4 and 8 fold symmetries may support the hypothesis that the microfibril consists of 8 fibrillin microfibrils arranged into 4 dimers {Wang, 2009 #335}. The 12 fold pseudosymmetry may therefore arise from 4 dimers folding back on themselves. The resolution of the bead region sub-model was estimated by calculating the FSC in SPIDER. Figure 3.7 shows the FSC curve for the bead region sub-model. The FSC drops off at a resolution of 31 Å but a reliable estimation could not be determined for the model as the FSC curve did not cross the 0.5 threshold before 20 Å (models were filtered to 20 Å).

Figure 3.6 3D reconstruction of the fibrillin microfibril bead region.

A) The top left panel shows aligned particles extracted from TEM micrographs manually using EMAN2 and aligned using FindEM. The middle left panel shows classum images of the aligned particles and the bottom left panel are 2D projections around the fibre axis of the final reconstructed 3D model shown in B. The right panel is a slice through the centre of the rotational average of the 3D bead region model. Highlighted by white arrows are two bright spots which correspond to a ring structure. The bead region is also highlighted. The box sizes are 102 x 102 nm. B(i) A 3D reconstruction of the bead region of fibrillin microfibrils. The model is refined with 2-fold symmetry imposed along the fibre axis. B(ii) An image of the model which has been segmented into two parts, a ring encircling the bead coloured blue and the bead core coloured in pink. The segmented bead model is shown at a 40% threshold to reveal more internal bead structure.

Figure 3.7 Symmetry analysis and resolution estimation of the bead region sub-model

(A) Shown is a graph of the mean cross correlation scores of particles to projections around the fibre axis of the bead region. The bead region sub-model was projected at 5° increments from 0- 355° and correlated against the particle stack. Particles which correlated most highly with 0° projections are shown. (B) Shown is a graph of the FSC for the bead region reconstruction plotted against resolution. The green line shows the 0.5 threshold for resolution estimation.

3.1.1.3 Arm region model

When the arm region of the microfibril is refined separately, the increase in detail shows that the arm region is comprised of four distinct arms highlighted in Figure 3.8. Using UCSF Chimera the arms were segmented into separate volumes. As there is two-fold symmetry applied along the microfibril the four arms can be categorised into two symmetric pairs and were coloured blue and red accordingly. The volumes of each arm were estimated using UCSF Chimera, see Table 3.2. Arm one (red) has a volume of 2.50x105 Å3 which corresponds to a mass of 210 kDa. Arm two (blue) had a volume of 1.40 x105 Å3 and mass of 120 kDa.

Figure 3.8 3D reconstruction of fibrillin microfibril arm region.

A) The top left panel shows aligned particles extracted from TEM micrographs, manually using EMAN2 and aligned using FindEM. The middle left panel shows classum images of the aligned particles and the bottom left panel are 2D projections around the fibre axis of the final reconstructed 3D model shown in B. The right panel is a slice through the centre of the rotational average of the 3D arm region model. The arm region is highlighted. The box sizes are 102 x 102 nm. B) A 3D reconstruction of the arm region of fibrillin microfibrils. The model is refined with 2-fold symmetry imposed along the fibre axis. The four separate arms are coloured blue and red according to symmetric pairs.

Rotational cross correlation analysis of the microfibril arm region when no symmetry has been applied shows that there is a second strong correlation at 180 ° (Figure 3.9) suggesting that the arm region is 2-fold symmetric. There are smaller peaks at approximately 45 ° and 90 ° intervals suggesting potential features with 4 and 8 fold pseudo-symmetries. The resolution of the arm region sub-model was estimated by calculating the FSC in SPIDER. Figure 3.9 shows the FSC curve for the arm region sub-model. The FSC for the arm region reconstruction gives an estimated resolution of 38 Å.

Figure 3.9 Symmetry analysis and resolution estimation of the arm region sub-model

(A) Shown is a graph of the mean cross correlation scores of particles to projections around the fibre axis of the bead region. The arm region sub-model was projected at 5° increments from 0- 355° and correlated against the particle stack. Particles which correlated most highly with 0° projections are shown. (B) Shown is a graph of the FSC for the arm region reconstruction plotted against resolution. The green line shows the 0.5 threshold for resolution estimation.

3.1.1.4 Interbead region model

Refinement of the interbead region led to an increase of detail and the classum images and the radial average image shown in Figure 3.10 reveal a structure with 3 distinct layers. The top layer has four separate stands, which connect to the arm region in the 3D model. The central layer has a more compact structure which splits into four globular densities in the bottom layer of the structure. The interbead region was set to a threshold value which resulted in a volume of 4.80x105 Å³ which corresponds to a mass of 400 kDa. Classum and projection images show that areas outside the bead region have become very poorly defined (Figure 3.10A). This averaging out of the regions adjacent to the interbead region could suggest that the interbead region is relatively rigid and the arm and shoulder regions are areas of flexibility in the microfibril.

Rotational cross correlation analysis of the microfibril interbead region when no symmetry has been applied shows that there is a second strong correlation at 180 ° (Figure 3.11) suggesting that the interbead region is 2-fold symmetric. There are smaller peaks at approximately 45 ° and 90 ° intervals suggesting potential features with 4 and 8 fold pseudo- symmetries. The resolution of the interbead region sub-model was estimated by calculating the FSC in SPIDER. Figure 3.11 shows the FSC curve for the interbead region sub-model. The FSC for the interbead region reconstruction gives an estimated resolution of 34 Å.

Figure 3.10 3D reconstruction of the fibrillin microfibril interbead region.

(A) The top left panel shows aligned particles extracted from TEM micrographs, manually using EMAN2 and aligned using FindEM. The middle left panel shows classum images of the aligned particles and the bottom left panel are 2D projections around the fibre axis of the final reconstructed 3D model shown in B. The right panel is a slice through the centre of the rotational average of the 3D interbead region model. The interbead region is highlighted. The box sizes are 102 x 102 nm. B) A 3D reconstruction of the interbead region of fibrillin microfibrils. The model is refined with 2-fold symmetry imposed along the fibre axis.

Figure 3.11 Symmetry analysis and resolution estimation of the interbead region sub-model

3.1.1.5 Shoulder region model

Aligning particles to the shoulder region led to more detailed classum images (Figure 3.12A). The shoulder region can be seen to be made of separate stands of density surrounding a stain accessible region (highlighted by white arrows in Figure 3.12A) which more closely resembles the reference free classums shown in Figure 3.2C. The shoulder region sub-model still contains noise which can be seen as lobe like densities attached to the model in Figure 3.12B.

Figure 3.12 3D reconstruction of the fibrillin microfibril shoulder region.

(A) The top left panel shows aligned particles extracted from TEM micrographs, manually using EMAN2 and aligned using FindEM. The middle left panel shows classum images of the aligned particles and the bottom left panel are 2D projections of the final reconstructed 3D model shown in B. White arrows highlight stands of density which can be seen to surround a stain including hollow. The right panel is a slice through the centre of the rotational average of the 3D shoulder region model. The shoulder region is highlighted. The box sizes are102 x 102 nm. (B) A 3D reconstruction of the shoulder region of fibrillin microfibrils. The model is refined with 2-fold symmetry imposed along the fibre axis.

Cross correlation analysis of the microfibril shoulder region when no symmetry has been applied shows that there is a second strong correlation at 180 ° (Figure 3.13) suggesting that the shoulder region is 2-fold symmetric. There are smaller peaks at approximately 45 ° and 90 ° intervals suggesting potential features with 4 and 8 fold pseudo-symmetries. The resolution of the shoulder region sub-model was estimated by calculating the FSC in SPIDER. Figure 3.13 shows the FSC curve for the shoulder region sub-model. The FSC for the shoulder region reconstruction gives an estimated resolution of 28 Å.

Figure 3.13 Symmetry analysis and resolution estimation of the shoulder region sub-model

Table 3.2 Predicted volumes of microfibril sub-models

Shows volumes of the different microfibril regions calculated using UCSF Chimera. Sub models were set to a threshold value which corresponded to the correct volume based on the microfibril masses published in Baldock et al., 2001.

3.1.2 Docking of molecular models

To determine how individual fibrillin molecules are arranged into mature microfibrils, SAXS structures of recombinant fibrillin-1 fragments (Baldock et al., 2006) (Figure 3.14) were fitted into the bead, arm and interbead sub-models based on the molecular pleated packing model (Baldock et al., 2006) and the half-staggered model (Kuo et al., 2007). The domains were not fit in the third-staggered model as this model does not support the observation that microfibrils can reversibly extended to ~100 nm {Jensen, 2012 #334}. A SAXS model of cbEGF domains 4-14 from LTBP-1 was used to model the fibrillin internal array of cbEGF domains 15-26. Models were placed into the density and their fit to the model densities were optimised using the fit in map program in UCSF Chimera. Fibrillin fragment models were fitted at the estimated resolution of the sub-region models.

Figure 3.14 SAXS structures and domain schematic of fibrillin fragments.

Molecular models of recombinant fibrillin fragments PF2, PF5, PF12 and PF13 and LTBP-1 cbEGF domains 4-14 are shown with a corresponding domain schematic, adapted from (Baldock et al., 2006). PF2 is composed of TB1 a proline rich region EGF6-10-TB2 domains, PF5 contains EGF11-15-hyb2-TB3 domains, PF12 is composed of domains TB6-EGF36-41-TB7 and PF13 contains domains TB7-EGF 41-47.

Fitting of SAXS models to the molecular pleated model is shown in Figure 3.15. The N- terminal PF2 fragment domain was fitted to the bead core structure. The TB1 domain fits into in the top layer of the bead structure. The model fits into the core of the bead with a fit 0.76. The TB2 domain of PF2 is predicted to fit into the arm region (Baldock et al., 2006). PF13 fragments fit into the bead ring region with a correlation value of 0.91 and the PF12 fragment

90 fits into the core of the bead adjacent to the PF13 model with a correlation of 0.91. In the arm region the PF5 fragment model fits with the TB3 domain and second hybrid domain fitting into the globular region in the bottom of the feature often referred to as the interbead striation. The model of a cbEGF domains 4-14 from LTBP-1 was fitted into the interbead model with a cross correlation of 0.81. The cbEGF domains 4-14 insert into the core of the interbead and fold back on themselves forming a compact structure in the globular bottom region of the interbead model. Fitting of molecular models and their cross correlation values are summarised in Table 3.3.

Figure 3.15 Docking of fibrillin molecular models according to the molecular pleated model.

(A) A schematic diagram of the molecular pleated fibrillin packing model adapted from (Baldock et al., 2006). SAXS structures of recombinant fibrillin and LTBP-1 fragments were fitted into the bead (B) arm (C) and interbead (D) sub-models using the UCSF Chimera fit in map program based on the molecule pleated model (Baldock et al., 2006). (B) The N-terminal fibrillin fragment PF2 was fitted into the core of the bead sub-model with a correlation of 0.76. C-terminal fragment PF12 was also fitted into the core of the bead with a correlation of 0.91. The very C-terminal fragment PF13 is fitted into the ring structure of the bead with a correlation value of 0.91. (C) PF5 is fitted into the arm region with a correlation of 0.82. The interbead striation is highlighted. (D) The cbEGF domains 4-14 from LTBP-1 are fitted to the interbead model with a correlation of 0.81.

Docking of SAXS models in the half-staggered model is shown in Figure 3.16. PF12 and PF13 models were fitted into the same area in the bead region as the molecular pleated model. The model of cbEGF domains 4-14 from LTBP-1 was fitted to the bead core structure with one end of the model fitting into the top layer of the bead structure and the opposite end into the bead ring structure with a cross correlation of 0.79. The N-terminal PF2 model was fitted to the arm region model. The TB1 domain fits into globular densities in the top of the arm structure. The TB2 domain of the PF2 model fits into the globular interbead striation region in the bottom of the arm-region. The PF2 model fits with a 0.76 cross correlation score. The PF5 fragment model was fitted into the interbead model with a correlation of 0.81. The cbEGF domains 11-13 fit into the top layer of the interbead with the TB3, cbEGF14 and the second hybrid domains fitting into the core of the model. Fitting of molecular models and their cross correlation values are summarised in Table 3.3

Negative stain TEM can cause artifacts due to specimen damage from drying and stain induced artifacts.To improve imaging of fibrillin microfibrils, collagenase extracted bovine microfibrils were imaged using cryo-TEM.

Figure 3.16 Docking of fibrillin molecular models according to the half-staggered model

(A) A schematic diagram of the half-staggered fibrillin packing model adapted from (Kuo et al., 2007). SAXS structures of recombinant fibrillin and LTBP-1 fragments were fitted into the bead (B) arm (C) and interbead (D) sub-models using the UCSF Chimera fit in map program based on the half-staggered fibrillin packing model (Kuo et al., 2007). (B) C-terminal fragment PF12 was fitted into the core of the bead with a correlation of 0.91. The very C-terminal fragment PF13 is fitted into the ring structure of the bead with a correlation value of 0.91. The cbEGF domains 4-14 from LTBP-1 are fitted into the core of the bead with a correlation score of 0.79 (C) The N- terminal fibrillin fragment PF2 was fitted into the arm sub-model with a correlation of 0.76. (D) PF5 is fitted into the interbead model with a correlation of 0.81.

Table 3.3 Docking of molecular models

The table summarises which domains were fitted into each sub-model in the molecular pleated or half-staggered fibrillin packing models.

3.1.3 Cryo-TEM

Cryo-TEM was used to image microfibrils in a hydrated state in the absence of stain to minimise specimen artifacts. Extracted bovine ciliary zonules were adsorbed onto carbon coated quantifoils and were plunge frozen in liquid ethane. Grids were imaged using a Tecnai G2 Polara at 110 °K under low dose conditions. A representative microfibril cryo-micrograph is shown in Figure 3.17A. Micrographs had a much lower contrast than negatively stained images but the characteristic bead-on-a-string pattern was visible. Micrographs were analysed using EMAN2 and a particle set of 70 particles were extracted. The preliminary dataset was iteratively rotationally and translationally aligned using FindEM before being averaged (Figure 3.17B). The average microfibril has a periodicity of 48.6 nm and a diameter of 16.8 nm see Table 3.4.

Figure 3.17 Cryo-TEM micrographs of extracted bovine ciliary zonule microfibrils

(A) A micrograph of a purified bovine fibrillin microfibril imaged using cryo-TEM; arrows highlight the bead regions of the microfibril; the contrast of the micrograph has been inverted. The boxed region shows a magnified single microfibril repeat (B) The left panel shows aligned particles extracted from TEM micrographs, using EMAN2 and aligned using FindEM, the box size is 154 x 154 nm. The right panel shows an average of the 70 aligned particles. The box size is 84 x 84 nm.

Table 3.4 Dimensions of fibrillin microfibril cryo-average

Shown are measured dimensions of the fibrillin microfibril average of aligned particles imaged using cryo-TEM.

3.2 Discussion and conclusions

The 3D model of the fibrillin repeating unit shown in Figure 3.3 has dimensions which are similar to previously published measurements of fibrillin microfibril models (Baldock et al., 2001). Fibrillin microfibrils viewed in 2D projection resemble beads-on-a-string but the 3D structure has a more hollow tubular appearance which has been described previously for microfibrils imaged in tissues (Davis et al., 2002, Wang et al., 2009).

The surface threshold of the 3D model cannot be set to a volume which corresponds to the measured mass by STEM (Baldock et al., 2001). This overestimation of volume in the full microfibril model could be caused by a number of factors. One of the reasons the model is too large could be due to heterogeneity in the dataset caused by flexibility along the length of the microfibril. As can be seen in several of the classum images in Figure 3.2C there is apparent flexibility in the arm and shoulder regions of the microfibril. This flexibility and the imposition of 2-fold symmetry could cause there to be a greater volume in the final model. Another possible cause of volume overestimation could be due to artifacts caused by imaging conditions. In negative stain TEM proteins are imaged in a non-hydrated state. Drying of microfibrils causes structures to collapse and flatten therefore the diameter would appear wider. The high negative charge of the carbon support film may also cause the microfibril to be distorted, as the charge of the substrate can have an effect on the microfibril structure (Sherratt et al., 2005).

To overcome potential heterogeneity caused by flexibility along the fibrillin microfibril, shorter sub-models which cover the bead, arm, interbead and shoulder regions of the microfibril were constructed. These shorter models were refined to a higher resolution than the model of the full repeat and more detailed structures could be determined. The sub-models could also have a surface threshold set which corresponded to the mass of a microfibril repeat measured by STEM (Baldock et al., 2001) without any loss of features. The sub-models have a combined volume which is consistent with eight fibrillin molecules per microfibril repeat. This suggests that the over estimation of the volume of the full fibrillin repeat model is likely due to the flexibility of microfibrils and imposing 2 fold symmetry on the full model.

FSC plots of the full fibrillin repeat model and the sub-models did not fall off in a smooth curve and instead fluctuated at higher resolutions making determination of resolution for some models difficult. As this occurred in all the fibrillin models, it is likely due to loss of some spatial frequencies due to zero points in the CTF. To fill in this lost information it would be

98 advantageous to collect more images over a range of defocus values to fill in the lost information.

Rotational correlation to the sub models has shown that there is a two-fold symmetry which runs down the fibril axis. This has been suggested previously from 2D averaging and tomographic reconstructions (Baldock et al., 2001). Potential higher order pseudo symmetries are also present in the microfibril based on correlation peaks suggesting pseudo four and eight-fold symmetries. This could also support the hypothesis that the fibrillin molecule is comprised of eight molecules per repeat (Baldock et al., 2001, Wang et al., 2009).

The higher resolution data in the reconstructed sub-models allowed for the fitting of molecular models of short fibrillin-1 (Baldock et al., 2006) and LTBP-1 fragments. The short molecular models were fitted into the sub-models based on the molecular pleated fibrillin packing model (Figure 3.15) and half-staggered model (Figure 3.16). Diagrams of the two fibrillin packing models overlaid on the 3D fibrillin microfibril model are shown in Figure 3.18.

As antibody labeling experiments have localised the N and C-termini to opposite sides of the bead region (Reinhardt et al., 1996, Baldock et al., 2001) both fibrillin packing models place the N and C-termini in the bead region. In the molecular pleated model the N-terminal fragment PF2 and the C-terminal fragments PF12 and PF13 were fitted into the bead model (Figure 3.15). PF13 was fitted into the bead ring region and PF12 fits into the core region of the microfibril in an area adjacent to PF13, with the TB6 domain fitting into the globular density in the upper band of the bead structure. The fragment PF2 also fitted into the core of the bead region with the TB1 domain fitting into the globular density in the upper band of the bead. Placement of these fibrillin fragments into the bead region allows the N- and C-termini to be in close proximity, this would potentially allow for the formation of the transglutaminase crosslink in a staggered arrangement (Qian and Glanville, 1997). The placement of the PF2 and PF13 in the bead core could also allow for the N and C-terminal heterotypic binding interactions which have been shown in vitro (Marson et al., 2005). Also if the C-terminal domains form the bead core stabilised by a ring cbEGF41-47 domains, this could support the ability of C-terminal fragments to form beaded structures without the N-terminus being present (Hubmacher et al., 2008). Fitting of these regions of fibrillin is also supported by previously published mass measurements. The molecular weight of PF2 is 42.7 kDa, PF12 42.7 kDa and PF13 is 43 kDa (Baldock et al., 2006) assuming there are eight fibrillin molecules per repeat these fragments would account for 94% of the bead mass as measured

99 by STEM (Table 3.2)(Baldock et al., 2001). Volume measurements of the segmented bead core and ring regions give further insight into the possible arrangements of fibrillin molecules in the bead. Fragments PF2 and PF12 would account for 101% of the mass of the core region and PF13 accounts for 78% of the ring region. The extra volume in the ring region not accounted for by PF13 could suggest that PF12 could form part of the ring structure or potentially this extra volume could be MAGP-1 which is constitutively present at the bead (Hanssen et al., 2004, Cain et al., 2006).

In the half staggered model (Figure 3.16) PF13 was fitted into the bead ring region and PF12 fits into the core region in the same position as the molecular pleated model but the N- terminal PF2 fragment was fitted into the arm region and the internal array of fibrillin cbEGF domains 15-26 were modeled using LTBP-1 domains 4-14 were fitted into the bead core. PF2 is 42.7 kDa, PF12 42.7 kDa and LTBP-1 domains 4-14 are 45 kDa assuming there are eight fibrillin molecules per repeat these fragments would account for 96% of the bead mass as measured by STEM (Table 3.2)(Baldock et al., 2001). However eight half-staggered fibrillin molecules would results in a mass for the full microfibril being half that of the mass measured by STEM (Baldock et al., 2001).

The arm region of the model has four separate arms. As there are predicted to be eight molecules per repeat each arm could correspond to a dimer. Recombinant fibrillin fragment have been shown to dimerise in vitro (Trask et al., 1999). In the molecular pleated model (Figure 3.15) the fibrillin fragment PF5 was fitted into the arm region sub-model. PF5 fits into the bottom of the arm region with the TB3 and second hybrid domain fitting into the globular density next to the interbead region (Figure 3.15). The pleated model predicts that the EGF domains and TB2 of the fragment PF2 form part of the top of the arm region (Figure 3.18). The two different arms coloured in blue or red have substantially different volumes. This could be due to flexibility in the arms regions causing the blue arm to average out and be underrepresented in the data or other potential imaging artifacts as previously described. Another explanation could be that the composition of the different arm region could be different supporting a staggered arrangement of fibrillin molecules. In the staggered model the N-terminal PF2 domain was fitted into the arm region. This model also predicts that the TB4, cbEGF26-27 and TB5 would also form part of the arm region (Figure 3.18).

In the molecular pleated model positioning of TB3 in the bottom of the arm regions placed the internal stretch of EGF domains cbEGF15-26 in the interbead region. LTBP-1 domains 4-14

100 were fitted into the interbead sub-model, forming a compact structure with bottom of the model fitting into the bottom globular layer of the interbead structure next to the shoulder region. This supports the molecular pleating model showing that molecules can fold back on themselves. Loss of the interbead structure has been observed on microfibril extension (Baldock et al., 2001, Wang et al., 2009), pleating in this region would support the ability of microfibrils to extend. The domains EGF15-26 are in a region known as the neonatal region (Robinson et al., 2006) as mutations in this region cause a severe neonatal form of MFS. Proper folding of the interbead region could be essential for microfibril function as disruption of the pleating may be a potential cause of the more severe MFS phenotype. In the half- staggered model the P5 fragment was fitted into the interbead region. The cbEGF domains 11-13 fit into the top layer of the interbead with the TB3, cbEGF14 and the second hybrid domains fitting into the core of the model.

The shoulder region is predicted to contain the N-terminus and domains TB5-EGF29-35 in the molecular pleated model see Figure 3.18 (Baldock et al., 2006). These regions have been shown to interact in in vitro binding experiments (Chaudhry et al., 2007). Epitope labeling of the microfibril suggests that the N-terminal is present at the side of the bead adjacent to the shoulder region (Reinhardt et al., 1996, Baldock et al., 2001). The shoulder region is less well defined than the other microfibril regions. Noise in the shoulder region model could be caused by heterogeneity from flexibility in the microfibril. Heterogeneity could also arise from irregular binding of ECM proteins as the N-terminal region is a binding site for several fibrillin associated proteins such as MAGP1 (Jensen et al., 2001) and LTBP-1,2 and 4 (Isogai et al., 2003, Hirani et al., 2007, Ono et al., 2009). The half-staggered model in contrast places the internal stretch of cbEGF domains EGF15-26 in the shoulder and bead region.

Fitting of molecular models to the fibrillin-1 sub regions gives an insight into how the fibrillin molecule could be arranged into a mature microfibril. Unfortunately the 3D microfibril reconstructions presented here are still too low resolution to resolve individual domains and therefore neither fibrillin packing model can be ruled out.

Images of hydrated fibrillin microfibril using cryo-TEM showed that microfibrils have beads on a string structure when imaged in the absence of stain. The bead region of the microfibril is also shown to be a highly electron dense structure. The average of the preliminary microfibril had a shorter periodicity and width. The difference between the periodicity measured from microfibrils imaged using negative stain and cryo-TEM is likely to be due to the cryo-TEM

101 average only representing a small preliminary data set which is most likely more poorly aligned due to the low SNR in cryo images making alignment of cryo-TEM data more difficult.

Figure 3.18 Schematic diagram of the fibrillin microfibril folding models

(A) Shown is a cartoon illustrating the domain organisation of fibrillin-1. (B) A diagram of the molecular pleated packing arrangement of fibrillin overlaid on the fibrillin microfibril model. The approximate binding sites of antibodies Mab26, Mab69, Mab201 and antibody PF2 are shown (Reinhardt et al., 1996, Baldock et al., 2001). (C) A diagram of the half-staggered arrangement of fibrillin overlaid on the fibrillin microfibril model.

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4 Results chapter 2: Fibrillin microfibril tissue micro-structure

To determine how fibrillin microfibrils are organised into larger networks in tissues, ciliary zonules from bovine and murine eyes were imaged in situ. The ciliary zonule is a complicated arrangement of ligament-like structures which connects the ciliary body to the lens of the eye shown in Figure 4.1. The zonule keeps the lens in the correct optical axis and also transmits force from the ciliary body to deform the lens during accommodation. Ciliary zonule fibres are mainly comprised of bundles of fibrillin microfibrils, these bundles of microfibrils are also organised into larger fascicle-like structures which are held together by circumferentially organised zonule fibres (Hiraoka et al., 2010). The main component of ciliary zonule fibres is fibrillin-1 (Cain et al., 2006), murine ciliary zonule microfibrils also contain fibrillin-2 (Beene et al., 2013). The ciliary zonule contains many other microfibrils associated proteins such as MAGP-1, ADAMTS-10 and ADAMTS-17, ADAMTSL-4, and LTBP-2 as previously discussed in section 1.1.7.1.

Previous studies of the 3D organisation of ciliary zonules using SEM, ultra-sound biomicroscopy (UBM) (Lutjen-Drecoll et al., 2010), environmental SEM (ESEM) (Sherratt et al., 2001, Nankivil et al., 2009) and immunofluorescence microscopy (Shi et al., 2013a) have provided a detailed understanding of the gross structure of the ciliary zonule but little is known about the 3D organisation of fibrillin microfibrils in ciliary zonule fibres and how these are organised in fascicle-like structures. To address this, ciliary zonules were imaged using SBF-SEM and electron tomography.

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Figure 4.1 Schematic diagram of a cross section of a human eye.

(A) A schematic diagram of the anterior half of the human eye is shown in (A). The coronal and sagittal planes of the eye are highlighted. B) A diagram of the arrangement of fibrillin microfibrils in the ciliary zonule. Individual microfibrils bundle together to form ciliary zonule fibres which are organised into fascicle-like structures. 4.1 Murine ciliary zonule structure

In order to determine how fibrillin microfibrils are organized into ciliary zonule fibres and fascicle-like structures, ciliary zonules from murine eyes were imaged in situ using SBF-SEM. SBF-SEM was used as it allows for the imaging of a large volume of tissue. The microscope

104 uses an electron beam to scan the surface of a sample and forms an image by detecting back scattered electrons emitted from the sample. The microscope then removes a section from the sample using an in-built microtome before imaging the surface again. This allows for serial imaging of volumes of tissues without the need for subsequent alignments of images which can be problematic in serial section TEM. The use of mice as a model organism for the organisation of fibrillin in ciliary zonules may not be ideal, as mice are nocturnal animals with comparitvley large lenses and little or no accommodative ability, however studies of the organisation and composition of mouse ciliary zonule have shown they are similar to that of the human {Shi, 2013 #315}.

Murine eyes from 27 day old mice were fixed and samples were stained using osmium tetroxide and UA before embedding in epoxy resin. Two data sets were collected, in the first data set the sample block was orientated so coronal sections of the eye (Figure 4.1) were taken resulting in imaging of the ciliary zonule in longitudinal cross section (Figure 4.2A). The second data set was collected by sectioning along the sagittal plane resulting in transverse sections of the ciliary zonule (Figure 4.2B). Figure 4.2A shows the block face before the SBF- SEM dataset was collected. An area of interest was selected encompassing the ciliary body on one side of the field of view and the lens capsule on the other side.

During sectioning of preliminary runs it was difficult to image the microfibril bundles at a high enough resolution to resolve individual fibrillin microfibrils. The murine ciliary zonule comprised a small proportion of the field of view and therefore a large proportion of the image was empty resin and extended imaging of these areas led to problems with charging of the resin. In heavily stained areas, the charge can be conducted through the metal stain but as there was less stain to conduct the charge, after a few images the sample resin could not be sectioned properly by the knife. Therefore to reduce charging, the beam was spread over a larger area and was imaged at a lower magnification. The final data set was imaged at magnification which resulted in a sampling of 26 nm/pixel. As microfibrils are ~20 nm in diameter at their widest point this sampling was insufficient to resolve individual microfibrils. The data set is composed of 500 images and a section thickness of 100 nm was removed after each image. A representative image from the dataset is shown in Figure 4.2A(ii).

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Figure 4.2 Murine ciliary zonule tissue imaged with SBF-SEM.

Ciliary zonule tissue was imaged using SBF-SEM. Ai) An image of the block face oriented in the coronal plane prior to sectioning. Aii) A representative image from the SBF-SEM data set. 500 images were taken with 100 nm section removed from the block face after each scan. The regions highlighted are the ciliary body CB, the ciliary zonule CZ and the lens capsule LC. N=1 Bi) An image of the block face oriented in the sagittal plane, the scan area is highlighted with a blue square. Bii) A representative image from the SBF-SEM dataset, 900 scans were taken with 100 nm sections removed after each scan. Black arrows in A and B highlight ciliary zonule fibres. N=1

The SBF-SEM data was visualised using Chimera as shown in Figure 4.3. Thin ciliary zonule fibres can be seen emerging from invaginations in the non-pigmented ciliary epithelium

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(NPCE) (Figure 4.3B). These thin bundles of fibres can be seen contacting the basement membrane of the NPCE before aggregating to form larger bundles. The larger bundles are contacted by several interweaving thin zonule fibres which run parallel to the ciliary body. At the lens, fibres fan out into smaller fibres before contacting the lens capsule (Figure 4.3C).

In the second dataset, the eye was imaged through the sagittal plane and the ciliary zonule was imaged in transverse cross section allowing for more accurate measurement of ciliary zonule fibre diameter as there is a potential for a loss of mass if fibres are imaged in longitudinal cross section. To calculate the diameter of fibres, one section was taken in every 100 and converted into a binary image using the threshold tool in ImageJ. Binary images were analysed using the particle analysis tool. Ellipses were fitted to individual particles to estimate the diameter of zonule fibres imaged in cross-section, the minor diameter was used as a measure of fibre diameter (Figure 4.4B). The distribution of fibre diameters is shown in Figure 4.4, the mean ciliary zonule fibre diameter was 0.50 ± 0.01 µm (standard error of the mean (SEM)).

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Figure 4.3 3D reconstructions of Murine ciliary zonule.

(A) The full SBF-SEM data set was rendered using Chimera; sample blocks were orientated so eyes were sectioned along the Coronal plane. B) Close up image of the ciliary body non- pigmented epithelium. C) Close up image of zonule fibres inserting into the lens capsule. Regions highlighted are the ciliary body (CB) ciliary zonule (CZ) and lens capsule (LC). Scale bars represent a distance of 10 µm. The data set is composed of 500 images with ~100 nm thick sections removed after each scan. Samples were imaged at a magnification resulting in a pixel size of 26 nm/pixel.

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Figure 4.4 Murine ciliary zonule fibre diameter

(A) The full SBF-SEM data set was rendered using Chimera; sample blocks were orientated so eyes were sectioned along the sagittal plane. Regions highlighted are the ciliary body (CB) ciliary zonule (CZ). Scale bars represent a distance of 10 µm. The data set is composed of 917 images with ~100 nm thick sections removed after each scan. Samples were imaged at a magnification resulting in a pixel size of 26 nm/pixel. (B) A representative image from the SBF-SEM dataset. Particles were selected using the ImageJ analyse particles tool and ellipses were fitted to the selected particles. Fitted ellipses are shown in red. (C) A histogram of zonule fibre diameters is shown. Diameters were measured using ImageJ particle analysis. N=1105 from 10 images

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4.2 Bovine ciliary zonule structure

As murine ciliary zonule tissue samples could not be imaged at a high enough resolution to image individual microfibrils, due to charging effects, bovine ciliary zonule was imaged using SBF-SEM. The larger ciliary zonules in bovine tissue have less empty resin in a field of view and more metal stain which can conduct charge away from the sample surface allowing samples to be imaged at higher resolution. Imaging microfibrils from bovine ciliary zonule would also allow for the comparison of microfibrils imaged in situ with extracted bovine microfibrils.

Ciliary zonules were dissected from bovine ocular tissue and the ciliary body to the lens capsule were fixed and stained before being embedded in epoxy resin. The sample block was orientated so sagittal sections through the ciliary zonule were taken to image ciliary zonules in transverse cross section. Figure 4.5 shows a representative image of a bovine ciliary zonule fascicle-like structure from the SBF-SEM data set. The data set of 1500 slices was collected at a resolution of 14 nm/pixel and sections of ~100 nm in thickness were removed from the surface of the sample block between scans. The fascicle-like structure can be seen to be comprised of a combination of well-defined ciliary zonule fibres and less well defined amorphous fibrillar masses. The diameter of the fibrils was measured by segmenting individual fibres in ImageJ and fitting ellipses to contours. As the images were quite noisy and the contrast difference between zonule fibres was not well defined fibres could not be automatically segmented. Fibres with defined perimeters were used for diameter measurements. Fibre measurements were taken every 100 images, the mean diameter of the fibres was 2.72 µm ± 0.1 µm (S.E.M.) a histogram of the data is shown in Figure 4.5B. The SBF- SEM data set of the ciliary zonule fascicle was rendered in 3D using Chimera (Figure 4.5C). The fascicle-like structure is formed of bundles of zonule fibres with circumferentially arranged zonule fibrils around the perimeter. Some ciliary zonule fibres throughout the SBF- SEM volume can be seen to have more darkly stained fibres around their perimeter shown as darker stained patches highlighted in Figure 4.5A. These darkly stained regions could be seen to wrap around the perimeter of the fibres.

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Figure 4.5 SBF-SEM of bovine ciliary zonule tissue.

A bovine ciliary zonule fascicle-like structure was imaged using SBF-SEM. (A) A representative image from the SBF-SEM data set. Samples were sectioned along the sagittal plane of the eye taking transverse cross sections through the bovine ciliary zonule. The fascicle-like structure is composed of ciliary zonule fibres which are highlighted by black arrows. Red arrows highlight darkly stained fibrous patches on the perimeters of zonule fibres. B) A histogram of zonule fibre diameters. Diameters were measured using the ImageJ measure tool. N=232, from 15 images. C) The SBF-SEM data set was rendered using Chimera, circumferentially organised ciliary zonule fibres are highlighted with black arrows. The scale bar represents a distance of 10 µm.

4.2.1 Correlative bovine ciliary zonule tomography

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The resolution of SBF-SEM was too low to resolve individual microfibrils therefore to determine how fibrillin microfibrils are arranged in ciliary zonule fibres, correlative thick section electron tomography was used. Thick sections ~300 nm were cut from the sample blocks which had been previously imaged using SBF-SEM. Sections were collected on copper slot grids and sections were supported on a formvar carbon support film. Sections were coated on both sides with gold fiducial markers which were used for subsequent alignment of tomograms. Sections of bovine ciliary zonule were imaged using a FEI G2 Polara operating at an accelerating voltage of 300 kV. A low magnification image is shown in Figure 4.6A showing the same zonule fascicle-like structure which was imaged by SBF-SEM. Areas of interest were selected and tilt series were collected from +65o to -65o at a magnification of 23000 x, a region of interest is highlighted in Figure 4.6A. Tilt series were aligned and tomograms were reconstructed using back projection in IMOD. A Representative virtual z-slice from a tomogram of the bovine ciliary zonule is shown in Figure 4.6B. A small bundle of microfibrils is highlighted in-between two ciliary zonule fibres. These small bundles of fibrillin microfibrils, which border the ciliary zonule fibres could potentially correspond to the darkly stained fibres circumferentially arranged around zonule fibres seen in the SBF-SEM data set (Figure 4.5). Tomograms were analysed using ImageJ particle analysis. Particles had a mean diameter of 11.38 nm ± 0.05 nm (SEM). A histogram of the data is shown in Figure 4.6C. The distance between each microfibril was determined by using nearest neighbor analysis in ImageJ. The mean distance between the centre of each microfibril was 28 nm ± 0.15 (SEM), a histogram of the data is shown in Figure 4.6D.

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Figure 4.6 Correlative bovine ciliary zonule tomography

Sample blocks from SBF-SEM were sectioned and imaged using TEM. (A) An image of the ciliary zonule fascicle-like structure is shown, highlighted in red is the area imaged in B. (B) Shown is a representative virtual z-slice from a reconstructed tomogram. Highlighted by an arrow is a darkly stained patch as previously seen in SBF-SEM. (C) A histogram of microfibril diameter. The ImageJ particle analysis tool was used to measure individual microfibril diameter from representative tomogram virtual z-slices. (D) A histogram of the shortest distance between the centres of individual microfibrils. Particle distance was measured between the centroids of particles using ImageJ. (C and D) N=7990 measured from N=4 representative tomograms.

Electron tomography revealed the detailed structure of ciliary zonule fibres. Individual microfibrils could be resolved, and had a very similar structure to the 3D structure determined in section 3.1, with a hollow tube like appearance and characteristic bead, arm, interbead and shoulder regions. Individual microfibril volumes were extracted from ciliary zonule tomograms. Microfibrils were aligned using Chimera fit in map tool and were averaged using SPIDER. The averaged microfibril is shown in Figure 4.7. The model has a periodicity of 49

113 nm and diameter of 32 nm. Microfibrils in tomograms could be seen to form contacts between microfibrils. Contacts appeared to be predominantly between bead regions (Figure 4.7C), but due to noise present in the tomograms it was often difficult to resolve.

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Figure 4.7 3D reconstructions of a ciliary zonule microfibrils

Ciliary zonule fibre tomograms were rendered using Chimera. (A) An image taken of an area from a ciliary zonule tomogram. (B) Shows representative microfibrils from a ciliary zonule bundle. Areas of density connecting two microfibrils are highlighted by black arrows. Red arrows highlight microfibril bead regions. (C) 30 microfibril repeats were extracted from the ciliary zonule tomogram, the densities were aligned using Chimera fit in map. Aligned microfibrils were averaged using SPIDER. In the right of the panel is a 3D model of the fibrillin repeat, reconstructed from negative stain TEM images of extracted bovine ciliary zonule microfibrils. The averaged microfibril images are not at the same scale. Scale bars in A and B represent a distance of 50 nm.

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4.2.2 Ciliary zonule fibres contact the basement membrane of non-pigmented ciliary epithelial cells

Fibrillin microfibrils can be seen contacting the surface of the NPCE basement membrane in Figure 4.3. To determine what regions of the fibrillin molecule may be involved in binding to the basement membrane, tomograms of bovine ciliary zonule fibres contacting the basement membrane were reconstructed. Figure 4.8 shows representative slices of two tomograms of the basement membrane of the NPCE in the ciliary body. A tomogram rendered in Chimera is shown in Figure 4.9. Fibrillin microfibrils can be seen contacting densities which connect to the basement membrane. To determine what regions are potentially involved in binding to the basement membrane, the single particle reconstruction from extracted microfibrils of the fibrillin repeat was fitted into microfibrils in the tomogram. Volumes emerging from the basement membrane were seen to interact with fibrillin microfibrils in the bead and arm region as shown in Figure 4.9.

Figure 4.8 Tomography of ciliary body non-pigmented epithelium.

Shown are representative virtual z-slices of two tomograms of the NPCE. Highlighted are ciliary zonule microfibrils (CZ) and the basement membrane (BM). Panel on left shows microfibrils/zonular fibres running perpendicular to basement membrane whereas in the right panel they are parallel.

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Figure 4.9 Ciliary zonule microfibrils associate with the basement membrane of the NPCE.

Tomograms were constructed from tilt series collected at the basement membrane of the NPCE. (A) Shows a representative tomogram visualised in Chimera. The red box highlights areas shown in B and C. The scale bar represents a distance of 500 nm. (B and C) are magnified regions of the tomogram highlighted in A. Red arrows show contact points between microfibrils and the basement membrane (BM). The plasma membrane (PM) of the NPCE is labelled. (C) The 3D model of extracted microfibrils (see section 3.1) was docked into the tomogram using Chimera fit in map. Black arrows highlight the bead regions of the microfibril. Scale bars in B and C represent a distance of 50 nm

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4.3 Discussion

To determine how individual fibrillin microfibrils are arranged in 3D networks in situ, murine and bovine ciliary zonule were imaged using SBF-SEM and electron tomography. Measurements of microfibrils in situ from tomograms of bovine ciliary zonule fibres showed that microfibrils had a mean diameter of 11 nm shown in Figure 4.6, which is similar to previously published measurements of tissue microfibrils (Sakai et al., 1986). The average periodicity of microfibrils from tomograms was shown to be approximately 49 nm which is also consistent to periodicity measured for microfibrils in ciliary zonule tissue (Davis et al., 2002). Microfibrils imaged in the tissue had the characteristic banding pattern with defined bead, arm, interbead and shoulder regions and had a hollow tube like structure which is consistent with the 3D structure of extracted microfibrils. The beads-on-a-string appearance of microfibrils has often been attributed to loss of protein during extraction (Davis et al., 2002) as traditionally microfibrils imaged in tissues have a tubular appearance. 3D reconstructions of microfibrils from ciliary zonule tissue presented here show microfibrils have a characteristic fibril banding pattern suggesting that the beads on a string appearance of extracted microfibrils is most likely due to them being represented in 2D projection and are not as a result of artifacts from extraction. Tomogram averaged microfibrils had a slightly shorter periodicity and a larger diameter than the 3D structure of extracted microfibrils (Baldock et al., 2001). This could be due to only a small number of microfibrils being aligned and averaged; also tomograms were only collected in one tilt axis so have a missing wedge of data which means microfibrils will appear to be stretched along the z-axis.

The average distance between the centre of fibrillin microfibrils was calculated as 28nm, this corresponds to the spacing between microfibrils in tensioned ciliary zonules measured by X- ray fibre diffraction (Wess et al., 1998a). This packing of microfibrils in ciliary zonule fibres may be maintained by cross-bridges which were seen between the bead regions of microfibrils. These protein cross-links have been imaged previously in ciliary zonule microfibrils and appeared to be between bead regions (Davis et al., 2002). Recent studies of murine models of ectopia lentis have shown that LTBP-2 plays an essential role in the maintenance of ciliary zonule fibre stability (Inoue et al., 2014), as previously mentioned in section 1.1.6, and could therefore be a candidate for forming links between microfibrils. LTBP-2 binds to the N-terminus of fibrillin-1 (Hirani et al., 2007) which has been predicted to located in the bead region of the microfibril (Reinhardt et al., 1996, Baldock et al., 2006). To better locate cross-links between microfibrils, sub-tomogram averaging and classification of

118 tomogram data could be able to determine if crosslinks occur in specific regions of microfibrils. Murine ciliary zonules fibres had a mean diameter of 0.50 ± 0.01 µm which was much smaller than the average diameter measured for bovine ciliary zonules (2.72 µm ± 0.1 µm). In addition, fascicle-like structures were not seen in murine ciliary zonules. Murine ciliary zonules contain fibrillin-2 microfibrils (Hubmacher et al., 2014), however LTBP-2 does not interact with fibrillin-2 (Hirani et al., 2007). Therefore, LTBP-2 stabilisation of ciliary zonules could allow formation of fascicle-like structures in bovine zonules, which do not contain fibrillin-2 (Cain et al., 2006, Beene et al., 2013)and the presence of fibrillin-2 could potentially prevent formation of fascicle-like structures in murine ciliary zonules. Fibrillin-2 knockout mice have disrupted zonules which appear to be thicker (Shi et al., 2013b). In this study murine tissue was collected from 27 day old mice to further determine the higher order organisation of fibrillin microfibrils in ciliary zonule tissue it would be beneficial to image ciliary zonule tissue collected at range of time points.

Ciliary zonule fibrils imaged with SBF-SEM often had darkly stain fibrous patches which could be seen around the perimeter of some zonule fibres in the ciliary zonule. Using correlative tomography, similar areas of the ciliary zonule fascicle-like structure were imaged. Tomograms revealed that the darkly stain patches may be small bundles of fibrillin microfibrils which may stabilise the structure of ciliary zonule fibres. Ciliary zonule fibres were also seen to be circumferentially organised around the fascicle-like structure shown in Figure 4.5, which has been shown previously (Hiraoka et al., 2010). Often areas of the ciliary zonule were amorphous and poorly defined. This could be because the zonule was relaxed during sample processing and also meant that fibres could not be tracked through the fascicle-like structure to determine their length. In future, tissues could be loaded during fixation to simulate the physiological tension forces they would experience in vivo.

Ciliary zonule fibres are anchored to the NPCE basement membrane. Tomography of the basement membrane showed that microfibrils can be seen contacting volumes connected to the basement membrane. This volume could potentially be the proteoglycan perlecan. Fibrillin has been shown previously to bind to perlecan in vitro and fibrillin-1 and perlecan have been shown to colocalise at the ciliary NPCE basement membrane (Tiedemann et al., 2005).The type I and II domains of perlecan have been shown to bind to fibrillin in an internal region of fibrillin (Tiedemann et al., 2005). Fibrillin may also interact with perlecan through interaction with its HS chains. Another potential candidate could be the CSPG versican which has been shown to form complexes with fibrillin in the ciliary body (Ohno-Jinno et al., 2008). Versican

119 interacts with microfibrils through its C-terminal lectin domain binding to the N-terminus of fibrillin (Isogai et al., 2002).

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5 Results chapter 3: Nano and micro-scale organisation of collagen VI microfibrils

Previous structural and biochemical studies have shown that collagen VI microfibrils have a complex hierarchical structure where three α-chains form a heterotrimeric monomer which subsequently forms anti-parallel disulphide linked dimers and tetramers before being assembled in an end-to-end fashion to form beaded microfibrils (Engvall et al., 1986). Previously a 3D reconstruction of collagen VI using negative stain-TEM has revealed the organisation of C- and N-terminal VWA domains in the bead region, where C-terminal VWA domains form a compact head structure with N-terminal VWA domains forming the more flexible tail regions (Beecher et al., 2011). However it is still unclear how the VWA domains are arranged in the globular bead region of collagen VI microfibrils. To further determine how the α-chains are organised in the bead structure tissue extracted microfibrils were imaged, in a frozen hydrated state without stain, using cryo-TEM and a 3D model was constructed using single particle reconstruction methods.

5.1 Collagen VI microfibril single particle averaging and 3D model reconstruction

Collagen VI microfibrils were extracted from dissected bovine corneal tissue. Corneas were mechanically disrupted before being enzymatically digested using type VII collagenase as described previously (Beecher et al., 2011)(see section 2.3.2). Microfibrils were purified from the corneal extract using size exclusion chromatography (Figure 5.1A). The central fraction from the void peak was separated using SDS-PAGE and collagen VI was detected by western blotting with a polyclonal rabbit anti collagen VI antibody. Two bands were detected by western blotting which had masses of ~250 kDa and ~130 kDa which were seen by SDS- PAGE (Figure 5.1B). Previous studies of extracted corneal collagen VI mass spectrometry had identified the more slowly migrating band as collagen VI α3 chain and the faster migrating band as collagen VI α1 and α2 chains (Beecher et al., 2011).

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Figure 5.1 Size exclusion chromatography of bovine corneal extracts

(A) An example chromatogram obtained from the size exclusion chromatography of collagenase extracted bovine corneal tissue using a sepharose Cl-2B column. The absorbance (mAu) at 280 nm is plotted against the elution volume (ml). The first peak represents the void volume of the column and the second peak represents the included volume. (B) The left hand panel shows an SDS-PAGE gel run under reducing conditions of the central fraction from the void peak. The right hand panel shows a western blot of the central fraction of the void peak. Collagen VI chains were detected using a polyclonal rabbit anti collagen VI antibody. Arrows highlight bands at approximately 250 kDa, which corresponds to the α3 chain, and at 120 kDa which corresponds to α1 and α2 chains.

Purified corneal microfibrils were imaged using cryo-TEM. Samples from the void volume of the sepharose CL-2B column were adsorbed onto Quantifoil grids which had a thin (~2nm)

122 carbon support film added. To create a thin layer of amorphous ice, grids were blotted and plunge frozen in liquid ethane before being transferred into a FEI Tecnai G2 Polara. Samples were imaged at cryo temperatures (-170 °C) and under low dose (~20 e-/Å2) to reduce the radiation damage to the sample. Due to the relatively small mass of collagen VI and the presence of a carbon support film, images were taken at a defocus range between -2 to -5 µm to allow enough contrast for visualisation. A representative micrograph is shown in Figure 5.2. Microfibrils had the characteristic globular bead structures separated by the collagen helical region as has been seen previously using negative stain TEM (Beecher et al., 2011).

Figure 5.2 A cryo-TEM micrograph of extracted bovine collagen VI microfibrils.

Bovine collagen VI was imaged under cryo conditions using a FEI Tecnai G2 Polara TEM operating at an accelerating voltage of 200KV. This image was taken with a defocus of ~5 µm. Beam damage which can be seen was a result of the image being part of an image series and therefore received a greater electron dose multiple exposures. Black arrows highlight the globular bead regions. A magnified image of a bead region is shown in the top right of the figure.

Micrographs were analysed and 1060 images of the bead region of the collagen VI microfibrils were selected with a 256 x 256 pixel box size using EMAN2. Particles were

123 normalised and CTF corrected before being iteratively rotationally and translationally aligned to an average image of the unaligned data set using FindEM. An initial cylindrical 3D model was generated and iteratively refined using a projection matching method using FINDEM before a stable model was generated. As microfibrils are formed from the end-to-end assembly of tetramers (dimer of dimers) (see Figure 1.7) two fold symmetry was applied along the fibre axis during refinement.

Corresponding class sum images and projections which represent 10° rotations around the fibre axis of the final model are shown in Figure 5.3. The two half-beads could be resolved and were joined by an interbead region. Well defined head, intermediate and tail regions can also be seen (Figure 5.3C). Class sum images and model reprojections are very similar in appearance suggesting that the final 3D reconstruction is representative of the data set. In several class sum images and the model radial average image, the second half-bead (highlighted with red arrows in Figure 5.3A) appears to be more-diffuse/less well-defined. This is most likely due to heterogeneity caused by flexibility from twisting between the two half- beads which has been seen previously (Beecher et al., 2011).

The 3D model of the collagen VI bead region is shown in Figure 5.4. The model surface is shown at a surface threshold which corresponds to a molecular mass of 750 kDa for each half-bead, which would correspond to VWA N1-C2 domains from α1 and α2 chains and VWA C1-N6+N8 from the α3 chain which has been shown to be the predominant α3 chain variant in corneal extracts (Beecher et al., 2011). The top bead can be seen to be made up 4 distinct layers which have been labelled the head, intermediate, tail and interbead regions. The head region is composed of four distinct lobe like densities and volume mapping (see Figure 5.4B) shows that the region is hollow. The head region has a length of 6.6 nm and a width of 13.2 nm. The intermediate region has a rectangular shape and measures 4.8 nm x 12.6 nm. The two tail regions can be seen emerging from the intermediate region and form a compact C- shape with the bottom of the tail regions joining back together at the interbead region; each tail measures 10.2 nm x 6.6 nm. Areas of the interbead region in the model are lost at this threshold value and the lower bead at the bottom of the panel has less density and appears to be less well-defined. The resolution of the collagen VI bead reconstruction was estimated from the FSC (see Figure 5.4C) as 50 Å at the 0.5 FSC threshold.

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Figure 5.3 3D reconstruction of the bead region from collagen VI microfibrils

(A) Shown are classum images of the collagen VI bead region. Red arrows highlight classum images where the second half-bead region appears to be less well-defined. (B) Representative reprojections of the collagen VI bead region reconstruction. Images in A and B represent 10 ° rotations around the fibre axis from 0-170 °. (C) The central slice from a radial average of the 3D bead region reconstruction. The head, intermediate, tail and interbead regions are labelled. The box size is 77x 77 nm.

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Figure 5.4 3D model of the bead region of bovine corneal extracted collagen VI microfibrils

(A) 3D reconstruction of the bead region of collagen VI. The head, intermediate, tail and interbead regions are labelled. (B) Shows a volume map of the bead model. Red represents areas with the highest density. (C) Collagen VI bead region resolution estimate. The FSC for the bead region reconstruction is plotted against spatial resolution. The red line shows the 0.5 threshold for resolution estimation. The estimated resolution of the reconstruction is 50 Å.

As the bottom half-bead in the collagen VI bead model is less well-defined, to improve the resolution a single half-bead was reconstructed separately, this approach was taken previously by Beecher et al., 2011. Each half-bead was extracted from the images of collagen VI bead regions and were aligned. Each half-bead was shifted to the centre of the image to form a data set of 2120 particles The better defined half-bead from Figure 5.4A of the bead model was used as the initial model for a model based refinement using the half-bead data

126 set. Figure 5.5 shows classum images and projections which represent 10° rotations around the fibre axis of the collagen VI half-bead. Classum images and the reprojections look similar to those of the top half of the full bead region (see Figure 5.3A). The 3D reconstruction of the half-bead is very similar to the top bead of the bead model (as shown in Figure 5.4A), with a head region composed of four lobe like structures, a rectangular intermediate region, and two C-shaped tail regions. The interbead region where the two tail regions converge is better defined in the half-bead than in the bead model (see Figure 5.6). The radial average of the half-bead model was analysed using ImageJ. The head region has dimensions of 6.6 nm x 13.2 nm; the intermediate region has dimensions of 4.1 nm x 12 nm and the tail regions 9 x 6 nm (Table 5.1) which are similar to the dimensions measured for the top bead of the bead model. The resolution of the collagen VI half-bead reconstruction was estimated by calculating the FSC (see Figure 5.6) and the model had an estimated resolution of 48 Å at the 0.5 FSC threshold.

To determine if the heterogeneity observed in the tail regions is due to flexibility of these regions, or potentially due to variability in the α-chain composition of the microfibril, the volume of extracted microfibril bead regions were analysed using AFM.

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Figure 5.5 Collagen VI half-bead 3D reconstruction

(A) Class-sum images of aligned particles. (B) Reprojections of the final half-bead model. Class- sum images and model reprojections represent 10° rotations around the collagen VI fibre axis. (C) The central slice from a radial average of the collagen VI half-bead model. The box size is 77 x 77 nm

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Figure 5.6 3D reconstruction of the collagen VI half-bead.

(A) 3D reconstruction of the bead region of collagen VI. The head, intermediate, tail and interbead regions are labelled. (B) Collagen VI half-bead resolution estimate. The FSC for the half-bead reconstruction is plotted against spatial resolution. The red line shows the 0.5 threshold for resolution estimation. The estimation resolution of the reconstruction is 48 Å.

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5.1.1 AFM of extracted bovine collagen VI microfibrils

In the collagen VI half-bead reconstruction the N-terminal tail regions seem to be less well defined then the head region. This region was also shown to be poorly defined in the negative stain TEM model of the half-bead of collagen VI (Beecher et al., 2011). To determine if the heterogeneity observed in the N-terminal tail regions is due to flexibility in the α3 chain N- terminal VWA domains or potentially due to different compositions of α-chains, the volume of collagen VI beads was measured using AFM. Collagen VI microfibrils are formed from heterotrimeric monomers of α1 + α2 and αX (where x can be one of the long α-chains 3, 4, 5, or 6), the α3 chain can also undergo alternative splicing where N-terminal VWA domains are spliced out (Dziadek et al., 2002).

Corneal extracted collagen VI was adsorbed onto glass coverslips and was imaged using AFM. Figure 5.7 shows an AFM image of a bovine collagen VI microfibril. The volume of 230 bead regions was measured using ImageJ and the background of the image was then subtracted. A histogram of the data is shown in Figure 5.7, the maximum bead height was 4.64 nm and the mean microfibril bead volume was 2663 nm³. The data fits a single Gaussian distribution suggesting that a single α-chain variant is present.

Figure 5.7 AFM analysis of collagen VI.

(A) AFM image of bovine extracted collagen VI adsorbed onto a glass cover slip. (B) A histogram of collagen VI bead region volumes. A Gaussian curve was fitted to the data with an R square of 0.969. N=230

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5.2 N6-C5 α3 chain collagen VI microfibril

Heterogeneity caused by flexibility in the tail regions of the collagen VI reconstruction leads to loss of density in these regions which makes it more difficult to determine how the N-terminal VWA domains are organised. To reduce heterogeneity in N-terminal tail regions, microfibrils were extracted from cell cultures which produce collagen VI microfibrils which are missing the N-terminal VWA domains N10-N7 in the α3 chain (N6-C5 microfibrils). The shorter α3 chain N6-C5 microfibrils may have a reduced flexibility in the tail region, also these microfibrils may provide insights into how the N-terminal VWA domains are organised in the bead region. By comparing the N6-C5 microfibril to tissue extracted microfibrils, any loss of densities could localise the N-terminal VWA domains in the half-bead.

SaOS-2 cells, which do not express the α3 chain endogenously and therefore do not secrete collagen VI microfibrils, had previously been transfected with a N6-C5 α3 chain construct (Fitzgerald et al., 2001). A schematic diagram of the domain organisation of the N6-C5 α3 chain is shown in Figure 5.8. The N6-C5 SaOS-2 cells were cultured for 2 weeks before cell layers were scraped from tissue culture dishes and were digested using type VII collagenase. Cell layer extracts were subjected to size exclusion chromatography using a sepharose CL- 2B column. A representative trace showing the purification of N6-C5 is shown in Figure 5.8. Fractions from the void volume of the column containing N6-C5 microfibrils were imaged using negative stain TEM, with a Tecnai Twin in low dose mode. A representative image of a N6-C5 microfibril is shown in Figure 5.9A. Initially 521 images of the bead region of the N6- C5 microfibrils was boxed using EMAN2 and aligned to an average of the unaligned image stack using FindEM. Class sum averages from a reference free classification using IMAGIC are shown in Figure 5.9B. Poorly stained particles were removed from the data set and half- beads were extracted from the bead region images. A final stack of 396 half-beads were aligned using FindEM. The aligned half-beads were classified using reference free classification using IMAGIC (see Figure 5.9C) The average image of the aligned N6-C5 dataset is shown in Figure 5.9D. The half-bead has a similar banding pattern as the cryo- reconstruction of the half-bead shown in Figure 5.6. The head region has dimensions of 5.6 nm x 12.8 nm the intermediate region is 4 nm x 12.8 nm and the tail regions are 8 nm x 6.4 nm (Table 5.1). As the N6-C5 microfibril measurements are very similar to the dimension of the cryo-TEM half-bead model this suggests that flexibility in the N-terminal tail regions is a likely cause for the loss in density in this region in the half-bead model. Unfortunately the low number of particles and the irregular staining meant that there were too few particles to

131 reconstruct the 3D volume of the N6-C5 half-bead region and it was not possible to see whether the arm regions were better defined from these data.

Figure 5.8 Collagen VI N6-C5 microfibril purification

(A) A schematic diagram of the domain organisation of collagen VI α3 chain and the N6-C5 construct. (B) An example chromatogram obtained from the size exclusion chromatography of collagenase cell culture extracted N6-C5 microfibril using a sepharose Cl-2B column. The absorbance (mAu) at 280 nm is plotted against elution volume (ml). The first peak represents the void volume of the column and the second peak represents the included volume.

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Figure 5.9 Cell culture extracted N6-C5 collagen VI microfibrils

(A) An image of a negatively stained purified N6-C5 collagen VI microfibril, arrows highlight the bead regions of the microfibril. The boxed region shows a magnified single microfibril repeat. B) Aligned class sum images of the bead region. (C) Class sum images of the aligned N6-C5 microfibril half- bead. (D) Average of the final aligned N6-C5 half-bead particle set.

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Table 5.1 Dimensions of the half-bead of bovine corneal and N6-C5 collagen VI microfibrils

Shown are the measured dimensions of the central slice of the half-bead model from extracted bovine corneal microfibrils and the average of the final aligned N6-C5 half-bead particle set.

5.3 3D reconstructions of murine chondrocyte PCM

Collagen VI is found in high concentrations in the PCM surrounding chondrocytes in articular cartilage, where it plays a key role in the PCM structure. The correct organisation of the PCM is essential for maintaining the mechanical properties of cartilage and for transducing biomechanical signals from the surrounding ECM to the chondrocytes (Zelenski et al., 2015). Although collagen VI has an important role in the PCM it is still not fully understood how microfibrils are organised into larger networks surrounding chondrocytes. To address this, SBF-SEM and electron tomography has been used to create 3D models of the chondrocyte PCM in murine articular cartilage.

5.3.1 Murine articular cartilage PCM structure

To determine how collagen VI is organised in the PCM, articular cartilage was dissected from murine femur and prepared for SBF-SEM as previously described (Starborg et al., 2013). Figure 5.10 shows an image of the sample block face which was imaged using SBF-SEM; the region where the data set was collected is highlighted. A data set of 313 images was collected with a sampling of 10 nm/pixel with sections of ~100 nm thickness removed after each scan. A representative image from the data set is shown in Figure 5.10B. The SBF-SEM data set was rendered in UCSF Chimera (see Figure 5.10C); a magnified region of the PCM between two chondrocytes can be seen in the right hand panel. The PCM can be seen to be organised into a mesh like network between the two chondrocytes.

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Figure 5.10 Murine articular cartilage imaged with SBF-SEM Murine articular cartilage tissue was imaged using SBF-SEM. (A) An image of the sample block face is shown, highlighted is the region where the SBF-SEM data set was collected. (B) A representative image from the SBF-SEM data set. (C) 3D reconstruction of a section of the SBF- SEM data-set, the right panel shows a 3D reconstruction of the PCM in between two chondrocytes. N=1.

As the diameter of a collagen VI microfibril is ~20 nm the SBF-SEM data set was sampled at too low resolution (10 nm/pixel) to resolve individual microfibrils. Also as sections removed were approximately 100 nm thick, the information lost would make it difficult to track individual collagen VI microfibrils which have a periodicity of 112 nm. Therefore to allow imaging of

135 individual collagen VI microfibrils in the PCM, thick section electron tomography was used to image the articular cartilage chondrocyte PCM.

Thick sections ~300 nm were cut from the sample blocks which had been previously imaged using SBF-SEM. Sections were collected on copper slot grids supported by a formvar carbon support film. Sections were coated on both sides with gold fiducial markers which were used for subsequent alignment of tomograms using IMOD (Kremer et al., 1996). Sections of murine articular cartilage were imaged using a FEI Tecnai G2 Polara operating at an accelerating voltage of 300 kV. A low magnification image is shown in Figure 5.11A. Areas of interest were selected and tilt series were collected from +65o to -65o at a magnification of 23000 x. Tilt series were aligned and tomograms were reconstructed using back projection in IMOD (Kremer et al., 1996). A representative z-slice from a tomogram of the murine PCM is shown in Figure 5.11B. Highlighted in the figure are globular structures which when rendered in 3D using UCSF Chimera (Figure 5.11C) appear to form a meshwork which spans the gap between the chondrocytes‟ cell membranes. ImageJ was used to analyse the diameter of these globular densities. As the tomograms had a low contrast and were relatively noisy, automated particle analysis could not be used so particles were hand segmented. 218 particles were measured from virtual z slices from a tomogram; a histogram of the particle diameters is shown in Figure 5.12. Globular densities had a mean diameter of 30.4 nm ± 0.5 nm (SEM).

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Figure 5.11 Murine articular cartilage electron tomography

Articular cartilage sample previously imaged with SBF-SEM was imaged using TEM. (A) A representative image of the PCM between two chondrocytes. (B) A virtual z-slice from a tomogram of the chondrocyte PCM. Highlighted are globular densities potentially bundles of collagen VI. (C) A tomogram rendered using UCSF Chimera. The right panel shows a magnified region of the tomogram. N=1.

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Figure 5.12 A Histogram of chondrocyte PCM globular-density diameters

A histogram of PCM globular densities diameters. Diameters were measured using ImageJ measure tool. The mean particle diameter was 30.4 nm ± 0.5 nm (SEM). A total of 218 particles were measured from one tomogram.

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5.4 Discussion and conclusions

Reconstructions of collagen VI microfibrils imaged using cryo-TEM are very similar to previously published negative stain TEM reconstructions (Beecher et al., 2011). However the cryo-TEM model reveals that the half-bead model has a compact head region which is composed of four lobe-like structures, also this model reveals the structure of the collagenous interbead region. The head region is likely to contain the C1 domains from the α1 and α2 α3 chain, with the intermediate region potentially being formed from the C2 domains from the α1 and α2 chains (see Figure 5.13). The N-terminal tail regions form a C-shape. Recombinant N- terminal domains N9-N1 or N7-N1 reconstructed using EM and SAXS also show these regions form a compact C-shape arrangement (Beecher et al., 2011, Maass et al., 2016). The tail regions are less well defined then the head and intermediate regions. This is likely caused by flexibility in the N-terminal tail regions. A contiguous array of N-terminal VWA domains have been shown to be flexible (Maass et al., 2016). The observed heterogeneity is less likely to be caused by different splice variants of the α3 chain as PAGE and AFM volume analysis of the bead region suggested that it was made up of a single species and previous studies of bovine corneal collagen identified that collagen VI microfibrils were composed of VWA C1- N6+N8 from the α3 chain (Beecher et al., 2011) and mass spectroscopy analysis did not identify α4, 5 or 6 chains. The N-terminal tail regions in the N6-C5 microfibril average has a similar length and diameter to the half-bead reconstruction from corneal microfibrils which further suggest that heterogeneity in this region is caused by flexibility in the N-terminal tail regions as these microfibrils contain a recombinant α3 chain of known composition. The interbead domain which could be resolved in this half-bead model is likely formed from the collagenous triple helical regions.

The resolution of the double-bead and the single bead models is lower than previously published negative stain models. This is potentially due to the relatively low number of particles used in the cryo-TEM reconstruction. As cryo-TEM images do not use stain to create contrast they have a lower SNR than negative stain images, this means that they are more difficult to align. Also as collagen VI has a relatively low mass it does not create much phase contrast, therefore to resolve collagen VI microfibrils images have to be taken further from focus. This defocusing causes the CTF to drop off at higher spatial frequencies which results in the loss of high frequency information. In future to create higher resolution reconstructions more images could be collected to create larger particle sets and particles can be imaged closer to focus using a DDD instead of CCD. Creating a 3D reconstruction of the N6-C5 will

139 also allow for better modelling of the collagen VI hierarchical organisation. Analysis of N6-C5 microfibrils was hampered by the low concentration of microfibrils in ECM extracts from SaOS-2 cells. This is most likely due to the low expression of the collagen VI in these cells. To improve the N6-C5 microfibrils newer vectors such as pHLsec (Aricescu et al., 2006) with stronger promoters such as β-actin promoter could be used in SaOS-2 cells.

Figure 5.13 A schematic model of the collagen VI bead region.

We predict that the head region is composed of six C1 VWA domains from a dimer containing α1, α2 and α3 chains and the intermediate region is formed from the C2 VWA domains from the α1 and α2 chains. The N-terminal VWA domains form a compact C shape with the N1 domain closest to the intermediate region. The interbead region is composed of intertwined collagenous regions from two tetramers.

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Due to the high concentration of collagen VI expressed in the PCM and staining which shows it is widely distributed in the PCM (Kuo et al., 1997, Wilusz et al., 2014) it is likely that these globular densities which form a meshwork between the chondrocytes are collagen VI. The mesh like network modelled using SBF-SEM and electron tomography is similar in appearance and diameter to the hexagonal arrangement which form in vitro when purified collagen VI is incubated in the presence of biglycan (Wiberg et al., 2002). To further determine if these structures are collagen VI immuno gold labelling EM could be used to specifically label collagen VI. Also tissue from mouse knockout models which do not produce collagen VI could be imaged to see if these structures were not present, which would support that these structures are composed of collagen VI. Sub-tomogram averaging could also be used to increase the SNR in the 3D models. This technique could also be used to create an average network model to see if there was a defined structure which could also be used to dock molecular models of collagen VI microfibrils.

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6 Final discussion and conclusions

Defining how microfibrils are organised in their different levels of hierarchy is critical to understanding how they function in the ECM of tissues. Microfibrils are important components of connective tissues with a wide range of mechanical and cellular signalling functions. In this thesis the nanoscale structure of the fibrillin microfibril has been reconstructed using negative stain TEM. Reconstructions of fibrillin have revealed a complex internal structure. SBF-SEM and electron tomography of murine and bovine ciliary zonule tissue has allowed for the in situ imaging of fibrillin microfibrils. This has shed light on the structure of ciliary zonule fibres and fascicle-like structures. The frozen hydrated structure of the collagen VI bead region has been reconstructed using cryo-TEM. SBF-SEM and electron tomography have also been used to show how collagen VI microfibrils are organised in the PCM of murine articular cartilage.

6.1 Fibrillin microfibril nanoscale structure

Negative stain TEM 3D reconstructions of the fibrillin microfibril have revealed that fibrillin microfibrils have a hollow tube-like structure which is similar to the appearance of microfibrils when they are imaged in tissues (Davis et al., 2002, Wang et al., 2009). When the reconstruction is viewed in 2D projection the microfibril model has a distinct beads on a string appearance and asymmetrical banding pattern as seen previously for extracted fibrillin microfibrils (Baldock et al., 2001, Sherratt et al., 2003, Wang et al., 2009). Microfibrils are flexible along the fibre axis and seem to be most flexible in the arm and shoulder regions flexing around the interbead region of the microfibril. Due to heterogeneity caused by this flexibility in the molecule the microfibril was broken down into specific regions which were modelled separately. The increase in the resolution in these sub-models allowed for docking of fibrillin fragment molecular models (Baldock et al., 2006) to test different fibrillin packing arrangements. SAXS models could be fitted in either the molecular pleated (Baldock et al., 2006) or half-staggered fibrillin packing models (Kuo et al., 2007). Unfortunately however, the sub-models were not of sufficient resolution to define individual domains and rule out either molecular packing model. Sub-models of the fibrillin regions presented in this study have volumes which are consistent with eight fibrillin monomers per microfibril repeat, which has been shown previously using STEM mass mapping of extracted microfibril and EM analysis of tissue sections (Baldock et al., 2001, Wang et al., 2009).

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The fibrillin microfibril bead region has a well-defined interwoven structure, which could be segmented into a bead core and ring structure. This region of the microfibril is well defined in the sub-model suggesting a rigid stable structure. Epitope labelling studies have shown that the N- and C-terminal regions form the bead region of microfibrils (Reinhardt et al., 1996, Baldock et al., 2001). The stable bead structure is likely stabilised by crosslinks (Qian and Glanville, 1997) and homo- and heterotypic interaction between the N- and C-termini (Trask et al., 1999, Marson et al., 2005). Mass mapping studies support the bead being a stable structure as the bead region does not lose mass until microfibrils are extended to ~90 nm (Baldock et al., 2001). The arm regions of the fibrillin microfibril can be seen to form four separate densities, this could suggest that the fibrillin microfibrils are arranged into four dimers in this region, as dimerisation of fibrillin has been shown in vitro (Trask et al., 1999). The interbead region forms a compact structure, potentially formed from cbEGF domains 15- 26 folding back on themselves. Folding in this region is supported by studies which shows the interbead region undergoes conformational change when microfibrils are extended (Baldock et al., 2001, Wang et al., 2009) suggesting this region has the ability to unfold. The shoulder region of the model was poorly defined which may potentially be due to flexibility in this region. Heterogeneity could also be caused by irregular binding of microfibril associated molecules. Fibrillin can bind several microfibrillar associated proteins which predominantly bind to the N-terminal region of fibrillin (Baldwin et al., 2013) and epitope labelling has shown the N-terminus of fibrillin is located on the shoulder side of the bead region. Mass spectroscopy studies of purified ciliary zonule microfibrils have shown the MAGP-1 co-purifies with extracted microfibrils (Cain et al., 2006) and extracted nucal ligament microfibrils can be labelled with anti MAGP-1 and MAGP-2 antibodies (Hanssen et al., 2004). It is therefore likely that MAGP-1 is bound to the microfibrils imaged here. Fibrillin can bind to a large number of fibrillin associated molecules (see section 1.1.7) such as; tropoelastin (Rock et al., 2004), fibulins-2, -4 and -5 (El-Hallous et al., 2007), ADAMTS-10, ADAMTSL-2, -3, -4 and -6 (Tsutsui et al., 2010, Kutz et al., 2011, Sengle et al., 2012) and LTBP-1,-2,-4 (Isogai et al., 2003, Hirani et al., 2007, Ono et al., 2009). The tissue specific expression of these associated molecules is likely to impact on the structure and function of microfibrils. Microfibrils extracted from tissues such as the aorta, vitreous and ciliary zonule have subtle differences in their structure (Lu et al., 2006), the mass of microfibril can also vary from ~1400 kDa in some early foetal tissues and cell culture to ~2500 kDa in adult tissues (Sherratt et al., 1997, Wess et al., 1998b, Baldock et al., 2001, Sherratt et al., 2010).

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6.2 Fibrillin microfibril microscale structure

The 3D organization of fibrillin microfibrils is highly dependent on tissue context. In the medial layer of the aorta and elastic arteries, elastic fibres form concentric fenestrated lamella, surrounded by circumferentially oriented smooth muscle cells. In skin, the reticular dermis contains a horizontal arrangement of thick elastic fibres which are connected to thinner perpendicular arranged microfibrils called elaunin fibres. These elaunin fibres then merge with bundles of microfibrils devoid of elastin called oxytalan fibres and intercalate in the dermal-epidermal junction. Part of this thesis has focused on the arrangement of fibrillin microfibrils in bovine and murine ciliary zonule tissue which is primarily formed from elastin- free fibrillin microfibrils.

The ciliary zonule keeps the lens in the correct optical axis and also transmits force from the ciliary body to deform the lens during accommodation. Ciliary zonule fibres are mainly comprised of bundles of fibrillin microfibrils (Cain et al., 2006), these bundles of microfibrils are also organised into larger fascicle-like structures which are held together by circumferentially organised zonule fibres (Hiraoka et al., 2010). Microfibrils associate with the basement membrane of the NPCE on one side of the ciliary zonule, and are anchored by insertion into the lens epithelium on the other.

Ciliary zonule fibrillin microfibrils imaged in situ using electron tomography had a structure very similar to that of extracted microfibrils and had the characteristic banding pattern with bead, arm, interbead and shoulder regions and had a hollow tube like structure. The spacing between microfibrils in a ciliary zonule fibre was measured as 28 nm which corresponds to the spacing between microfibrils in tensioned ciliary zonules measured by X-ray fibre diffraction (Wess et al., 1998a). This packing of microfibrils in ciliary zonule fibres may be maintained by cross-bridges which were seen between the bead regions of microfibrils. Ciliary zonule fibrils imaged with SBF-SEM often had darkly stained fibrous patches which could be seen around the perimeter of some zonule fibres in the ciliary zonule. Using correlative tomography similar areas of the ciliary zonule fascicle-like structure were imaged. Tomograms revealed that the darkly stained patches may be small bundles of fibrillin microfibrils which may stabilise the structure of ciliary zonule fibres. Ciliary zonule fibres were also seen to be circumferentially organised around the fascicle-like structures which has been shown previously (Hiraoka et al., 2010).

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The formation of fibrillin microfibrils into bundles and ciliary zonule fascicle-like structures is potentially supported by protein cross-links which have been imaged previously in ciliary zonule microfibrils and appeared to be between bead regions (Davis et al., 2002). Davis et al suggested MAGP-1 could be forming these crosslinks, however MAGP-1 mice develop normally (Weinbaum et al., 2008). LTBP-2 has been shown to be involved in ciliary zonule stability as LTBP-2 mutations cause the disease microspherophakia (Kumar et al., 2010) and studies of murine models of ectopia lentis have shown that LTBP-2 plays an essential role in the maintenance of ciliary zonule fibre stability (Inoue et al., 2014). LTBP-2 could therefore be a candidate for forming links between microfibrils. LTBP-2 binds to the N-terminus of fibrillin-1 (Hirani et al., 2007) which has been predicted to be located in the bead region of the microfibril (Reinhardt et al., 1996, Baldock et al., 2006). However, LTBP-2 knock-out mice can still form normal elastic fibres and live to adulthood showing that LTBP-2 is not essential for fibrillin organisation in other tissues (Inoue et al., 2014). ADAMTS proteins have also been implicated in maintaining the structure of the ciliary zonule, as mutations in ADAMTS-10, ADAMTS-17 and ADAMTSL-4 cause ectopia lentis (Dagoneau et al., 2004, Morales et al., 2009, Collin et al., 2015). ADAMTS-10 has been localized across the length of the ciliary zonule (Kutz et al., 2011). ADAMSTS-10 has also been shown to cause WMS (Dagoneau et al., 2004, Kutz et al., 2008) showing that ADAMTS-10 is important in fibrillin organisation in multiple tissue types.

Tomography of the ciliary body showed ciliary zonule fibres are anchored to the NPCE basement membrane. Microfibrils can be seen contacting volumes connected to the basement membrane. These volumes could potentially be the proteoglycan perlecan. Fibrillin has been shown previously to bind to perlecan in vitro and fibrillin-1 and perlecan have been shown to colocalise at the ciliary NPCE basement membrane as well as in skin and retinal blood vessels (Tiedemann et al., 2005). Binding of fibrillin may also be facilitated through interaction with versican, which has been also been shown to form complexes with fibrillin in the ciliary body (Ohno-Jinno et al., 2008). Versican interacts with microfibrils through its C- terminal lectin domain binding to the N-terminus of fibrillin (Isogai et al., 2002). Assembly of these anchoring complexes may also involve ADAMSTSL-4. ADAMTSL-4 has been shown to be important in anchoring the ciliary zonule into the NPCE basement membrane, as ciliary zonules in ADAMTSL-4 knockout mice do not connect to the NPCE (Collin et al., 2015).

Fibrillin molecules have a complicated hierarchical organisation, polymerising to from individual microfibrils, which then form larger organisations from nanometre to millimetre

145 length scales. Like fibrillin, collagen VI also forms complex microfibrillar assemblies which form large networks in tissues.

6.3 Collagen VI nanoscale structure

Collagen VI microfibrils are heterotrimers of three α-chains (α1+ α2+ αX where X can be α3, 4, 5 or 6). Collagen VI monomers, associate to form a disulphide bonded anti-parallel homo- dimer, which in turn forms dimers then homo-tetramers which are secreted into the extracellular space (Furthmayr et al., 1983, Engvall et al., 1986). Heteromeric microfibrils can form from different α3, α4, α5 or α6 chain homo-tetramers (Maass et al., 2016).

Reconstructions of collagen VI imaged using cryo-TEM presented in this study are very similar to previously published negative stain TEM reconstructions (Beecher et al., 2011). The half-bead model has a compact hollow head region composed of four lobe-like structures which likely contain the C1 VWA domains from the three α-chains. The intermediate region had a more rectangular shape and is potentially formed from the C2 VWA domains from the α1 and α2 chains. The N-terminal tail regions have a compact C-shape which is similar to the structures of recombinant N-terminal VWA domains N1-N9 reconstructed using EM and SAXS (Beecher et al., 2011, Maass et al., 2016). Reconstructions presented here could resolve part of the collagenous region which connects the two half-beads which was poorly defined in previous negative stain studies (Beecher et al., 2011). Due to flexibility in this region it is still not clear how the N-terminal VWA domains are arranged in the bead region of the microfibrils. It is also unclear how the collagenous regions are organised in the globular bead region.

SDS-PAGE and AFM analysis suggests that microfibrils imaged here were formed from a single α3 chain variant, most likely α3 N8+N6-C1 as mass spectroscopy analysis of corneal microfibrils (Beecher et al., 2011) showed that microfibrils were comprised of the α3 chain as the α4, 5 and 6 chains were not detected. SAXS structures of the α4, 5 and 6 globular regions have shown they have a similar structure to the α3 chain (Maass et al., 2016). If microfibrils with the α4, 5 or 6 chains could be produced recombinantly this would allow for comparison with α3 chain containing microfibrils to determine what effect these chains have on the bead structure. Microfibrils could not be produced in SaOS cells transfected with the α5 and 6 chains (Fitzgerald et al., 2008) however this could have been due to the constructs missing the C2 VWA domains. The different α-chain composition could also have an impact on the higher order structure of collagen VI networks.

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6.4 Collagen VI microscale structure

Like fibrillin, collagen VI forms a number of different higher order structures. In skin, collagen VI forms an irregular web-like network of fibrils which associate with collagen II and III fibrils. (Keene et al., 1988). In tissue culture, collagen VI can form bundles of aligned filaments (Bruns, 1984). These large aggregate-like structures can also be seen in diseased tissue such as the Bruch‟s membrane of the eye from patients with age-related macular degeneration (AMD) and Sorby‟s Fundus Dystrophy (Knupp et al., 2000, Knupp et al., 2002, Knupp et al., 2006). In vitro assays using purified collagen VI tetramers have demonstrated that collagen VI can assemble to form large hexagonal networks when incubated with biglycan (Wiberg et al., 2002). Hexagonal arrangements of collagen VI networks can also be observed in tissue culture (Engvall et al., 1986). Collagen VI is also highly expressed and widely distributed in the PCM surrounding chondrocytes in the articular cartilage (Kuo et al., 1997, Wilusz et al., 2014) where it plays an important role in chondrocyte swelling and mechanotransduction (Zelenski et al., 2015). The PCM has a structure which resembles a meshwork of beaded filaments which surround the chondrocyte (Vanden Berg-Foels et al., 2012). The PCM between two chondrocytes in murine articular cartilage was imaged using SBF-SEM and electron tomography which allows for the 3D reconstruction of the tissue, unlike previous EM studies which have used SEM (Raya et al., 2011) and helium ion microscopy (Vanden Berg-Foels et al., 2012).

The PCM was formed from a dense meshwork of globular densities which due to its distribution most likely consists of collagen VI microfibrils. The mesh-like network had a similar appearance to the meshwork observed using helium ion microscopy (Vanden Berg- Foels et al., 2012) and diameter to the hexagonal arrangements which form in vitro when purified collagen VI are incubated in the presence of biglycan (Wiberg et al., 2002). The globular densities had a much larger diameter than a single collagen VI microfibril and are likely to be multiple microfibrils complexed with other PCM molecules and adaptor proteins. Collagen VI interacts with a large range of other ECM proteins such as collagen II, aggrecan, fibronectin and hyaluronan (Poole, 1997). These interactions may occur through direct binding but may also be organised by adaptor complexes of matrilins or SLRPs biglycan and decorin. Perlecan has also been shown to colocalise with collagen VI (Vincent et al., 2007) and mapping of the PCM mechanical properties show that perlecan and collagen VI colocalise in areas where the matrix is less stiff next to the chondrocyte (Wilusz et al., 2012). So it is likely that perlecan also forms part of these assemblies in the PCM.

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The main form of collagen VI expressed in the PCM is thought to contain the α3 chain. The α6 chain is also expressed in articular cartilage but has a different expression pattern as it is expressed further into the territorial matrix and less in the PCM (Fitzgerald et al., 2008). This raises the possibility that the α-chain composition of the collagen VI microfibril could be what defines the diameter of the PCM meshwork structure. The α6 chain like the α3 chain is widely expressed and is also found in tissues such as skin, lung and blood vessels, so it would be interesting to see how the overlapping expression of multiple α-chain variants affects collagen VI microfibril hierarchical structure in tissues.

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7 Future work

7.1 Fibrillin microfibril organisation

An important question which still needs to be resolved is how fibrillin molecules are organised in a microfibril. 3D reconstructions presented here are of insufficient resolution to distinguish between proposed fibrillin packing models. Significantly increasing the resolution of microfibril 3D reconstructions would allow for fitting of individual domains which could be used to determine how the fibrillin molecule packs into a microfibril. Higher resolution reconstructions can be obtained by imaging extracted microfibrils using cryo-TEM with a DDD instead of CCD. DDD have created a “resolution revolution” (Kuhlbrandt, 2014) and can be used to reconstruct protein structures at near atomic resolution ~ 2 Å (Bartesaghi et al., 2015). DDDs can create images with greater contrast SNR which makes alignment of images less difficult, also increasing the contrast in images allows for imaging of microfibrils closer to focus. As increasing the defocus is needed to create contrast in cryo-TEM, using a DDD allows for higher resolution features to be imaged as a lower defocus creates a less steep fall off of the CTF. Additionally cryo-TEM images microfibrils in a hydrated state so microfibrils would be less prone to specimen artefacts inherent in negative stain TEM such as flattening of structures and stain artefacts. Also unlike negative stain TEM, cryo-TEM is not limited by the stain grain size used to visualise the microfibrils which limits resolution to ~20 Å. However, a challenge of cryo-TEM will be collecting the larger dataset required for cryo-TEM, as images have much lower SNR then negatively stain-TEM images. Flexibility of the microfibril will still be an issue in cryo-EM but using a sub-modelling approach as described in this thesis as well as collecting larger datasets will allow for 3D classification of reconstructions which can reduce heterogeneity and increase the resolution of reconstructions. 3D classification may also distinguish microfibrils in different conformational states, for example changes in the microfibril structure during extension.

Another technique which can be used to determine how fibrillin molecules pack into a microfibril is cross-linking mass spectrometry (CXMS). CXMS can be used to determine the folding of protein assemblies by using cross-links to impose distance constraints on models (Tabb, 2012). Cross-linking experiments could be used to determine which domains are in close proximity to each other and therefore could determine which molecular fibrillin packing model was correct. Interdomain crosslinking could also be used to validate domain fitting into EM reconstructions.

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Truncated microfibrils extracted from the heterozygous GT8 mice (Charbonneau et al., 2010) can also be used to determine packing within a microfibril. GT8 mice express a truncated fibrillin construct with an N-terminal eGFP tag. Heterozygous GT8 mice develop normally but suffer from progressive fragmentation of aortic elastic lamellae and disruption of microfibril networks in the skin. Microfibrils in the GT8 mice can be imaged by fluorescence suggesting that the mutant fibrillin can be incorporated into microfibrils. In theory microfibrils from heterozygous GT8 mice should contain 50 % wild type fibrillin and 50 % GFP tagged truncated fibrillin. If arranged in the folding model these microfibrils will have a reduction of 50 % mass/density in the interbead region of the microfibril. Whereas in a half-staggered arrangement the microfibrils would have a loss of mass/density of ~25 % in the bead region. Single particle EM imaging and STEM mass mapping could then be used to distinguish between these two different arrangements of fibrillin molecules.

In this study the ciliary zonule microfibrils were imaged in situ in the ciliary zonule. To determine how fibrillin is arranged into suprafibrillar structures it will be key to understand what microfibrillar structures are common in different tissues which have fibrillin microfibrils. Microfibrils from different tissues can differ in structure (Lu et al., 2006) and mass (Sherratt et al., 1997, Wess et al., 1998b, Baldock et al., 2001, Sherratt et al., 2010). Microfibrils from a range of different tissues should therefore be extracted and reconstructed using TEM and single particle averaging so their high resolution structures can be compared. The higher order structures that these microfibrils form can also be compared by imaging these tissues using SBF-SEM and electron tomography as discussed in this thesis. Fibrillin microfibrils are multicomponent assemblies containing many microfibrillar associated molecules such as MAGP-1 (Cain et al., 2006) determining the composition of microfibrils in different tissues will also be key in understanding fibrillin structure and assembly.

7.2 Collagen VI microfibril organisation

Reconstructions of collagen VI bead regions imaged using cryo-TEM have been determined, but these reconstructions are still of insufficient resolution to determine how the VWA domains and collagenous regions are organised in the bead region. To achieve higher resolution, cryo-TEM using a DDD and collecting a larger cryo-TEM dataset as previously described would increase the resolution of the collagen VI microfibril structure. These higher resolution models could potentially distinguish individual VWA domains and determine how collagenous regions are organised in the bead region. So far reconstructions of collagen VI

150 have focused on microfibrils containing α1, 2 and 3 chains. To determine what affect the collagen VI α4, 5 and 6 constructs had on microfibrils structure these α-chains could be transfected into SaOS-2 cells. This has been attempted previously but the α5 and α6 constructs did not contain the C2 VWA domain which may have been necessary for microfibril assembly (Fitzgerald et al., 2008). If these cells can produce microfibrils they could be used to determine what contribution the α4, α5 and α6 chains have on microfibril structure using techniques such as AFM, STEM and TEM.

In this study the 3D organisation of collagen VI was investigated in the PCM of articular cartilage. This revealed a meshwork-like organisation which was likely to be assemblies of collagen VI and other PCM proteins. To understand how collagen VI is arranged in these densities, AFM could be used to determine the arrangement of collagen VI microfibrils using periodicity measurements as previously used to determine the arrangement of collagen VI aggregates in AMD and Sorby‟s Fundus Dystrophy (Knupp et al., 2000, Knupp et al., 2002, Knupp et al., 2006). The globular densities could also be averaged using sub-tomogram averaging to create an average network model to see if there was a defined structure which could be used to dock molecular models of collagen VI microfibrils. The techniques described in this thesis could also be applied to other tissues which contain microfibrils with different compositions of α3, 4, 5 and 6 chains to determine how different α-chain composition affects the higher order networks which collagen VI forms.

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7.3 Summary

The overall aim of this project has been to determine the hierarchical organisation of fibrillin and collagen VI microfibrils. To achieve this, the nanoscale 3D structure of bovine ciliary zonule extracted fibrillin microfibrils has been reconstructed using negative stain TEM and single particle reconstruction techniques. To investigate the higher order organisation of fibrillin microfibrils murine and bovine ciliary zonules were imaged in 3D using SBF-SEM and electron tomography. This allowed for the in situ imaging of individual microfibrils, showing that microfibrils form ciliary zonule fibres stabilised by cross-bridges, these ciliary zonule fibres are then further organised into fascicle-like structures held together by circumferentially organised ciliary zonule fibres. The structure of the bead region of bovine corneal extracted collagen VI microfibrils was determined using cryo-TEM. To determine how collagen VI microfibrils are organised into networks in tissues the PCM surrounding chondrocytes in murine articular cartilage was imaged in 3D using SBF-SEM and electron tomography. This revealed that the PCM surrounding chondrocytes is formed from a meshwork like arrangement of globular densities.

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Appendix 1: Initial fibrillin microfibril processing

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