Journal of Molecular Biology
Volume 332, Issue 1, 5 September 2003, Pages 183-193
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Fibrillin Microfibrils are Stiff Reinforcing Fibres in Compliant Tissues

https://doi.org/10.1016/S0022-2836(03)00829-5Get rights and content

Abstract

Fibrillin-rich microfibrils have endowed tissues with elasticity throughout multicellular evolution. We have used molecular combing techniques to determine Young's modulus for individual microfibrils and X-ray diffraction of zonular filaments of the eye to establish the linearity of microfibril periodic extension. Microfibril periodicity is not altered at physiological zonular tissue extensions and Young's modulus is between 78 MPa and 96 MPa, which is two orders of magnitude stiffer than elastin. We conclude that elasticity in microfibril-containing tissues arises primarily from reversible alterations in supra-microfibrillar arrangements rather than from intrinsic elastic properties of individual microfibrils which, instead, act as reinforcing fibres in fibrous composite tissues.

Introduction

Fibrillin-rich microfibrils are evolutionarily ancient elastic polymers, conserved in cnidarians, deuterostomes and protostomes, which are thought to endow tissues with long-range elasticity.1., 2. These microfibrils account for the elasticity of invertebrate low-pressure closed circulatory systems3 and provide essential elastic recoil to tissues such as sea cucumber (Cucumaria frondosa) dermis.4 In vertebrates, fibrillin microfibrils are abundant in tissues with anchoring roles such as ocular zonular filaments that hold the lens in dynamic suspension. They also act as a template for the deposition of elastin, a vertebrate protein, and are components of the mature elastic fibres that endow lungs, ligaments, skin and elastic cartilage with elasticity and resilience, and crucially reinforce high-pressure blood circulatory systems. Despite clear evidence of a role for tissue microfibrils in elasticity, the molecular basis of the biomechanical properties of individual microfibrils remains unresolved. A major issue is whether elasticity arises from the intrinsic properties of individual microfibrils, from the packing arrangement of parallel microfibrils within bundles, and/or from higher-order bundle architectures.

Isolated microfibrils have a complex ultrastructure with a ∼56 nm periodic “beads-on-a-string” appearance in the presence of bound calcium.5 They are based on a polymerised scaffold of fibrillin molecules, which are large glycoproteins with a multidomain structure dominated by contiguous disulphide-bonded, calcium-binding epidermal growth factor-like (cbEGF) domain arrays.2., 6. In addition to regular untensioned 56 nm periodicities, highly stretched sections of microfibrils (periodicities up to ∼150 nm) have sometimes been observed, giving rise to suggestions that individual microfibrils are elastic.7., 8. Examination of this phenomenon by scanning transmission electron microscopy (STEM) mass mapping revealed that such highly stretched periodic regions were stable in physiological buffers and did not exhibit elastic recoil. Periodicities within the range 56–90 nm were observed less commonly, and interbead mass changes alone accounted for periodic changes within this range.8 Raman spectroscopy of ciliary zonules has further suggested that molecular conformational changes occur on stretching of microfibrils. These changes may involve realignment of the flexible cbEGF linker regions of the fibrillin molecule.9 Three-dimensional reconstructions, using automated electron tomography and antibody epitope mapping, were used to study how fibrillin molecules (∼150 nm in length) are packed in microfibrils with a 56 nm periodicity. We developed a model, based on a parallel head-to-tail fibrillin-1 molecular alignment,10 which predicted multiple folding events in which molecules form a one-third stagger stabilised by transglutaminase cross-links (periodicity ∼90 nm) and further flex at certain transforming growth factor β-binding protein-like domain to cbEGF domain linkages (to 56 nm).1., 8. By this model, microfibrils could extend between 56 nm and 90 nm by reversible interbead unfolding and/or conformation changes, but microfibril extensions >90 nm would require cleavage of the covalent cross-links that maintain the microfibril elastic conformation.

The elastic properties of invertebrate tissue microfibrils, and vertebrate elastin and elastic fibres have all been studied. Microfibril networks extracted from echinoderm tissue using guanidine and collagenase had long-range elasticity that was dependent on the presence of ions in solution and on disulphide bonds. These networks were reversibly extensible up to ∼300% of their initial length, and behaved like viscoelastic solids with a long-range component as well as a time-dependent viscous component.4 Young's modulus for microfibrils within assembled arrays in lobster aorta wall was approximately 1 MPa; this work indicated that this microfibril-rich tissue displays linear deformation behaviour.11 Young's modulus of sea cucumber dermis microfibrils, based on mechanical testing of guanidine and collagenase-extracted tissue was 0.2 MPa.4 These values are in the same range as elastin, which is 1.1 MPa.12 Short recombinant elastin peptides comprising alternating hydrophobic and lysine-rich domains, when coacervated and cross-linked, had a Young's modulus of 0.2 MPa.13 Mammalian microfibril studies have focussed on the zonular filaments of the eye. X-ray fibre diffraction of whole bovine zonules,14 and biomechanical testing of microfibril bundles teased out from zonules15 have shown that these tissue microfibrils are reversibly extensible. Preliminary studies using bovine zonular filaments showed that at tissue extensions of 100% from rest length, microfibril periodicity increased to approximately 80 nm but returned in the relaxed state to 56 nm.14 With extensions of up to 50% rest length, microfibril periodicity remained unchanged. X-ray diffraction has provided evidence for a specific alignment or one-third staggering of adjacent microfibrils.16 This arrangement is recoverable even after 150% tissue extension, which is probably beyond the physiological range of microfibril elasticity.8., 9., 17.

Section snippets

The effect of applied force on tissue microfibril periodicity

In order to determine whether microfibrils exhibit linear elasticity within the predicted elastic range8 when the whole tissue is extended, we conducted small-angle X-ray diffraction on mammalian zonular filaments at increasing strains of 0–2 (Figure 1(a)–(c)). X-ray diffraction provides information on the structure and behaviour of zonular microfibrils in the hydrated state and, since the molecular organisation is examined using an X-ray beam that covers a macroscopic area of the specimen (300 

Small-angle X-ray scattering

Previous small-angle X-ray scattering studies of mammalian zonular filaments were conducted using bovine ocular tissue.14., 16., 29. UK restrictions on the handling of bovine nervous tissue have restricted the availability of this tissue. This study, therefore, was conducted using red deer eye as an alternative source of zonular filaments. The X-ray diffraction patterns obtained were remarkably similar to those observed using zonular filaments extracted from bovine eye. Zonular filaments were

Acknowledgements

The authors thank Miss Amanda Morgan for expert technical assistance and Dr Adrian Mann for advice on biomechanical aspects of the study. M.J.S. and C.M.K. acknowledge the support of the Medical Research Council (UK). J.L.H. acknowledges the support of the BBSRC (grant reference number 98/S15326). D.F.H. acknowledges the support of the Wellcome Trust. C.B. has a Royal Society Olga Kennard research fellowship. T.J.W. acknowledges the ESRF for providing funding and access.

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