Three-dimensional reconstructions of extracellular matrix polymers using automated electron tomography

https://doi.org/10.1016/S1047-8477(02)00028-XGet rights and content

Abstract

The extracellular matrix is an intricate network of macromolecules which provides support for cells and a framework for tissues. The detailed structure and organisation of most matrix polymers is poorly understood. These polymers have a complex ultrastructure, and it has proved a major challenge both to define their structural organisation and to relate this to their biological function. However, new approaches using automated electron tomography are beginning to reveal important insights into the molecular assembly and structural organisation of two of the most abundant polymer systems in the extracellular matrix. We have generated three-dimensional reconstructions of collagen fibrils from bovine cornea and fibrillin microfibrils from ciliary zonules. Analysis of these data has provided new insights into the organisation and function of these large macromolecular assemblies.

Introduction

The extracellular matrix (ECM) is a dynamic entity, constantly being remodelled, that provides support for cells and a framework for tissues. Cell–matrix interactions play critical roles in influencing almost all aspects of cellular behaviour, including development, cell migration, proliferation, tissue formation, and repair. The ECM is composed of four main classes of macromolecules: collagens, proteoglycans, glycoproteins, and elastin. This paper describes electron tomography reconstructions of macromolecular assemblies from two of these classes, namely collagen fibrils (collagen) and fibrillin microfibrils (glycoprotein).

Collagen is the most abundant protein in the animal kingdom. It assembles to form fibrils with an axial periodicity of ∼67 nm and is the principal source of the tensile strength leading to tissue integrity. The axial structure of collagen fibrils is well known (see review by Chapman et al., 1990), but elucidation of the three-dimensional structure has been largely restricted to the large type I collagen fibrils of rat tail tendon which are unique in possessing sufficient crystalline order to allow X-ray diffraction analysis (Orgel et al., 2001; Wess et al., 1998a). Corneal collagen fibrils have a uniform diameter (30–35 nm) and are heterotypic, composed of both type I and type V collagen molecules in the ratio of ∼10:1 (Birk, 2001). Importantly, the type V collagen has a retained globular N-propeptide which has been immunolocalised on the fibril surface. FACIT (fibril associated collagens with interrupted triple helices) collagens and proteoglycans, including leucine-rich repeat proteoglycans, are also surface components and have important roles in modifying fibril assembly, spatial organisation of fibrils, and the mechanical properties of the fibrillar matrix.

Fibrillin microfibrils are essential structural elements of matrix, with widespread distributions in elastic and nonelastic tissues (Sherratt et al., 2001). They are critically important in maintaining the integrity of tissues such as blood vessels, lung, and skin, both in terms of their key roles in linking cells and matrix macromolecules and in the specific biomechanical properties they impart (Kielty and Shuttleworth, 1995). Linkage of mutations in the gene encoding fibrillin-1 to Marfan syndrome confirmed both the key role of fibrillin in microfibril formation and the importance of microfibrils to connective tissue integrity (Robinson and Godfrey, 2000).

The unique elastic properties of fibrillin-rich microfibrils have recently become apparent. X-ray diffraction, tensile testing, and stress–relaxation tests demonstrated that hydrated mammalian ciliary zonules and microfibril bundles are reversibly extensible in the presence or absence of calcium (Wess et al., 1998b). Mass mapping has shown that, in solution, microfibrils with periodicities of <70 nm and >140 nm are stable, but periodicities of ∼100 nm are rare (Baldock et al., 2001).

The arrangement of fibrillin molecules that generates the beaded fibrillin-rich microfibrils remains poorly defined (Handford et al., 2000). The complexity of microfibrils is further enhanced by the fact that they are probably multicomponent polymers. Several other matrix molecules colocalise with microfibrils, including microfibril-associated glycoproteins (Jenson et al., 2001; Trask et al., 2000), latent TGF-β binding proteins (Dallas et al., 2000; Sinha et al., 1998), and proteoglycans (Sherratt et al., 1997).

Collagen fibrils and fibrillin-rich microfibrils have a complex ultrastructure, and until now it has proved a major challenge both to define their structural organisation and to relate this to biological function. These extracellular matrix assemblies are refractory to conventional structural biology techniques because of their size, complexity, noncrystallinity, and nonidentical nature. Factors causing heterogeneity include associated molecules, splice variants, and glycosylation. These problems can now be circumvented with automated electron tomography (AET), since all data for a 3D reconstruction are collected from one region of interest. By collecting a number of data sets on different fibrils, individual repeating units can be compared to find common features.

Here, we have used AET to generate three-dimensional reconstructions of extracellular matrix polymers. The reconstructions of collagen fibrils from cornea have revealed the filamentous substructure and specific surface macromolecules. For fibrillin–microfibrils we have, in addition to three-dimensional reconstructions, also localised fibrillin antibody binding epitopes, and together these data provide evidence for a fibrillin alignment model. This study also highlights the applicability of AET approaches to the ultrastructural analysis of complex isolated polymers.

Section snippets

Collagen fibrils

Collagen fibrils isolated from bovine cornea (Graham et al., 2000) and negatively stained with uranyl acetate were examined by AET using a uniaxis tilt range of −70° to +70° at 2° increments (Holmes et al., 2001). The reconstructions from six tomography data sets revealed a filamentous (i.e., microfibrillar) substructure (Fig. 1) and globular surface components. The visibility of the filaments in the longitudinal virtual sections was enhanced by Fourier filtering (Fig. 2), and they were found

Conclusions

This study has demonstrated the capability of AET approaches to reveal crucial new structural details of complex isolated polymers such as fibrillin-rich microfibrils and collagen fibrils. This structural information provides a basis for understanding the mechanisms of regulated assembly and the mechanical properties of these fibrous components of the extracellular matrix at the molecular level.

Acknowledgements

C.B. has a Royal Society Olga Kennard research fellowship, C.M.K. is funded by the Medical Research Council, K.E.K. and D.F.H. are funded by the Wellcome Trust. The research of A.J.K. has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences (KNAW).

Cited by (0)

1

Present address: Department of Cell Biology, University of Texas, Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.

View full text