Correspondence to: Clair Baldock, Wellcome Trust Centre for Cell-Matrix Research, Schools of Biological Sciences and Medicine, 2.205 Stopford Building, University of Manchester, Manchester, M13 9PT, UK. Tel:0161 275 5756 Fax:0161 275 5752 E-mail:clair.baldock{at}man.ac.uk.
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Abstract |
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We propose a new model for the alignment of fibrillin molecules within fibrillin microfibrils. Automated electron tomography was used to generate three-dimensional microfibril reconstructions to 18.6-Å resolution, which revealed many new organizational details of untensioned microfibrils, including heart-shaped beads from which two arms emerge, and interbead diameter variation. Antibody epitope mapping of untensioned microfibrils revealed the juxtaposition of epitopes at the COOH terminus and near the proline-rich region, and of two internal epitopes that would be 42-nm apart in unfolded molecules, which infers intramolecular folding. Colloidal gold binds microfibrils in the absence of antibody. Comparison of colloidal gold and antibody binding sites in untensioned microfibrils and those extended in vitro, and immunofluorescence studies of fibrillin deposition in cell layers, indicate conformation changes and intramolecular folding. Mass mapping shows that, in solution, microfibrils with periodicities of <70 and >140 nm are stable, but periodicities of 100 nm are rare. Microfibrils comprise two in-register filaments with a longitudinal symmetry axis, with eight fibrillin molecules in cross section. We present a model of fibrillin alignment that fits all the data and indicates that microfibril extensibility follows conformation-dependent maturation from an initial head-to-tail alignment to a stable approximately one-third staggered arrangement.
Key Words: three-dimensional reconstruction, automated electron tomography, fibrillin microfibrils, molecular alignment, scanning transmission electron microscopy mass mapping
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Introduction |
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Fibrillins form the structural framework of an essential class of extracellular microfibrils that endow dynamic connective tissues with long-range elasticity (
Fibrillin-rich microfibrils have a complex ultrastructure with a repeating "beads-on-a-string" appearance, with a number of "arms" extending from globular bead structures (56 nm. By this approach, bead diameters of
23 nm have been recorded (
The two isoforms, fibrillin-1 and fibrillin-2, are large glycoproteins (350 kD) with multidomain structures dominated by 43 calcium-binding consensus sequences (cbEGF domains) (
2.75 and 2.02.4 nm, respectively. The TB module-cbEGF linkage is predicted to have flexibility. Ultrastructural and x-ray diffraction studies have shown that bound calcium profoundly influences packing and periodicity of isolated microfibrils and hydrated microfibril arrays (
The unique elastic properties of fibrillin-rich microfibrils have recently become apparent. The extensibility of lobster aorta was accounted for by microfibril arrays that intersperse medial smooth muscle cells (165 nm (
Several models of fibrillin alignment in microfibrils have been proposed. A model based on antibody epitope mapping and measured molecular dimensions suggested a parallel head-to-tail alignment of unstaggered fibrillin monomers with amino and carboxy termini at, or close to, the beads (
Here, we have used automated electron tomography (AET) to develop the first three-dimensional reconstructions to define molecular organization within microfibrils. We have also localized fibrillin antibody and colloidal gold-binding epitopes in directionally oriented untensioned zonular microfibrils, and mapped bead and interbead mass changes on extension. These data provide strong new evidence for a fibrillin alignment model that indicates that initial fibrillin assemblies undergo conformational maturation to a reversibly extensible beaded polymer, and thus suggests a molecular explanation for microfibril extensibility. The study also highlights, for the first time, the applicability of AET approaches to the ultrastructural analysis of complex isolated polymers.
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Materials and Methods |
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Sample Preparation for Reconstructions
All microfibrils were isolated in native, nondenaturing conditions from bovine and human ciliary zonules using modifications of a previously described methodology (
Purified microfibrils were allowed to absorb for 30 s onto glow-discharged carbon-coated copper grids with 5 nm colloidal gold particles on. The grids were washed three times with water, and then negatively stained with 2% (wt/vol) uranyl acetate, pH 4.7. Immediately after wicking off the stain, the grids were snap-frozen in liquid nitrogen (-196°C), freeze dried at -90°C for 2 h in a Cressington CFE50B, and then slowly brought to room temperature.
Data Collection and Reconstruction
We employed a Philips CM200 FEG transmission electron microscope operating at 200 kV at the University of Utrecht. Data was collected at 20,000x nominal magnification and 1 µm defocus. The microscope was equipped with a computer-controllable goniometer and CCD camera for image collection (TVIPS GmbH). The calibrated pixel size at specimen plane was 0.625 nm. A suitable area containing microfibrils with good deposition of gold particles was identified in the electron microscope. Electron tomographic data sets were collected by tilting the specimen over a tilt range of typically ±70° with 2° increments in a high tilt holder. The digital data sets were recorded by automatic correction of image shift and focus variation during the collection of the tilt series with the EM Menu software (TVIPS GmbH). The IMOD software ( significance threshold (
Microfibril Binding Studies
Preparations of human or bovine zonular microfibrils were absorbed for 30 s onto glow discharged carbon-coated copper grids. Grids were washed three times with deionized water before a drop of colloidal gold (British BioCell Int.) was placed on each grid for 1 min. Grids were blotted, washed twice with water, negatively stained, and then air dried.
The following antibodies were used in binding studies. Monoclonal antibodies 11C1.3 and 12A5.18 (Neomarkers; Lab Vision Corp.) each recognize epitope(s) within fibrillin-1 residues 451909 (exons 1122). Since 11C1.3 does not recognize a fibrillin-1 minigene (exons 115 spliced onto exons 5065) that we produced in a mammalian cell system (
Purified microfibrils were incubated with primary antibody (1:20) for 15 min on ice. Microfibrils were then pelleted by centrifuging at 60,000 g for 1 h at 4°C. Supernatants were discarded and pellets resuspended in buffer (400 mM NaCl, 50 mM Tris-HCl, pH 7.4, 10 mM CaCl2). Samples were absorbed onto carbon-coated copper grids, air-dried, and then viewed in an electron microscope (EM 1200EX; JEOL) at 100 kV accelerating voltage.
Cell Layer Immunofluorescence
Normal human dermal fibroblasts were plated at hyperconfluence and grown for up to 3 wk in Dulbecco's minimum essential medium containing 10% fetal calf serum and antibiotics (penicillin/ streptomycin, 100 IU/ml-1). Cell layers were fixed in 95% ethanol, and then processed for immunofluorescence, as previously performed (
Microfibril Extension Studies
Several approaches were investigated. Isolated bovine or human zonular microfibrils in buffer were centrifuged at 4°C for 1 h at 60,000 g, and pellets were resuspended in 50 µl buffer. Aliquots (500 µl) of the same microfibril preparations were drawn 200x through a hypodermic needle 40-mm long of 0.8-mm diameter bore into a disposable 1-ml plastic syringe (1 stroke/s). Microfibrils were adsorbed directly from droplet surfaces (20100 µl microfibril solution) onto carbon-coated grids that had not been glow discharged. Grids were floated on sample drops for 30 s, washed three times in purified deionized water, negatively stained, and then air-dried. Canine (Jack Russell) eyes with dislocated lenses were obtained from the Animal Medical Centre (Manchester, UK) at the time of euthanasia for medical reasons. All preparations were visualized by electron microscopy after negative staining.
STEM Mass Mapping
STEM of unstained unshadowed canine zonular microfibrils provided quantitative data on microfibril mass and periodicity, as previously described (
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Results |
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Analysis of Untensioned Microfibrils
Automated Electron Tomography: Data Sets.
Six automated electron tomography data sets were collected on negatively stained isolated untensioned microfibrils from bovine ciliary zonules, in the presence of calcium. Each data set consists of a region of 10 repeating units from a microfibril. The resolution of a test data set was calculated to be 18.6 Å by Fourier shell correlation using a 3
significance threshold (
Automated Electron Tomography: Overall Microfibril Dimensions.
Negatively stained microfibrils showed repeating units of "beads" and "interbeads," a diameter of 1518 nm, and a mean periodicity of 57.5 nm (Fig 1, ad, and Table 1). The interbeads often appeared to bow out between beads. Average bead height was 9 nm, calculated using 5 nm gold for comparison.
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Automated Electron Tomography: Structural Details Revealed by AET.
Three-dimensional reconstruction of microfibrils to 18.6-Å resolution revealed new structural features and dimensions (Fig 1, BD). Table 1 describes the mean distances and SD within a microfibril repeating unit. Beads appear as dense masses that are more "heart-shaped" than spherical, with undulating surfaces. Bead diameter varies axially between 14.8 and 18.7 nm. Some bead morphological variability occurred in all data sets. In most repeats, two prominent arms emerge from the broader bead face, meeting at a fixed position 43% of bead-to-bead distance (14.7 nm from bead edge,
29% of the interbead). They appear as stain-excluding regions that bow out between the beads, between which is stain-accessible space. Fig 1 B shows six slices through a microfibril and highlights that the stain-penetrating space occurs throughout all Z sections and is therefore a 3-D cavity. In some repeats, the arms are less clearly defined, appearing as a number of fine filaments. The point where the arms terminate within the interbead may correspond to an interbead "striation" detected by rotary shadowing (
Twisting along isolated microfibrils within the interbead may occur (Fig 1b and Fig c). In many repeats, the two interbead arms appear to cross over between consecutive beads, which emphasizes stain pooling between them and gives a "bow-tie" appearance. Twisting supports the concept that the isolated microfibrils are in an untensioned, relaxed conformation.
Binding Studies: Antibody Mapping.
Fibrillin-1 antibody epitopes accessible on isolated untensioned microfibrils were mapped after incubation, centrifugation, and ultrastructural examination (Fig 2 and Table 2). Antibody 2502, which recognizes an NH2-terminal fibrillin-1 sequence within residues 45450 (referred to as antibody 26 in
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Immunofluorescence analysis using PF2 revealed abundant fibrillin networks in hyperconfluent fibroblast cell layers from 3 d in culture (Fig 2 E). However, 11C1.3 only detected microfibril arrays after 2 wk in culture. Even when 11C1.3 was used at 1:20 dilution, which is five times the concentration of PF2, it still did not detect any extracellular fibrillin-rich microfibrils until 14 d in culture. A polyclonal antifibrillin-2 antibody failed to detect microfibrils in the cell layers at any time point (not shown).
Binding Studies: Gold Binding.
Colloidal gold particles, used as fiducial markers in alignment of the AET data, associated periodically with untensioned microfibrils at the interbead ends of the arms (Fig 1 E). In many fields, the gold bound periodically in an aligned pair-wise manner. A second lower-affinity gold binding site at the bead was sometimes occupied.
Analysis of Extended Microfibrils
Microfibril Extension Studies.
Several approaches were investigated to generate extended isolated microfibrils. When microfibril preparations were centrifuged at 60,000 g for 1 h, a small proportion of microfibrils appeared stretched in the range 70110 nm, but most retained untensioned periodicity. When microfibril preparations were repeatedly drawn through narrow bore needles, the majority of microfibrils retained untensioned (56 nm) periodicity, although a few were extended to
70 nm. Interbead morphology of many of these microfibrils appeared diffuse, suggesting conformation change (not shown). However, a significant number of microfibrils in extended state (70110-nm range) were captured from sample dropair interfaces directly onto carbon-coated grids (Fig 3). Interbead morphology of these microfibrils was diffuse, indicating major conformational changes. By contrast, canine zonules associated with dislocated lenses contained numerous stable highly extended microfibrils (see Fig 4).
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Binding Studies: Antibody Binding.
The 12A5.18 antibody-treated bovine zonular microfibrils were adsorbed directly onto grids to locate the binding site in extended microfibrils (Fig 3 B). In these preparations, some microfibrils remained in untensioned state and displayed in-register array banding patterns similar to untreated preparations (see Fig 2 B). In other cases, antibody-banded microfibril arrays were partially extended to 70 nm, and in these the position of 11C1.3/12A5.18 remained at the end of the interbead arms (23 nm from bead centre) (Fig 3 B shows a comparison of partially extended and untensioned microfibrils). This corresponds to 41.1% of bead-to-bead distance for untensioned 56-nm microfibrils, but 32% of bead-to-bead distance for 70-nm microfibrils. Thus, microfibrils can only extend for
10 nm before this epitope has to move. At higher extensions, all antibody banding was lost.
Binding Studies: Colloidal Gold Binding.
On microfibrils extended to 100 nm after adsorption onto grids directly from sample dropair interfaces, colloidal gold particles bound at the beads and, in some cases, at the interbead ends of the arms (Fig 3 A).
Scanning Transmission Electron Microscopy Analysis
Untensioned (56 nm) and extended microfibrils isolated in native state using nondenaturing conditions, from canine zonules were examined by STEM to investigate how mass distribution changes on extension (Fig 4). Within any single microfibril, the vast majority of repeats were either
56 or
160 nm, but there was always a short sharp periodicity transition (Fig 4a and Fig b). While total mass per repeat remained unchanged irrespective of microfibril periodicity, there was both a progressive reduction in interbead mass per unit length and loss of the shoulder at periodicities from
56 to 100 nm, and then a rapid reduction in bead mass at higher periodicities (Fig 4 C). These data show for the first time that interbead unfolding accounts for extension between
56 and 100 nm, and that bead unraveling occurs at periodicities >100 nm. The highly extended microfibrils (
160 nm) appear to be irreversibly stretched since they are stable in solution. The mean MUL within the interbead of highly extended microfibrils was 14.25 kD/nm.
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Discussion |
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We have derived a model of fibrillin alignment in microfibrils based on (a) AET-generated three-dimensional reconstructions of untensioned microfibrils that define microfibril dimensions and molecular organization, (b) mapping of antibody and colloidal gold binding sites in directionally orientated untensioned microfibrils, which demonstrate intramolecular folding, (c) mass changes on microfibril extension showing that interbead unfolding precedes bead unraveling, (d) immunofluorescence studies of extracellular fibrillin deposition that show a major conformational change, and (e) published observations (100-nm form (Fig 5). This model accounts for all microfibril structural features, suggests that inter- and intramolecular interactions drive conformation changes to form extensible microfibrils, and defines the number of molecules in cross section.
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Fibrillin Alignment Model
We propose that the first step in microfibril assembly is NH2- and COOH-terminal association at the cell surface, probably regulated by terminal processing (160 nm. Predicted on the basis of domain dimension (
118.25 nm), 4 EGFs (
10 nm), 7 TB modules (
15.4 nm), 2 hybrid domains (
5 nm), NH2 and COOH termini, and the proline-rich region (>7.5 nm); thus,
160 nm in calcium-loaded form. Actual molecular measurements range between 148 nm in PBS-treated preparations (
130 nm (based on
60 nm for peptides encoded by exons 136, and
70 nm for peptides encoded by exons 3765;
20 nm less than predicted from domain structure. Free molecules may fold at the proline-rich region since NH2-terminal peptides up to the proline-rich region are predicted to be
20 nm (
100 nm (32 cbEGFs, 1 hybrid motif, 5 TB modules); we have observed this periodicity in most of the isolated intact microfibrils that were trapped in partially extended form at solutionair interfaces, but it is not stable in solution. This arrangement thus provides the structural context for reversible extension between 56 and
100 nm. Microfibril "recoil" could involve intramolecular folding at flexible TB-cbEGF linkages (
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Five such TB-cbEGF junctions exist between the cross-link sites (exons 1458), and several folding arrangements could thus occur to generate the 56-nm untensioned microfibrils. TB3, which precedes the central 12 cbEGF array, has the longest linker region (19 residues) and may be particularly flexible. Of these possibilities, only one fits the formation of interbead arms of observed dimensions (14.7 nm or approximately six domains) and antibody binding sites. This arrangement would involve hinging at the TB3-cbEGF junction so that the central 12 cbEGF array folds back, juxtaposing the center of this array with the core bead, thereby enhancing its mass and reducing periodicity to
56 nm (18 cbEGFs, 3 TB modules). Characteristic diameter variations within each untensioned repeat (Fig 1 C) shown in our reconstructions also indicate complex interbead molecular folding. N-Glycosylation sites, all accommodated within the interbead, would protect exposed interbead domain arrays from proteolytic attack (
Our proposed overlap is further supported by localization of the COOH terminus (antibody 2499) close to 12A5.18 but on the opposite side of the bead to the NH2 terminus (antibody 2502) (Fig 2). The binding site of antibody PF2 in the center of the interbead is just 5 nm (approximately two domains) away from 12A5.18 (Fig 2), whereas in an extended molecule they would be at least 42 nm apart. Substantial molecular folding must thus occur to place the PF2 epitope in such close proximity to the interbead ends of the arms. Twisting in the 56 nm microfibrils (Fig 1 C) could reflect the flexibility in the absence of tension at interbead TB-cbEGF junctions, and may explain why the PF2 epitope appears to encircle the microfibril (Fig 2 C). Twisting may not occur in physiological arrays due to packing constraints, and there is no evidence of regular helical symmetry in fiber-diffraction patterns (
Our immunofluorescence studies of human dermal fibroblast cell layers show that PF2 detects abundant extensive fibrillin "microfibrils" from 3 d onwards, whereas antibody 11C1.3 does not detect extracellular microfibrils until 2 wk in culture (Fig 2 E). The gradual pericellular appearance of 11C1.3-reactive microfibrils provides strong evidence of a physiological conformational rearrangement consistent with our folding model, exposing or unmasking a cryptic 11C1.3 epitope. Since the epitope for antibody 11C1.3 is highly surface accessible in 56 nm untensioned microfibrils (Fig 2 B), this is presumably the mature native microfibril configuration recognized in the
2-wk cell layers. Since microfibrils containing fibrillin-2 were not detected in these cell layers, it is unlikely that these observations reflect any potential preference for fibrillin-2 by either 11C1.3 or PF2. These observations also indicate that more than one type of fibrillin polymer may occur in cell layers and tissues.
The effects on microfibrils of adsorption directly from the sample solutionair interface onto grids may be explained in part by the effects of surface tensional forces, and in part by the behavior of proteins at aqueousair interfaces, which are arranged to decrease the surface tension. As with other proteins, fibrillin-rich microfibrils may be concentrated at the bufferair interface with exposed or exposable hydrophobic regions migrating to, and partly through, the waterair interface, and their hydrophilic regions directed to the water phase (100 nm are only observed when isolated directly from sample solutionair interfaces (Fig 3A and Fig B) and that there is such a sharp periodic transition between <70 and >140 nm in canine zonular microfibrils (Fig 4a and Fig b) shows that this periodicity range is energetically unfavorable, and predicts that molecular folding to
56 nm is an inevitable consequence of the one-third stagger.
Reversible extension (56100 nm) would involve unfolding of the TB3-cbEGF fold. Irreversible extension could involve disruption of the molecular association at the transglutaminase cross-link site. Our STEM studies also show that the
100-nm microfibrils have no measurable shoulder of mass in contrast to the 56-nm form (
Colloidal gold particles bind proteins through charge, hydrophobic interaction, or dative binding to sulphur-containing groups (
Number of Molecules in Cross Section and Longitudinal Symmetry Axis
Our data provide evidence that there are approximately eight fibrillin molecules in a microfibril cross-sectional diameter. The 3-D reconstructions contain volumetric information indicating how many linearly aligned fibrillin molecules are packed within the interbead. The arms emerging from the bead each measure 6 x 5 nm (30 nm2) in cross section. Since the cross-sectional diameter of calcium-bound cbEGF-like domains, as determined by nuclear magnetic resonance (NMR) (
3.6 nm2, this corresponds to approximately eight domain arrays per arm. In our model, since each molecule in the arms is folded back, there would be four molecules per arm, possibly arranged as dimer pairs or tetramers. The cross-sectional diameter of the narrow interbead region measures a minimum of
10 x 6 nm (60 nm2), so at least 16 cbEGF domain arrays may be aligned within this region, which is consistent with predicted domain array folding. STEM has also established here that the MUL of the extended interbead is 14.25 kD/nm. Determination of the MUL of a fibrillin molecule was based on exons 2336 (13 cbEGF domains and 1 TB module). The predicted mass of this peptide is 67.18 kD. Its length is 37.4 nm, based on domain dimensions determined by NMR (37.8 nm) (
1.80 kD/nm, which indicates 7.92 molecules in an extended interbead cross section. Moreover, the actual mass per repeat is
2,4902,510 kD compared with a predicted 2,510 kD for eight aligned molecules.
We have outlined above how fibrillin monomers may be aligned and have provided evidence that there are eight molecules in cross-sectional diameter. We have also shown that microfibrils have a longitudinal axis of symmetry and the interbead has two symmetrical arms. Previous biochemical studies have suggested that fibrillin dimers may be a physiological intermediate of assembly (
Summary
This study has demonstrated, for the first time, the power of AET approaches to reveal crucial new structural details of complex isolated polymers such as fibrillin-rich microfibrils. Our model of fibrillin alignment in microfibrils accounts for the structural features of microfibrils and predicts that molecular interactions drive conformation changes that, in turn, underpin reversible microfibril extensibility. The critical importance of the sequences involved in the conformational changes is supported by the clustering on either side of predicted bead position in our model, of mutations that cause the severe neonatal form of Marfan syndrome.
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Footnotes |
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1 Abbreviations used in this paper: 3-D, three dimensional; AET, automated electron tomography; MUL, mass per unit length; STEM, scanning transmission electron microscopy; TB, TGF-ß binding protein like.
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Acknowledgements |
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Canine zonules were kindly provided by Dr. J.L. Ashworth. We thank Dr. D.F. Holmes and Mr. C. Gilpin for assistance with the Fourier shell correlation.
This work was supported by Medical Research Council grant G117/268 (C.M. Kielty and C. Baldock). The research of A.J. Koster has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences (KNAW).
Submitted: 25 September 2000
Revised: 11 December 2000
Accepted: 18 January 2001
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References |
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