Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility.

Decorin is a member of the expanding group of widely distributed small leucine-rich proteoglycans that are expected to play important functions in tissue assembly. We report that mice harboring a targeted disruption of the decorin gene are viable but have fragile skin with markedly reduced tensile strength. Ultrastructural analysis revealed abnormal collagen morphology in skin and tendon, with coarser and irregular fiber outlines. Quantitative scanning transmission EM of individual collagen fibrils showed abrupt increases and decreases in mass along their axes. thereby accounting for the irregular outlines and size variability observed in cross-sections. The data indicate uncontrolled lateral fusion of collagen fibrils in the decorindeficient mice and provide an explanation for the reduced tensile strength of the skin. These findings demonstrate a fundamental role for decorin in regulating collagen fiber formation in vivo.

Electron microscope 3D reconstruction of branched collagen fibrils in vivo.

The ability of tendon to withstand tensile forces is largely attributable to an extracellular matrix containing parallel collagen fibrils organized into fascicles. A major belief is that force is transmitted between collagen fibrils via interactions of molecules at the fibril surface. However, there is existing evidence (reviewed here) for persistent connections between fibrils formed by interfibrillar fusion. Furthermore, in vitro studies have shown the ability of the ends of fibrils to fuse together. In this study, we show using serial section electron microscopy of embryonic mouse-tail tendon further evidence for interfibril fusion in vivo. We showed: (1) fibrils fused via Y-shaped branches without disruption of the 67 nm D-periodicity, (2) the frequency of the branches was approximately 1:20 000 D-periods, and (3) the small angle of the Y ranged from 4 degrees to 10 degrees, indicating a structure-based mechanism of branch formation. The regular occurrence of Y-shaped branches between collagen fibrils suggests direct force transmission between fibrils. Furthermore, the formation of the Y-shaped branches by tip-to-shaft fusion would explain the paucity of fibril tips in vivo.

Crystalline regions in collagen fibrils.

A new image processing technique, content-dependent anisotropic spatial frequency filtering, has been developed to visualize the location and orientation of crystalline regions in collagen fibril cross-sections. The results show that most crystalline regions are oriented with their approximately 4 nm periodicity directed radially from the fibril centre. This periodicity corresponds to the separation between rows of molecular ends in the quasi-hexagonal molecular packing scheme. The extent of crystallinity increases with radius and frequently the lattice is either continuously distorted or interrupted by sharp discontinuities.

Morphology of sheet-like assemblies of pN-collagen, pC-collagen and procollagen studied by scanning transmission electron microscopy mass measurements.

At high concentrations, type I pN-collagen, pC-collagen and procollagen (the first 2 generated from procollagen by enzymic cleavage of C-propeptides and N-propeptides, respectively) can all be made to assemble in vitro into thin D-periodic sheets or tapes. Scanning transmission electron microscopy mass measurements show that the pN-collagen sheets and procollagen tapes have a mass per unit area corresponding to that of approximately 6.8 monolayers of close-packed molecules. pN-collagen sheets are extensive and remarkably uniform in mass thickness (fractional S.D. 0.035); procollagen tapes are neither as extensive nor as uniform in thickness. The mean thickness of pC-collagen tapes is less and the variability is greater. In pN-collagen sheets, the overlap: gap mass contrast in a D-period is increased from 5:4 (the ratio in a native collagen fibril) to 6:4, showing that the N-propeptides do not project into the gap but are folded back over the overlap zone. Assuming all N-propeptides to be constrained to the two surfaces of a sheet, their surface density can be found from the mass thickness of the sheet. In a lateral direction (i.e. normal to the axial direction where the spacing is D-periodic), the N-propeptide domains are calculated to be spaced, centre to centre, by 2.23 (+/- 0.1) nm on both surfaces. This value (approx. 1.5 x the triple-helix diameter) implies close-packing laterally with adjacent domains in contact. Sheet formation and the "surface-seeking" behaviour of propeptides can be understood in terms of the dual character of the molecules, evident from solubility data, with propeptides possessing interaction properties very different from those displayed by the rest of the molecule. The form and stability of sheets (and of first-formed fibrils assembling in vivo) could, it is suggested, depend on the partially fluid-like nature of lateral contacts between collagen molecules.

Vertebrate (chick) collagen fibrils formed in vivo can exhibit a reversal in molecular polarity.

A reversal in molecular polarity can occur in vertebrate collagen fibrils. This has been demonstrated using a method for isolating, from chick embryo tendon, entire collagen fibrils 2 to 14 microns in length and suitable for electron-optical examination. A polarity reversal is present in some, but not all, of these fibrils. Such fibrils have two N-ends. The transition region, occupying several D-periods in which the reversal occurs, is not restricted to a central location in a fibril. Analysis of the fibril banding pattern through the transition region shows that the relative axial alignment of antiparallel molecules brings oppositely-directed C-telopeptides into axial register. This could allow antiparallel molecules to be covalently linked via polymeric cross-links involving these C-telopeptides.

Echinoderm collagen fibrils grow by surface-nucleation-and-propagation from both centers and ends.

Collagen fibrils from sea cucumber (class Holothuroidea) dermis were previously found to grow by coordinated monomer addition at both centers and ends. This analysis of sea urchin (class Echinoidea) collagen fibrils was undertaken to compare the growth characteristics of fibrils from two classes of echinoderms, and to determine whether a single growth model could account for the main features of fibrils from these two taxa. Native collagen fibrils (37-431 micrometer long) from the spine ligaments of the sea urchin Eucidaris tribuloides were studied by scanning transmission electron microscopy and image analysis. The analyses revealed the mass per unit length, and hence the number of molecules in cross-section, along the entire length of each fibril. The fibrils were symmetrically spindle shaped. The maximum mass per unit length occurred in the center of each fibril, where the fibril contains anti-parallel molecules in equal numbers. The two pointed tips of each fibril showed similar linear axial mass distributions, indicating that the two tips retain shape and size similarity throughout growth. The linear axial mass distributions showed that the tips were paraboloidal, similar to those of vertebrate and sea cucumber fibrils. The computed maximum diameters of the fibrils increased linearly with fibril length. The overall shapes of the fibrils showed that they retain geometric similarity throughout growth. Computer modeling showed that the simplest self-assembly mechanism that can account for the features of these fibrils, and of the sea cucumber fibrils that have been described, is one in which the fibril tips produce independent axial growth, while lateral growth takes place through a surface nucleation and propagation mechanism. This mechanism produces coordinated growth in length and diameter as well as geometric similarity, characteristic features of echinoderm collagen fibrils.

Collagen fibrils forming in developing tendon show an early and abrupt limitation in diameter at the growing tips.

The formation of very long and near-uniform diameter collagen fibrils is fundamental to the assembly of the extracellular matrix of animals. However, how growth in length and diameter is regulated, and how fibrils increase in diameter during development, are poorly understood. The approach in this study was to examine the tips and central shaft regions of fibrils from 12 and 18-day embryonic chick metatarsal tendon using quantitative mass mapping electron microscopy. We found that the fibrils had smoothly tapered C and N-terminal tips, which had linear axial mass distributions and were consequently parabolic in shape. An invariant feature of all tips (N and C) was an abrupt stop in lateral growth leading to a local plateau in diameter. The distance from the end of the fibril to the abrupt stop occurred at multiples of five D-periods (where D=67 nm). This implies that D-periods at the ends of fibrils are not equivalent sites for accretion, and that diameter regulation relies on surface structural features, which repeat every 5D. Mass mapping of entire fibrils at day 12 showed that, on average, the coarseness of the fibril tips was independent of fibril length, consistent with individual fibrils growing at constant tip shape. Comparison of diameters in the plateau (close to the tips) and shaft regions of the fibril showed that fibrils in day 12 tendons grow in length at constant diameter. Analysis of tendons from day 18 embryos showed that the increase in diameter at this stage of development was the result of both increases in the coarseness of the tips and continued lateral accretion of mass onto the central shafts at distances away from the growing tips. Regulated tip growth provides an attractive explanation for how cells are able to synthesise very long fibrils during the organisation of the extracellular matrix.

D-periodic assemblies of type I procollagen.

The solubility limit of purified chick type I procollagen, incubated at 37 degrees C in phosphate-buffered saline, was found to be in the range 1 to 1.5 mg/ml. At higher concentrations large aggregates formed. These comprised: (1) D-periodic assemblies; (2) narrow filaments with no apparent periodicity; and (3) segment-long-spacing-like aggregates. The D-periodic assemblies, which predominated at high concentrations, were separated from the other types of aggregate and found to be ribbon-like. Ribbons were uniform in thickness (approximately 8 nm) and up to 1 micron wide. Staining patterns showed features similar to those in native-type collagen fibrils. Immunolabelling indicated that the carboxyl-terminal propeptide domains were close to the carboxyl-terminal gap-overlap junction, and that the amino-terminal propeptide domains were folded over into the amino-terminal side of the overlap zone. Both propeptide domains appeared to be located on the surface of the assemblies. These observations show that intact propeptide domains hinder, but do not prevent, the formation of D-periodic assemblies. The presence of the propeptide domains on the surface of a growing assembly could restrict its lateral growth and limit its final thickness.

Axial structure of the heterotypic collagen fibrils of vitreous humour and cartilage.

We have compared the axial structures of negatively stained heterotypic, type II collagen-containing fibrils with computer-generated staining patterns. Theoretical negative-staining patterns were created based upon the "bulkiness" of the individual amino acid side-chains in the primary sequence and the D-staggered arrangement of the triple-helices. The theoretical staining pattern of type II collagen was compared and cross-correlated with the experimental staining pattern of both reconstituted type II collagen fibrils, and fibrils isolated from adult and foetal cartilage and vitreous humour. The isolated fibrils differ markedly in both diameter and composition. Correlations were significantly improved when a degree of theoretical hydroxylysine glycosylation was applied, showing for the first time that this type of glycosylation influences the negative-staining pattern of collagen fibrils. Increased correlations were obtained when contributions from types V/XI and IX collagen were included in the simulation model. The N-propeptide of collagen type V/XI and the NC2 domain of type IX collagen both contribute to prominent stain-excluding peaks in the gap region. With decreasing fibril diameter, an increase of these two peaks was observed. Simulations of the fibril-derived staining patterns with theoretical patterns composed of proportions of types II, V/XI and IX collagen confirmed that the thinnest fibrils (i.e. vitreous humour collagen fibrils) have the highest minor collagen content. Comparison of the staining patterns showed that the organisation of collagen molecules within vitreous humour and cartilage fibrils is identical. The simulation model for vitreous humour, however, did not account for all stain-excluding mass observed in the staining pattern; this additional mass may be accounted for by collagen-associated macromolecules.

Identification of collagen fibril fusion during vertebrate tendon morphogenesis. The process relies on unipolar fibrils and is regulated by collagen-proteoglycan interaction.

The synthesis of an extracellular matrix containing long (approximately mm in length) collagen fibrils is fundamental to the normal morphogenesis of animal tissues. In this study we have direct evidence that fibroblasts synthesise transient early fibril intermediates (approximately 1 micrometer in length) that interact by tip-to-tip fusion to generate long fibrils seen in older tissues. Examination of early collagen fibrils from tendon showed that two types of early fibrils occur: unipolar fibrils (with carboxyl (C) and amino (N) ends) and bipolar fibrils (with two N-ends). End-to-end fusion requires the C-end of a unipolar fibril. Proteoglycans coated the shafts of the fibrils but not the tips. In the absence of proteoglycans the fibrils aggregated by side-to-side interactions. Therefore, proteoglycans promote tip-to-tip fusion and inhibit side-to-side fusion. This distribution of proteoglycan along the fibril required co-assembly of collagen and proteoglycan prior to fibril assembly. The study showed that collagen fibrillogenesis is a hierarchical process that depends on the unique structure of unipolar fibrils and a novel function of proteoglycans.

Enzymic control of collagen fibril shape.

The shape of collagen fibrils growing in vitro in a cell-free enzyme/substrate system is shown to be dependent on the enzyme/substrate (E/S) ratio. Long fibrils with tapered ends were generated by exposing pCcollagen (procollagen from which the N-propeptides had been removed) to procollagen C-proteinase (which acts by cleaving the C-propeptides from the pCcollagen, converting it to insoluble fibril-forming collagen). Tip shape profiles, established quantitatively by scanning transmission electron microscopy, depended critically on the C-proteinase/pCcollagen ratio. The finest tips occurred at low ratios, the coarsest at high ratios. All fibrils had molecules oriented with amino termini closest to the pointed ends, i.e. N,N-bipolar fibrils in which molecules change orientation abruptly at one location along the fibril. Fibrils had maximal diameter at this molecular switch region. Shape asymmetric fibrils occurred at low E/S ratios, near-shape symmetric fibrils occurred at high ratios. Fibrils generated at low E/S ratios bore the closest resemblance to those formed in vivo except that the central shaft regions of fibrils formed in vitro showed no tendency to be limited to a uniform diameter.

Surface located procollagen N-propeptides on dermatosparactic collagen fibrils are not cleaved by procollagen N-proteinase and do not inhibit binding of decorin to the fibril surface.

Dermatosparaxis is a recessive disorder of animals (including man) which is caused by mutations in the gene for the enzyme procollagen N-proteinase and is characterised by extreme skin fragility. Partial loss of enzyme activity results in accumulation of pNcollagen (collagen with N-propeptides) and abnormal collagen fibrils in the fragile skin. How the N-propeptides persist in the tissue and how abnormal fibril morphology results in fragile skin is poorly understood. Using biochemical and quantitative mass mapping electron microscopy we showed that the collagen fibrils in the skin of a dermatosparactic calf contained 57% type I pNcollagen and 43% type I collagen and the fibrils were irregularly arranged in bundles and hieroglyphic in cross-section. Image analysis of the fibril cross-sections suggested that the deviation from circularity of dermatosparactic fibrils was caused by N-propeptides of pNcollagen being located at the fibril surface. Comparison of experimental and theoretical axial mass distributions of the fibrils showed that the N-propeptides were located to the overlap zone of the fibril D-period (where D=67 nm, the characteristic axial periodicity of collagen fibrils). Treatment of the dermatosparactic fibrils with N-proteinase did not remove the N-propeptides from the fibrils, although the N-propeptides were efficiently removed by trypsin and chymotrypsin. However, the N-propeptides were efficiently cleaved by the N-proteinase when the pNcollagen molecules were extracted from the fibrils. These results are consistent with close packing of N-propeptides at the fibril surface which prevented cleavage by the N-proteinase. Long-range axial mass determination along the fibril length showed gross non-uniformity with multiple mass bulges. Of note is the skin fragility in dermatosparaxis, and also the appearance of mass bulges along the fibril long axis symptomatic of the fragile skin of mice which lack decorin. Western blot analysis showed that the dermatosparactic fibrils bound elevated levels of the proteoglycan, compared with normal skin fibrils. The results showed that N-propeptides can distort the morphology of fibrils, that they do not inhibit binding of gap-associated macromolecules (such as decorin) and that the normal mechanical properties of skin are strongly dependent on the close association of near-cylindrical fibrils, thereby enabling maximal fibril-fibril interactions.

Growth of sea cucumber collagen fibrils occurs at the tips and centers in a coordinated manner.

Collagen fibrils are the principle source of mechanical strength in the mutable dermis of the sea cucumber Cucumaria frondosa. To obtain information about the mechanism by which collagen molecules self-assemble into fibrils, we have isolated single intact fibrils with lengths in the range 14-444 microm. These fibrils have been studied by scanning transmission electron microscopy, yielding data that show how cross-sectional mass, and hence the number of molecules in the cross-section, depend on axial location. In an individual fibril, the two ends always display similar mass distributions. The two tips of each fibril must therefore maintain identity in shape and size throughout growth. The linear relationship between cross-sectional mass and distance from the adjacent end shows that a growing tip is (like the tip of a vertebrate collagen fibril) paraboloidal in shape. Comparison of data from many different fibrils, over a wide range of lengths, however, revealed that the paraboloidal tip becomes blunter as the fibril grows in length. In contrast to vertebrate fibrils, those from C. frondosa do not have a central shaft region of constant cross-sectional mass. Rather, the cross-sectional mass increases to a maximum in the center of each fibril. The maximum cross-sectional mass of the fibrils increases exponentially with increasing fibril length. The centrosymmetry, the paraboloidal shape of the tips, and the hyperbolic increase in maximum cross-sectional mass with fibril length, is evidence for a co-ordinated regulation of length and diameter, which differs from the kind of regulation that gives rise to collagen fibrils in vertebrates (chickens and mice).

Pleomorphism in type I collagen fibrils produced by persistence of the procollagen N-propeptide.

The assembly of type I collagen and type I pN-collagen was studied in vitro using a system for generating these molecules enzymatically from their immediate biosynthetic precursors. Collagen generated by C-proteinase digestion of pC-collagen formed D-periodically banded fibrils that were essentially cylindrical (i.e. circular in cross-section). In contrast, pN-collagen generated by C-proteinase digestion of procollagen formed thin, sheet-like structures that were axially D-periodic in longitudinal section, of varying lateral widths (up to several microns) and uniform in thickness (approximately 8 nm). Mixtures of collagen and pN-collagen assembled to form a variety of pleomorphic fibrils. With increasing pN-collagen content, fibril cross-sections were progressively distorted from circular to lobulated to thin and branched structures. Some of these structures were similar to fibrils observed in certain heritable disorders of connective tissue where N-terminal procollagen processing is defective. The observations are considered in terms of the hypothesis that the N-propeptides are preferentially located on the surface of a growing assembly. The implications for normal diameter control of collagen fibrils in vivo are discussed.

Scanning transmission electron microscopy mass analysis of fibrillin-containing microfibrils from foetal elastic tissues.

We have applied scanning transmission electron microscopy to intact native fibrillin-containing microfibrils isolated from foetal bovine elastic tissues in order to derive new insights into microfibril organisation. This technique provides quantitative data on the mass per unit length and axial mass distribution of unstained, unshadowed macromolecules. Scanning transmission electron microscopy of microfibrils from aorta, skin and nuchal ligament revealed that the beads corresponded to peaks of mass and the interbead regions to troughs of mass. These major features of axial mass distribution were characteristic of all microfibrils examined. Tissue-specific and age-dependent variations in mass were identified in microfibrils that were structurally comparable by rotary shadowing electron microscopy. Increased microfibril mass correlated with increasing gestational age. The additional mass was associated predominantly at, or close to, the bead. Some microfibril populations exhibited pronounced assymetry in their axial mass distribution. These data indicate that intact native microfibrillar assemblies from developing elastic tissues are heterogeneous in composition. Loss of mass following chondroitinase ABC or AC lyase treatment confirmed the presence of chondroitin sulphate in nuchal ligament microfibrillar assemblies.

Tip-mediated fusion involving unipolar collagen fibrils accounts for rapid fibril elongation, the occurrence of fibrillar branched networks in skin and the paucity of collagen fibril ends in vertebrates.

Collagen fibrils are the principal source of mechanical strength of connective tissues such as tendon, skin, cornea, cartilage and bone. The ability of these tissues to withstand tensile forces is directly attributable to the length and diameter of the fibrils, and to interactions between individual fibrils. Although electron microscopy studies have provided information on fibril diameters, little is known about the length of fibrils in tissue and how fibrils interact with each other. The question of fibril length has been difficult to address because fibril ends are rarely observed in cross-sections of tissue. The paucity of fibril ends, or tips, has led to controversy about how long individual fibrils might be and how the fibrils grow in length and diameter. This review describes recent discoveries that are relevant to these questions. We now know that vertebrate collagen fibrils are synthesised as short (1-3 microm) early fibrils that fuse end-to-end in young tissues to generate very long fibrils. The diameter of the final fibril is determined by the diameter of the collagen early fibrils. During a late stage of tissue assembly fibril tips fuse to fibril shafts to generate branched networks. Of direct relevance to fibril fusion is the fact that collagen fibrils can be unipolar or bipolar, depending on the orientation of collagen molecules in the fibril. Fusion relies on: (1) specific molecular interactions at the carboxyl terminal ends of unipolar collagen fibrils; and (2) the insulator function of small proteoglycans to shield the surfaces of fibrils from inappropriate fusion reactions. The fusion of tips to shafts to produce branched networks of collagen fibrils is an elegant mechanism to increase the mechanical strength of tissues and provides an explanation for the paucity of fibril tips in older tissue.

Experimental coonhound paralysis: animal model of Guillain-Barré syndrome.

Coonhound paralysis (CHP), a polyradiculoneuritis of dogs that resembles the human Guillain-Barré syndrome, was experimentally reproduced by inoculating a dog with raccoon saliva. The test animal was a coonhound that had previously sustained two naturally occurring attacks of CHP. Success in inducing the disease strengthened the notion that raccoon saliva contains the etiologic factor for CHP and that only specifically susceptible dogs are at risk of developing CHP when exposed to this factor.

STEM/TEM studies of collagen fibril assembly.

Quantitative scanning transmission electron microscopy (STEM), implemented on a conventional transmission electron microscope with STEM-attachment, has been a primary tool in our laboratory for the quantitative analysis of collagen fibril assembly in vivo and in vitro. Using this technique, a precise measurement of mass per unit length can be made at regular intervals along a fibril to generate an axial mass distribution (AMD). This in turn allows the number of collagen molecules to be calculated for every transverse section of the fibril along its entire length. All fibrils show a near-linear AMD in their tip regions. Only fibrils formed in tissue environments, however, show a characteristic abrupt change in mass slope along their tips. It appears that this tip growth characteristic is common to fibrils from evolutionarily diverse systems including vertebrate tendon and the mutable tissues of the echinoderms. Computer models of collagen fibril assembly have now been developed based on interpretation of the STEM data. Two alternative models have so far been generated for fibril growth by accretion; one is based on diffusion limited aggregation (DLA) and the other based on an interface-limited growth mechanism. Inter-fibrillar fusion can also contribute to the growth of fibrils in vertebrate tissues and STEM data indicates the presence of a tight regulation in this process. These models are fundamental for the hypotheses regarding how cells synthesise and spatially organise an extracellular matrix (ECM), rich in collagen fibrils.

Collagen fibril organisation in mammalian vitreous by freeze etch/rotary shadowing electron microscopy.

Mammalian vitreous gel contains two major network-forming polymeric systems: long, thin fibrils comprising predominantly type II collagen and a meshwork of hyaluronan. The gel structure is maintained primarily by the collagen component, but little is known about the mechanisms of spacing of the collagen fibrils and of interactions between fibrils to form a stable network. In this study we have applied the technique of freeze etching/rotary shadowing electron microscopy in order to reveal the fibrillar network in central, cortical and basal vitreous and to understand the structural relationship between the collagen fibrils. The fibrils were arranged side by side in narrow bundles that frequently branched to link one bundle to another. Only a minor part of the fibrillar network consisted of segments that had a diameter of a single fibril (16.4nm mean diameter). In addition, three morphologically distinct filamentous structures were observed that appeared to form links within the collagen fibrillar network: short, single interlinking filaments of 7.0nm mean diameter, network-forming filaments of 6.7nm mean diameter, and longer filaments of 8.2nm mean diameter. All three types of filamentous structure were removed by digestion of the vitreous gels with Streptomyces hyaluronan lyase prior to freeze etching, indicating that these structures contain or are stabilised by hyaluronan. These filamentous structures may contribute to the structural stability of the vitreous gel.

Body temperature and behaviour of mares during the last two weeks of pregnancy.

Average daily core body temperature and behavioural patterns of pregnant mares were studied, in search of definitive signs of parturition within 24 h of the event. Nineteen pony mares were sampled twice daily for core body temperature. A significant temperature drop, averaging 0.1 degrees C (0.2 degrees F) was observed during the day prior to parturition. Between 18.00 h and 06.00 h, during the two weeks before parturition, Thoroughbred and Standardbred mares (n = 52) spent an average 66.8 per cent of their time standing, 27.0 per cent eating, 4.9 per cent lying in sternal recumbency, 1.0 per cent lying in lateral recumbency, and 0.3 per cent walking. On the night before parturition, mares spent significantly less time lying in sternal recumbency than on previous nights and on the night of parturition all behaviour patterns except eating were significantly different from the nights of the two weeks before parturition. There was an increase in walking (5.3 per cent), lying in sternal recumbency (8 per cent) and lying in lateral recumbency (5.3 per cent) whereas standing (53.3 per cent) was decreased. In 58 observed pregnancies, 54 mares (97 per cent) foaled in a recumbent position and 50 mares (86 per cent) foaled between 18.00 h and 06.00 h.