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.     

Modeling the effect of collagen fibril alignment on ligament mechanical behavior

Ligament mechanical behavior is primarily regulated by fibrous networks of type I collagen. Although these fibrous networks are typically highly aligned, healthy and injured ligament can also exhibit disorganized collagen architecture. The objective of this study was to determine whether variations in the collagen fibril network between neighboring ligaments can predict observed differences in mechanical behavior. Ligament specimens from two regions of bovine fetlock joints, which either exhibited highly aligned or disorganized collagen fibril networks, were mechanically tested in uniaxial tension. Confocal microscopy and FiberFit software were used to quantify the collagen fibril dispersion and mean fibril orientation in the mechanically tested specimens. These two structural parameters served as inputs into an established hyperelastic constitutive model that accounts for a continuous distribution of planar fibril orientations. The ability of the model to predict differences in the mechanical behavior between neighboring ligaments was tested by (1) curve fitting the model parameters to the stress response of the ligament with highly aligned fibrils and then (2) using this model to predict the stress response of the ligament with disorganized fibrils by only changing the parameter values for fibril dispersion and mean fibril orientation. This study found that when using parameter values for fibril dispersion and mean fibril orientation based on confocal imaging data, the model strongly predicted the average stress response of ligaments with disorganized fibrils (\(R^{2}=0.97\)); however, the model only successfully predicted the individual stress response of ligaments with disorganized fibrils in half the specimens tested. Model predictions became worse when parameters for fibril dispersion and mean fibril orientation were not based on confocal imaging data. These findings emphasize the importance of collagen fibril alignment in ligament mechanics and help advance a mechanistic understanding of fibrillar networks in healthy and injured ligament.     

Collagen fibril formation in a wound healing model

Control of tissue composition and organization will be a key feature in the development of successful products through tissue engineering. However, the mechanism of collagen fibril formation, growth, and organization is not yet fully understood. In this study we have examined collagen fibril formation in a wound healing model in which the newly formed fibrils were kept distinct from preexisting tissue through use of a porous tubular biomaterial implant. Samples were examined after 4, 6, 14, and 28 days by light microscopy, in situ hybridization, and immunofluorescence microscopy. These showed a normal wound healing response, with significant collagen formation at 14 and 28 days. Individual collagen fibrils were isolated from these samples by gentle extraction in a gentamicin-containing buffer which allowed extraction of a large proportion of intact fibrils. Examination by transmission electron microscopy showed that approximately 80% of the intact fibrils showed a single polarity reversal, with both ends of each fibril comprising collagen amino-terminal domains; the remaining fibrils had no polarity reversal. All fibrils had similar diameters at both time points. Immunoelectron microscopy showed that all labeled fibrils contained both type I and III collagens. These data indicate that this wound healing model provides a system in which collagen fibril formation can be readily followed.     

Murine model of the Ehlers-Danlos syndrome. col5a1 haploinsufficiency disrupts collagen fibril assembly at multiple stages

The most commonly identified mutations causing Ehlers-Danlos syndrome (EDS) classic type result in haploinsufficiency of proalpha1(V) chains of type V collagen, a quantitatively minor collagen that co-assembles with type I collagen as heterotypic fibrils. To determine the role(s) of type I/V collagen interactions in fibrillogenesis and elucidate the mechanism whereby half-reduction of type V collagen causes abnormal connective tissue biogenesis observed in EDS, we analyzed mice heterozygous for a targeted inactivating mutation in col5a1 that caused 50% reduction in col5a1 mRNA and collagen V. Comparable with EDS patients, they had decreased aortic stiffness and tensile strength and hyperextensible skin with decreased tensile strength of both normal and wounded skin. In dermis, 50% fewer fibrils were assembled with two subpopulations: relatively normal fibrils with periodic immunoreactivity for collagen V where type I/V interactions regulate nucleation of fibril assembly and abnormal fibrils, lacking collagen V, generated by unregulated sequestration of type I collagen. The presence of the aberrant fibril subpopulation disrupts the normal linear and lateral growth mediated by fibril fusion. Therefore, abnormal fibril nucleation and dysfunctional fibril growth with potential disruption of cell-directed fibril organization leads to the connective tissue dysfunction associated with EDS.     

D-periodic distribution of collagen type IX along cartilage fibrils

It has recently become apparent that collagen fibrils may be composed of more than one kind of macromolecule. To explore this possibility, we developed a procedure to purify fibril fragments from 17-d embryonic chicken sternal cartilage. The fibril population obtained shows, after negative staining, a uniformity in the banding pattern and diameter similar to the fibrils in situ. Pepsin digestion of this fibril preparation releases collagen types II, IX, and XI in the proportion of 8:1:1. Rotary shadowing of the fibrils reveals a d-periodic distribution of 35-40-nm long projections, each capped with a globular domain, which resemble in form and dimensions the aminoterminal globular and collagenous domains, NC4 and COL3, of type IX collagen. The monoclonal antibody (4D6) specific for an epitope close to the amino terminal of the COL3 domain of type IX collagen bound to these projections, thus confirming their identity. Type IX collagen is therefore distributed in a regular d-periodic arrangement along cartilage fibrils, with the chondroitin sulfate chain of type IX collagen in intimate contact with the fibril.     

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.     

Copolymerization of normal type I collagen with three mutated type I collagens containing substitutions of cysteine at different glycine positions in the alpha 1 (I) chain.

Previous observations with type I collagen from a proband with lethal osteogenesis imperfecta demonstrated that type I collagen containing a substitution of cysteine for glycine alpha 1-748 copolymerized with normal type I collagen (Kadler, K. E., Torre-Blanco, A., Adachi, E., Vogel, B. E., Hojima, Y., and Prockop, D. J. (1991) Biochemistry 30, 5081-5088). Here, three preparations containing normal type I procollagen and type I procollagen with a substitution of cysteine for glycine alpha 1-175, glycine alpha 1-691, or glycine alpha 1-988 were purified from cultured skin fibroblasts from probands with osteogenesis imperfecta. The procollagens were then used as substrates in a system for assaying the self-assembly of type I collagen into fibrils. The cysteine-substituted collagens in all three preparations were incorporated into fibrils. The cysteine alpha 1-175 and cysteine alpha 1-691 collagens were shown to increase the lag time and decrease the propagation rate constant for fibril assembly. All three preparations containing cysteine-substituted collagens formed fibrils with diameters that were two to four times the diameter of fibrils formed under the same conditions by normal type I collagen. Also, the three preparations containing cysteine substituted collagens had higher solubilities than normal type I collagen. The results, therefore, demonstrated that the three cysteine-substituted collagens copolymerized with normal type I collagen. The effects of the mutated collagens on fibril assembly can be understood in terms of a recently proposed model of fibril growth from symmetrical tips by assuming that the mutated monomers partially inhibit tip growth but not lateral growth of the fibrils. Of special interest was the observation that the Cys alpha 1-175 collagen from a proband with a non-lethal variant of osteogenesis imperfecta had quantitatively less effect on several parameters of fibril assembly at 37 degrees C than cysteine-substituted collagens from three probands with lethal variants of the disease.     

Reduction of type V collagen using a dominant-negative strategy alters the regulation of fibrillogenesis and results in the loss of corneal- specific fibril morphology

A number of factors have been implicated in the regulation of tissue- specific collagen fibril diameter. Previous data suggest that assembly of heterotypic fibrils composed of two different fibrillar collagens represents a general mechanism regulating fibril diameter. Specifically, we hypothesize that type V collagen is required for the assembly of the small diameter fibrils observed in the cornea. To test this, we used a dominant-negative retroviral strategy to decrease the levels of type V collagen secreted by chicken corneal fibroblasts. The chicken alpha 1(V) collagen gene was cloned, and retroviral vectors that expressed a polycistronic mRNA encoding a truncated alpha 1(V) minigene and the reporter gene LacZ were constructed. The efficiency of viral infection was 30-40%, as determined by assaying beta- galactosidase activity. To assess the expression from the recombinant provirus, Northern analysis was performed and indicated that infected fibroblasts expressed high steady-state levels of retroviral mRNA. Infected cells synthesized the truncated alpha 1(V) protein, and this was detectable only intracellularly, in a distribution that colocalized with lysosomes. To assess endogenous alpha 1(V) protein levels, infected cell cultures were assayed, and these consistently demonstrated reductions relative to control virus-infected or uninfected cultures. Analyses of corneal fibril morphology demonstrated that the reduction in type V collagen resulted in the assembly of large- diameter fibrils with a broad size distribution, characteristics similar to fibrils produced in connective tissues with low type V concentrations. Immunoelectron microscopy demonstrated the amino- terminal domain of type V collagen was associated with the small- diameter fibrils, but not the large fibrils. These data indicate that type V collagen levels regulate corneal fibril diameter and that the reduction of type V collagen is sufficient to alter fibril assembly so that abnormally large-diameter fibrils are deposited into the matrix.     

Interaction of bilirubin with reconstituted collagen fibrils.

1. Interaction of bilirubin with collagen fibrils was explored in a two-phase system where collagen was present as an opaque rigid gel composed of striated fibrils, and bilirubin as an aqueous solution. 2. The Ka value of the binding of bilirubin to collagen fibrils is 5.4 X 10(3)M-1. The interaction of bilirubin with collagen fibrils depends on temperature. Below 5 degrees C, the binding is greatly diminished and denaturation of collagen fibril aggregates at 52--53 degrees C into a dissolution state abolishes binding of bilirubin. 3. Salicylate and sulphanilamide do not affect the binding of bilirubin to reconstituted collagen fibrils. 4. Serum albumin (40--80mM), known to reverse the binding of bilirubin to lipids, dissociates only 50% of the bilirubin bound to collagen fibrils. This suggests that sites located on collagen participate in some tight binding of bilirubin and the corresponding binding sites on albumin do not compete with them. 5. Urea (4M) abolishes more than 70% of the binding of bilirubin to collagen. Urea and thermal denaturation studies indicate the importance of conformation and organization of collagen fibrillar aggregates for the binding of bilirubin.     

Particular structure of the anterior third of the human true vocal cord.

The histological aspects of the true vocal cord mucosa change in the anterior third compared with the posterior two thirds. The anterior third is characterized by an epithelium where the ridges, marked in the posterior two thirds, are very slight or even absent. The underlying basement membrane, which is thin in the posterior two thirds, here appears particularly thick. At the ultrastructural level in this area, beneath a normally thickened basal lamina, a thick layer of finely granulated electron-dense material, interspersed with thin and randomly scattered collagen fibrils and proteoglycan filaments, is detectable. Beneath this thickened basement membrane, a layer of small undulated collagen fibril bundles with very numerous interspersed oxytalan fibres is found. The collagen fibrils, small in diameter (30-40 nm), seem to continue with the collagen fibrils of the basement membrane. In this layer numerous blood vessels with a very thick, delaminated basement membrane are also observed. The underlying area is characterized by the vocal cord ligament, composed by large compact collagen fibril bundles with interspersed elastic fibres. The particular features of the thick basement membrane, the thick-walled and delaminated vessels and the modular distribution of the elastic system together may well form the basic structure enabling the functional integration of the vocal ligament into the overlying mucosa and the underlying vocal muscle.     

Structure of type I and type III heterotypic collagen fibrils: an X-ray diffraction study

The molecular packing arrangement within collagen fibrils has a significant effect on the tensile properties of tissues. To date, most studies have focused on homotypic fibrils composed of type I collagen. This study investigates the packing of type I/III collagen molecules in heterotypic fibrils of colonic submucosa using a combination of X-ray diffraction data, molecular model building, and simulated X-ray diffraction fibre diagrams. A model comprising a 70-nm-diameter D- (approximately 65 nm) axial periodic structure containing type I and type III collagen chains was constructed from amino acid scattering factors organised in a liquid-like lateral packing arrangement simulated using a classical Lennard-Jones potential. The models that gave the most accurate correspondence with diffraction data revealed that the structure of the fibril involves liquid-like lateral packing combined with a constant helical inclination angle for molecules throughout the fibril. Combinations of type I:type III scattering factors in a ratio of 4:1 gave a reasonable correspondence with the meridional diffraction series. The attenuation of the meridional intensities may be explained by a blurring of the electron density profile of the D period caused by nonspecific or random interactions between collagen types I and III in the heterotypic fibril.     

Collagen type I and type V are present in the same fibril in the avian corneal stroma

The distribution, supramolecular form, and arrangement of collagen types I and V in the chicken embryo corneal stroma were studied using electron microscopy, collagen type-specific monoclonal antibodies, and a preembedding immunogold method. Double-label immunoelectron microscopy with colloidal gold-tagged monoclonal antibodies was used to simultaneously localize collagen type I and type V within the chick corneal stroma. The results definitively demonstrate, for the first time, that both collagens are codistributed within the same fibril. Type I collagen was localized to striated fibrils throughout the corneal stroma homogeneously. Type V collagen could be localized only after pretreatment of the tissue to partially disrupt collagen fibril structure. After such pretreatments the type V collagen was found in regions where fibrils were partially dissociated and not in regions where fibril structure was intact. When pretreated tissues were double labeled with antibodies against types I and V collagen coupled to different size gold particles, the two collagens colocalized in areas where fibril structure was partially disrupted. Antibodies against type IV collagen were used as a control and were nonreactive with fibrils. These results indicate that collagen types I and V are assembled together within single fibrils in the corneal stroma such that the interaction of these collagen types within heterotypic fibrils masks the epitopes on the type V collagen molecule. One consequence of the formation of such heterotypic fibrils may be the regulation of corneal fibril diameter, a condition essential for corneal transparency.     

Ehlers Danlos syndrome type VIIB. Incomplete cleavage of abnormal type I procollagen by N-proteinase in vitro results in the formation of copolymers of collagen and partially cleaved pNcollagen that are near circular in cross-section.

We have shown that a child with Ehlers Danlos syndrome (EDS) type VII has a G to A transition at the first nucleotide of intron 6 in one of her COL1A2 alleles. Half of the cDNA clones prepared from the proband's pro alpha 2(I) mRNA lacked exon 6. The type I procollagen secreted by the proband's dermal fibroblasts in culture was purified, and collagen fibrils were generated in vitro by cleavage of the procollagen with the procollagen N- and C-proteinases. Incubation of the procollagen with N-proteinase resulted in a 1:1 mixture of pCcollagen and uncleaved procollagen. Incubation of this mixture with C-proteinase generated collagen and abnormal pNcollagen (pNcollagen-ex6) that readily copolymerized into fibrils. By electron microscopy these fibrils resembled the hieroglyphic fibrils seen in the N-proteinase-deficient skin of dermatosparactic animals and humans and were distinct from the near circular cross-section fibrils seen in the tissues of individuals with EDS type VII. Further incubation of the hieroglyphic fibrils with N-proteinase resulted in partial cleavage of the pNcollagen-ex6 in which the abnormal pN alpha 2(I) chains remained intact. These fibrils were not hieroglyphic but were near circular in cross-section. Fibrils formed from collagen and pNcollagen-ex6 that had been partially cleaved with elevated amounts of N-proteinase prior to fibril formation were also near circular in cross-section. The results are consistent with a model of collagen fibril formation in which the intact N-propeptides are located exclusively at the surface of the hieroglyphic fibrils. Partial cleavage of the pNcollagen-ex6 by N-proteinase allows the N-propeptides to be incorporated within the body of the fibrils. The model provides an explanation for the morphology and molecular composition of collagen fibrils in the tissues of patients with EDS type VII.     

Type V collagen controls the initiation of collagen fibril assembly

Vertebrate collagen fibrils are heterotypically composed of a quantitatively major and minor fibril collagen. In non-cartilaginous tissues, type I collagen accounts for the majority of the collagen mass, and collagen type V, the functions of which are poorly understood, is a minor component. Type V collagen has been implicated in the regulation of fibril diameter, and we reported recently preliminary evidence that type V collagen is required for collagen fibril nucleation (Wenstrup, R. J., Florer, J. B., Cole, W. G., Willing, M. C., and Birk, D. E. (2004) J. Cell. Biochem. 92, 113-124). The purpose of this study was to define the roles of type V collagen in the regulation of collagen fibrillogenesis and matrix assembly. Mouse embryos completely deficient in pro-alpha1(V) chains were created by homologous recombination. The col5a1-/- animals die in early embryogenesis, at approximately embryonic day 10. The type V collagen-deficient mice demonstrate a virtual lack of collagen fibril formation. In contrast, the col5a1+/- animals are viable. The reduced type V collagen content is associated with a 50% reduction in fibril number and dermal collagen content. In addition, relatively normal, cylindrical fibrils are assembled with a second population of large, structurally abnormal collagen fibrils. The structural properties of the abnormal matrix are decreased relative to the wild type control animals. These data indicate a central role for the evolutionary, ancient type V collagen in the regulation of fibrillogenesis. The complete dependence of fibril formation on type V collagen is indicative of the critical role of the latter in early fibril initiation. In addition, this fibril collagen is important in the determination of fibril structure and matrix organization.     

Cartilage Fibrils of Mammals are Biochemically Heterogeneous: Differential Distribution of Decorin and Collagen IX

Cartilage fibrils contain collagen II as the major constituent, but the presence of additional components, minor collagens, and noncollagenous glycoproteins is thought to be crucial for modulating several fibril properties. We have examined the distribution of two fibril constituents—decorin and collagen IX—in samples of fibril fragments obtained after bovine cartilage homogenization. Decorin was preferentially associated with a population of thicker fibril fragments from adult articular cartilage, but was not present on the thinnest fibrils. The binding was specific for the gap regions of the fibrils, and depended on the decorin core protein. Collagen IX, by contrast, predominated in the population with the thinnest fibrils, and was scarce on wider fibrils. Double-labeling experiments demonstrated the coexistence of decorin and collagen IX in some fibrils of intermediate diameter, although most fibril fragments from adult cartilage were strongly positive for one component and lacked the other. Fibril fragments from fetal epiphyseal cartilage showed a different pattern, with decorin and collagen IX frequently colocalized on fragments of intermediate and large diameters. Hence, the presence of collagen IX was not exclusive for fibrils of small diameter. These results establish that articular cartilage fibrils are biochemically heterogeneous. Different populations of fibrils share collagen II, but have distinct compositions with respect to macromolecules defining their surface properties.     

Structure of corneal scar tissue: an X-ray diffraction study.

Full-thickness corneal wounds (2 mm diameter) were produced in rabbits at the Schepens Eye Research Institute, Boston. These wounds were allowed to heal for periods ranging from 3 weeks to 21 months. The scar tissue was examined using low- and wide-angle x-ray diffraction from which average values were calculated for 1) the center-to-center collagen fibril spacing, 2) the fibril diameter, 3) the collagen axial periodicity D, and 4) the intermolecular spacing within the collagen fibrils. Selected samples were processed for transmission electron microscopy. The results showed that the average spacing between collagen fibrils within the healing tissue remained slightly elevated after 21 months and there was a small increase in the fibril diameter. The collagen D-periodicity was unchanged. There was a significant drop in the intermolecular spacing in the scar tissues up to 6 weeks, but thereafter the spacing returned to normal. The first-order equatorial reflection in the low-angle pattern was visible after 3 weeks and became sharper and more intense with time, suggesting that, as healing progressed, the number of nearest neighbor fibrils increased and the distribution of nearest neighbor spacings reduced. This corresponded to the fibrils becoming more ordered although, even after 21 months, normal packing was not achieved. Ultrastructural changes in collagen fibril density measured from electron micrographs were consistent with the increased order of fibril packing measured by x-ray diffraction. The results suggest that collagen molecules have a normal axial and lateral arrangement within the fibrils of scar tissue. The gradual reduction in the spread of interfibrillar spacings may be related to the progressive decrease in the light scattered from the tissue as the wound heals.     

SLRP interaction can protect collagen fibrils from cleavage by collagenases

Decorin, fibromodulin and lumican are small leucine-rich repeat proteoglycans (SLRPs) which interact with the surface of collagen fibrils. Together with other molecules they form a coat on the fibril surface which could impede the access to collagenolytic proteinases. To address this hypothesis, fibrils of type I or type II collagen were formed in vitro and treated with either collagenase-1 (MMP1) or collagenase-3 (MMP13). The fibrils were either treated directly or following incubation in the presence of the recombinant SLRPs. The susceptibility of the uncoated and SLRP-coated fibrils to collagenase cleavage was assessed by SDS/PAGE. Interaction with either recombinant decorin, fibromodulin or lumican results in decreased collagenase cleavage of both fibril types. Thus SLRP interaction can help protect collagen fibrils from cleavage by collagenases.     

THE STRUCTURE OF THE ZONULA OCCLUDENS : A Single Fibril Model Based on Freeze-Fracture

Replicas of freeze-fractured toad urinary bladder and gallbladder were analysed in an attempt to determine the fracturing properties and structure of the zonula occludens (tight junction). Chalcroft and Bullivant have proposed that the junction has a double set of fibrils with one set associated with each of the adjacent cell membranes. However, the fracturing pattern that is observed might also result from only a single set of fibrils which is shared by the adjacent membranes if fracturing occurred around either side of the fibrils. These two models predict quite different structures at regions of the junction where tranl sitions are made between face A and face B. The relative heights of face A and face B and the shape of the transition from face A to face B do not agree with that expected according to the two fibril model but agree exactly with that expected if only a single set of fibrils existed. Further evidence for the single fibril model is derived from fractures of the mucosa membrane which cross the junction to the membrane of the adjacent cell without deflection. Such fractures reveal a single ridge which appears to be identical to the juxtaluminal fibril of the junction. In addition, small ridges are occasionally found in place of the grooves on face B which, although not consistent with the double fibril model, is expected if the single fibril model were correct. Although alternative explanations might account for these observations, we believe that the simplest and most consistent explanation is that the zonula occludens fractures as would be expected of a single set of fibrils shared by adjacent cells.     

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.