The twisted collagen network of the box-fish scutes

The present article describes the three-dimensional arrangement of collagen fibrils in dermal plates of different species of Ostraciidae. These dermal plates or 'scutes' are transformed scales, which have a polygonal shape and form a rigid tiling. They are natural composites, associating a fibrous network with a mineral deposit lying at two different levels of the scute, the 'ceiling' and the 'floor', plus a set of similarly mineralized walls joining the two levels. The three-dimensional structure of the collagen network can be compared to that of 'plywood': fibrils align parallel within superposed layers of uniform thickness, and their direction changes from layer to layer. In the dermal plate, two types of plywood have been evidenced: (1) one lying between the two mineralized plates, where the orientation of fibrils rotates continuously, and (2) one under the lower plate, with thick layers of fibrils, each showing a constant orientation, but abrupt angular changes are observed at the transition from one layer to the following one. In oblique sections, both types of plywood reveal large series of arced patterns, testifying to a twisted arrangement of collagen fibrils, analogous to the arrangement of molecules or polymers in cholesteric liquid crystals. The network is reinforced by some collagen fibrils running unidirectionally and almost normally to the lamellate structure. Moreover in the overall organization of the scute, these plywood systems form a set of nested boxes. This original architecture is compared to the arrangement of the collagenous network previously described in most fish scales and in other extracellular matrices.     

Development of the basement membrane and formation of collagen fibrils below the placodes in the head of anuran larvae

The development of the basement membrane and collagen fibrils below placodes, including the corneal region of the ectoderm, lens epithelium, nasal plate, and auditory vesicle in anuran larvae was observed by transmission electron microscopy and compared with that in nonplacodal regions such as the epidermis, neural tube, and optic vesicle. In the corneal region the lamina densa becomes thick concomitantly with the development of the connecting apparatuses such as hemidesmosomes and anchoring fibrils. The collagen fibrils increase in number and form a multilayered structure, showing similar morphology to the connective tissues below the epidermis. These two areas, i.e., the corneal region and epidermis, possess much collagenous connective tissue below them. On the other hand, the neural tube and ophthalmic vesicle that originated from the neural tube each have a thin lamina densa and a small number of underlying collagen fibrils. The lamina densa does not thicken and the number of collagen fibrils do not significantly increase during development. These two areas possess little extracellular matrix. The nasal plate and auditory vesicle show intermediate characteristics between the epidermis-type and the neural tube-type areas. In these areas, the lamina densa becomes thick and hemidesmosomes and anchoring fibrils develop. The number of collagen fibrils increases during development, but does not show an orderly arrangement; rather, they are randomly distributed. It is thought that the difference in the arrangement of collagen fibrils in different tissues is due to differences in the extracellular matrix around the collagen fibrils. Placodal epithelia have the same origin as epidermis, but during development their morphological characteristics differ and they are not associated with the pattern of extracellular matrix with characteristics of epidermal and corneal multilayered collagen fibril areas.     

Characterization of the cytoplasmic fibrils of Treponema refringens (Nichols)

The cytoplasmic fibrils of Treponema refringens were studied in situ by electron microscopy of thin sectioned and negatively stained cells. From 5 to 21 parallel fibrils ran through the cell in a band adjacent to the inner side of the cytoplasmic membrane, on the inner sides of the curves of the spirochete. The nuclear areas of cells were adjacent to the fibrils. Cross sections of fibrils isolated from cells which had been lysed were polygonal and not uniformly electron dense. Polyacrylamide gel electrophoresis of partially purified fibril preparations indicated their main component to be a protein with a molecular weight of 97,000. Fibrils were solubilized by 1% trypsin, 1% pronase, 6 M urea, 1 N HCl, 0.005 N NaOH or 1.3% sodium dodecyl sulfate. By electron microscopy of negatively stained isolated fibrils, each fibril was found to be a complex arrangement of strands rather than a single tubule.Abbreviations CM Cytoplasmic membrane - PTA Phosphotungstic acid - UOx Uranyl oxalate - SDS sodium dodecyl sulfate This communication is Journal Acticle No. 7644 from the Michigan Agricultural Experiment Station     

Distinguishing the cross-beta spine arrangements in amyloid fibrils using FRET analysis

The recently published microcrystal structures of amyloid fibrils from small peptides greatly enhanced our understanding of the atomic-level structure of the amyloid fibril. However, only a few amyloid fibrils can form microcrystals. The dansyl-tryptophan fluorescence resonance energy transfer (FRET) pair was shown to be able to detect the inter-peptide arrangement of the Transthyretin (105-115) amyloid fibril. In this study, we combined the known microcrystal structures with the corresponding FRET efficiencies to build a model for amyloid fibril structure classification. We found that fibrils with an antiparallel structural arrangement gave the largest FRET signal, those with a parallel arrangement gave the lowest FRET signal, and those with a mixed arrangement gave a moderate FRET signal. This confirms that the amyloid fibril structure patterns can be classified based on the FRET efficiency.     

Analysis of the crystal arrangement in collagen fibrils of mineralizing turkey tibia tendon

Summary Mineralized pieces of tendons from the tibio-tarsus of turkeys were (i) shock-frozen, freeze-dried, embedded and cut without staining, or (ii) fixed, embedded and stained after sectioning. Micrographs were taken with an electron microscope on longitudinally cut sections. The center-to-center distances of neighboring apatitic needles within collagen fibrils were measured. For shock-frozen and freeze-dried specimens, the average of these distances is 4.7 nm and the most frequent value 4.2 nm; for fixed and stained specimens, 3.8 nm and 3.6 nm, respectively. Laser diffraction of the electron micrographs showed a dumbbell-like intensity pattern (two diffuse maxima of intensity on the equator, one on each side of the central spot), giving an average distance of about 6 nm. This value represents the upper range of the direct measurements. The measurements demonstrate that the arrangement of the collagen microfibrils is mainly preserved during mineralization. However, using laser diffraction, distances of 9–11 nm were also observed. Such large distances can also be demonstrated by X-ray diffraction on collagen fibrils stained under special conditions. This may indicate that special conditions of apatitic mineralization or staining may alter the arrangement of the microfibrils.The authors thank the Deutsche Forschungsgemeinschaft for financial support     

X-ray diffraction studies of the corneal stroma.

A low-angle diffraction pattern has been obtained from corneal stroma. This pattern arises both from the arrangement of the collagen fibrils and from the packing of the tropocollagen molecules along the axes of the fibrils. The spacing arising from the packing of the fibrils increases homogeneously on swelling although the tissue as a whole swells only radially referred to the intact eye. The necessary rearrangement of the fibrils for this type of swelling to occur might result in the formation of regions devoid of collagen fibrils and the water not in the lattice of collagen fibrils could be synonymous with the lakes postulated by Benedek (1971) to explain the loss of transparency on swelling.The spacings due to the packing of the tropocollagen molecules are unusual in that, although they index as the third and fifth orders of the well-known 66 nm repeat, the first order of this spacing is absent. Calculation of the Patterson function for corneal collagen leads to peaks in electron density separated by distances of 0.38 and 0.24 of the repeat distance.     

Interaction of collagen with hydrophobic protein granules in the egg capsule of the dogfish scyliorhinus canicula

The egg capsule of the dogfish is a composite material containing collagenous fibrils and 2 mum spherical hydrophobic protein granules. The latter appear to owe much of their hydrophobicity to an exceptionally high tyrosine content (approximately 20% of total amino acid residues). The hydrophobic component appears to form as an emulsion in the secretory granules of the D and E zone gland cells of the nidamental gland. Droplets of the hydrophobic material appear to become coated with remarkably regular layers of radially-arranged collagen molecules which form a series of concentric, evenly spaced layers around each hydrophobic granule. Numerous disclinations were seen where the layers around adjacent granules interfered with one another. The layers are thought to represent a lamellar liquid crystalline phase previously described for this collagen (Knight et al., 1993). The fine structural appearance of the concentric layers and evidence for radial arrangement of collagen molecules within them is compatible with the suggestion that the layers are built from a dumbbell-shaped unit approximately 35 nm long with hydrophobic groups concentrated at the ends. This unit may represent a dumbbell-shaped molecule or an oligomer of two or more molecules lying parallel with one another in a head-to-tail arrangement. Such a unit can be readily incorporated into models for the micellar, hexagonal columnar and final fibrillar phases previously described for this collagen (Knight et al., 1993). Evidence from the TEM study of stretched egg capsule wall suggests that there is a mechanical interaction between the hydrophobic granules and the collagen fibrils in the fully formed material. We suggest that the radial, concentric layered arrangement of collagen molecules is established by hydrophobic interactions within the liquid crystalline material and locked into place by oxidative covalent cross-linking to give a 3-dimensional cross-linked meshwork of collagen fibrils and hydrophobic granules. The latter arrangement helps to account for the high tensilestrength and toughness of this material.     

Different architectures of the collagen fibril: morphological aspects and functional implications

Several tissues known to contain collagen fibrils with a ‘helical’ arrangement were studied by t.e.m. and freeze-fracture. In all the tissues examined, the diameter of the collagen fibrils appeared to be tissue-specific and fairly constant within the same tissue. No statistical differences, on the contrary, were detectable in the coiling angle which appeared similar in all the tissues and independent of both diameter and age of the fibril. Rat tail tendon was also examined under the same technical conditions and showed collagen fibrils of large and very heterogeneous diameter and with a consistent ‘straight’ arrangement. These data seem to suggest that the ‘helical’ and ‘straight’ arrangements may actually identify different types of collagen fibrils. The authors discuss the possible functional significance of these arrangements and present two hypotheses on the three-dimensional structure of the ‘helical’ fibril.     

The relationship of the biophysical and biochemical characteristics of type VII collagen to the function of anchoring fibrils

Type VII collagen is a major component of anchoring fibrils, which are 800-nm-long centrosymmetrically cross-banded fibrils that are believed to secure the attachment of certain epithelial basement membranes to the underlying stromal matrix. The ultrastructure of the anchoring fibrils is highly variable, suggesting that the fibrils are flexible. Flexibility measurements along the length of the triple-helical domain of type VII procollagen indicate that major flexible sites correlate well with known discontinuities in the (Gly-X-Y)n repeating sequence. Therefore, the helical disruptions may account for the tortuous shapes of anchoring fibrils observed ultrastructurally. The centrosymmetrical banding pattern observed for anchoring fibrils results from the unstaggered lateral packing of antiparallel type VII collagen dimers that form these structures. This antiparallel arrangement is specified by disulfide bonds formed at the margins of a 60-nm overlap of the amino termini. As long as these disulfide bonds remain intact, they protect the amino-terminal overlapping triple helices from collagenase digestion. This disulfide-bonded pair of triple helices is termed C-1. Large nonhelical domains (NC-1) extend from both ends of the anchoring fibrils and are believed to interact with the basement membrane or with anchoring plaques. Rotary shadowing of the NC-1 domains showed trident-like shapes, suggesting that a single alpha-chain contributed the structure of each arm and that the three arms were extended. Biochemical and biophysical analyses of NC-1 domains independently confirm these suggestions and imply that the arms of NC-1 domains are identical and individually capable of interactions with basement membrane components, potentially allowing trivalent interaction of type VII collagen with various macromolecules.     

Characterization of the cytoplasmic fibrils of Treponema refringens (Nichols).

The cytoplasmic fibrils of Treponema refringens were studied in situ by electron microscopy of thin sectioned and negatively stained cells. From 5 to 21 parallel fibrils ran through the cell in a band adjacent to the inner side of the cytoplasmic membrane, on the inner sides of the curves of the spirochete. The nuclear areas of cells were adjacent to the fibrils. Cross sections of fibrils isolated from cells which had been lysed were polygonal and not uniformly electron dense. Polyacrylamide gel electrophoresis of partially purified fibril preparations indicated their main component to be a protein with a molecular weight of 97,000. Fibrils were solubilized by 1% trypsin, 1% pronase, 6 M urea, 1 N HCl, 0.005 N NaOH or 1.3% sodium dodecyl sulfate. By electron microscopy of negatively stained isolated fibrils, each fibril was found to be a complex arrangement of strands rather than a single tubule.     

Fibrillar Array in the Cell Wall of a Gliding Filamentous Cyanobacterium

The cell walls of a number of filamentous, gliding cyanobacteria of the genus Oscillatoria were examined by transmission electron microscopy of ultrathin sections, of freeze-etched replicas, and of whole cells crushed between glass slides and negatively stained. All three techniques revealed the presence of a highly ordered array of parallel fibrils, seen in transverse sections to be situated between the peptidoglycan and the outer membrane. Approximately 200 individual fibrils, each 25 to 30 nm in width, form a parallel, helical array that completely surrounds each cyanobacterial filament, running at an angle of 25 to 30° to its long axis. This highly regular arrangement of the fibrillar layer may imply some underlying symmetry responsible for its organization. A possible source of such symmetry would be the peptidoglycan, and some form of interaction between this layer and the fibrils might provide the necessary scaffolding for the fibrillar array. In crushed, negatively stained samples of fresh cells, individual fibrils were seen outside the filament, released from the cell wall. These released fibrils were of the same width as those observed in situ but were in short lengths, mostly of 100 to 200 nm, and were invariably bent, sometimes even into U shapes, implying great flexibility. Negative staining of released fibrils showed no evidence that they were hollow tubes but did give some indication of a substructure, implying that they were composed of many subunits. The function of this fibrillar array is unknown, although its position in the cell wall, as well as the correspondence between the angle of the fibrils with respect to the long axis of the filament and the rotation of the filament during gliding, may imply an involvement in gliding motility.     

Hierarchical modeling of the elastic properties of bone at submicron scales: the role of extrafibrillar mineralization

We model the elastic properties of bone at the level of mineralized collagen fibrils via step-by-step homogenization from the staggered arrangement of collagen molecules up to an array of parallel mineralized fibrils. A new model for extrafibrillar mineralization is proposed, assuming that the extrafibrillar minerals are mechanically equivalent to reinforcing rings coating each individual fibril. Our modeling suggests that no more than 30% of the total mineral content is extrafibrillar and the fraction of extrafibrillar minerals grows linearly with the overall degree of mineralization. It is shown that the extrafibrillar mineralization considerably reinforces the fibrils’ mechanical properties in the transverse directions and the fibrils’ shear moduli. The model predictions for the elastic moduli and constants are found to be in a good agreement with the experimental data reported in the literature.     

Infrared linear dichroism spectroscopy on amyloid fibrils aligned by molecular combing

We report the use of molecular combing as an alignment method to obtain macroscopically oriented amyloid fibrils on planar surfaces. The aligned fibrils are studied by polarized infrared spectroscopy. This gives structural information that cannot be definitively obtained from standard infrared experiments on isotropic samples, for example, confirmation of the characteristic cross-β amyloid core structure, the side-chain orientation from specific amino acids, and the arrangement of the strands within the fibrils, as we demonstrate here. We employed amyloid fibrils from hen egg white lysozyme (HEWL) and from a model octapeptide. Our results demonstrate molecular combing as a straightforward method to align amyloid fibrils, producing highly anisotropic infrared linear dichroism (IRLD) spectra.     

An X-ray scattering investigation of corneal structure in keratocan-deficient mice.

The transparency of the cornea has been closely linked with the characteristic size and arrangement of its constituent collagen fibrils. This arrangement, in turn, is thought to depend on interactions with intervening matrix proteoglycans. The purpose of this investigation was to examine fibrillar collagen organisation in the corneas of mice homozygous for a null mutation in keratocan, a keratan sulfate-containing proteoglycan. Low-angle synchrotron X-ray scattering techniques were used. We found that keratocan-deficient mice had corneal collagen fibrils with significantly larger diameters than those in wild-type littermates. Furthermore, there was an increase in the centre-to-centre spacing of the collagen fibrils that was accompanied by a decrease in nearest-neighbour fibrillar order. We hypothesise that a lack of keratocan might lower the number of keratan sulfate proteoglycans that associate with collagen, leading to alterations in their diameters and spatial arrangements. Alternatively, it might change the osmotic balance between the inside and outside of fibrils, causing them to swell and move further apart.     

Some observations on the collagen fibrils of the egg capsule of the dogfish, Scyliorhinus canicula

The egg capsule of the dogfish Scyliorhinus canicula is a collagenous material with a laminated, plywood (orthogonal) construction. The collagen fibrils which constitute the bulk of the egg capsule wall have a unique, highly ordered structure (Knight and Hunt, 1974; 1976, 1986; Gathercole et al., 1993) which is thought to represent a smectic A liquid crystalline phase (Knight et al., 1993). The egg capsule is extremely strong and chemically inert (Hunt, 1985). It is stored, secreted and formed by the nidamental gland (Rusaou?n 1976, 1990 a, b; Knight and Feng, 1992). During intracellular storage, secretion and fibrillogenesis, the dogfish egg capsule collagen appears to pass through a remarkable series of textures within a lyotropic liquid crystalline phase diagram (Knight et al., 1993). In the present communication, further observations on the ultrastructure of the collagen fibrils and their arrangement within the laminae of the fully-formed egg capsule are reported. The effect of tilting ultrathin sections of fibrils in the goniometer stage of a transmission electron microscope are described, demonstrating that the crystalline lattice within the fibril appeared twisted more or less regularly into a long pitch helix. Other observations indicated that some of the fibrils were in turn twisted round one another to form fibres which therefore had a coiled-coil structure. The fibres are arranged parallel to one another in the laminae which are stacked to give an orthogonal plywood construction. The effects of staining fibrils with cuprolinic blue and with tannic acid are reported. Reduction in the water content of the fibrils before fixation appeared to move some of the fibrils through the part of the lyotropic phase transition diagram converting them from smectic A to smectic C. Finally, evidence is presented that the fibrils shrank, but remarkably, still retained a longitudinally-ordered but modified, molecular arrangement even after boiling in water for periods of up to 10 min. These observations are discussed in relation to other collagens.     

Spatial contrast sensitivity of birds

Contrast sensitivity (CS) is the ability of the observer to discriminate between adjacent stimuli on the basis of their differences in relative luminosity (contrast) rather than their absolute luminances. In previous studies, using a narrow range of species, birds have been reported to have low contrast detection thresholds relative to mammals and fishes. This was an unexpected finding because birds had been traditionally reported to have excellent visual acuity and color vision. This study reports CS in six species of birds that represent a range of visual adaptations to varying environments. The species studied were American kestrels (Falco sparverius), barn owls (Tyto alba), Japanese quail (Coturnix coturnix japonica), white Carneaux pigeons (Columba livia), starlings (Sturnus vulgaris), and red-bellied woodpeckers (Melanerpes carolinus). Contrast sensitivity functions (CSFs) were obtained from these birds using the pattern electroretinogram and compared with CSFs from the literature when possible. All of these species exhibited low CS relative to humans and most mammals, which suggests that low CS is a general characteristic of birds. Their low maximum CS may represent a trade-off of contrast detection for some other ecologically vital capacity such as UV detection or other aspects of their unique color vision.     

Crystallization and dissolution of collagen fibrils during the histogenesis of the intervertebral disk]

We studied the modifications of collagen fibrils during the histogenesis of the intervertebral disc of cats. In connexion with these studies it is necessary to distinguish between the fibrillar (functional) structure, the arrangement of fibrils, and the nature of fibrils, their diameter, period and other properties. Collagen fibrils (40--50 nm) of the anulus fibrosus enter in hyaline cartilage and split off in thin fibrils (8--10 nm). In this area the cartilage fibrils have a diameter of 20--22 nm, in a greater distance the diameter is diminished to 7--8 nm. Analogous to the changing of the nature of fibrils, the number of the cells related to the sectional area is diminished. The cells of the anulus fibrosus resemble those of tendons. In the transition area their shape becomes roundish, the number of granular membranes is increased, a voluminous Golgi-Apparatus appears for a short time. Finally, the cells are once transformed in cartilage cells with a small reticulum or cells of fibrocartilage with a capsule and a decreased cytoplasm; some cells are disintegrated. In the capsule of the fibrocartilage cells, parallel orientated filaments exhibit a periodical arrangement. In the border of the capsule, filaments change into periodical fibrils. Therefore, we must regard cells and their surrounding intercellular substance as metabolic unity which in the cartilage may be characterized as the chondron.     

Existence of Different Structural Intermediates on the Fibrillation Pathway of Human Serum Albumin

The fibrillation propensity of the multidomain protein human serum albumin (HSA) was analyzed under different solution conditions. The aggregation kinetics, protein conformational changes upon self-assembly, and structure of the different intermediates on the fibrillation pathway were determined by means of thioflavin T (ThT) fluorescence and Congo Red absorbance; far- and near-ultraviolet circular dichroism; tryptophan fluorescence; Fourier transform infrared spectroscopy; x-ray diffraction; and transmission electron, scanning electron, atomic force, and microscopies. HSA fibrillation extends over several days of incubation without the presence of a lag phase, except for HSA samples incubated at acidic pH and room temperature in the absence of electrolyte. The absence of a lag phase occurs if the initial aggregation is a downhill process that does not require a highly organized and unstable nucleus. The fibrillation process is accompanied by a progressive increase in the β-sheet (up to 26%) and unordered conformation at the expense of α-helical conformation, as revealed by ThT fluorescence and circular dichroism and Fourier transform infrared spectroscopies, but changes in the secondary structure contents depend on solution conditions. These changes also involve the presence of different structural intermediates in the aggregation pathway, such as oligomeric clusters (globules), bead-like structures, and ring-shaped aggregates. We suggest that fibril formation may take place through the role of association-competent oligomeric intermediates, resulting in a kinetic pathway via clustering of these oligomeric species to yield protofibrils and then fibrils. The resultant fibrils are elongated but curly, and differ in length depending on solution conditions. Under acidic conditions, circular fibrils are commonly observed if the fibrils are sufficiently flexible and long enough for the ends to find themselves regularly in close proximity to each other. These fibrils can be formed by an antiparallel arrangement of β-strands forming the β-sheet structure of the HSA fibrils as the most probable configuration. Very long incubation times lead to a more complex morphological variability of amyloid mature fibrils (i.e., long straight fibrils, flat-ribbon structures, laterally connected fibers, etc.). We also observed that mature straight fibrils can also grow by protein oligomers tending to align within the immediate vicinity of the fibers. This filament + monomers/oligomers scenario is an alternative pathway to the otherwise dominant filament + filament manner of the protein fibril's lateral growth. Conformational preferences for a certain pathway to become active may exist, and the influence of environmental conditions such as pH, temperature, and salt must be considered.     

Noncore residues influence the kinetics of functional TTR(105-115)-based amyloid fibril assembly

Mutations in the polypeptide sequence that forms the core structure of amyloid fibrils are known to impact on fibril assembly and stability but the effect of changes on noncore residues, particularly relating to functionalized fibrils where the fibril core is preserved, has not been systematically examined. In this study, the short peptide sequence TTR(105-115) (also known as TTR1) and the functionalized variants TTR1-RGD and TTR1-RAD are used as a model system to investigate the effect of noncore residues on the kinetics of fibril assembly. The noncore residues in TTR1-RGD and TTR1-RAD influence the rate of fibril assembly in non-seeded samples with the glycine residue at position 15 increasing the rate of aggregation compared to alanine. Mature TTR1-RGD fibrils were also found to fragment more readily, indicating possible differences in mechanical properties. Fragments of each type of fibril are capable of self- and cross-seeding, generating fibrils with a highly similar cross-β core structure. The similar rates of assembly observed for self-seeded samples reflect the similar free energy of elongation calculated for these peptides, while the morphology of cross-seeded fibrils is determined by the properties of the monomeric peptide and its macromolecular arrangement within the protofilaments and fibrils. These findings illustrate that noncore residues impact on fibril formation and fibril properties and demonstrate that the influence of noncore residues should be considered when designing sequences for the production of self-assembling functional fibrillar materials.     

A microbeam X-ray diffraction study of insulin spherulites

The peptide hormone insulin forms a spherical aggregate, called a spherulite, at low pH and high temperature. A spherulite is composed of a core and many fibrils extending from it. These fibrils are thought to be amyloid fibers with a beta-sheet structure. In the present study, spherulites with a diameter of 50-100 microm were examined by X-ray fiber diffraction using a 6 microm beam. When a spherulite was scanned with the microbeam and the observed diffraction patterns were arranged in a two-dimensional array, the direction of the scatter was centrosymmetric, demonstrating a symmetric growth of fibrils. There were diffraction peaks at Bragg spacings of 23 nm, 3.3 nm and 1.2 nm in the direction perpendicular to the fibrils and 0.48 nm along the fibrils. The 0.48 nm reflection shows that the hydrogen bonds between beta-strands are along the fibril. The 23 nm reflection corresponds to the separation between fibrils, the 3.3 nm reflection is due to the arrangement of protofilaments, and the 1.2 nm reflection arises from the arrangement of peptide chains. On the basis of these results, a model of a fibril with an extended insulin molecule is proposed.