Article pubs.acs.org/Biomac
Evidence of a Discrete Axial Structure in Unimodal Collagen Fibrils Mario Raspanti,*,† Marcella Reguzzoni,† Marina Protasoni,† and Désirée Martini‡ †
Department of Human Morphology, Insubria University, Via Monte Generoso 71, 21100 Varese, Italy Department of Human Anatomical Sciences, Bologna University, Via Irnerio 48, 40126 Bologna, Italy
‡
ABSTRACT: The collagen fibrils of cornea, blood vessel walls, skin, gut, interstitial tissues, the sheath of tendons and nerves, and other connective tissues are known to be made of helically wound subfibrils winding at a constant angle to the fibril axis. A critical aspect of this model is that it requires the axial microfibrils to warp in an implausible way. This architecture lends itself quite naturally to an epitaxial layout where collagen microfibrils envelop a central core of a different nature. Here we demonstrate an axial domain in collagen fibrils from rabbit nerve sheath and tendon sheath by means of transmission electron microscopy after a histochemical reaction designed to evidence all polysaccharides and by tapping-mode atomic force microscopy. This axial domain was consistently found in fibrils with helical microfibrils but was not observed in tendon, whose microfibrils run longitudinal and parallel.
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and p tend to zero, the microfibrils should warp to unrealistic values. This architecture lends itself quite naturally to an epitaxial layout where layers of collagen microfibrils envelop a central core of a different nature, as hypothesized in Figure 1. Collagen fibrils with an electrolucent central core are not an uncommon finding in invertebrates and in lower vertebrates, 10 but they were never investigated in detail, and apparently they were unheard of in mammals. In the present research, we looked for evidence of an epitaxial structure in collagen fibrils from Achilles tendon and from nerve sheath. This latter tissue was chosen because its typical small, uniform collagen fibrils follow a highly regular course, running prevalently aligned to the nerve axis.11,12
INTRODUCTION As early as 1949 transmission electron microscopy (TEM) revealed a helical arrangement within the collagen fibrils of certain tissues.1 Subsequent research2−4 confirmed that this aspect was observable in a wide range of tissues as different as the cornea, blood vessel walls, skin, gut, interstitial tissues, and the sheath of tendons and nerves. The winding angle of the subfibrils was invariably close to 18°; the precise value varied slightly with the technique used to measure it, but it always was clearly different from the longitudinal layout observed in tendons, bone, and ligaments, and it was invariably associated with a small and extremely uniform fibril diameter. These findings were subsequently confirmed by several unrelated techniques.5 Accurate measurements6 demonstrated that in these small, uniform fibrils the usual 67 nm collagen banding was reduced to 64 nm because of the winding angle of the subfibrils, being 64 ≈ 67 × cos(18°), and that the whole array of the intraperiod bands was equally and proportionally shortened. This implied that all subfibrils projected the same length along the fibril axis, a condition that was shown to be compatible with two alternative architectures, indicated as “constant pitch” and “constant angle”.4 A few years later, the electron-tomography technique provided undisputable evidence of the constant angle model, showing that the collagen fibrils of cornea are made of concentric layers of Smith-type microfibrils,7 which in each layer follow a helical course with a constant angle of ∼17° to the fibril axis.8 In this model, subsequently confirmed also by diffraction studies,9 the pitch p of the helix that each microfibril describes around the fibril axis depends on its position and is equal to p = 2πr/tan(θ), where r is the distance from the axis and θ is the winding angle. A critical shortcoming of this structure that was never addressed is that in the proximity of the fibril axis, where both r © 2011 American Chemical Society
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EXPERIMENTAL PROCEDURES
Adult New Zealand rabbits were humanely killed by excess Pentothal injection. Small specimens of ischiatic nerve and of Achilles tendon were dissected and immediately fixed in 2.5% glutaraldehyde plus 4% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) at 4 °C, dehydrated in graded ethanol series, and embedded in Araldite. Thin sections were cut with a diamond knife on a Reichert OM-U3 ultramicrotome and stained with the Seligman variant of the periodic acid-thiosemicarbazide-silver proteinate method of Thiery.13 This latter is an old technique (1967) for the revelation of glycogen and other polysaccharides, seldom used nowadays but very efficient. The Seligman variant is a cleaner, simpler alternative which in the last step substitutes the silver proteinate solution with osmium vapors. In brief, the grids were floated for 1 h in 1% periodic acid at room temperature, then for another hour in 1% thiosemicarbazide in 10% acetic acid, thoroughly rinsed in distilled water, and exposed to vapors of osmium tetroxide for another hour at room temperature. No other staining was Received: September 20, 2011 Revised: November 1, 2011 Published: November 9, 2011 4344
dx.doi.org/10.1021/bm201314e | Biomacromolecules 2011, 12, 4344−4347
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Article
RESULTS
Because the Thiéry−Seligman staining reaction was carried out in the vapor phase, the sections were substantially clean and free from precipitates. Because they received no conventional staining the contrast was low and the pictures were of relatively poor quality. The collagen fibrils, however, were anyway easily recognizable, and, in thin sections of the nerve sheath, transected collagen fibrils consistently showed a distinctive axial spot (Figure 2a) with an average diameter of 8.7 ± 2.0 nm,
Figure 1. Schematic model of the proposed architecture of unimodal collagen fibrils. The slender filamentous units represent five-stranded collagen microfibrils; some of them were cut out to show the inner structure of the fibril and the axial core. The red arrow highlights the winding angle, equal to 17° for all microfibrils. Figure parts are not to scale. used. The sections were then observed with an FEI Morgagni TEM equipped with a SIS MegaView II CCD camera and operated at 80 kV. Images were directly acquired in digital format as grayscale TIFF files with a resolution of 1280 × 1024 pixels. Measurements were taken on selected micrographs with ImageJ 1.45 (http://rsbweb.nih.gov/ij/). Other unstained ultrathin sections were also observed with a Digital Instruments MultiMode atomic force microscope fitted with a Nanoscope IIIa controller and phase extender. All observations were carried out in air, using Nanoscope TESP or Olympus OTESPA probes (k ≈ 42 N m−1 and f ≈ 300 kHz, for both). Because of the very high Z-axis resolution of tapping-mode atomic force microscopy (TMAFM), even ultrathin sections intended for TEM have enough surface detail to allow an easy recognition of the embedded structures. All three simultaneous channels available in our instrument were used and were assigned to the height, amplitude, and phase signals; the interaction force was kept to the bare minimum (light tapping) to ensure that the phase modulation depended only on the superficial friction or adhesiveness.14
Figure 2. TEM micrographs of transected collagen fibrils. An axial domain is readily appreciable in the fibrils of the nerve and tendon sheaths but not in the tendon proper. (top) Nerve sheath, (middle) tendon, (bottom) tendon sheath. The field of view is always 1 × 1 μm.
n = 100. Even in the low contrast of our micrographs, this central feature stood out clearly as a small dark dot on an otherwise unstained fibril; its diminutive size and fuzzy aspect are, however, responsible for a relatively high uncertainty in its measurement. This spot became less, if at all, visible where the fibrils were cut obliquely. Because the technique used involved prolonged exposure to strong oxidants and other chemicals, the 4345
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Figure 3. AFM micrographs of transected collagen fibrils. The picture depicts a simultaneous view of three corresponding channels (height, amplitude, and phase) of collagen bundles from nerve sheath (A, above), tendon (B, middle), and tendon sheath (C, below). In the nerve sheath, in particular, all three channels clearly show the axial domain, which never appears in tendon fibrils and is barely appreciable in the tendon sheath. The field of view is always 1 × 1 μm.
fibrils are gathered in slender fascicles winding sinuously in random directions. In the occasional circumstance where the fibrils were cut orthogonally, they showed an axial core identical to that observed in nerve sheath (Figure 2c) with an average size of 8.9 ± 1.4 nm, n = 50. TMAFM observations were substantially consistent with TEM micrographs. In cross sections of nerve sheath, all three channels consistently revealed within collagen fibrils a single
size of the spot may not represent the actual size of the structure it corresponds to, but its consistency and position are unambiguous. Thin sections of tendon were of comparable quality and showed large and multimodal fibrils interspersed with rare cells. Intrafibrillar dark spots, however, were never observed on transected fibrils (Figure 2b). An unexpected additional finding came from the tendon sheath, whose thin, uniform collagen 4346
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distinctive axial spot (Figure 3a), which was never observed in tendon fibrils (Figure 3b). In the flexuous bundles of tendon sheath it was almost impossible to find a suitably cross-cut fascicle, so in this tissue the axial spot remained just barely detectable (Figure 3c).
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are the most widespread form among connective tissues. This also suggests a glycosaminoglycan as the most plausible candidate for our inner core, and perhaps hyaluronic acid, which can form long, antiparallel supercoils. Collagen type V is already known to be segregated into the fibril interior20 and was also proposed as a fibril nucleator,21,22 but it is unlikely to contain enough sugars to justify our findings. Most of our knowledge on the fibrillogenesis process was gained on embryonic tendon and does not apply to these fibrils. New data are required, and further research on the precise nature and function of the axial domain is underway.
DISCUSSION
In contrast with other authors15 who reported multiple axial domains within the collagen fibrils of tendons, in our observations not only was the axial domain present exclusively in fibrils known to have a helical architecture but also it was always single. This may be related to the mechanism of fibrils formation. Collagen fibrils with approximately parallel subfibrils,4,5 such as those of tendon, are supposed to grow by a cooperative process involving lateral fusion of smaller protofibrils, but in contrast with this mechanism fibrils with a helical architecture cannot possibly undergo lateral fusion: 16 for a helically wound fibril to merge with another similar fibril it would have to be entirely unwound and rewound, a process made implausible by the energy variation required. The consistent finding of a single axial domain confirms that no lateral fusion of subfibrils took place during the assembly of these fibrils. In other words, at variance with the large heterogeneous fibrils of tendon, which emerge as the result of a stochastic lateral fusion of protofibrils, the slender fibrils of cornea, blood vessels, and sheaths can grow only by progressive addition of collagen microfibrils from a supersaturated solution. Under these conditions each growing fibril competes with its neighbors for the available subunits until these have all been depleted. Because the extracellular environment is the same for all forming fibrils in a given location, they all end up of a similar size. A similar phenomenon can be observed, for instance, in snowflakes and in hailstones which, albeit variable from place to place, tend to have the same size in the same location. The remarkable uniformity in size of collagen fibrils of cornea, sheaths, and blood vessels could therefore emerge spontaneously as the result of this single structural feature. The helical architecture of these fibrils could be both a necessary and a sufficient condition for their diameter uniformity, whereas collagen fibrils with straight microfibrils still need proteoglycans or some other mechanism to terminate their lateral aggregation.17 As a consequence, a decorin −/− organism, for instance, can be expected to show altered fibrils in tendons (fibrils with parallel microfibrils) but not in cornea (fibrils with helical microfibrils), and this is exactly what was found in experimental observations.18
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AUTHOR INFORMATION
Corresponding Author *Tel: +39-0332-217451. Fax: +39-0332-217459. E-mail: mario.
[email protected].
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ACKNOWLEDGMENTS We thank the Large Instruments Centre of the Insubria University for making available the TEM and the scanning probe microscope.
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REFERENCES
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CONCLUSIONS Small, uniform collagen fibrils show an axial structure, rich in sugars but of otherwise unknown nature and function, distinct from the surrounding collagen microfibrils. Nothing similar has been found in the collagen fibrils of tendon, which are known to have a different inner architecture. An intriguing, although entirely speculative, hypothesis is that the axial domain may have a role in determining the helical course of the surrounding microfibrils, not unlike the organizing role that sulphated glycosaminoglycans were reported to have toward paired helical filaments19 in Alzheimer’s disease. This may explain why highly purified monomeric collagen, which is obviously devoid of any other component, when precipitated in vitro always form fibrils with parallel, straight subfibrils, whereas in vivo helical subfibrils 4347
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