Coalignment of plasma membrane channels and protrusions (fibripositors) specifies the parallelism of tendon

The functional properties of tendon require an extracellular matrix (ECM) rich in elongated collagen fibrils in parallel register. We sought to understand how embryonic fibroblasts elaborate this exquisite arrangement of fibrils. We show that procollagen processing and collagen fibrillogenesis are initiated in Golgi to plasma membrane carriers (GPCs). These carriers and their cargo of 28-nm-diam fibrils are targeted to previously unidentified plasma membrane (PM) protrusions (here designated "fibripositors") that are parallel to the tendon axis and project into parallel channels between cells. The base of the fibripositor lumen (buried several microns within the cell) is a nucleation site of collagen fibrillogenesis. The tip of the fibripositor is the site of fibril deposition to the ECM. Fibripositors are absent at postnatal stages when fibrils increase in diameter by accretion of extracellular collagen, thereby maintaining parallelism of the tendon. Thus, we show that the parallelism of tendon is determined by the late secretory pathway and interaction of adjacent PMs to form extracellular channels.     

Collagen fibril biosynthesis in tendon: a review and recent insights

The development and evolution of multicellular animals relies on the ability of certain cell types to synthesise an extracellular matrix (ECM) comprising very long collagen fibrils that are arranged in very ordered 3-dimensional scaffolds. Tendon is a good example of a highly ordered ECM, in which tens of millions of collagen fibrils, each hundreds of microns long, are synthesised parallel to the tendon long axis. This review highlights recent discoveries showing that the assembly of collagen fibrils in tendon is hierarchical, and involves the formation of fairly short "collagen early fibrils" that are the fusion precursors of the very long fibrils that occur in mature tendon.     

Tenocyte contraction induces crimp formation in tendon-like tissue

Tendons are composed of longitudinally aligned collagen fibrils arranged in bundles with an undulating pattern, called crimp. The crimp structure is established during embryonic development and plays a vital role in the mechanical behaviour of tendon, acting as a shock-absorber during loading. However, the mechanism of crimp formation is unknown, partly because of the difficulties of studying tendon development in vivo. Here, we used a 3D cell culture system in which embryonic tendon fibroblasts synthesise a tendon-like construct comprised of collagen fibrils arranged in parallel bundles. Investigations using polarised light microscopy, scanning electron microscopy and fluorescence microscopy showed that tendon constructs contained a regular pattern of wavy collagen fibrils. Tensile testing indicated that this superstructure was a form of embryonic crimp producing a characteristic toe region in the stress–strain curves. Furthermore, contraction of tendon fibroblasts was the critical factor in the buckling of collagen fibrils during the formation of the crimp structure. Using these biological data, a finite element model was built that mimics the contraction of the tendon fibroblasts and monitors the response of the Extracellular matrix. The results show that the contraction of the fibroblasts is a sufficient mechanical impulse to build a planar wavy pattern. Furthermore, the value of crimp wavelength was determined by the mechanical properties of the collagen fibrils and inter-fibrillar matrix. Increasing fibril stiffness combined with constant matrix stiffness led to an increase in crimp wavelength. The data suggest a novel mechanism of crimp formation, and the finite element model indicates the minimum requirements to generate a crimp structure in embryonic tendon.     

Extracellular compartments in tendon morphogenesis: collagen fibril, bundle, and macroaggregate formation

The formation of collagen fibrils, fibril bundles, and tissue-specific collagen macroaggregates by chick embryo tendon fibroblasts was studied using conventional and high voltage electron microscopy. During chick tendon morphogenesis, there are at least three extracellular compartments responsible for three levels of matrix organization: collagen fibrils, bundles, and collagen macroaggregates. Our observations indicate that the initial extracellular events in collagen fibrillogenesis occur within narrow cytoplasmic recesses, presumably under close cellular regulation. Collagen fibrils are formed within these deep, narrow recesses, which are continuous with the extracellular space. Where these narrow recesses fuse with the cell surface, it becomes highly convoluted with folds and processes that envelope forming fibril bundles. The bundles laterally associate and coalesce, forming aggregates within a third cell-defined extracellular compartment. Our interpretation is that this third compartment forms as cell processes retract and cytoplasm is withdrawn between bundles. These studies define a hierarchical organization within the tendon, extending from fibril assembly to fascicle formation. Correlation of different levels of extracellular compartmentalization with tissue architecture provides insight into the cellular controls involved in collagen fibril and higher order assembly and a better understanding of how collagen fibrils are collected into structural groups, positioned, and woven into functional tissue-specific collagen macroaggregates.     

Collagen fibril assembly and deposition in the developing dermis: segmental deposition in extracellular compartments

The hierarchy of extracytoplasmic compartmentalization and fibrillar organization as well as the assembly and deposition of collagen fibrils was characterized in the 15-day chick embryo dermis using transmission electron microscopy. At least two levels of extracellular compartmentalization are recognizable at this stage of dermal development. The first compartment consists of a series of narrow channels containing single or small groups (less than 5) of collagen fibrils. These channels course deep within the cell and are open to the extracellular space. The second extracellular compartment consists of fibrils grouped as small bundles in close association with the cell surface and is most often defined by a single fibroblast. A third level of fibril organization and compartmentalization is sometimes apparent at this stage of dermal development consisting of laterally associated bundles, more characteristic of the mature dermis. This compartment is associated with the fibroblast surface, but is less well defined than the fibril channels or bundle-forming compartments. Dermal collagen fibrils within bundles are discontinuous. Numerous fibrils ends are identified from serial sections and the ends gradually taper. These data indicate that the dermal fibroblast compartmentalizes the extracellular space and deposits collagen fibril segments during dermal morphogenesis. A model for the genesis of the extracellular compartments and their role in collagen fibrillogenesis and development of regularly arranged connective tissues, tendon, and cornea has been proposed. Dermal development conforms to this model and we suggest that extracytoplasmic compartmentalization of the steps in matrix assembly and segmental deposition of collagen fibrils are important mechanisms in the development of a wide variety of connective tissues.     

Tension is required for fibripositor formation

Embryonic tendon cells (ETCs) have actin-rich fibripositors that accompany parallel bundles of collagen fibrils in the extracellular matrix. To study fibripositor function, we have developed a three-dimensional cell culture system that promotes and maintains fibripositors. We show that ETCs cultured in fixed-length fibrin gels replace the fibrin during ~6 days in culture with parallel bundles of narrow-diameter collagen fibrils that are uniaxially aligned with fibripositors, thereby generating a tendon-like construct. Fibripositors occurred simultaneously with onset of parallel collagen fibrils. Interestingly, the constructs have a tendon-like crimp. In initial experiments to study the effects of tension, we showed that cutting the constructs resulted in loss of tension, loss of fibripositors and the appearance of immature fibrils with no preferred orientation.     

Coalignment of microtubules, cytokeratin intermediate filaments, and collagen fibrils in a collagen-secreting cell system

The distribution of microtubules and intermediate filaments in the collagen-secreting scleroblasts of the goldfish scale was investigated by immunofluorescence and electron microscopy. Many of the microtubules and cytokeratin type intermediate filaments formed bundles that were aligned with the underlying, parallel collagen fibrils. The intermediate filament bundles were evenly spaced and located adjacent to the basal plasma membrane. The microtubules, on the other hand, were located further away from the membrane, although many were found very close to the intermediate filament bundles. No detectable change was observed in scleroblast microtubules when cells on scales were treated with colchicine or cooled (greater than or equal to 0 degrees C) for up to 1 h. Cells had to be cooled overnight before the microtubules were affected. The final number and length of the microtubules in the cell depended only on the final steady-state temperature and not the temperature history of the scale cell, and steady state was reached more slowly at colder temperatures. The microtubules but not the intermediate filaments rapidly (within 5 min) and reversibly depolymerized when cells were chilled to -2 approximately -4 degrees C. When chilled cells were warmed, the microtubules polymerized back, within 15 min at room temperature, to the same pattern of parallel coalignment with the underlying collagen. They appeared to repolymerize via two different pathways: (1) a radial growth outwards from the microtubule organizing center followed by a progressive realignment with the underlying collagen and (2) a gradual and simultaneous polymerization along cold-stable, antitubulin staining fibers. These fibers were also aligned with the collagen fibrils and may be related to the aligned intermediate filaments.     

Dynamic testicular adhesion junctions are immunologically unique. II. Localization of classic cadherins in rat testis

In the seminiferous epithelium, morphologically diverse junctions mediate inter-Sertoli and Sertoli-germ cell adhesive contact and likely transmit signals between contacting cells. Defining the molecular composition of testicular cell-cell junctions is an important step in determining their function. Proteins belonging to the cadherin superfamily are important mediators of cell-cell adhesion, as well as cell signaling. Here, we determined the spatial and temporal protein expression of four classic cadherins in rat testis: N-cadherin, cadherin-6, cadherin-11, and a cadherin defined by an antiserum generated against a conserved classic cadherin peptide (L4). Through Western blot analysis, all antibodies recognized unique proteins. Similarly, each cadherin displayed unique, cell-type specific immunostaining patterns. Whereas N-cadherin, cadherin-11, and L4-positive cadherin were expressed from Postnatal Day 7 through adulthood, cadherin-6 protein was not present at Postnatal Day 7 and first appeared at Day 21. Immunostaining of testis cryosections on Postnatal Days 7, 21, 31, 43, and those of adults indicated that cadherin-11 localized to peritubular cell junctions. N-cadherin immunostaining localized to basal inter-Sertoli junctions, Sertoli-spermatocyte junctions, and at about stages I-VII in Sertoli-elongate spermatid junctions. Cadherin-6 immunostaining was restricted to Sertoli-round spermatid and in Sertoli-elongate spermatid junctions at approximately stages XII-I. Finally, L4-positive immunostaining also detected Sertoli-round spermatid junctions in addition to Sertoli-elongate spermatid junctions at approximately stages XII-I. These data show that the various testicular cell-cell junctions are molecularly unique and dynamic complexes.     

Actin filaments are required for fibripositor-mediated collagen fibril alignment in tendon

Cells in tendon deposit parallel arrays of collagen fibrils to form a functional tissue, but how this is achieved is unknown. The cellular mechanism is thought to involve the formation of intracellular collagen fibrils within Golgi to plasma membrane carriers. This is facilitated by the intracellular processing of procollagen to collagen by members of the tolloid and ADAMTS families of enzymes. The carriers subsequently connect to the extracellular matrix via finger-like projections of the plasma membrane, known as fibripositors. In this study we have shown, using three-dimensional electron microscopy, the alignment of fibripositors with intracellular fibrils as well as an orientated cable of actin filaments lining the cytosolic face of a fibripositor. To demonstrate a specific role for the cytoskeleton in coordinating extracellular matrix assembly, cytochalasin was used to disassemble actin filaments and nocodazole or colchicine were used to disrupt microtubules. Microtubule disruption delayed procollagen transport through the secretory pathway, but fibripositor numbers were unaffected. Actin filament disassembly resulted in rapid loss of fibripositors and a subsequent disappearance of intracellular fibrils. Procollagen secretion or processing was not affected by cytochalasin treatment, but the parallelism of extracellular collagen fibrils was altered. In this case a significant proportion of collagen fibrils were found to no longer be orientated with the long axis of the tendon. The results suggest an important role for the actin cytoskeleton in the alignment and organization of the collagenous extracellular matrix in embryonic tendon.     

Evidence of a discrete axial structure in unimodal collagen fibrils

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.     

Extracellular compartments in matrix morphogenesis: collagen fibril, bundle, and lamellar formation by corneal fibroblasts

The regulation of collagen fibril, bundle, and lamella formation by the corneal fibroblasts, as well as the organization of these elements into an orthogonal stroma, was studied by transmission electron microscopy and high voltage electron microscopy. Transmission and high voltage electron microscopy of chick embryo corneas each demonstrated a series of unique extracellular compartments. Collagen fibrillogenesis occurred within small surface recesses. These small recesses usually contained between 5 and 12 collagen fibrils with typically mature diameters and constant intrafibrillar spacing. The lateral fusion of the recesses resulted in larger recesses and consequent formation of prominent cell surface foldings. Within these surface foldings, bundles that contained 50-100 collagen fibrils were formed. The surface foldings continued to fuse and the cell surface retracted, forming large surface-associated compartments in which bundles coalesced to form lamellae. High voltage electron microscopy of 0.5 micron sections cut parallel to the corneal surface revealed that the corneal fibroblasts and their processes had two major axes at approximately right angles to one another. The surface compartments involved in the production of the corneal stroma were aligned along the fibroblast axes and the orthogonality of the cell was in register with that of the extracellular matrix. In this manner, corneal fibroblasts formed collagen fibrils, bundles, and lamellae within a controlled environment and thereby determined the architecture of the corneal stroma by the configuration of the cell and its associated compartments.     

Two forms of apatite deposited during mineralization of the hen tendon.

Old hen tendon provides a model suitable for the study of calcification in an extracellular matrix. In the present study, we observed the mineralizing substances of hen tendon by scanning electron microscopy of plasma-osmium-coated specimens and by transmission electron microscopy of those processed by a plasma-polymerization film replica method. The mineralizing front area revealed a number of elliptical particles fused to each other and forming rod-like structures oriented parallel to collagen fibrils. The area of advanced mineralization possessed non-mineralizing cavities, in which tendon cells were likely to exist. At this site, we recognized a second form of mineral structure, one in which the crystals had a scale-like morphology and were deposited onto the major first-form mineral component. This crystal form was similar to hydroxyapatite synthesized under wet reaction conditions. These findings strongly suggest that the second form of mineral formed independent of collagen fibrils existed together with the predominant, collagen-dependent form of mineral. We speculate that cell membranes and an extremely slow mineralization process may contribute to the formation of this form of mineral during the mineralization process in the hen tendon.     

The myotendinous junction of the smooth feather muscles (mm. pennati)

Summary Smooth feather muscles (mm. pennati) consist of bundles of smooth muscle cells which are attached to the feather follicles by short elastic tendons. In addition, some muscle bundles are interrupted by elastic tendons. The elastic tendon is composed of longitudinally arranged elastic fibers which branch and wavy bundles of collagen fibrils. Smooth muscle cells of the muscle bundles are attached to each other by desmosome-like junctions and by fusion of the basal laminae. The cytoplasm of the muscle cells is characterized by conspicuous thick filaments and abundant thin and intermediate filaments. These are attached to band-like dense patches (dense bands) at the plasma membrane which are particularly broad at the tapering end of the muscle cell. The contact surface between smooth muscle cells and their elastic tendon is considerably increased (i) by deep finger-like invaginations and indentations located at the tapering muscle end, and (ii) by branching of the coarse elastic fibers into slender processes, which are attached to the richly folded surface of the muscle cell endings by peripheral microfibrils. This intimate interlocking closely resembles the myotendinous junctions in skeletal muscle. In addition to fibroblasts and fibrocytes, the myotendinous junction of the young growing chicks contains numerous so-called myofibroblasts, which are suggested to represent smooth muscle cells differentiating into fibroblasts of the developing tendon.Dedicated to Professor Dr. Helmut Leonhardt on the occasion of his 60th birthdaySupported by a grant from the Deutsche Forschungsgemeinschaft (Dr. 91/1)     

Three-dimensional ultrastructure of human tendons.

The three-dimensional ultrastructure of human tendons has been studied. Epitenon and peritenon consist of a dense network of longitudinal, oblique and transversal collagen fibrils crossing the tendon fibres. The internal structure of tendon fibres is also complex. The collagen fibrils are oriented not only longitudinally but also transversely and horizontally. The longitudinal fibrils do not run only parallel but also cross each other forming spirals (plaits). These fibril bundles are bound together by a three-dimensional collagen fibril network of endotenon. In the myotendinous junction the surface of the muscle cells form processes. A network of tendineal collagen fibrils fills the recesses between the muscle cell processes penetrating the basement membrane of these processes. This complex ultrastructure of human tendons most likely offers a good buffer system against longitudinal, transversal, horizontal as well as rotational forces during movement and activity.     

The association of microtubules with the plasmalemma in epidermal tendon cells of the river crab

The mode of association of microtubules (MTs) with the plasmalemma in epidermal tendon cells of the river crab, Potamon dehaani was studied by thin-section electron microscopy. In the leg muscle, the tendon cells connect striated muscle cells with the cuticle, forming specialized junctions at both ends. At the muscle-tendon cell junction, the apposed plasmalemmas are interdigitated in a zig-zag pattern separated by a uniform space of about 50 nm, where the basal lamina is shared by two cells. At the tendon cell-cuticle junction, the plasmalemma of the tendon cell forms many conical invaginations, into which dense fibrous material extends from the cuticle. Inside the tendon cell, numerous microtubules run parallel to the direction of tension transmission and are arranged into parallel bundles of various sizes. Within such bundles, fine filamentous structures cross-link adjacent MTs. MTs span the entire length of the cell and attach at their both ends to the junctional domains of the plasmalemma. The junctional plasmalemma is characterized by formation of an electron-dense undercoat, through which MTs are connected with the plasmalemma proper. The ultrastructural features of MT association with the plasmalemma are basically the same at both junctions. At the junctions, MTs usually terminate with free ends and are linked laterally to the plasmalemmal undercoat with fine filamentous structures. These observations emphasize the role of the plasmalemmal undercoat as a device of the attachment of MTs to the plasmalemma.     

Matrix loading: assembly of extracellular matrix collagen fibrils during embryogenesis

Nothing in biology stimulates the imagination like the development of a single fertilized egg into a newborn child. Consequently, a major focus of biomedical research is aimed at understanding cell differentiation, proliferation, and specialization during child health and human development. However, the fact that the increase in size and shape of the growing embryo has as much to do with the extracellular matrix (ECM) as with the cells themselves, is largely overlooked. Cells in developing tissues are surrounded by a fiber-composite ECM that transmits mechanical stimuli, maintains the shape of developing tissues, and functions as a scaffold for cell migration and attachment. The major structural element of the ECM is the collagen fibril. The fibrils, which are indeterminate in length, are arranged in different tissues in exquisite supramolecular architectures, including parallel bundles, orthogonal lamellae, and concentric weaves. This article reviews our current understanding of the synthesis and assembly of collagen fibrils, and discusses challenging questions about how cells assemble an organized ECM during embryogenesis.     

Interpretation of the meridional X-ray diffraction pattern from collagen fibrils in corneal stroma

The low angle X-ray diffraction pattern from corneal stroma can be interpreted as arising from the equivalent of sharp meridional reflections due to the packing of molecules along the collagen fibrils and an equatorial pattern due to the packing of these fibrils within lamellae.Axial electron density profiles for corneal collagen fibrils have been produced by combining intensity data from the meridional pattern with two independent sets of phases. The first set was obtained using an electron microscopical technique, whereas the second set consisted of calculated tendon collagen phases given in the literature. Substantial agreement between the two electron density profiles was found.A quantitative analysis of the difference between the electron density profiles of rat tail tendon and corneal collagen showed that the step between the gap and overlap regions is smaller in cornea than in tendon. This is probably due to the binding of non-collagenous material in the gap region as occurs in bone and other tissue. Two peaks corresponding to regions where electron density is greater in the cornea are situated at the gap/overlap junctions. A third region where the corneal collagen is more electron dense is located near the centre of the gap region. The proximity of these peaks to the positions of hydroxylysine residues along the fibril axis suggests that they may be the major sites at which sugars are bound to corneal collagen.     

Three-dimensional organization of the collagen fibrils in the rat sciatic nerve as revealed by transmission- and scanning electron microscopy

Summary The organization of collagen fibrils in the rat sciatic nerve was studied by scanning electron microscopy after digestion of cellular elements by sodium hydroxide treatment, and by conventional transmission electron microscopy. The epineurium consisted mainly of thick bundles of collagen fibrils measuring about 10–20 m in width; they were wavy and ran slightly obliquely to the nerve axis. Between these collagen bundles, a very coarse meshwork of randomly oriented collagen fibrils was present. In the perineurium, collagen fibrils occupied the interspaces between the concentrically arranged perineurial cells; in each interspace, they formed a sheet of characteristic lacework elaborately interwoven by thin (about 3 m or less in width) bundles of collagen fibrils. In the subperineurial region, there was a distinct sheet of densely woven collagen fibrils between the perineurium and underlying endoneurial fibroblasts. In the endoneurium, collagen fibrils surrounded individual nerve fibers in two layers as scaffolds: the inner layer was made up of a delicate meshwork of very fine collagen fibrils, and the outer one consisted of longitudinally oriented bundles of about 1–3 m in width. The collagen fibril arrangement described above may protect the nerve fibers against external forces.     

Collagen fibril bundles: a branching assembly unit in tendon morphogenesis

The assembly, deposition and organization of collagen fibril bundles and their composite fibrils were studied during morphogenesis of the chick embryo tendon using electron microscopy, serial sections and computer-assisted three-dimensional reconstruction techniques. The 14-day chick embryo is a stage when tendon architecture is being established and rapid changes in the mechanical properties occur between days 14 and 17 of development. Tendon matrix structure develops from discrete subunits, bundles of collagen fibrils. The bundles branch; undergo a gradual rotation over several micrometers; are intimately associated with the cellular elements of the developing tendon; and form arborizing networks within and among fascicles. The organization of discrete fibril segments into bundles, during the establishment of tendon architecture and function, where the segmental fibrillar components could interact with the interfibrillar matrix as well as with adjacent fibrils would contribute to the stabilization of this structure. The observed gradual rotation of the bundles would serve to stabilize the immature bundle through the physical twining of the composite fibrils while the extensive branching of the bundles observed at 14-days of development and their intimate association with the cellular elements would provide a higher order of structure stabilization.     

Neural crest cell migration in relation to extracellular matrix organization in the embryonic axolotl trunk

Temporal and regional aspects of early neural crest cell migration in relation to extracellular matrix (ECM) organization and distribution in the embryonic axolotl trunk were studied by light microscopy, TEM, and SEM. The dominating structure of the interstitial ECM is a complex network of fibrils, which are indicated by ruthenium red staining to consist of collagen in association with ruthenium red-positive components, probably including glycosaminoglycans. The ECM fibrils, which are largely used as substratum for locomotion by the crest cells, have a temporally and regionally specific organization and distribution. Increase in ECM fibrils on the neural tube, ahead of the crest cell front, is correlated with initiation of crest cell emigration, and it is suggested that the fibrils may stimulate this process by providing a suitable substratum for cell locomotion. An increase in ECM fibrils in extracellular spaces surrounding the crest cell population is correlated with an expansion of these spaces and with progressing crest cell migration into them. It is proposed that the spatial organization of the ECM fibrils influences crest cell shape and orientation during early migration.