Collagen fibrillogenesis in tendon development: current models and regulation of fibril assembly

Tendons are collagen-based fibrous tissues that connect and transmit forces from muscle to bone. These tissues, which are high in collagen type I content, have been studied extensively to understand collagen fibrillogenesis. Although the mechanisms have not been fully elucidated, our understanding has continued to progress. Here, we review two prevailing models of collagen fibrillogenesis and discuss the regulation of the process by candidate cellular and extracellular matrix molecules. Although numerous molecules have been implicated in the regulation of collagen fibrillogenesis, we focus on those that have been suggested to be particularly relevant to collagen type I fibril formation during tendon development, including members of the collagen and small leucine-rich proteoglycan families, as well as other molecules, including scleraxis, cartilage oligomeric matrix protein, and cytoskeletal proteins.     

Matricellular hevin regulates decorin production and collagen assembly

Matricellular proteins such as SPARC, thrombospondin 1 and 2, and tenascin C and X subserve important functions in extracellular matrix synthesis and cellular adhesion to extracellular matrix. By virtue of its reported interaction with collagen I and deadhesive activity on cells, we hypothesized that hevin, a member of the SPARC gene family, regulates dermal extracellular matrix and collagen fibril formation. We present evidence for an altered collagen matrix and levels of the proteoglycan decorin in the normal dermis and dermal wound bed of hevin-null mice. The dermal elastic modulus was also enhanced in hevin-null animals. The levels of decorin protein secreted by hevin-null dermal fibroblasts were increased by exogenous hevin in vitro, data indicating that hevin might regulate both decorin and collagen fibrillogenesis. We also report a decorin-independent function for hevin in collagen fibrillogenesis. In vitro fibrillogenesis assays indicated that hevin enhanced fibril formation kinetics. Furthermore, cell adhesion assays indicated that cells adhered differently to collagen fibrils formed in the presence of hevin. Our observations support the capacity of hevin to modulate the structure of dermal extracellular matrix, specifically by its regulation of decorin levels and collagen fibril assembly.     

Modulation of collagen fibrillogenesis by tenascin-X and type VI collagen

Tenascin-X (TNX) is an extracellular matrix glycoprotein. We previously demonstrated that TNX regulates the expression of type VI collagen. In this study, we investigated the binding of TNX to type I collagen as well as to type VI collagen and the effects of these proteins on fibrillogenesis of type I collagen. Full-length recombinant TNX, which is expressed in and purified from mammalian cell cultures, and type VI collagen purified from bovine placenta were used. Solid-phase assays revealed that TNX or type VI collagen bound to type I collagen, although TNX did not bind to type VI collagen, fibronectin, or laminin. The rate of collagen fibril formation and its quantity, measured as increased turbidity, was markedly increased by the presence of TNX, whereas type VI collagen did not increase the quantity but accelerated the rate of collagen fibril formation. Combined treatment of both had an additive effect on the rate of collagen fibril formation. Furthermore, deletion of the epidermal growth factor-like (EGF) domain or fibrinogen-like domain of TNX attenuated the initial rate of collagen fibril formation. Finally, we observed abnormally large collagen fibrils by electron microscopy in the skin from TNX-deficient (TNX-/-) mice during development. These findings demonstrate a fundamental role for TNX and type VI collagen in regulation of collagen fibrillogenesis in vivo and in vitro.     

COMP acts as a catalyst in collagen fibrillogenesis

We have previously reported that COMP (cartilage oligomeric matrix protein) is prominent in cartilage but is also present in tendon and binds to collagens I and II with high affinity. Here we show that COMP influences the fibril formation of these collagens. Fibril formation in the presence of pentameric COMP was much faster, and the amount of collagen in fibrillar form was markedly increased. Monomeric COMP, lacking the N-terminal coiled-coil linker domain, decelerated fibrillogenesis. The data show that stimulation of collagen fibrillogenesis depends on the pentameric nature of COMP and not only on collagen binding. COMP interacts primarily with free collagen I and II molecules, bringing several molecules to close proximity, apparently promoting further assembly. These assemblies further join in discrete steps to a narrow distribution of completed fibril diameters of 149 +/- 16 nm with a banding pattern of 67 nm. COMP is not found associated with the mature fibril and dissociates from the collagen molecules or their early assemblies. However, a few COMP molecules are found bound to more loosely associated molecules at the tip/end of the growing fibril. Thus, COMP appears to catalyze the fibril formation by promoting early association of collagen molecules leading to increased rate of fibrillogenesis and more distinct organization of the fibrils.     

Mapping the ligand-binding sites and disease-associated mutations on the most abundant protein in the human, type I collagen.

Type I collagen is the most abundant protein in humans, and it helps to maintain the integrity of many tissues via its interactions with cell surfaces, other extracellular matrix molecules, and growth and differentiation factors. Nearly 50 molecules have been found to interact with type I collagen, and for about half of them, binding sites on this collagen have been elucidated. In addition, over 300 mutations in type I collagen associated with human connective tissue disorders have been described. However, the spatial relationships between the known ligand-binding sites and mutation positions have not been examined. To this end, here we have created a map of type I collagen that includes all of its ligand-binding sites and mutations. The map reveals the existence of several hot spots for ligand interactions on type I collagen and that most of the binding sites locate to its C-terminal half. Moreover, on the collagen fibril some potentially relevant relationships between binding sites were observed including the following: fibronectin- and certain integrin-binding regions are near neighbors, which may mechanistically relate to fibronectin-dependent cell-collagen attachment; proteoglycan binding may potentially impact upon collagen fibrillogenesis, cell-collagen attachment, and collagen glycation seen in diabetes and aging; and mutations associated with osteogenesis imperfecta and other disorders show apparently nonrandom distribution patterns within both the monomer and fibril, implying that mutation positions correlate with disease phenotype. These and other observations presented here may provide novel insights into evaluating type I collagen functions and the relationships between its binding partners and mutations.     

Fibrillogenesis of collagen types I, II, and III with small leucine-rich proteoglycans decorin and biglycan

Collagen has found use as a scaffold material for tissue engineering as well as a coating material for implants with a view to enhancing osseointegration through mimicry of the bone extracellular matrix in vivo. The aim of this study was to compare the collagen types I, II, and III with regard to their ability to bind the small leucine-rich proteoglycans (SLRPs) decorin and biglycan during fibrillogenesis in vitro in phosphate buffer. In addition, the influence of SLRPs on the proportion of collagen molecules incorporated into fibrils during fibrillogenesis in vitro at high and low ionic strength was investigated, as were their effects on the morphology of collagen fibrils and the speed of fibrillogenesis. Considerably more biglycan than decorin was bound by all three collagen types. Collagen II bound significantly more SLRPs in fibrils than collagen I and III. Decorin and biglycan decreased the proportion of collagen molecules of all three collagen types incorporated into fibrils in similar fashion. Biglycan affected neither fibril diameter nor the speed of fibrillogenesis. Decorin reduced the fibril diameter of all three collagen types. The differences in SLRP-binding ability between collagen types could be of significance when selecting collagen type and/or SLRPs as scaffold materials for tissue engineering or implant coatings.     

Binding Properties of the Neural Cell Adhesion Molecule to Different Components of the Extracellular Matrix

A soluble form of the neural cell adhesion molecule (N-CAM) was obtained from 100,000-g supernatants of crude brain membrane fractions by incubation for 2 h at 37 degrees C. The isolated N-CAM, consisting of one polypeptide chain with a molecular mass of 110 kilodaltons (N-CAM 110), was studied for its binding specificity to different components of the extracellular matrix (ECM). N-CAM 110 bound to different types of collagen (collagen types I-VI and IX). The binding efficiency was dependent on salt concentration and could be called specific according to the following criteria: (a) Binding showed substrate specificity (binding to collagens, but not to other ECM components, such as laminin or fibronectin). (b) Binding of N-CAM 110 to heat-denatured collagens was absent or substantially reduced. (c) Binding was saturable (Scatchard plot analyses were linear with KD values in the range of 9.3-2.0 X 10(-9) M, depending on the collagen type and buffer conditions). Binding of N-CAM 110 to collagens could be prevented in a concentration-dependent manner by the glycosaminoglycans heparin and chondroitin sulfate. N-CAM 110 also interacted with immobilized heparin, and this interaction could be prevented by heparin and chondroitin sulfate. Thus, in addition to its role in cell-cell adhesion, N-CAM is a binding partner for different ECM components, an observation suggesting that it also serves as a substrate adhesion molecule in vivo.     

Mapping the heparin-binding sites on type I collagen monomers and fibrils

The glycosaminoglycan chains of cell surface heparan sulfate proteoglycans are believed to regulate cell adhesion, proliferation, and extracellular matrix assembly, through their interactions with heparin-binding proteins (for review see Ruoslahti, E. 1988. Annu. Rev. Cell Biol. 4:229-255; and Bernfield, M., R. Kokenyesi, M. Kato, M. T. Hinkes, J. Spring, R. L. Gallo, and E. J. Lose. 1992. Annu. Rev. Cell Biol. 8:365-393). Heparin-binding sites on many extracellular matrix proteins have been described; however, the heparin-binding site on type I collagen, a ubiquitous heparin-binding protein of the extracellular matrix, remains undescribed. Here we used heparin, a structural and functional analogue of heparan sulfate, as a probe to study the nature of the heparan sulfate proteoglycan-binding site on type I collagen. We used affinity coelectrophoresis to study the binding of heparin to various forms of type I collagen, and electron microscopy to visualize the site(s) of interaction of heparin with type I collagen monomers and fibrils. Using affinity coelectrophoresis it was found that heparin has similar affinities for both procollagen and collagen fibrils (Kd's approximately 60-80 nM), suggesting that functionally similar heparin- binding sites exist in type I collagen independent of its aggregation state. Complexes of heparin-albumin-gold particles and procollagen were visualized by rotary shadowing and electron microscopy, and a preferred site of heparin binding was observed near the NH2 terminus of procollagen. Native or reconstituted type I collagen fibrils showed one region of significant heparin-gold binding within each 67-nm period, present near the division between the overlap and gap zones, within the "a" bands region. According to an accepted model of collagen fibril structure, our data are consistent with the presence of a single preferred heparin-binding site near the NH2 terminus of the collagen monomer. Correlating these data with known type I collagen sequences, we suggest that the heparin-binding site in type I collagen may consist of a highly basic triple helical domain, including several amino acids known sometimes to function as disaccharide acceptor sites. We propose that the heparin-binding site of type I collagen may play a key role in cell adhesion and migration within connective tissues, or in the cell- directed assembly or restructuring of the collagenous extracellular matrix.     

Characterization of binding properties of the myelin-associated glycoprotein to extracellular matrix constituents.

The myelin-associated glycoprotein (MAG) can be obtained from adult mouse brain from detergent-lysates of a crude membrane fraction as a 96-100 kd form (detergent solubilized MAG), and from 100,000 g supernatants of homogenates as a 90-96 kd form (soluble MAG). The soluble form distributes into the Triton X-114-poor aqueous phase, while detergent-solubilized MAG predominantly enters the Triton X-114-rich phase. Both molecular forms bind to heparin in hypo- and isotonic buffers. Soluble MAG binds to several collagens (type G, I, II, III, IV, V, VI, IX) with a kd of 5.7 X 10(-8) M for collagen type IX and 2.0 X 10(-7) for collagen type IV. Binding of 125I-labeled MAG to collagen G can be completely inhibited by unlabeled MAG and collagen G, but not by heat-denatured collagen. MAG does not bind to itself, laminin, fibronectin, or the neural cell adhesion molecules L1 and N-CAM. Binding of MAG to collagen G is most effectively blocked by a high molecular weight dextran sulfate, heparan sulfate and heparin, with chondroitin sulfate and a low molecular weight dextran sulfate being less potent blockers. These findings are in agreement with previous observations on the localization of MAG in basal lamina and interstitial collagens of the sciatic nerve in situ.     

Trabecular meshwork's collagen network formation is inhibited by non-pigmented ciliary epithelium-derived extracellular vesicles

The present research aims to determine whether the application of non-pigmented ciliary epithelium cells derived extracellular vesicles to human trabecular meshwork cells affects the formation and secretion of collagen type I to the extracellular matrix formation. Following the extraction of non-pigmented ciliary epithelium derived extracellular vesicles by a precipitation method, their size and concentration were determined using tunable resistive pulse sensing technology. Extracellular vesicles were incubated with trabecular meshwork cells for 3 days. Morphological changes of collagen type I in the extracellular matrix of trabecular meshwork cells were visualized using confocal microscopy and scanning electron microscopy. A Sirius Red assay was used to determine the total amount of collagen. Finally, collagen type I expression levels in the extracellular matrix of trabecular meshwork cells were quantified by cell western analysis. We found that non-pigmented ciliary epithelium extracellular vesicles were very effective at preventing collagen fibres formation by the trabecular meshwork cells, and their secretion to the extracellular matrix was significantly reduced (P < .001). Morphological changes in the extracellular matrix of trabecular meshwork cells were observed. Our study indicates that non-pigmented ciliary epithelium extracellular vesicles can be used to control collagen type I fibrillogenesis in trabecular meshwork cells. These fibrils net-like structure is responsible for remodelling the extracellular matrix. Moreover, we suggest that targeting collagen type I fibril assembly may be a viable treatment for primary open-angle glaucoma abnormal matrix deposition of the extracellular matrix.     

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.     

Integrin-mediated cell adhesion to type I collagen fibrils

In the integrin family, the collagen receptors form a structurally and functionally distinct subgroup. Two members of this subgroup, alpha(1)beta(1) and alpha(2)beta(1) integrins, are known to bind to monomeric form of type I collagen. However, in tissues type I collagen monomers are organized into large fibrils immediately after they are released from cells. Here, we studied collagen fibril recognition by integrins. By an immunoelectron microscopy method we showed that integrin alpha(2)I domain is able to bind to classical D-banded type I collagen fibrils. However, according to the solid phase binding assay, the collagen fibril formation appeared to reduce integrin alpha(1)I and alpha(2)I domain avidity to collagen and to lower the number of putative alphaI domain binding sites on it. Respectively, cellular alpha(1)beta(1) integrin was able to mediate cell spreading significantly better on monomeric than on fibrillar type I collagen matrix, whereas alpha(2)beta(1) integrin appeared still to facilitate both cell spreading on fibrillar type I collagen matrix and also the contraction of fibrillar type I collagen gel. Additionally, alpha(2)beta(1) integrin promoted the integrin-mediated formation of long cellular projections typically induced by fibrillar collagen. Thus, these findings suggest that alpha(2)beta(1) integrin is a functional cellular receptor for type I collagen fibrils, whereas alpha(1)beta(1) integrin may only effectively bind type I collagen monomers. Furthermore, when the effect of soluble alphaI domains on type I collagen fibril formation was tested in vitro, the observations suggest that integrin type collagen receptors might guide or even promote pericellular collagen fibrillogenesis.     

Role of decorin on in vitro fibrillogenesis of type I collagen

Tendon and corneal decorins are differently iduronated dermatan sulphate/proteoglycan (DS/PG) and the biochemical parameter that differentiates type I collagens is the hydroxylysine glycoside content. We have examined the effect of tendon and corneal decorins on the individual phases (tlag, dA/dt) of differently glycosylated type I collagens fibril formation, at molar ratios PG:collagen monomer ranging from 0.15 : 1 to 0.45 : 1. The results obtained indicate that decorins exert a different effect on the individual phases of fibril formation, correlated to the degree of glycosylation of collagen: at the same PG:collagen ratio the fibril formation of highly glycosylated corneal collagen is more efficiently inhibited than that of the poorly glycosylated one (tendon). Moreover tendon and corneal decorins exert a higher control on the fibrillogenesis of homologous collagen with respect to the heterologous one. These data suggest a possible tissue-specificity of the interaction decorin/type I collagen correlated to the structure of the PG and collagen present in extracellular matrices. This revised version was published online in November 2006 with corrections to the Cover Date.     

Identification of the fibronectin sequences required for assembly of a fibrillar matrix

During extracellular matrix assembly, fibronectin (FN) binds to cell surface receptors and initiates fibrillogenesis. As described in this report, matrix assembly has been dissected using recombinant FN polypeptides (recFNs) expressed in mammalian cells via retroviral vectors. RecFNs were assayed for incorporation into the detergent-insoluble cell matrix fraction and for formation of fibrils at the cell surface as detected by indirect immunofluorescence. Biochemical and immunocytochemical data are presented defining the minimum domain requirements for FN fibrillogenesis. The smallest functional recFN is half the size of native FN and contains intact amino- and carboxy-terminal regions with a large internal deletion spanning the collagen binding domain and the first seven type III repeats. Five type I repeats at the amino terminus are required for assembly and have FN binding activity. The dimer structure mediated by the carboxy-terminal interchain disulfide bonds is also essential. Surprisingly, recFNs lacking the RGDS cell binding site formed a significant fibrillar matrix. Therefore, FN-FN interactions and dimeric structure appear to be the major determinants of fibrillogenesis.     

Interactions of the chondroitin sulfate proteoglycan phosphacan, the extracellular domain of a receptor-type protein tyrosine phosphatase, with neurons, glia, and neural cell adhesion molecules

Phosphacan is a chondroitin sulfate proteoglycan produced by glial cells in the central nervous system, and represents the extracellular domain of a receptor-type protein tyrosine phosphatase (RPTP zeta/beta). We previously demonstrated that soluble phosphacan inhibited the aggregation of microbeads coated with N-CAM or Ng-CAM, and have now found that soluble 125I-phosphacan bound reversibly to these neural cell adhesion molecules, but not to a number of other cell surface and extracellular matrix proteins. The binding was saturable, and Scatchard plots indicated a single high affinity binding site with a Kd of approximately 0.1 nM. Binding was reduced by approximately 15% after chondroitinase treatment, and free chondroitin sulfate was only moderately inhibitory, indicating that the phosphacan core glycoprotein accounts for most of the binding activity. Immunocytochemical studies of embryonic rat spinal phosphacan, Ng-CAM, and N-CAM have overlapping distributions. When dissociated neurons were incubated on dishes coated with combinations of phosphacan and Ng-CAM, neuronal adhesion and neurite growth were inhibited. 125I-phosphacan bound to neurons, and the binding was inhibited by antibodies against Ng-CAM and N-CAM, suggesting that these CAMs are major receptors for phosphacan on neurons. C6 glioma cells, which express phosphacan, adhered to dishes coated with Ng-CAM, and low concentrations of phosphacan inhibited adhesion to Ng-CAM but not to laminin and fibronectin. Our studies suggest that by binding to neural cell adhesion molecules, and possibly also by competing for ligands of the transmembrane phosphatase, phosphacan may play a major role in modulating neuronal and glial adhesion, neurite growth, and signal transduction during the development of the central nervous system.     

Collagen fibrillogenesis in vitro: comparison of types I, II, and III

The self-assembly of pepsin-extracted types I, II, and III collagen was studied to determine how differences in the triple-helical structure between collagen types influence in vitro collagen fibrillogenesis. Collagen types I, II, and III were extracted and purified from bovine sources, and were studied in solution by laser light scattering, pH titration, and determination of turbidity-time curves. The molecular weights were between 280,000 and 289,000, while the translational diffusion coefficients and particle scattering factors at 175.5 degrees were consistent with those expected for single collagen molecules. Titration of collagen types I, II, and III between pH 7.0 and 2.0 using HCl indicated that type I collagen had the most titratable carboxylic groups with type II and III having significantly fewer titratable groups. The self-assembly of these collagens was studied in vitro in phosphate-buffered saline. The time course and extent of fibril formation were studied turbidimetrically, and were found to be dependent on collagen type. Apparent rate constants were determined for both the lag and growth phases of fibril formation. The rates of both phases were greater for type III than for type I collagen, with the rates for type II collagen being intermediate. The extent of fibril formation was based on the turbidity per unit concentration (specific turbidity) extrapolated to zero concentration (intrinsic turbidity), which was found to be greater for type I than for type III collagen. Type II collagen had the smallest intrinsic turbidity. The specific and intrinsic turbidity values were consistent with the relative fibril diameters seen in dermis and cartilage by transmission electron microscopy. These observations indicate that helix-helix interactions are important in the regulation of the rate and extent of collagen fibrillogenesis and may be involved in the determination of fibril structure.     

Roles of lumican and keratocan on corneal transparency

Lumican and keratocan are members of the small leucine-rich proteoglycan (SLRP) family, and are the major keratan sulfate (KS) proteoglycans in corneal stroma. Both lumican and keratocan are essential for normal cornea morphogenesis during embryonic development and maintenance of corneal topography in adults. This is attributed to their bi-functional characteristic (protein moiety binding collagen fibrils to regulate collagen fibril diameters, and highly charged glycosaminoglycan (GAG) chains extending out to regulate interfibrillar spacings) that contributes to their regulatory role in extracellular matrix assembly. The absence of lumican leads to formation of cloudy corneas in homozygous knockout mice due to altered collagenous matrix characterized by larger fibril diameters and disorganized fibril spacing. In contrast, keratocan knockout mice exhibit thin but clear cornea with insignificant alteration of stromal collaegenous matrix. Mutations of keratocan cause cornea plana in human, which is often associated with glaucoma. These observations suggest that lumican and keratocan have different roles in regulating formation of stromal extracellular matrix. Experimental evidence indicates that lumican may have additional biological functions, such as modulation of cell migration and epithelium-mesenchyme transition in wound healing and tumorgenesis, besides regulating collagen fibrillogenesis. Published in 2003.     

Development of tendon structure and function: regulation of collagen fibrillogenesis

In the tendon, the development of mature mechanical properties is dependent on the assembly of a tendon-specific extracellular matrix. This matrix is synthesized by the tendon fibroblasts and composed of collagen fibrils organized as fibers, as well as fibril-associated collagenous and non-collagenous proteins. All of these components are integrated, during development and growth, to form a functional tissue. During tendon development, collagen fibrillogenesis and matrix assembly progress through multiple steps where each step is regulated independently, culminating in a structurally and functionally mature tissue. Collagen fibrillogenesis occurs in a series of extracellular compartments where fibril intermediates are assembled and mature fibrils grow through a process of post-depositional fusion of the intermediates. Linear and lateral fibril growth occurs after the immature fibril intermediates are incorporated into fibers. The processes are regulated by interactions of extracellular macromolecules with the fibrils. Interactions with quantitatively minor fibrillar collagens, fibril-associated collagens and proteoglycans influence different steps in fibrillogenesis and the extracellular microdomains provide a mechanism for the tendon fibroblasts to regulate these extracellular interactions.     

Fibronectin binding site in type I collagen regulates fibronectin fibril formation

Mov13 fibroblasts, which do not express endogenous alpha 1(I) collagen chains due to a retroviral insertion, were used to study the role of type I collagen in the process of fibronectin fibrillogenesis. While Mov13 cells produced a sparse matrix containing short fibronectin fibrils, transfection with a wild type pro alpha 1(I) collagen gene resulted in the production of an extensive matrix containing fibronectin fibrils of normal length. To study the amino acids involved in the fibronectin-collagen interaction, mutations were introduced into the known fibronectin binding region of the pro alpha 1(I) collagen gene. Substitution of Gln and Ala at positions 774 and 777 of the alpha 1(I) chain for Pro resulted in the formation of short fibronectin fibrils similar to what was observed in untransfected Mov13 cells. Type I collagen carrying these substitutions bound weakly to fibronectin- sepharose and could be eluted off with 1 M urea. The effect of this mutation on fibronectin fibrillogenesis could be rescued by adding either type I collagen or a peptide fragment (CB.7) which contained the wild type fibronectin binding region of the alpha 1(I) chain to the cell culture. These results suggest that fibronectin fibrillogenesis in tissue culture is dependent on type I collagen synthesis, and define an important role for the fibronectin binding site in this process.     

Absence of the myelin-associated glycoprotein (MAG) and the neural cell adhesion molecule (N-CAM) interferes with the maintenance,but not with the formation of peripheral myelin

We have previously shown that mice deficient in the gene for the myelin-associated glycoprotein (MAG) develop normal myelin in the peripheral nerves, but show axon and myelin degeneration at eight months of age, suggesting that MAG is involved in the maintenance of axon-Schwann cell integrity. The search for molecules that might replace MAG during myelination revealed an overexpression of the neural cell adhesion molecule (N-CAM) at those aspects where MAG is detectable in wild type mice. To test whether N-CAM might compensate for MAG during myelination in MAG-deficient mice, double mutants deficient in both MAG and N-CAM (MAG⁻/N-CAM⁻mice) were generated by cross-breeding the single mutants. Whereas alterations of myelin development were not detectable in either of the single or double mutants, degeneration of myelin and axons occurred approximately 4 weeks earlier in MAG⁻/N-CAM⁻than in MAG⁻mutants. Furthermore, at 8 weeks of age, single fiber preparation and electron microscopy revealed that the number of profiles indicative of degeneration was substantially increased in MAG⁻/N-CAM⁻mutants when compared to MAG⁻mice. These data suggest that in MAG-deficient mice N-CAM does not compensate for MAG in myelin formation but partially substitutes for it in the maintenance of axon-myelin integrity. Received: 20 May 1996 / Accepted: 19 July 1996