Fibroblasts that proliferate near denervated synaptic sites in skeletal muscle synthesize the adhesive molecules tenascin(J1), N-CAM, fibronectin, and a heparan sulfate proteoglycan

Four adhesive molecules, tenascin(J1), N-CAM, fibronectin, and a heparan sulfate proteoglycan, accumulate in interstitial spaces near synaptic sites after denervation of rat skeletal muscle (Sanes, J. R., M. Schachner, and J. Covault. 1986. J. Cell Biol. 102:420-431). We have now asked which cells synthesize these molecules, and how this synthesis is regulated. Electron microscopy revealed that mononucleated cells selectively accumulate in perisynaptic interstitial spaces beginning 2 d after denervation. These cells were identified as fibroblasts by ultrastructural and immunohistochemical criteria; [3H]thymidine autoradiography revealed that their accumulation results from local proliferation. Electron microscopic immunohistochemistry demonstrated that N-CAM is associated with the surface of the fibroblasts, while tenascin(J1) is associated with collagen fibers that abut fibroblasts. Using immunofluorescence and immunoprecipitation methods, we found that fibroblasts isolated from perisynaptic regions of denervated muscle synthesize N-CAM, tenascin(J1), fibronectin, and a heparan sulfate proteoglycan in vitro. Thus, fibroblasts that selectively proliferate in interstitial spaces near synaptic sites are likely to be the cellular source of the interstitial deposits of adhesive molecules in denervated muscle. To elucidate factors that might regulate the accumulation of these molecules in vivo, we analyzed the expression of tenascin(J1) and fibronectin by cultured fibroblasts. Fibroblasts from synapse-free regions of denervated muscle, as well as skin, lung, and 3T3 fibroblasts accumulate high levels of tenascin(J1) and fibronectin in culture, showing that perisynaptic fibroblasts are not unique in this regard. However, when they are first placed in culture, fibroblasts from denervated muscle bear more tenascin(J1) than fibroblasts from innervated muscle, indicating that expression of this molecule by fibroblasts is regulated by the muscle's state of innervation; this difference is no longer apparent after a few days in culture. In 3T3 cells, accumulation of tenascin(J1) is high in proliferating cultures, depressed in confluent cultures, and reactivated in cells stimulated to proliferate by replating at low density or by wounding a confluent monolayer. Thus, synthesis of tenascin(J1) is regulated in parallel with mitotic activity. In contrast, levels of fibronectin, which increase less dramatically after denervation in vivo, are similar in fibroblasts from innervated and denervated muscle and in proliferating and quiescent 3T3 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of N-CAM in synaptic and extrasynaptic portions of developing and adult skeletal muscle

Previous studies of denervated and cultured muscle have shown that the expression of the neural cell adhesion molecule (N-CAM) in muscle is regulated by the muscle's state of innervation and that N-CAM might mediate some developmentally important nerve-muscle interactions. As a first step in learning whether N-CAM might regulate or be regulated by nerve-muscle interactions during normal development, we have used light and electron microscopic immunohistochemical methods to study its distribution in embryonic, perinatal, and adult rat muscle. In embryonic muscle, N-CAM is uniformly present on the surface of myotubes and in intramuscular nerves; N-CAM is also present on myoblasts, both in vivo and in cultures of embryonic muscle. N-CAM is lost from the nerves as myelination proceeds, and from myotubes as they mature. The loss of N-CAM from extrasynaptic portions of the myotube is a complex process, comprising a rapid rearrangement as secondary myotubes form, a phase of decline late in embryogenesis, a transient reappearance perinatally, and a more gradual disappearance during the first two postnatal weeks. Throughout embryonic and perinatal life, N-CAM is present at similar levels in synaptic and extrasynaptic regions of the myotube surface. However, N-CAM becomes concentrated in synaptic regions postnatally: it is present in postsynaptic and perisynaptic areas of the muscle fiber, both on the surface and intracellularly (in T-tubules), but undetectable in portions of muscle fibers distant from synapses. In addition, N-CAM is present on the surfaces of motor nerve terminals and of Schwann cells that cap nerve terminals, but absent from myelinated portions of motor axons and from myelinating Schwann cells. Thus, in the adult, N-CAM is present in synaptic but not extrasynaptic portions of all three cell types that comprise the neuromuscular junction. The times and places at which N-CAM appears are consistent with its playing several distinct roles in myogenesis, synaptogenesis, and synaptic maintenance, including alignment of secondary along primary myotubes, early interactions of axons with myotubes, and adhesion of Schwann cells to nerve terminals.     

Molecular forms of N-CAM and its RNA in developing and denervated skeletal muscle

The neural cell adhesion molecule (N-CAM) is present in both embryonic and perinatal muscle, but its distribution changes as myoblasts form myotubes and axons establish synapses (Covault, J., and J. R. Sanes, 1986, J. Cell Biol., 102:716-730). Levels of N-CAM decline postnatally but increase when adult muscle is denervated or paralyzed (Covault, J., and J. R. Sanes, 1985, Proc. Natl. Acad. Sci. USA., 82:4544-4548). To determine the molecular forms of N-CAM and N-CAM-related RNA during these different periods we used immunoblotting and nucleic acid hybridization techniques to analyze N-CAM and its RNA in developing, cultured, adult, and denervated adult muscle. As muscles develop, the extent of sialylation of muscle N-CAM decreases, and a 140-kD desialo form of N-CAM (generated by neuraminidase treatment) is replaced by a 125-kD form. This change in the apparent molecular weight of desialo N-CAM is paralleled by a change in N-CAM RNA: early embryonic muscles express a 6.7-kb RNA species which hybridizes with N-CAM cDNA, whereas in neonatal muscle this form is largely replaced by 5.2- and 2.9-kb species. Similar transitions in the desialo form of N-CAM, but not in extent of sialylation, accompany differentiation in primary cultures of embryonic muscle and in cultures of the clonal muscle cell lines C2 and BC3H-1. Both in vivo and in vitro, a 140-kD desialo form of N-CAM and a 6.7-kb N-CAM RNA are apparently associated with myoblasts, whereas a 125-kD desialo form and 5.2- and 2.9-kb RNAs are associated with myotubes and myofibers. After denervation of adult muscle, a approximately 12-15-fold increase in the levels of N-CAM is accompanied by a approximately 30-50-fold increase in N-CAM RNA, suggesting that N-CAM expression is regulated at a pretranslational level. Forms of N-CAM and its RNA in denervated muscle are similar to those seen in perinatal myofibers.     

A heparin-binding domain from N-CAM is involved in neural cell- substratum adhesion

Cell-substratum adhesion in the embryonic chicken nervous system has been shown to be mediated in part by a 170,000-mol-wt polypeptide that is a component of adherons. Attachment of retinal cells to the 170,000-mol-wt protein is inhibited by the C1H3 monoclonal antibody and by heparan sulfate (Cole, G. J., D. Schubert, and L. Glaser, 1985, J. Cell Biol., 100:1192-1199). In the present study we have demonstrated that the 170,000-mol-wt C1H3 polypeptide is immunologically identical to the neural cell adhesion molecule N-CAM, and that the 170,000-mol-wt component of N-CAM is preferentially secreted by cells as a component of adherons. We have identified a monoclonal antibody, designated B1A3, that inhibits heparin binding to N-CAM and cell-to-substratum adhesion. A 25,000-mol-wt heparin (heparan sulfate)-binding domain of N-CAM has been identified by limited proteolysis, and this fragment promotes cell attachment when bound to glass surfaces. The fragment also partially inhibits cell binding to adherons when bound to retinal cells, and the B1A3 monoclonal antibody inhibits retinal cell attachment to substrata composed of intact N-CAM or the heparin-binding domain. These data are the first evidence that N-CAM is a multifunctional protein that contains both cell-and heparin (heparan sulfate)-binding domains.     

Effects of antibodies to neural cell adhesion molecule (N-CAM) on the differentiation of neuromuscular contacts between ciliary ganglion neurons and myotubes in vitro

Previous experiments have suggested that the neural cell adhesion molecule (N-CAM) may have a role in initial nerve-muscle adhesion. To determine whether N-CAM might be involved in synaptic differentiation, we grew ciliary ganglion neurons and embryonic myotubes together in the presence and absence of monovalent antibodies to N-CAM. In normal cultures, undifferentiated neurites contact myotubes, and the nerve at some of these neurite-myotube contacts acquires concentrations of synaptic vesicle antigens. Most of these vesicle antigen-positive contacts become associated with patches of acetylcholine receptor (AChR) on the surface of the underlying myotube. Contacts without concentrations of vesicle antigens do not become associated with AChR patches. In the presence of antibodies to N-CAM, adhesion between neuronal somata and myotubes was reduced, but neurites contacted myotubes with near-normal frequency. The subsequent differentiation of nerve and muscle at these contacts, as assayed by the localization of vesicle antigens and AChR, proceeded normally in the presence of anti-N-CAM antibodies. The results suggest that N-CAM-mediated adhesion between neurite and myotube is not required for synaptic differentiation.     

Expression of the adhesion molecules L1, N-CAM and J1/tenascin during development of the murine small intestine

We have previously studied the immunohistological localization of the three adhesion molecules L1, N-CAM and J1/tenascin in adult mouse small intestine and shown that L1 expression in epithelial crypt cells underlies the adhesion of these cells to one another [63]. To obtain further insight into the functional roles of L1, N-CAM and J1/tenascin in this organ we studied their expression starting at embryonic day 14 during embryonic and early postnatal morphogenesis and during epithelial cell migration in the adult. Expression of L1 was restricted to neural cells until approximately postnatal day 5, when L1 started to be detectable on crypt but not on villus cells, predominantly on the basolateral membrane infoldings. As in brain, L1-specific mRNA was approximately 6 kb in size. L1 from intestine appears to differ from the brain-derived equivalent in possessing a higher level of glycosylation. N-CAM was detectable from embryonic day 14 onward in neural and also in mesenchymal cells. Expression by smooth muscle cells decreased during development. In the villus core, N-CAM was strongly detectable at contact sites between smooth muscle cells forming the cellular scaffold of the villus. From embryonic day 14 onward, N-CAM appeared in both 180- and 140-kDa forms. J1/tenascin was present in both neural and mesenchymal cells from embryonic day 14 onward. Starting at embryonic day 17, J1/tenascin appeared concentrated at the boundary between mesenchyme and epithelium in an increasing gradient from the crypt base to the villus top. From embryonic day 14 onward J1/tenascin consisted of the 190- and 220-kDa components. J1/tenascin from intestine differed from brain-derived J1 in its carbohydrate composition. These observations show that the three adhesion molecules are expressed by distinct cell populations and may serve as cell-type-specific markers in pathologically altered intestinal tissue.     

Neural cell adhesion molecule mediates initial interactions between spinal cord neurons and muscle cells in culture

Previous studies in this laboratory have described a cell surface glycoprotein, called neural cell adhesion molecule or N-CAM, that appears to be a ligand in the adhesion between neural membranes. N-CAM antigenic determinants were also shown to be present on embryonic muscle and an N-CAM-dependent adhesion was demonstrated between retinal cell membranes and muscle cells in short-term assays. The present studies indicate that these antigenic determinants are associated with the N-CAM polypeptide, and that rapid adhesion mediated by this molecule occurs between spinal cord membranes and muscle cells. Detailed examination of the effects of anti-(N-CAM) Fab' fragments in cultures of spinal cord with skeletal muscle showed that the Fab' fragments specifically block adhesion of spinal cord neurites and cells to myotubes. The Fab' did not affect binding of neurites to fibroblasts and collagen substrate, and did not alter myotube morphology. These results indicate that N-CAM adhesion is essential for the in vitro establishment of physical associations between nerve and muscle, and suggest that binding involving N-CAM may be an important early step in synaptogenesis.     

Altered expression of neuronal cell adhesion molecules induced by nerve injury and repair

Peripheral nerve injury results in short-term and long-term changes in both neurons and glia. In the present study, immunohistological and immunoblot analyses were used to examine the expression of the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) within different parts of a functionally linked neuromuscular system extending from skeletal muscle to the spinal cord after peripheral nerve injury. Histological samples were taken from 3 to 150 d after crushing or transecting the sciatic nerve in adult chickens and mice. In unperturbed tissues, both N-CAM and Ng-CAM were found on nonmyelinated axons, and to a lesser extent on Schwann cells and myelinated axons. Only N-CAM was found on muscles. After denervation, the following changes were observed: The amount of N-CAM in muscle fibers increased transiently on the surface and in the cytoplasm, and in interstitial spaces between fibers. Restoration of normal N-CAM levels in muscle was dependent on reinnervation; in a chronically denervated state, N-CAM levels remained high. After crushing or cutting the nerve, the amount of both CAMs increased in the area surrounding the lesion, and the predominant form of N-CAM changed from a discrete Mr 140,000 component to the polydisperse high molecular weight embryonic form. Anti-N-CAM antibodies stained neurites, Schwann cells, and the perineurium of the regenerating sciatic nerve. Anti-Ng-CAM antibodies labeled neurites, Schwann cells and the endoneurial tubes in the distal stump. Changes in CAM distribution were observed in dorsal root ganglia and in the spinal cord only after the nerve was cut. The fibers within affected dorsal root ganglia were more intensely labeled for both CAMs, and the motor neurons in the ventral horn of the spinal cord of the affected segments were stained more intensely in a ring pattern by anti-N-CAM and anti-Ng-CAM than their counterparts on the side contralateral to the lesion. Taken together with the previous studies (Rieger, F., M. Grumet, and G. M. Edelman, J. Cell Biol. 101:285-293), these data suggest that local signals between neurons and glia may regulate CAM expression in the spinal cord and nerve during regeneration, and that activity may regulate N-CAM expression in muscle. Correlations of the present observations are made here with established events of nerve degeneration and suggest a number of roles for the CAMs in regenerative events.(ABSTRACT TRUNCATED AT 400 WORDS)     

Syndecan-4 and β1 integrin are regulated by electrical activity in skeletal muscle: Implications for cell adhesion

Syndecan-4 and integrins are involved in the cell migration and adhesion processes in several cell types. Syndecan-4, a transmembrane heparan sulfate proteoglycan, is associated to focal adhesions in adherent cells and has been described as a marker of satellite cells in skeletal muscle. In this tissue, β1 integrin forms heterodimers with α5 and α6 during myoblast differentiation and with α7 in adult muscle. Here, we show that the levels of these two cell surface membrane molecules are regulated by spontaneous electrical activity during the differentiation of rat primary myoblasts. Syndecan-4 and β1 integrin protein levels decrease after the inhibition of electrical activity using tetrodotoxin (TTX). Syndecan-4 also decreases substantially in denervated rat tibialis anterior muscle. Indirect immunofluorescence analysis shows that syndecan-4 and β1 integrin co-localize with vinculin, a molecular marker of costameres in skeletal muscle myofibers. Co-localization is lost in inactive myotubes adopting a diffuse pattern, suggesting that the costameric organization is disrupted in TTX-treated myotubes. Moreover, the inhibition of spontaneous electrical activity decreases myotube cell adhesion. In summary, this work shows that syndecan-4 and β1 integrin protein levels and their localization in costameric structures are regulated by electrical activity and suggests that this regulatory mechanism influences the adhesion properties of skeletal myotubes during differentiation.     

Development of basal lamina in synaptic and extrasynaptic portions of embryonic rat muscle

Each vertebrate skeletal muscle fiber is ensheathed by a basal lamina (BL) which passes through the synaptic cleft of the neuromuscular junction. In the adult, the synaptic portion of the BL is both functionally and chemically specialized. We have used an immunofluorescence method to compare the development of synaptic and extrasynaptic portions of BL in embryonic rat intercostal muscles. Immunohistochemical staining of adult muscle fibers with monoclonal and serum antibodies defines "synaptic" antigens (including acetylcholinesterase) that are concentrated in synaptic BL, "extrasynaptic" antigens that are concentrated in extrasynaptic regions, and "shared" antigens (including collagen IV, fibronectin, laminin, and a heparan sulfate proteoglycan) that are present in both synaptic and extrasynaptic BL ( Sanes and Chiu , 1983). Synapses appear on newly formed myotubes on embryonic Day 14 (E14; birth is on E22 ). Patches of BL that contain shared and extrasynaptic antigens are present on myotube surfaces by E15, and BL forms a continuous sheath by E17. Shared antigens are present at but not confined to synaptic areas by E15. Two synaptic antigens appear in synaptic areas a day later, and are not detectable extrasynaptically . At least one extrasynaptic antigen is present at immature synapses, and lost or masked by E19 . Thus synaptic BL is not assembled as a unit; rather, components are added, lost, or modified as synaptogenesis proceeds.     

Perlecan inhibits smooth muscle cell adhesion to fibronectin: role of heparan sulfate

Smooth muscle cell migration, proliferation, and deposition of extracellular matrix are key events in atherogenesis and restenosis development. To explore the mechanisms that regulate smooth muscle cell function, we have investigated whether perlecan, a basement membrane heparan sulfate proteoglycan, modulates interaction between smooth muscle cells and other matrix components. A combined substrate of fibronectin and perlecan showed a reduced adhesion of rat aortic smooth muscle cells by 70-90% in comparison to fibronectin alone. In contrast, perlecan did not interfere with cell adhesion to laminin. Heparinase treated perlecan lost 60% of its anti-adhesive effect. Furthermore, heparan sulfate as well as heparin reduced smooth muscle cell adhesion when combined with fibronectin whereas neither hyaluronan nor chondroitin sulfate had any anti-adhesive effects. Addition of heparin as a second coating to a preformed fibronectin matrix did not affect cell adhesion. Cell adhesion to the 105- and 120 kDa cell-binding fragments of fibronectin, lacking the main heparin-binding domains, was also inhibited by heparin. In addition, co-coating of fibronectin and (3)H-heparin showed that heparin was not even incorporated in the substrate. Morphologically, smooth muscle cells adhering to a substrate prepared by co-coating of fibronectin and perlecan or heparin were small, rounded, lacked focal contacts, and showed poorly developed stress fibers of actin. The results show that the heparan sulfate chains of perlecan lead to altered interactions between smooth muscle cells and fibronectin, possibly due to conformational changes in the fibronectin molecule. Such interactions may influence smooth muscle cell function in atherogenesis and vascular repair processes.     

Patterns of N-CAM expression during myogenesis in Xenopus laevis

The neural cell adhesion molecule (N-CAM) is seen in the membrane of nerves and muscles from several vertebrate species. Using indirect immunofluorescence, we have examined the expression of this protein during embryonic and postembryonic myogenesis in the African clawed frog, Xenopus laevis. While good staining for N-CAM was seen in neuronal tissues at all stages examined, no staining of embryonic muscle was observed, including both mononucleated and polynucleated myoblasts. In contrast, limb muscles formed at metamorphosis showed strong expression of N-CAM. The developing limb muscles eventually lose their N-CAM, but will reexpress it dramatically when denervated. These observations suggest that myogenesis programs executed at different stages of development can display distinct patterns of N-CAM expression.     

Correlation between cell substrate attachment in vitro and cell surface heparan sulfate affinity for fibronectin and collagen

Heparan sulfate glycosaminoglycan, isolated from the cell surface of nonadhering murine myeloma cells (P3X63-Ag8653), does not bind to plasma fibronectin, but binds partially to collagen type I, as assayed by affinity chromatography with proteins immobilized on cyanogen bromide-activated Sepharose 4B. Identical results were obtained when myeloma heparan sulfate was cochromatographed, on the same fibronectin and collagen columns, with cell surface heparan sulfates collagen columns, with cell surface heparan sulfates from adhering Swiss mouse 3T3 and SV3T3 cells. These latter heparan sulfates do, however, bind to both fibronectin and collagen, as reported earlier (Stamatoglou, S.C., and J.M. Keller, 1981, Biochim. Biophys. Acta., 719:90-97). Cell adhesion assays established that hydrated collagen substrata can support myeloma cell attachment, but fibronectin cannot. Saturation of the heparan sulfate binding sites on the collagen substrata with heparan sulfate or heparin, prior to cell inoculation, abolished the ability to support cell adhesion, whereas chondroitin 4 sulfate, chondroitin 6 sulfate, and hyaluronic acid had no effect.     

Specific regulation of N-CAM/D2-CAM cell adhesion molecule during skeletal muscle development

The expression of the N-CAM/D2-CAM cell adhesion molecule was studied in skeletal muscle. In cell cultures derived from adult human muscle N-CAM/D2-CAM was found at the cell surface of myoblasts and myotubes but not fibroblasts, showing that N-CAM/D2-CAM is a specific gene product of muscle. Western blots showed that the anti N-CAM/D2-CAM antibody reacted with a single protein band of 180 000 daltons in these cultures that differed in mobility from the broad band of 150 000-200 000 daltons found in brain. N-CAM/D2-CAM is also expressed by muscle at certain stages of development. Human foetal muscle of 10 and 20 weeks gestation showed N-CAM/D2-CAM around developing myofibres while both fast and slow adult muscle fibres did not express N-CAM/D2-CAM, suggesting that the protein is down regulated during myofibre maturation. This was studied further in developing rat muscle where N-CAM/D2-CAM was found on myofibres in the day 1 neonate, but had disappeared by day 9. N-CAM/D2-CAM is, however, re-expressed in human muscle disease where there is muscle regeneration such as in polymyositis, and here is associated with classic regenerating myofibres. N-CAM/D2-CAM expression is temporally regulated and is expressed only at times of synapse formation consistent with the idea that it may be involved in early nerve-muscle interactions.     

Identification of a heparin binding domain of the neural cell adhesion molecule N-CAM using synthetic peptides

The neural cell adhesion molecule (N-CAM) plays an integral role in cell interactions during neural development, with the binding of heparan sulfate proteoglycan to the amino-terminal region of N-CAM being required for N-CAM function. In the present study we have used synthetic peptides (HBD-1 and HBD-2), derived from the primary amino acid sequence of rat N-CAM, to identify the region of N-CAM that binds heparan sulfate. The 28 amino acid HBD-1 synthetic peptide was shown to bind both [3H]heparin and dissociated retinal cells. Retinal cells also attach to a substratum of HBD-2 peptide, but fail to bind to a control peptide containing a scrambled amino acid sequence of HBD-2. The HBD-2 peptide also inhibits retinal cell adhesion to N-CAM, demonstrating the physiological importance of the amino acid sequence encoded by the HBD peptide. These data therefore permit the localization of a heparin binding domain to a 17 amino acid region of immunoglobulin-like loop 2.     

Topographic localization of the heparin-binding domain of the neural cell adhesion molecule N-CAM

Previous studies have reported that the cell-binding region of the neural cell adhesion molecule (N-CAM) resides in a 65,000-D amino-terminal fragment designated Frl (Cunningham, B. A., S. Hoffman, U. Rutishauser, J. J. Hemperly, and G. M. Edelman, 1983, Proc. Natl. Acad. Sci. USA, 80:3116-3120). We have reported the presence of two functional domains in N-CAM, each identified by a specific mAb, that are required for cell-cell or cell-substratum adhesion (Cole, G. J., and L. Glaser, 1986, J. Cell Biol., 102:403-412). One of these domains is a heparin (heparan sulfate)-binding domain. In the present study we have determined the topographic localization of the heparin-binding fragment from N-CAM, which has been identified by our laboratory. The B1A3 mAb recognizes a 25,000-D heparin-binding fragment derived from chicken N-CAM, and also binds to a 65,000-D fragment, presumably Frl, produced by digestion of N-CAM with Staphylococcus aureus V8 protease. Amino-terminal sequence analysis of the isolated 25,000-D heparin-binding domain of N-CAM yielded the sequence: Leu-Gln-Val-Asp-Ile-Val-Pro-Ser-Gln-Gly. This sequence is identical to the previously reported amino-terminal sequence for murine and bovine N-CAM. Thus, the 25,000-D polypeptide fragment is the amino-terminal region of the N-CAM molecule. We have also shown that the B1A3 mAb recognizes not only chicken N-CAM but also rat and mouse N-CAM, indicating that the heparin-binding domain of N-CAM is evolutionarily conserved among different N-CAM forms. Additional peptide-mapping studies indicate that the second cell-binding site of N-CAM is located in a polypeptide region at least 65,000 D from the amino-terminal region. We conclude that the adhesion domains on N-CAM identified by these antibodies are physically distinct, and that the previously identified cell-binding domain on Frl is the heparin-binding domain.     

Cell-substratum adhesion in chick neural retina depends upon protein- heparan sulfate interactions

Embryonic chick neural retina cells in culture release complexes of proteins and glycosaminoglycans, termed adherons, which stimulate cell-substratum adhesion when adsorbed to nonadhesive surfaces. Two distinct retinal cell surface macromolecules, a 170,000-mol-wt glycoprotein and a heparan sulfate proteoglycan; are components of adherons that can independently promote adhesion when coated on inert surfaces. The 170,000-mol-wt polypeptide contains a heparin-binding domain, as indicated by its retention on heparin-agarose columns and its ability to bind [3H]heparin in solution. The attachment of embryonic chick retinal cells to the 170,000-mol-wt protein also depends upon interactions between the protein and the heparan sulfate proteoglycan, since heparan sulfate in solution disrupts adhesion of chick neural retina cells to glass surfaces coated with the 170,000-mol-wt protein. This adhesion is not impaired by chondroitin sulfate or hyaluronic acid, which indicates that inhibition by heparan sulfate is specific. Polyclonal antisera directed against the cell surface heparan sulfate proteoglycan also inhibit attachment of retinal cells to the 170,000-mol-wt protein, which suggests that cell-adheron binding is mediated in part by interactions between cell surface heparan sulfate proteoglycan and 170,000-mol-wt protein contained in the adheron particles. Previous studies have indicated that this type of cell-substratum adhesion is tissue-specific since retina cells do not attach to muscle adherons. Schubert D., M. LaCorbiere, F. G. Klier, and C. Birdwell, 1983, J. Cell Biol. 96:990-998.     

Expression of N-cadherin, N-CAM, fibronectin and tenascin is stimulated by TGF-beta1, beta2, beta3 and beta5 during the formation of precartilage condensations

Cell surface adhesion and extracellular matrix proteins are known to play a key role in the formation of cell condensations during skeletal development, and their formation is crucial for the expression of cartilage-specific genes. However, little is known about the relationship between adhesion molecules (N-cadherin and N-CAM), extracellular matrix proteins (fibronectin and tenascin) and TGF-beta1, TGF-beta2 and TGF-beta3 during in vitro precartilage condensations in mouse chondrogenesis. On these bases, we determined the participation of mammalian TGF-beta1, TGF-beta2 and TFG-beta3 and Xenopus TGF-beta5 on the expression of cell surface adhesion and extracellular matrix proteins during the formation of precartilage condensations. Also, we characterized the effects of TGF-betas on proteoglycan metabolism at different cellular densities in mouse embryonic limb bud mesenchymal cells. In TGF-beta1 and TGF-beta5-treated cultures, proteoglycan biosynthesis was higher than in controls, while there were no differences in proteoglycan catabolism, which caused the accumulation of cartilage extracellular matrix. When mesenchymal cells were seeded at three different cellular densities in the presence of TGF-betas, only high density cultures presented increased stimulation of proteoglycan biosynthesis, compared to low and intermediate densities. To determine whether the effect of TGF-betas on precartilage condensations is mediated through the expression of N-cadherin, N-CAM, fibronectin and tenascin, we evaluated their expression. Results showed that TGF-beta1, TGF-beta2, TGF-beta3, and TGF-beta5 differentially enhanced the expression of N-cadherin, N-CAM, fibronectin and tenascin in precartilage condensations, suggesting that TGF-beta isoforms play an important role in the establishment of cell-cell and cell-extracellular matrix interactions during precartilage condensations.     

N-CAM at the vertebrate neuromuscular junction

We have detected the neural cell adhesion molecule, N-CAM, at nerve-muscle contacts in the developing and adult mouse diaphragm. Whereas we found N-CAM staining with fluorescent antibodies consistently to overlap with the pattern of alpha-bungarotoxin staining at nerve-muscle contacts both during development and in the adult, we observed N-CAM staining on the surfaces of developing myofibers and at much lower levels on adult myofibers. Consistent with its function, N-CAM was also detected on axons and axon terminals. Immunoblotting experiments with anti-N-CAM antibodies on detergent extracts of embryonic (E) diaphragm muscle revealed a polydisperse polysialylated N-CAM polypeptide, which in the adult (A) was converted to a discrete form of Mr 140,000; this change, called E-to-A conversion, was previously found to occur in different neural tissues at different rates. The Mr 140,000 component was not recognized by monoclonal antibody anti-N-CAM No. 5, which specifically recognizes antigenic determinants associated with N-linked oligosaccharide determinants on N-CAM from neural tissue. The relative concentration of the Mr 140,000 component prepared from diaphragm muscle increased during fetal development and then decreased sharply to reach adult values. Nevertheless, expression of N-CAM in muscle could be induced after denervation: one week after the sciatic nerve was severed, the relative amount of N-CAM increased dramatically as detected by immunoblots of extracts of whole muscle. Immunofluorescent staining confirmed that there was an increase in N-CAM, both in the cell and at the cell surface; at the same time, however, staining at the motor endplate was diminished. Our findings indicate that, in muscle, in addition to chemical modulation, cell-surface modulation of N-CAM occurs both in amount and distribution during embryogenesis and in response to denervation.     

Mapping of three carbohydrate attachment sites in embryonic and adult forms of the neural cell adhesion molecule

The sialic-rich carbohydrate moiety of the neural cell adhesion molecule (N-CAM) undergoes major structural changes during development and plays a significant role in altering the homophilic binding of the molecule. In order to understand the mechanism of these changes, a cyanogen bromide (CNBr) fragment that contained 90% of the sialic acid of N-CAM was isolated and characterized according to the number of carbohydrate attachment sites and reactivity with specific monoclonal antibodies. The CNBr sialopeptide migrated on SDS PAGE as a broad zone of Mr 42,000-60,000. Upon treatment with neuraminidase, it was converted to a single component of Mr 42,000, and subsequent, limited treatment with endoglycosidase F gave four evenly spaced components of Mr 35,000-42,000, suggesting that it contained three attachment sites for N-linked oligosaccharides. The fragment reacted with monoclonal antibody 15G8, which detects the sialic acid in embryonic N-CAM, and with a monoclonal antibody, anti-(N-CAM) No. 2. Treatment with neuraminidase or with endoglycosidase F destroyed reactivity with 15G8 but not with anti-(N-CAM) No. 2. A similar CNBr sialopeptide was obtained from adult N-CAM; it contained sialic acid, had three N-linked oligosaccharides and reacted with anti-(N-CAM) No. 2 but not with 15G8 monoclonal antibodies. A peptide fragment, Fr2, comprising the NH2 terminal and middle regions of the molecule yielded a CNBr fragment closely similar to the fragment obtained from the whole molecule. The CNBr fragment from Fr2 reacted with monoclonal antibody anti-(N-CAM) No. 2. Fr1, comprising the NH2 terminal region alone, failed to react. These data confirm that the majority of the sialic acid is localized in the middle region of the N-CAM molecule and support the hypothesis that embryonic to adult conversion of N-CAM is the result of differences in sialidase or sialytransferase activity.