Chick myotendinous antigen. I. A monoclonal antibody as a marker for tendon and muscle morphogenesis

Extracellular matrix components are likely to be involved in the interaction of muscle with nonmuscle cells during morphogenesis and in adult skeletal muscle. With the aim of identifying relevant molecules, we generated monoclonal antibodies that react with the endomysium, i.e., the extracellular matrix on the surface of single muscle fibers. Antibody M1, which is described here, specifically labeled the endomysium of chick anterior latissimus dorsi muscle (but neither the perimysium nor, with the exception of blood vessels and perineurium, the epimysium ). Endomysium labeling was restricted to proximal and distal portions of muscle fibers near their insertion points to tendon, but absent from medial regions of the muscle. Myotendinous junctions and tendon fascicles were intensely labeled by M1 antibody. In chick embryos, " myotendinous antigen" (as we tentatively call the epitope recognized by M1 antibody) appeared first in the perichondrium of vertebrae and limb cartilage elements, from where it gradually extended to the premuscle masses. Around day 6, tendon primordia were clearly labeled. The other structures labeled by M1 antibody in chick embryos were developing smooth muscle tissues, especially aorta, gizzard, and lung buds. In general, tissues labeled with M1 antibody appeared to be a subset of the ones accumulating fibronectin. In cell cultures, M1 antibody binds to fuzzy, fibrillar material on the substrate and cell surfaces of living fibroblast and myogenic cells, which confirms an extracellular location of the antigenic site. The appearance of myotendinous antigen during limb morphogenesis and its distribution in adult muscle and tendon are compatible with the idea that it might be involved in attaching muscle fibers to tendon fascicles. Its biochemical characterization is described in the accompanying paper ( Chiquet , M., and D. Fambrough , 1984, J. Cell Biol. 98:1937-1946).     

Immunochemical and biochemical characterization of a glioma-associated extracellular matrix glycoprotein

A novel human glioma-associated extracellular matrix (ECM) glycoprotein has been identified by murine monoclonal antibody 81C6. The glycoprotein, designated GMEM, is expressed in the ECM of glioma and mesenchymal cell cultures, in the perivascular matrix of endothelial proliferations of human gliomas, and in the stroma of human glioma xenografts in athymic mice, where it has been used as a target antigen for monoclonal antibody tumor localization and radioimaging. We report here on the immunochemical and biochemical characterization of GMEM. Polyacrylamide gel analysis of immunoprecipitated [3H]-leucine- and [3H]-glucosamine-labeled ECM from the human glioma cell line U-251MG has shown that GMEM is a high-molecular-weight macromolecule (Mr approximately 1,000,000) composed of Mr approximately 230,000 disulfide-bonded glycoprotein subunits. Immunoprecipitation, immunoblot, and one-dimensional peptide map analysis have shown that GMEM is distinct from human fibroblast and plasma fibronectin. These results support previous immunohistology and absorption analysis findings, indicating that GMEM is distinct from fibronectin, laminin, and glycosaminoglycans secreted by U-251MG.     

Monoclonal antibody MT2 identifies an extracellular matrix glycoprotein that is co-localized with tenascin during adult newt limb regeneration

Using immunohistochemical techniques and mAb MT2, we describe here a novel extracellular matrix (ECM) molecule that is developmentally regulated during limb regeneration in adult newts. The MT2 antigen appears during preblastema stages, is most abundant during blastema stages, and persists, near undifferentiated cells, until digit stages. The MT2 antigen is located in an acellular layer under the wound epithelium and throughout the ECM of the undifferentiated mesenchyme as a thick, cord-like component. In unamputated limbs mAb MT2 reactivity is restricted to tendons, myotendinous junctions, periosteum and to a layer of material beneath the epidermis. In both unamputated limbs and regenerating limbs, the reactivity to mAb MT2 colocalizes closely with urodele tenascin. Immunoblot analysis of blastema extracts showed that the unreduced form of the MT2 antigen is a large, polydispersed protein of approximately the same size as tenascin. However, based upon (a) molecular weights of reduced subunits, (b) competition experiments on tissue sections, and (c) analysis of molecules immunoprecipitated by mAb MT2, we conclude that the MT2 substance is unrelated biochemically to tenascin. The results from immunoblots, enzyme digestions and DEAE-Sephacell binding studies suggest that the unreduced MT2 antigen is a large protein composed of subunits which are connected by disulfide bonds. Reduction of the MT2 antigen results in three components recognized by mAb MT2. The largest of these reduced components is a chondroitin sulfate-like glycoprotein with a molecular weight (Mr) of 310-325 x 10(3). A second component (Mr, 285-300 x 10(3)) is the core protein of the 310-325 x 10(3) glycoprotein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Collagen synthesis by normal and bromodeoxyuridine-modulated cells in myogenic culture

Connective tissue appears to play an important role in the development, function, and pathology of vertebrate skeletal muscle. In order to analyze the source of the extracellular matrix in developing muscle, hydroxyproline assays and fine-structure analyses were made on clonal cultures derived from embryonic musculature. The cells in fibroblast cultures synthesized and accumulated an extensive extracellular matrix of nonstriated reticular-like microfibrils. In myoblast cultures, no extracellular matrix was evident by electron microscopy, although hydroxyproline assays demonstrated that the cells synthesized and released a soluble collagenous protein into the medium. When grown with fibroblasts, multinucleated myofibers exhibited a complete basement membrane consisting of both basal and reticular laminae. Bromodeoxyuridine-treated myoblasts, in addition to exhibiting morphological and cytological alterations, synthesized and elaborated an extracellular matrix identical to that in fibroblast cultures. The potential for a similar alteration or “modulation” of myoblast expression in situ is discussed in regard to its possible relation to fibrosis in muscular dystrophy.     

Dynamic formation of microenvironments at the myotendinous junction correlates with muscle fiber morphogenesis in zebrafish

Muscle development involves the specification and morphogenesis of muscle fibers that attach to tendons. After attachment, muscles and tendons then function as an integrated unit to transduce force to the skeletal system and stabilize joints. The attachment site is the myotendinous junction, or MTJ, and is the primary site of force transmission. We find that attachment of fast-twitch myofibers to the MTJ correlates with the formation of novel microenvironments within the MTJ. The expression or activation of two proteins involved in anchoring the intracellular cytoskeleton to the extracellular matrix, Focal adhesion kinase (Fak) and beta-dystroglycan is up-regulated. Conversely, the extracellular matrix protein Fibronectin (Fn) is down-regulated. This degradation of Fn as fast-twitch fibers attach to the MTJ results in Fn protein defining a novel microenvironment within the MTJ adjacent to slow-twitch, but not fast-twitch, muscle. Interestingly, however, Fak, laminin, Fn and beta-dystroglycan concentrate at the MTJ in mutants that do not have slow-twitch fibers. Taken together, these data elucidate novel and dynamic microenvironments within the MTJ and indicate that MTJ morphogenesis is spatially and temporally complex.     

Location and Distribution of Non-collagenous Matrix Proteins in Musculoskeletal Tissues of Rat

The study assessed immunohistochemically the location and distribution of various non-collagenous matrix proteins (fibronectin, laminin, tenascin-C, osteocalcin, thrombospondin-1, vitronectin and undulin) in musculoskeletal tissues of rat. Fibronectin and thrombospondin-1 were found to be ubiquitous in the studied tissues. High immunoreactivity of these proteins was found in the extracellular matrix of the anatomical sites where firm bindings are needed, i.e. between muscle fibres and fibre bundles, between the collagen fibres of a tendon and at myotendinous junctions, osteotendinous junctions and articular cartilage. Tenascin-C was found in the extracellular matrix of regions where especially high forces are transmitted from one tissue component to the other, such as myotendinous junctions and osteotendinous junctions. Laminin was demonstrated in the basement membranes of the muscle cells and capillaries of the muscle–tendon units. Osteocalc in immunoreactivity concentrated in the extracellular matrix of areas of newly formed bone tissue, i.e. in the subperiosteal and subchondral regions, osteoid tissue and mineralized fibrocartilage zone of the osteotendinous junction. Mild vitronectin activity could be seen in the extracellular matrix of the osteotendinous and myotendinous junctions, and high activity around the bone marrow cells. Undulin could be demonstrated in the extracellular matrix (i.e. on the collagen fibres) of the tendon and epimysium only. However, it was co-distributed with fibronectin and tenascin-C. Together, these findings on the normal location and distribution of these non-collagenous proteins in the musculoskeletal tissues help to form the basis of knowledge against which the location and distribution of the these proteins in various pathological processes could be compared.     

Formation of high molecular weight dermatan sulfate proteoglycan in bovine aortic endothelial cell cultures. Evidence for transglutaminase-catalyzed cross-linking to fibronectin

Three glucuronate-rich dermatan sulfate proteoglycan (DS-PG) subclasses were isolated and previously characterized from bovine aortic endothelial cell cultures (Kinsella, M. G., and Wight, T. N. (1988) J. Biol. Chem. 263, 19222-19231). In the present study, pulse-chase experiments indicate that the DS-PG of highest apparent Mr (approximately 1 x 10(6)), denoted previously as HMW-DS, is a relatively stable component of the endothelial extracellular matrix and is formed at the expense of lower Mr DS-PG species. The formation of HMW-DS is reduced in a dose-dependent manner in the presence of dansylcadaverine, an inhibitor of transglutaminase-catalyzed protein cross-linking, but not when the activity of other cross-linking enzymes such as lysyl oxidase is inhibited. The putative DS-PG precursor to HMW-DS accumulates during inhibition of cross-linking only when lysosomal degradation is also inhibited by ammonium chloride, suggesting that the precursor is degraded rapidly in the absence of cross-linking. HMW-DS is precipitable from endothelial cell monolayer extracts with antibodies against fibronectin, a known transglutaminase substrate. Thus, we conclude that the stability of HMW-DS in the subendothelial matrix in culture depends upon the cross-linking of a low Mr DS-PG precursor to matrical protein(s), including fibronectin, resulting in the formation of a DS-PG subclass of high apparent molecular mass.     

Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular collagen and fibronectin fibril deposition. Matrix fibronectin and collagen deposition remained decreased in cultures containing anti-60k Fab' until cells became bilayered or more dense, when fibronectin and collagen began to appear in lower cell layers. Anti-60k Fab' added to confluent cultures 24 h before fixation and staining had no effect on matrix fibronectin or collagen, so anti- 60k Fab' did not simply block immunostaining. Confluent cultures grown in anti-60k Fab' and labeled for 24 h with [3H]proline incorporated identical amounts of [3H]proline and [3H]hydroxyproline, but [3H]hydroxyproline deposition in the cell layer was significantly decreased by anti-60k Fab' (P less than 0.01). Extracellular matrix collagen does not appear to form a scaffold for fibronectin deposition, as neither gelatin nor a gelatin-binding fragment of plasma fibronectin inhibited deposition of matrix fibronectin. Our results suggest that interstitial collagens and fibronectin interact to form a fibrillar component of the extracellular matrix, and that fibronectin is required for normal collagen organization and deposition by fibroblasts in vitro. Domain-specific antibodies to fibronectin are powerful tools to study the biological role of fibronectin in extracellular matrix organization and other processes.     

An extracellular matrix molecule of newt and axolotl regenerating limb blastemas and embryonic limb buds: immunological relationship of MT1 antigen with tenascin.

Several well-characterized extracellular matrix (ECM) components have been localized to the amphibian limb regenerate, but the identification and characterization of novel ECM molecules have received little attention. Here we describe, using mAb MT1 and immunocytochemistry, an ECM molecule expressed during limb regeneration and limb development. In limb stumps, mAb MT1 reactivity was restricted to tendons, myotendinous junctions, granules in the basal layers of epidermis, periosteum (newts) and perichondrium (axolotls). In regenerating limbs, reactivity in the distal limb stump was first detected 5 days and 1 day after amputation of newt and axolotl limbs, respectively. In both species, mAb MT1 recognized what appeared to be an abundant blastema matrix antigen, localized in both thin and thick cords between and sometimes closely associated with blastema cells. Reactivity was generally uniform throughout the blastema except for a particularly thick layer that was present immediately beneath the wound epithelium. During redifferentiation stages, mAb MT1 reactivity persisted among blastema cells and redifferentiating cartilage but was lost proximally in areas of muscle and connective tissue differentiation. During the entire period of embryonic limb development, mAb MT1 reactivity was seen in the ECM of the mesenchyme and in a layer beneath the limb bud ectoderm, similar to its distribution during regeneration. Considerable mAb MT1 reactivity was also associated with the developing somites. The reactivity of mAb MT1 in blastema and limb bud was similar if not identical to that of a polyclonal Ab against tenascin (pAbTN), a large, extracellular matrix glycoprotein implicated in growth control, inductive interactions, and other developmental events. This pAbTN effectively competed against mAb MT1 binding on blastema sections. In immunoblots, both mAb MT1 and pAbTN recognized a very high molecular weight (approximately Mr 1000 x 10(3)) protein in blastema extracts of both newts and axolotls. mAb MT1 immunoprecipitated a protein of Mr 1000K size which reacted to both mAb MT1 and pAbTN in immunoblots. These data show that tenascin is in the matrix of the urodele blastema and limb bud, and suggest that mAb MT1 identifies urodele tenascin.     

Arterial chondroitin sulfate proteoglycan: localization with a monoclonal antibody

We generated a monoclonal antibody (Mab) against a large chondroitin sulfate proteoglycan (CSPG) isolated from bovine aorta. This Mab (941) immunoprecipitates a CSPG synthesized by cultured monkey arterial smooth muscle cells. The immunoprecipitated CSPG is totally susceptible to chondroitinase ABC digestion and possesses a core glycoprotein of Mr approximately 400-500 KD. By use of immunofluorescence light microscopy and immunogold electron microscopy, the PG recognized by this Mab was shown to be deposited in the extracellular matrix of monkey arterial smooth muscle cell cultures in clusters which were not part of other fibrous matrix components and not associated with the cell's plasma membrane. With similar immunolocalization techniques, the CSPG antigen was found enriched in the intima and present in the medial portions of normal blood vessels, as well as in the interstitial matrix of thickened intimal lesions of atherosclerotic vessels. Immunoelectron microscopy revealed that this CSPG was confined principally to the space within the extracellular matrix not occupied by other matrix components, such as collagen and elastic fibers. These results indicate that this particular proteoglycan has a specific but restricted distribution in the extracellular matrix of arterial tissue.     

Tenascin variants: differential binding to fibronectin and distinct distribution in cell cultures and tissues.

In the chicken, three tenascin variants have been characterized that are generated by alternative splicing of 3 of its 11 fibronectin type III repeats. Using monoclonal antibodies that react with common regions versus extra repeats of tenascin, we could distinguish and separate tenascin variants and investigate their interaction with fibronectin using multiple experimental procedures. Interestingly, in all assays used the smallest tenascin variant bound more strongly to fibronectin than the larger ones. These biochemical data were paralleled by the observation that in chick embryo fibroblast cultures only the smallest form of tenascin could be detected in the fibronectin-rich extracellular matrix network laid down by the cells. Furthermore, each tissue present in adult chicken gizzard contained a distinct set of tenascin variants. Those tissues particularly rich in extracellular matrix, such as the tendon, contained the smallest tenascin only. Intermediate-sized tenascin was present in smooth muscle, whereas the largest form was exclusively detectable underneath the epithelial lining of the villi. Thus it appears that cell type-specific forms of tenascin exist that are appropriate for the functional requirements of the respective extracellular matrices.     

Transforming growth factor beta increases cell surface binding and assembly of exogenous (plasma) fibronectin by normal human fibroblasts.

Transforming growth factor beta (TGF-beta) enhances the cell surface binding of 125I-fibronectin by cultured human fibroblasts. The effect of TGF-beta on cell surface binding was maximal after 2 h of exposure to TFG-beta and did not require epidermal growth factor or protein synthesis. The enhancement was dose dependent and was found with the 125I-labeled 70-kilodalton amino-terminal fragment of fibronectin as well as with 125I-fibronectin. Treatment of cultures with TGF-beta for 6 h resulted in a threefold increase in the estimated number of fibronectin binding sites. The increase in number of binding sites was accompanied by an increased accumulation of labeled fibronectin in detergent-insoluble extracellular matrix. The effect of TGF-beta was biphasic; after 6 h of exposure, less labeled fibronectin bound to treated cultures than to control cultures. Exposure of cells to TGF-beta for greater than 6 h caused a two- to threefold increase in the accumulation of cellular fibronectin in culture medium as detected by a quantitative enzyme-linked immunosorbent assay. The second phase of the biphasic effect and the increase in soluble cellular fibronectin were blocked by cycloheximide. Immunofluorescence staining of fibroblast cultures with antifibronectin revealed that TGF-beta caused a striking increase in fibronectin fibrils. The 70-kilodalton amino-terminal fragment of fibronectin, which blocks incorporation of fibronectin into extracellular matrix, blocked anchorage-independent growth of NRK-49F cells in the presence of epidermal growth factor. Our results show that an increase in the binding and rate of assembly of exogenous fibronectin is an early event preceding the increase in expression of extracellular matrix proteins. Such an early increase in cell surface binding of exogenous fibronectin may be a mechanism whereby TGF-beta can modify extracellular matrix characteristics rapidly after tissue injury or during embryonic morphogenesis.     

Rapid isolation of metaphase chromosomes containing high molecular weight DNA

Normal rat kidney cells were cultured in medium supplemented with normal fetal bovine serum (FBS) or FBS depleted of fibronectin. The cell surface fibronectin of these cultures was visualized by indirect immunofluorescence using species-specific antisera for either rat fibronectin or bovine fibronectin. Anti-rat-fibronectin revealed fibrillar structures on the cells grown in either normal medium or fibronectin-depleted medium. Anti-bovine fibronectin revealed similar fibrillar networks, but only on the cells grown in medium containing bovine fibronectin. Staining in each case was abolished by absorption with the homologous antigen. It appears that exogenous fibronectin was incorporated into the same structures as endogenous fibronectin. This finding suggests that circulating fibronectin may serve as a building block for the assembly of extracellular matrix, possibly by cells which are incapable of synthesizing it.     

Mesosecrin: a secreted glycoprotein produced in abundance by human mesothelial, endothelial, and kidney epithelial cells in culture

Human mesothelial cells, endothelial cells, and type II kidney epithelial cells growing in culture devote approximately 3% of their total protein synthesis to the production of an Mr approximately 46-kD, pI 7.1, secreted glycoprotein (designated Sp46). Fibroblasts make about 1/10th as much Sp46 as these cell types, and their synthesis is dependent upon hydrocortisone. Keratinocytes, urothelial cells, conjunctival epithelial cells, and mammary epithelial cells do not make detectable amounts of Sp46. Mesothelial cells secrete Sp46 onto the substratum, and from there it is subsequently released into the medium. Immunofluorescence analysis using specific antisera discloses that Sp46 is deposited beneath cells as a fine coating on the substratum. In sparse cultures, Sp46 is detected in trails behind motile cells. In contrast, secreted fibronectin coalesces into fibers, most of which remain in contact with and on top of the cells; thus Sp46 does not preferentially bind to fibronectin. About 6 kD of the mass of human Sp46 is N-linked oligosaccharide, which is terminally sialated before secretion. Sp46 has a low glycine content, indicating that it is not a collagenlike protein. Its NH2-terminal sequence over the first 40 amino acids does not resemble any protein for which sequence information is available. Sp46 appears to be a novel extracellular glycoprotein, high-level constitutive expression of which is restricted to mesoderm-derived epithelial and endothelial cells. We therefore propose for it the name "mesosecrin."     

Stimulation of tenascin expression in mesenchyme by epithelial-mesenchymal interactions

Tenascin is an extracellular matrix glycoprotein with an unusually restricted tissue distribution in the developing embryo. The protein was independently discovered by several investigators, and has been given many different names. Synonyms of tenascin include cytotactin, J1, hexabrachion and glioma-mesenchymal extracellular matrix antigen. Whereas fibronectin is expressed rather uniformly in matrices of embryonic mesenchyme, tenascin is found in the mesenchyme at sites of epithelial-mesenchymal interactions. Tenascin is thus found close to epithelial basement membranes but it is probably not an integral basement membrane component. The distribution suggests that developing epithelial cells may produce locally active factors that stimulate tenascin synthesis in the nearby mesenchyme. Tenascin is composed of disulfide-bonded subunits of approximate Mr between 200-280 kD. Using monoclonal antibodies to mouse tenascin, we find two major subunits of Mr 260 and 200 kD from mouse fibroblasts. Work from many laboratories suggests that the different subunits arise by differential splicing of one mRNA. Rotary shadowing electron microscopy of the intact molecule suggests a six-armed structure connected by a central region. However, the different subunits are not co-ordinately expressed during embryogenesis, suggesting that tenascin can exist as different isoforms. The different isoforms may serve distinct functions. The function of tenascin is not well known, but it has been suggested that it alters the adhesive properties of cells and causes cell rounding.     

Bone marrow extracellular matrix induces HL-60 cells to produce an autonomous differentiation factor.

Conditioned medium from cultures of HL-60 myeloid leukemia cells grown on extracellular bone marrow matrix induces macrophage-like differentiation of fresh HL-60 cells. The active medium component is sensitive to protease treatment, indicating that it is a protein, but it is heat stable. Conditioned medium from HL-60 cells grown on protease-treated bone marrow matrix still contains the active component. Thus, it appears that the differentiation-inducing protein is produced by HL-60 cells and is not released from the bone marrow matrix. To identify this differentiation factor, RNA was isolated from HL-60 cells grown on bone marrow matrix and assayed by Northern analysis for expression of mRNA for human differentiation factor, tumor necrosis factor, and macrophage colony-stimulating factor, all inducers of monocyte/macrophage differentiation. Expression of differentiation factor, tumor necrosis factor, or macrophage colony-stimulating factor mRNA was not enhanced in HL-60 cells grown on matrix compared to cells grown on uncoated plastic flasks. Thus, the maturation factor does not appear to be differentiation factor, tumor necrosis factor, or macrophage colony-stimulating factor within the limits of detection of Northern analysis. Elution of the active conditioned medium fraction on a Sephacryl S-200 column revealed a molecular weight of approximately 40,000. The active protein eluted on a DEAE-cellulose ion-exchange column at an ionic strength of 0.3 M NaCl, indicating that it is fairly anionic. Thus, bone marrow matrix is able to induce HL-60 cells to produce a maturation-inducing 40 kilodalton protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular matrix organization in developing muscle: correlation with acetylcholine receptor aggregates

Monoclonal antibodies recognizing laminin, heparan sulfate proteoglycan, fibronectin, and two apparently novel connective tissue components have been used to examine the organization of extracellular matrix of skeletal muscle in vivo and in vitro. Four of the five monoclonal antibodies are described for the first time here. Immunocytochemical experiments with frozen-sectioned muscle demonstrated that both the heparan sulfate proteoglycan and laminin exhibited staining patterns identical to that expected for components of the basal lamina. In contrast, the remaining matrix constituents were detected in all regions of muscle connective tissue: the endomysium, perimysium, and epimysium. Embryonic muscle cells developing in culture elaborated an extracellular matrix, each antigen exhibiting a unique distribution. Of particular interest was the organization of extracellular matrix on myotubes: the build-up of matrix components was most apparent in plaques overlying clusters of an integral membrane protein, the acetylcholine receptor (AChR). The heparan sulfate proteoglycan was concentrated at virtually all AChR clusters and showed a remarkable level of congruence with receptor organization; laminin was detected at 70-95% of AChR clusters but often was not completely co-distributed with AChR within the cluster; fibronectin and the two other extracellular matrix antigens occurred at approximately 20, 8, and 2% of the AChR clusters, respectively, and showed little or no congruence with AChR. From observations on the distribution of extracellular matrix components in tissue cultured fibroblasts and myogenic cells, several ideas about the organization of extracellular matrix are suggested. (a) Congruence between AChR clusters and heparan sulfate proteoglycan suggests the existence of some linkage between the two molecules, possibly important for regulation of AChR distribution within the muscle membrane. (b) The qualitatively different patterns of extracellular matrix organization over myotubes and fibroblasts suggest that each of these cell types uses somewhat different means to regulate the assembly of extracellular matrix components within its domain. (c) The limited co-distribution of different components within the extracellular matrix in vitro and the selective immune precipitation of each antigen from conditioned medium suggest that each extracellular matrix component is secreted in a form that is not complexed with other matrix constituents.     

Role of fibronectin as a growth factor for fibroblasts

Fibroblast replication is regulated by exogenous signals provided by growth factors, mediators that interact with the target cell surface and signal the cell to proliferate. A useful model of growth regulation, the "dual control model," suggests that growth factors can be grouped either as competence factors or as progression factors, and that optimal replication of fibroblasts requires the presence of both types of growth factors. Although most growth factors are soluble mediators, recent studies have demonstrated that, for some cell types, the extracellular matrix can replace the requirement for a competence factor. Since fibronectin is an important constituent of the extracellular matrix that interacts with specific domains on the fibroblast surface, we examined the ability of fibronectin to act as a competence factor to promote the growth of human diploid fibroblasts. To accomplish this, fibronectins purified from two sources, human plasma and human alveolar macrophages, were tested for their ability to (a) stimulate fibroblast replication in serum-free medium containing characterized progression factors (insulin or alveolar macrophage-derived growth factor); (b) provide a growth-promoting signal early in G1. Fibronectin stimulated fibroblast replication in a dose-dependent manner in the presence of a fixed dose of a progression factor. Conversely, fibronectin conferred on previously unresponsive fibroblasts the ability to replicate in a dose-dependent manner when cultured with increasing amounts of a progression factor. Moreover, fibronectin signaled growth-arrested fibroblasts to traverse G1 approximately 4 h closer to S phase. No differences were observed in the ability of plasma or macrophage fibronectins to provide a competence signal for fibroblast replication. Since fibronectin is a major component of the extracellular matrix, these observations suggest that it may provide at least one of the signals by which the matrix conveys the "competence" that permits fibroblasts to replicate in the presence of an appropriate progression signal.     

Integrin on developing and adult skeletal muscle

Avian integrin is a complex of integral membrane glycoproteins that appears to function as a dual receptors for both intracellular cytoskeletal and extracellular matrix components. Antibodies were raised against this complex and used to (1) immunolocalize integrin on cryosections of developing and adult muscle tissue and on developing myotube cultures in vitro and (2) immunoaffinity purify integrin from various fiber-type specific muscles. Integrin localization was compared with that of its putative cytoskeletal-associated and extracellular matrix ligands, talin and vinculin and fibronectin and laminin, respectively. The goal was to identify putative sites of interaction between the muscle sarcolemma and the cytoskeleton and the extracellular matrix and to reveal any differences in the molecular composition at these sites. Integrin's distribution on the sarcolemma of early (Day 12) embryonic limb muscle was random and punctate. On late embryonic (Days 17-19) limb muscle tissue its distribution was generally uniform but with occasional increased densities at specific sites along the sarcolemma. Posthatch (greater than 3 weeks) fast twitch muscle showed a highly regionalized distribution. These regions of integrin concentration coincided with densities of acetylcholine receptors, revealed by TRITC alpha-bungarotoxin labeling, and regions of muscle-tendon interaction, identified by morphological criteria. Tissue culture studies also demonstrated integrin densities at analogous sites in vitro, e.g., acetylcholine receptor clusters and sites at which myofibrils terminate at the sarcolemma. These integrin-rich sites were also shown to be Triton X-100 insoluble and therefore presumably are linked to the cytoskeleton or extracellular matrix. The localization of integrin on developing and adult muscle tissue was compared with that of fibronectin, laminin, vinculin, and talin using double, immunofluorescently labeled cryosections. In general, integrin did not colocalize exclusively with any one of its putative ligands. In the embryo, discrete densities of both talin and vinculin were observed at the myotendinous junction, whereas integrin immunoreactivity was widely distributed on muscle, vasculature, nerve, and connective tissue with no discernible sites of increased density. Laminin was primarily associated with muscle and nerve whereas fibronectin was prominent on connective tissue. On posthatch tissue, the distributions of talin, vinculin, laminin, and fibronectin were similar to those in the embryo, whereas the distribution of integrin was restricted to specific sites. The distribution of integrin was also examined for fiber-type specific differences on adu     

Increase in rat soleus myotendinous interface after a 14-d spaceflight

Myotenclinous junctions (MTJs) transmit contractile force from skeletal muscles to tendons. The effects of a 14-d spaceflight on MTJ were studied in the soleus muscle of male adult Sprague Dawley rats by transmission electron microscopy and histomorphometric techniques. We showed that the length of the junctional membrane relative to the muscle fiber diameter increased by 58% after 14 d of spaceflight. This increase accompanies morphological changes at MTJs. The flight MTJs appeared more shredded. The ends of the muscle fibers exhibited T tubule dilatation, swollen mitochondria, Z-disk streaming, loss of myofilaments, a thinning down of subplasmalemmal densities, multivesicular bodies and signs of junctional membrane and basal lamina remodelling. The ultrastructural observations suggest that the increase in myotendinous interface could result from the extracellular matrix spreading into remodelling muscle fiber, whereas the constraints related to unloading were reduced by spaceflight conditions.