Myotendinous junctions of tonic muscle cells: structure and loading

Summary Regions within frog semitendinosus muscle that are rich in tonic muscle cells were identified histochemically by myosin adenosine triphosphatase- and succinic dehydrogenase-staining procedures. Bundles of cells still attached to tendinous insertions were removed from those sites, prepared for electron microscopy and sectioned longitudinally through their myotendinous junctions. Tonic cells were identified by electron-microscopic criteria and their myotendinous junctions' morphology evaluated by morphometry. Although junctional components appear identical to those in twitch cells, the degree of membrane folding increases tonic junction area by a factor of 50.2 whereas twitch cells' junctional area is increased 22.2 times by folding relative to cells terminating as right circular cylinders. Calculations show that the tonic cell junction bears average loads of 3.4×10³ N · m^-2 during maximum force generation and that nearly all of the load is borne as shear stress at the junction. The junctions of twitch cells bear average loads of 1.6×10⁴ N · m^-2 during peak tension. The findings indicate that the magnitude of loading does not alone determine the degree of junctional membrane folding. Interpretation of the data in view of viscoelastic behavior of membranes indicates that duration of loading may be a functionally important correlate to degree of membrane folding at myotendinous junctions.     

Desmin at myotendinous junctions

Myofibrils are linked to the cell membrane at myotendinous junctions located at the ends of muscle fibers, and at costameres, sites positioned periodically along lateral surfaces of muscle cells. Both of these sites are enriched in proteins that link active components of myofibrils to the cell membrane. Costameres are also enriched in desmin intermediate filaments that link passive components of myofibrils to the lateral surfaces of muscle cells. In this study, the possibility that desmin is also found between the terminal Z-disk of myofibrils and the myotendinous junction membrane is examined by immunocytochemistry and by KI-extraction procedures. Data presented show that desmin is located in the filamentous core of cellular processes at myotendinous junctions at sites 30 nm or more from the membrane. This core lies deep to subsarcolemmal material previously shown to contain talin, vinculin, and dystrophin. The distance from desmin to the membrane suggests desmin does not interact directly with membrane proteins at the junction. Immunoblots and indirect immunofluorescence of junctional regions of muscle compared to nonjunctional regions show no apparent enrichment of desmin at junctional sites, although vinculin, another costameric and junctional component, is significantly enriched at junctional regions. These findings show that passive elements of myofibrils may be continuous from myotendinous junctions of muscle origin to insertion via desmin filaments located between terminal Z-disks and the junctional membrane. This can provide a system in parallel to that involving thin filaments, vinculin, and talin for linking myofibrils to the cell membrane at myotendinous junctions.     

A novel marker of tissue junctions, collagen XXII

Here we describe a novel specific component of tissue junctions, collagen XXII. It was first identified by screening an EST data base and subsequently expressed as a recombinant protein and characterized as an authentic tissue component. The COL22A1 gene on human chromosome 8q24.2 encodes a collagen that structurally belongs to the FACIT protein family (fibril-associated collagens with interrupted triple helices). Collagen XXII exhibits a striking restricted localization at tissue junctions such as the myotendinous junction in skeletal and heart muscle, the articular cartilage-synovial fluid junction, or the border between the anagen hair follicle and the dermis in the skin. It is deposited in the basement membrane zone of the myotendinous junction and the hair follicle and associated with the extrafibrillar matrix in cartilage. In situ hybridization of myotendinous junctions revealed that muscle cells produce collagen XXII, and functional tests demonstrated that collagen XXII acts as a cell adhesion ligand for skin epithelial cells and fibroblasts. This novel gene product, collagen XXII, is the first specific extracellular matrix protein present only at tissue junctions.     

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

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

Nestin is expressed during development and in myotendinous and neuromuscular junctions in wild type and desmin knock-out mice.

Desmin, the main component of intermediate filaments (IFs) in mature skeletal muscle, forms an interlinking scaffold around myofibrils with connections to the sarcolemma and the nuclear membrane. Desmin is enriched in neuromuscular and myotendinous junctions. Mice lacking the desmin gene develop normally and reproduce. However, postnatally they develop a cardiomyopathy and a dystrophy in highly used muscles. We have investigated whether and how neuromuscular and myotendinous junctions are affected and whether nestin compensates for the lack of desmin in the knock-out (K/O) mice. We show that neither neuromuscular nor myotendinous junctions were markedly affected in the desmin K/O mice. In neuromuscular junctions nestin was present between the postjunctional folds and the subneural nuclei and between the nucleus and the myofibrillar cytoskeleton. In myotendinous junctions nestin was present between myofibrils at the Z-disc level and in longitudinal strands close to and at the junction. Nestin expression at these specialized sites, as well as during myogenesis and myofibrillogenesis, is independent of the presence of desmin. In desmin K/O mice nestin was also found in regenerating myofibers. The presence of nestin at neuromuscular and myotendinous junctions might provide enough strength for preservation and organization of the junctional areas, although desmin is lacking.     

Talin at myotendinous junctions

Junctions formed by skeletal muscles where they adhere to tendons, called myotendinous junctions, are sites of tight adhesion and where forces generated by the cell are placed on the substratum. In this regard, myotendinous junctions and focal contacts of fibroblasts in vitro are analogues. Talin is a protein located at focal contacts that may be involved in force transmission from actin filaments to the plasma membrane. This study investigates whether talin is also found at myotendinous junctions. Protein separations on SDS polyacrylamide gels and immunolabeling procedures show that talin is present in skeletal muscle. Immunofluorescence microscopy using anti-talin indicates that talin is found concentrated at myotendinous junctions and in lesser amounts in periodic bands over nonjunctional regions. Electron microscopic immunolabeling shows talin is a component of the digitlike processes of muscle cells that extend into tendons at myotendinous junctions. These findings indicate that there may be similarities in the molecular composition of focal contacts and myotendinous junctions in addition to functional analogies.     

Characterization of the column and autocellular junctions that define the vasculature of gill lamellae.

Novel adhesion junctions have been characterized that are formed at the interface between pillar cells and collagen columns, both of which are essential constituents of the gill lamellae in fish. We termed these junctions the "column junction" and "autocellular junction" and determined their molecular compositions by immunofluorescence microscopy using pufferfish. We visualized collagen columns by concanavalin A staining and found that the components of integrin-mediated cell-matrix adhesion, such as talin, vinculin, paxillin, and fibronectin, were concentrated on plasma membranes surrounding collagen columns (column membranes). This connection is analogous to the focal adhesion of cultured mammalian cells, dense plaque of smooth muscle cells, and myotendinous junction of skeletal muscle cells. We named this connection the "column junction." In the cytoplasm near the column, actin fibers, actinin, and a phosphorylated myosin light chain of 20 kDa are densely located, suggesting the contractile nature of pillar cells. The membrane infoldings surrounding the collagen columns were found to be connected by the autocellular junction, whose components are highly tyrosine-phosphorylated and contain the tight junction protein ZO-1. This study represents the first molecular characterization and fluorescence visualization of the column and autocellular junctions involved in both maintaining structural integrity and the hemodynamics of the branchial lamellae.     

Injury to nerves and the initiation of amphibian limb regeneration

The force produced within skeletal muscle fibers is transmitted to the bone via a myotendinous junction. This junctional region was examined by light and electron microscopy in the sartorius muscles of three Rana temporaria. The muscle fibers tapered and inserted at an angle of about 25 degrees with the connective tissue fascia near the bone. The composition of the structures within the last 100 microns of the fiber was analyzed morphometrically. The T-system, terminal cisternae, and caveolae were the same as in the central region of the muscle fiber. However, the mitochondrial content was higher and the volume of longitudinal sarcoplasmic reticulum was lower than elsewhere in the fiber. The membrane at the end of the fiber had extensive villiform processes interdigitating with the tendon. The surface area of the membrane around the villiform processes was estimated with point-counting techniques and calculated from the stereological equations appropriate for partially anisotropic structures. The extra membrane involved in the myotendinous junction was about 32 times that of the cross-sectional area of the fiber. Part of this additional membrane contained specialized adherens junctions through which the contractile proteins of the muscle are anchored to collagen. The increased area at the myotendinous junction presumably provides greater mechanical strength than a flat termination. The high values of membrane capacitance and specific resistance measured electrophysiologically at the end of the fiber also can be attributed to the characteristics of the terminal membrane structure.     

The passive mechanical properties of the extensor digitorum longus muscle are compromised in 2- to 20-mo-old mdx mice

Muscle rigidity and myotendinous junction (MTJ) deficiency contribute to immobilization in Duchenne muscular dystrophy (DMD), a lethal disease caused by the absence of dystrophin. However, little is known about the muscle passive properties and MTJ strength in a diseased muscle. Here, we hypothesize that dystrophin-deficient muscle pathology renders skeletal muscle stiffer and MTJ weaker. To test our hypothesis, we examined the passive properties of an intact noncontracting muscle-tendon unit in mdx mice, a mouse model for DMD. The extensor digitorum longus (EDL) muscle-tendon preparations of 2-, 6-, 14-, and 20-mo-old mdx and normal control mice were strained stepwisely from 110% to 160% of the muscle optimal length. The stress-strain response and failure position were analyzed. In support of our hypothesis, the mdx EDL preparation consistently developed higher stress before muscle failure. Postfailure stresses decreased dramatically in mdx but not normal preparations. Further, mdx showed a significantly faster stress relaxation rate. Consistent with stress-strain assay results, we observed significantly higher fibrosis in mdx muscle. In 2- and 6-mo-old mdx and 20-mo-old BL10 mice failure occurred within the muscle (2- to 14-mo-old BL10 preparations did not fail). Interestingly, in ≥14-mo-old mdx mice the failure site shifted toward the MTJ. Electron microscopy revealed substantial MTJ degeneration in aged but not young mdx mice. In summary, our results suggest that the passive properties of the EDL muscle and the strength of MTJ are compromised in mdx in an age-dependent manner. These findings offer new insights in studying DMD pathogenesis and developing novel therapies.     

Myomuscular junctions in the gill-sac muscle of the Atlantic hagfish,Myxine glutinosa: analogy with myotendinous junctions

Summary Myomuscular junctions between muscle fibers in the gill sacs of the Atlantic hagfish, Myxine glutinosa, were examined by electron microscopy. According to the presence of sarcolemmal differentiations typical of myotendinous junctions, the myomuscular junctions can be described as a symmetric myotendinous junction     

Transgenic isolation of skeletal muscle and kidney defects in laminin beta2 mutant mice: implications for Pierson syndrome

Pierson syndrome is a recently defined disease usually lethal within the first postnatal months and caused by mutations in the gene encoding laminin beta2 (LAMB2). The hallmarks of Pierson syndrome are congenital nephrotic syndrome accompanied by ocular abnormalities, including microcoria (small pupils), with muscular and neurological developmental defects also present. Lamb2(-/-) mice are a model for Pierson syndrome; they exhibit defects in the kidney glomerular barrier, in the development and organization of the neuromuscular junction, and in the retina. Lamb2(-/-) mice fail to thrive and die very small at 3 weeks of age, but to what extent the kidney and neuromuscular defects each contribute to this severe phenotype has been obscure, though highly relevant to understanding Pierson syndrome. To investigate this, we generated transgenic mouse lines expressing rat laminin beta2 either in muscle or in glomerular epithelial cells (podocytes) and crossed them onto the Lamb2(-/-) background. Rat beta2 was confined in skeletal muscle to synapses and myotendinous junctions, and in kidney to the glomerular basement membrane. In transgenic Lamb2(-/-) mice, beta2 deposition in only glomeruli prevented proteinuria but did not ameliorate the severe phenotype. By contrast, beta2 expression in only muscle restored synaptic architecture and led to greatly improved health, but the mice died from kidney disease at 1 month. Rescue of both glomeruli and synapses was associated with normal weight gain, fertility and lifespan. We conclude that muscle defects in Lamb2(-/-) mice are responsible for the severe failure to thrive phenotype, and that renal replacement therapy alone will be an inadequate treatment for Pierson syndrome.     

Structural changes at the myogenic cell surface during the formation of myotendinous junctions

Summary Myotendinous junctions are sites which are morphologically and molecularly specialized for force transmission between intracellular and extracellular structural proteins. In the present investigation, the formation of these specialized junctions is studied in chicken embryos from 9 days following fertilization to 1 day posthatching, using light and electron microscopy. Observations indicate that the first discernible event in myotendinous junction formation is the appearance of basement membrane at the incipient junction at 9–10 days postfertilization, concomitant with the aggregation of fibroblasts at the junctional regions of myogenic cells. Subsequently, subsarcolemmal densities appear at sites opposite basement membrane locations by 13 days postfertilization. Myofibrils insert into subsarcolemmal densities by day 15 and invaginations of the cell membrane are initiated at those insertions. Type I collagen fibers appear at the cell surface at day 17. Junctional structure at day 17 qualitatively resembles that of adult myotendinous junctions. Changes in junctional structure following day 17 are primarily increases in the amount of subsarcolemmal densities, myofibril-membrane associations, and amount of junctional membrane folding.     

Location of the integrin complex and extracellular matrix molecules at the chicken myotendinous junction

Summary The distribution of several extracellular matrix macromolecules was investigated at the myotendinous junction of adult chicken gastrocnemius muscle. Localization using monoclonal antibodies specific for 3 basal lamina components (type IV collagen, laminin, and a basement membrane form of heparan sulfate proteoglycan) showed strong fluorescent staining of the myotendinous junction for heparan sulfate proteoglycan and laminin, but not for type IV collagen. In addition, a strong fluorescent stain was observed at the myotendinous junction using a monoclonal antibody against the subunit of the chicken integrin complex (antibody JG 22). Neither fibronectin nor tenascin were concentrated at the myotendinous junction, but instead were present in a fibrillar staining pattern throughout the connective tissue which was closely associated with the myotendinous junction. Tenascin also gave bright fluorescent staining of tendon, but no detectable staining of the perimysium or endomysium. Type I collagen was observed throughout the tendon and in the perimysium, but only faintly in the endomysium. In contrast, type III collagen was present brightly in the endomysium and in the perimysium, but could not be detected in the tendon except when associated with blood vessels and in the epitendineum, which stained intensely. Type VI collagen was found throughout the tendon and in all connective tissue partitions of skeletal muscle. The results indicate that one or more molecules of the integrin family may play an important role in the attachment of muscle to the tendon. This interaction does not appear to involve extensive binding to fibronectin or tenascin, but may involve laminin and heparan sulfate proteoglycan.     

Moleskin is essential for the formation of the myotendinous junction in Drosophila

It is the precise connectivity between skeletal muscles and their corresponding tendon cells to form a functional myotendinous junction (MTJ) that allows for the force generation required for muscle contraction and organismal movement. The Drosophila MTJ is composed of secreted extracellular matrix (ECM) proteins deposited between integrin-mediated hemi-adherens junctions on the surface of muscle and tendon cells. In this paper, we have identified a novel, cytoplasmic role for the canonical nuclear import protein Moleskin (Msk) in Drosophila embryonic somatic muscle attachment. Msk protein is enriched at muscle attachment sites in late embryogenesis and msk mutant embryos exhibit a failure in muscle–tendon cell attachment. Although the muscle–tendon attachment sites are reduced in size, components of the integrin complexes and ECM proteins are properly localized in msk mutant embryos. However, msk mutants fail to localize phosphorylated focal adhesion kinase (pFAK) to the sites of muscle–tendon cell junctions. In addition, the tendon cell specific proteins Stripe (Sr) and activated mitogen-activated protein kinase (MAPK) are reduced in msk mutant embryos. Our rescue experiments demonstrate that Msk is required in the muscle cell, but not in the tendon cells. Moreover, muscle attachment defects due to loss of Msk are rescued by an activated form of MAPK or the secreted epidermal growth factor receptor (Egfr) ligand Vein. Taken together, these findings provide strong evidence that Msk signals non-autonomously through the Vein-Egfr signaling pathway for late tendon cell late differentiation and/or maintenance.     

The transmembrane protein Perdido interacts with Grip and integrins to mediate myotube projection and attachment in the Drosophila embryo

The molecular mechanisms underlying muscle guidance and formation of myotendinous junctions are poorly understood both in vertebrates and in Drosophila. We have identified a novel gene that is essential for Drosophila embryonic muscles to form proper projections and stable attachments to epidermal tendon cells. Loss-of-function of this gene - which we named perdido (perd)-results in rounded, unattached muscles. perd is expressed prior to myoblast fusion in a subset of muscle founder cells, and it encodes a conserved single-pass transmembrane cell adhesion protein that contains laminin globular extracellular domains and a small intracellular domain with a C-terminal PDZ-binding consensus sequence. Biochemical experiments revealed that the Perd intracellular domain interacts directly with one of the PDZ domains of the Glutamate receptor interacting protein (Grip), another factor required for formation of proper muscle projections. In addition, Perd is necessary to localize Grip to the plasma membrane of developing myofibers. Using a newly developed, whole-embryo RNA interference assay to analyze genetic interactions, perd was shown to interact not only with Grip but also with multiple edematous wings, which encodes one subunit of the alpha PS1-beta PS integrin expressed in tendon cells. These experiments uncovered a previously unrecognized role for the alpha PS1-beta PS integrin in the formation of muscle projections during early stages of myotendinous junction development. We propose that Perd regulates projection of myotube processes toward and subsequent differentiation of the myotendinous junction by priming formation of a protein complex through its intracellular interaction with Grip and its transient engagement with the tendon cell-expressed laminin-binding alpha PS1-beta PS integrin.     

The subcellular distribution of dystrophin in mouse skeletal, cardiac, and smooth muscle

We use a highly specific and sensitive antibody to further characterize the distribution of dystrophin in skeletal, cardiac, and smooth muscle. No evidence for localization other than at the cell surface is apparent in skeletal muscle and no 427-kD dystrophin labeling was detected in sciatic nerve. An elevated concentration of dystrophin appears at the myotendinous junction and the neuromuscular junction, labeling in the latter being more intense specifically in the troughs of the synaptic folds. In cardiac muscle the distribution of dystrophin is limited to the surface plasma membrane but is notably absent from the membrane that overlays adherens junctions of the intercalated disks. In smooth muscle, the plasma membrane labeling is considerably less abundant than in cardiac or skeletal muscle and is found in areas of membrane underlain by membranous vesicles. As in cardiac muscle, smooth muscle dystrophin seems to be excluded from membrane above densities that mark adherens junctions. Dystrophin appears as a doublet on Western blots of skeletal and cardiac muscle, and as a single band of lower abundance in smooth muscle that corresponds most closely in molecular weight to the upper band of the striated muscle doublet. The lower band of the doublet in striated muscle appears to lack a portion of the carboxyl terminus and may represent a dystrophin isoform. Isoform differences and the presence of dystrophin on different specialized membrane surfaces imply multiple functional roles for the dystrophin protein.     

Zasp is required for the assembly of functional integrin adhesion sites

The integrin family of heterodimeric transmembrane receptors mediates cell–matrix adhesion. Integrins often localize in highly organized structures, such as focal adhesions in tissue culture and myotendinous junctions in muscles. Our RNA interference screen for genes that prevent integrin-dependent cell spreading identifies Z band alternatively spliced PDZ-motif protein (zasp), encoding the only known Drosophila melanogaster Alp/Enigma PDZ-LIM domain protein. Zasp localizes to integrin adhesion sites and its depletion disrupts integrin adhesion sites. In tissues, Zasp colocalizes with βPS integrin in myotendinous junctions and with α-actinin in muscle Z lines. Zasp also physically interacts with α-actinin. Fly larvae lacking Zasp do not form Z lines and fail to recruit α-actinin to the Z line. At the myotendinous junction, muscles detach in zasp mutants with the onset of contractility. Finally, Zasp interacts genetically with integrins, showing that it regulates integrin function. Our observations point to an important function for Zasp in the assembly of integrin adhesion sites both in cell culture and in tissues.     

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.     

Organization of the nematocyst battery in the tentacle of hydra: Arrangement of the complex anchoring junctions between nematocytes,epithelial cells,and basement membrane

Summary Nematocytes (stinging cells) of hydra tentacles are anchored to the basement membrane by peculiar complex junctions in which a flattened tongue of an epithelial cell is interposed between the nematocyte and the basement membrane. In this paper we describe the arrangement of these junctions with emphasis on how they are related to the architecture of the epithelial cell. Each epithelial cell, called a battery cell, harbors 10–20 nematocytes and bears muscle processes that extend along the basement membrane. The epithelial cell component of the complex junction is usually a lateral extension of a muscle process. All nematocytes within a battery cell make junctions with muscle processes of the same (resident) epithelial battery cell despite the presence of numerous muscle processes from adjacent (foreign) cells. Some nematocytes make junctions with several resident processes, spanning the foreign processes to do so. Most junctions reside near the proximal ends of the muscle processes. New findings are reported on the substructure of the junctions. They are composed of aggregates of smaller elements, and the cytoskeleton within the complexes has a pronounced longitudinal organization. These observations are consistent with a suggestion that the complex junctions develop by aggregation of smaller junctional units originating elsewhere on the cells.