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.     

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.     

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.     

Integrins modulate the Egfr signaling pathway to regulate tendon cell differentiation in the Drosophila embryo

Changes in the extracellular matrix (ECM) govern the differentiation of many cell types during embryogenesis. Integrins are cell matrix receptors that play a major role in cell-ECM adhesion and in transmitting signals from the ECM inside the cell to regulate gene expression. In this paper, it is shown that the PS integrins are required at the muscle attachment sites of the Drosophila embryo to regulate tendon cell differentiation. The analysis of the requirements of the individual alpha subunits, alphaPS1 and alphaPS2, demonstrates that both PS1 and PS2 integrins are involved in this process. In the absence of PS integrin function, the expression of tendon cell-specific genes such as stripe and beta1 tubulin is not maintained. In addition, embryos lacking the PS integrins also exhibit reduced levels of activated MAPK. This reduction is probably due to a downregulation of the Epidermal Growth Factor receptor (Egfr) pathway, since an activated form of the Egfr can rescue the phenotype of embryos mutant for the PS integrins. Furthermore, the levels of the Egfr ligand Vein at the muscle attachment sites are reduced in PS mutant embryos. Altogether, these results lead to a model in which integrin-mediated adhesion plays a role in regulating tendon cell differentiation by modulating the activity of the Egfr pathway at the level of its ligand Vein.     

Progressive myopathy and defects in the maintenance of myotendinous junctions in mice that lack talin 1 in skeletal muscle

The development and function of skeletal muscle depend on molecules that connect the muscle fiber cytoskeleton to the extracellular matrix (ECM). beta1 integrins are ECM receptors in skeletal muscle, and mutations that affect the alpha7beta1 integrin cause myopathy in humans. In mice, beta1 integrins control myoblast fusion, the assembly of the muscle fiber cytoskeleton, and the maintenance of myotendinous junctions (MTJs). The effector molecules that mediate beta1 integrin functions in muscle are not known. Previous studies have shown that talin 1 controls the force-dependent assembly of integrin adhesion complexes and regulates the affinity of integrins for ligands. Here we show that talin 1 is essential in skeletal muscle for the maintenance of integrin attachment sites at MTJs. Mice with a skeletal muscle-specific ablation of the talin 1 gene suffer from a progressive myopathy. Surprisingly, myoblast fusion and the assembly of integrin-containing adhesion complexes at costameres and MTJs advance normally in the mutants. However, with progressive ageing, the muscle fiber cytoskeleton detaches from MTJs. Mechanical measurements on isolated muscles show defects in the ability of talin 1-deficient muscle to generate force. Collectively, our findings show that talin 1 is essential for providing mechanical stability to integrin-dependent adhesion complexes at MTJs, which is crucial for optimal force generation by skeletal muscle.     

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.     

“Importin” signaling roles for import proteins

The formation of a mature myotendinous junction (MTJ) between a muscle and its site of attachment is a highly regulated process that involves myofiber migration, cell-cell signaling, and culminates with the stable adhesion between the adjacent muscle-tendon cells. Improper establishment or maintenance of muscle-tendon attachment sites results in a decrease in force generation during muscle contraction and progressive muscular dystrophies in vertebrate models. Many studies have demonstrated the important role of the integrins and integrin-associated proteins in the formation and maintenance of the MTJ. We recently demonstrated that moleskin (msk), the gene that encodes for Drosophila importin-7 (DIM-7), is required for the proper formation of muscle-tendon adhesion sites in the developing embryo. Further studies demonstrated an enrichment of DIM-7 to the ends of muscles where the muscles attach to their target tendon cells. Genetic analysis supports a model whereby msk is required in the muscle and signals via the secreted epidermal growth factor receptor (Egfr) ligand Vein to regulate tendon cell maturation. These data demonstrate a novel role for the canonical nuclear import protein DIM-7 in establishment of the MTJ.     

“Importin” signaling roles for import proteins: The function of Drosophila importin-7 (DIM-7) in muscle-tendon signaling

The formation of a mature myotendinous junction (MTJ) between a muscle and its site of attachment is a highly regulated process that involves myofiber migration, cell-cell signaling, and culminates with the stable adhesion between the adjacent muscle-tendon cells. Improper establishment or maintenance of muscle-tendon attachment sites results in a decrease in force generation during muscle contraction and progressive muscular dystrophies in vertebrate models. Many studies have demonstrated the important role of the integrins and integrin-associated proteins in the formation and maintenance of the MTJ. We recently demonstrated that moleskin (msk), the gene that encodes for Drosophila importin-7 (DIM-7), is required for the proper formation of muscle-tendon adhesion sites in the developing embryo. Further studies demonstrated an enrichment of DIM-7 to the ends of muscles where the muscles attach to their target tendon cells. Genetic analysis supports a model whereby msk is required in the muscle and signals via the secreted epidermal growth factor receptor (Egfr) ligand Vein to regulate tendon cell maturation. These data demonstrate a novel role for the canonical nuclear import protein DIM-7 in establishment of the MTJ.     

Adhesive strength of single muscle cells to basement membrane at myotendinous junctions

Whole muscles loaded to failure frequently fail at or near myotendinous junctions. The present investigation was directed toward determining the breaking stress and failure site of intact and injured myotendinous junction preparations consisting of muscle cells dissected free from surrounding parallel structures but still attached to tendon collagen fibers. These tests show that the breaking stress for intact myotendinous units is 2.7 x 10(5) N/m2, expressed relative to cell cross-sectional area. Failure occurs immediately external to the junction membrane between the cell membrane and lamina densa of the basement membrane. Site and stress at failure are independent of strain and strain rate over a biologically relevant range. Breaking stress in the plane of the membrane, corrected for membrane folding, is 1.2 X 10(4) N/m2. This value is not significantly greater than stress at maximum isometric tension for these cells at these sarcomere lengths. After compression injury, cells fail within the compression site at significantly lower stress (1.9 X 10(5) N/m2). These findings suggest that, in muscle strain injuries that occur under conditions simulated here, failure occurs at myotendinous junctions unless the muscle has suffered previous compression injury leading to failure within the muscle.     

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)     

Modulation of Integrin Activity is Vital for Morphogenesis

Cells can vary their adhesive properties by modulating the affinity of integrin receptors. The activation and inactivation of integrins by inside-out mechanisms acting on the cytoplasmic domains of the integrin subunits has been demonstrated in platelets, lymphocytes, and keratinocytes. We show that in the embryo, normal morphogenesis requires the α subunit cytoplasmic domain to control integrin adhesion at the right times and places. PS2 integrin (α_PS2β_PS) adhesion is normally restricted to the muscle termini, where it is required for attaching the muscles to the ends of other muscles and to specialized epidermal cells. Replacing the wild-type α_PS2 with mutant forms containing cytoplasmic domain deletions results in the rescue of the majority of defects associated with the absence of the α_PS2 subunit, however, the mutant PS2 integrins are excessively active. Muscles containing these mutant integrins make extra muscle attachments at aberrant positions on the muscle surface, disrupting the muscle pattern and causing embryonic lethality. A gain- of-function phenotype is not observed in the visceral mesoderm, showing that regulation of integrin activity is tissue-specific. These results suggest that the α_PS2 subunit cytoplasmic domain is required for inside-out regulation of integrin affinity, as has been seen with the integrin α_IIbβ₃.     

Migration of the Drosophila primordial midgut cells requires coordination of diverse PS integrin functions.

Cell migration during embryogenesis involves two populations of cells: the migrating cells and the underlying cells that provide the substratum for migration. The formation of the Drosophila larval midgut involves the migration of the primordial midgut cells along a visceral mesoderm substratum. We show that integrin adhesion receptors are required in both populations of cells for normal rates of migration. In the absence of the PS integrins, the visceral mesoderm is disorganised, the primordial midgut cells do not display their normal motile appearance and their migration is delayed by 2 hours. Removing PS integrin function from the visceral mesoderm alone results in visceral mesoderm disorganization, but only causes a modest delay in migration and does not affect the appearance of the migrating cells. Removing PS integrin function from the migrating cells causes as severe a delay in migration as the complete loss of PS integrin function. The functions of PS1 and PS2 are specific in the two tissues, endoderm and mesoderm, since they cannot substitute for each other. In addition there is a partial redundancy in the function of the two PS integrins expressed in the endoderm, PS1 (alphaPS1betaPS) and PS3 (alphaPS3betaPS), since loss of just one alpha subunit in the midgut results in either a modest delay (alphaPS1) or no effect (alphaPS3). We have also examined the roles of small GTPases in promoting migration of the primordial midgut cells. We find that dominant negative (N17) versions of Rac and Cdc42 cause a very similar defect in migration as loss of integrins, while those of Rho and Ras have no effect. Thus integrins are involved in mediating migration by creating an optimal substratum for adhesion, adhering to that substratum and possibly by activating Rac and Cdc42.     

Intracellular signals direct integrin localization to sites of function in embryonic muscles

In the Drosophila embryo, the alphaPS2betaPS integrin heterodimer is localized tightly at the termini of the multinucleate muscles where they attach to the alphaPS1betaPS-containing epidermal tendon cells. Here we examine the basis for alphaPS2betaPS integrin subcellular localization. We show that the betaPS cytoplasmic tail is sufficient to direct the localization of a heterologous transmembrane protein, CD2, to the muscle termini in vivo. This localization does not occur via an association with structures set up by the endogenous betaPS integrins, since it can occur even in the absence of the betaPS protein. Furthermore, the subcellular localization of the alphaPS2betaPS integrin is not dependent on any other interactions between the muscles and the tendon cells. In embryos that lack the segmental tendon cells, due to a mutation removing the related segment polarity genes engrailed and invected, alphaPS2betaPS is still localized to the muscle termini even though the ventral longitudinal muscles are not attached to the epidermis, but instead are attached end to end. Thus the alphaPS2betaPS integrin can be localized by an intracellular mechanism within the muscles. Our results challenge the view that the transmission of signals from the extracellular environment via integrins is required for the organization of the cytoskeleton and the resultant cellular polarity.     

Tenectin is a novel αPS2βPS integrin ligand required for wing morphogenesis and male genital looping in Drosophila

Morphogenesis of the adult structures of holometabolous insects is regulated by ecdysteroids and juvenile hormones and involves cell-cell interactions mediated in part by the cell surface integrin receptors and their extracellular matrix (ECM) ligands. These adhesion molecules and their regulation by hormones are not well characterized. We describe the gene structure of a newly described ECM molecule, tenectin, and demonstrate that it is a hormonally regulated ECM protein required for proper morphogenesis of the adult wing and male genitalia. Tenectin's function as a new ligand of the PS2 integrins is demonstrated by both genetic interactions in the fly and by cell spreading and cell adhesion assays in cultured cells. Its interaction with the PS2 integrins is dependent on RGD and RGD-like motifs. Tenectin's function in looping morphogenesis in the development of the male genitalia led to experiments that demonstrate a role for PS integrins in the execution of left-right asymmetry.     

Tendon-muscle crosstalk controls muscle bellies morphogenesis, which is mediated by cell death and retinoic acid signaling

Vertebrate muscle morphogenesis is a complex developmental process, which remains quite yet unexplored at cellular and molecular level. In this work, we have found that sculpturing programmed cell death is a key morphogenetic process responsible for the formation of individual foot muscles in the developing avian limb. Muscle fibers are produced in excess in the precursor dorsal and ventral muscle masses of the limb bud and myofibers lacking junctions with digital tendons are eliminated via apoptosis. Microsurgical experiments to isolate the developing muscles from their specific tendons are consistent with a role for tendons in regulating survival of myogenic cells. Analysis of the expression of Raldh2 and local treatments with retinoic acid indicate that this signaling pathway mediates apoptosis in myogenic cells, appearing also involved in tendon maturation. Retinoic acid inhibition experiments led to defects in muscle belly segmentation and myotendinous junction formation. It is proposed that heterogeneous local distribution of retinoids controlled through Raldh2 and Cyp26A1 is responsible for matching the fleshy and the tendinous components of each muscle belly.     

Muscle mechanosensitive receptors close to the myotendinous junction of the Achilles tendon elicit a pressor reflex.

Feedback regulation by activation of mechanosensitive afferents in the exercising muscle causes the cardiovascular and sympathetic nerve responses, which follow tension development and are almost identical between static contraction and passive stretch. The precise location of the mechanoreceptors contributing to the exercise pressor reflex, however, remained unknown. To test the hypothesis that the mechanoreceptors will be located around the myotendinous junction to monitor a change in muscle tension than a change in muscle length, we examined the reflex cardiovascular responses to passive stretch of the triceps surae muscle in anesthetized rats with three interventions; systemic injection of gadolinium, cutting the Achilles tendon, and local injection of lidocaine into the myotendinous junction. Gadolinium (42 micromol/kg iv) blunted the increases in heart rate and mean arterial blood pressure during passive stretch by 36 and 22-26%, respectively, suggesting that the reflex cardiovascular responses were evoked by stimulation of muscle mechanosensitive receptors. The cardiovascular responses to passive stretch were not different between the cut Achilles tendon and the intact tendon in the same rats, suggesting that any mechanoreceptors, terminated in the more distal part of the tendon, did not contribute to the reflex cardiovascular responses. Lidocaine (volume, 0.04-0.1 ml) injected into the myotendinous junction blunted the stretch-induced increases in heart rate and mean arterial blood pressure by 37-49 and 27-34%, respectively. We conclude that the muscle mechanosensitive receptors evoking the reflex cardiovascular responses at least partly locate at or close to the myotendinous junction of the Achilles tendon.     

Repeated contractions alter the geometry of human skeletal muscle.

The aim of this study was to investigate the effect of repeated contractions on the geometry of human skeletal muscle. Six men performed two sets (sets A and B) of 10 repeated isometric plantarflexion contractions at 80% of the moment generated during plantarflexion maximal voluntary contraction (MVC), with a rest interval of 15 min between sets. By use of ultrasound, the geometry of the medial gastrocnemius (MG) muscle was measured in the contractions of set A and the displacement of the MG tendon origin in the myotendinous junction was measured in the contractions of set B. In the transition from the 1st to the 10th contractions, the fascicular length at 80% of MVC decreased from 34 +/- 4 (means +/- SD) to 30 +/- 3 mm (P < 0.001), the pennation angle increased from 35 +/- 3 to 42 +/- 3 degrees (P < 0.001), the myotendinous junction displacement increased from 5 +/- 3 to 10 +/- 3 mm (P < 0.001), and the average fascicular curvature remained constant (P > 0.05) at approximately 4.3 m(-1). No changes (P > 0.05) were found in fascicular length, pennation angle, and myotendinous junction displacement after the fifth contraction. Electrogoniometry showed that the ankle rotated by approximately 6.5 degrees during contraction, but no differences (P > 0.05) were obtained between contractions. The present results show that repeated contractions induce tendon creep, which substantially affects the geometry of the in-series contracting muscles, thus altering their potential for force and joint moment generation.