Acetylcholine receptor distribution on regenerating mammalian muscle fibers at sites of mature and developing nerve-muscle junctions

When rat soleus muscles fibers regenerated after notexin-induced damage, AChRs were present at high density on the surface of the new muscle fibers at the sites of the original NMJs, even if the intact motor axons were not present during regeneration. Some AChR molecules which were labelled with R-BgTx before notexin-induced damage persisted for some days at junctional sites after new muscle fibres had regenerated. During muscle fiber degeneration, components of the muscle fiber plasma membrane appeared to remain longer in the junctional region than elsewhere. When muscles on which new "ectopic" NMJs had been forming for at least 2 weeks were damaged, AChR clusters together with sites of high AChE activity were present 2 weeks later on the regenerated muscles in the region of new NMJ formation, even if the "foreign" nerve was not intact during the period of regeneration. If ectopic NMJs had been forming for only 4 days at the time of muscle and nerve damage, neither AChR clusters nor AChE activity were detected on the regenerated muscle fibers.     

Basal lamina directs acetylcholinesterase accumulation at synaptic sites in regenerating muscle

In skeletal muscles that have been damaged in ways which spare the basal lamina sheaths of the muscle fibers, new myofibers develop within the sheaths and neuromuscular junctions form at the original synaptic sites on them. At the regenerated neuromuscular junctions, as at the original ones, the muscle fibers are characterized by junctional folds and accumulations of acetylcholine receptors and acetylcholinesterase (AChE). The formation of junctional folds and the accumulation of acetylcholine receptors is known to be directed by components of the synaptic portion of the myofiber basal lamina. The aim of this study was to determine whether or not the synaptic basal lamina contains molecules that direct the accumulation of AChE. We crushed frog muscles in a way that caused disintegration and phagocytosis of all cells at the neuromuscular junction, and at the same time, we irreversibly blocked AChE activity. New muscle fibers were allowed to regenerate within the basal lamina sheaths of the original muscle fibers but reinnervation of the muscles was deliberately prevented. We then stained for AChE activity and searched the surface of the new muscle fibers for deposits of enzyme they had produced. Despite the absence of innervation, AChE preferentially accumulated at points where the plasma membrane of the new muscle fibers was apposed to the regions of the basal lamina that had occupied the synaptic cleft at the neuromuscular junctions. We therefore conclude that molecules stably attached to the synaptic portion of myofiber basal lamina direct the accumulation of AChE at the original synaptic sites in regenerating muscle. Additional studies revealed that the AChE was solubilized by collagenase and that it remained adherent to basal lamina sheaths after degeneration of the new myofibers, indicating that it had become incorporated into the basal lamina, as at normal neuromuscular junctions.     

Vitronectin at sites of cell-substrate contact in cultures of rat myotubes

Affinity-purified antibodies to the serum glycoprotein, vitronectin, were used to study sites of cell-substrate contact in cultures of rat myotubes and fibroblasts. Cells were removed from the substrate by treatment with saponin, leaving fragments of plasma membrane attached to the glass coverslip. When stained for vitronectin by indirect immunofluorescence, large areas of the substrate were brightly labeled. The focal contacts of fibroblasts and the broad adhesion plaques of myotubes appeared black, however, indicating that the antibodies had failed to react with those areas. Contact sites within the adhesion plaque remained unlabeled after saponin-treated samples were extracted with Triton X-100, or after intact cultures were sheared with a stream of fixative. These procedures expose extracellular macromolecules at the cell-substrate interface, which can then be labeled with concanavalin A. In contrast, when samples were sheared and then sonicated to remove all the cellular material from the coverslip, the entire substrate labeled extensively and almost uniformly with anti-vitronectin. Extracellular molecules associated with substrate contacts were also studied after freeze-fracture, using a technique we term "post-release fracture labeling." Platinum replicas of the external membrane were removed from the glass with hydrofluoric acid to expose the extracellular material. Anti-vitronectin, bound to the replicas and visualized by a second antibody conjugated to colloidal gold, labeled the broad areas of close myotube-substrate attachment and the nearby glass equally well. Our results are consistent with the hypothesis that vitronectin is present at all sites of cell-substrate contact, but that its antigenic sites are obscured by material deposited by both myotube and fibroblast cells.     

The influence of basal lamina on the accumulation of acetylcholine receptors at synaptic sites in regenerating muscle

If skeletal muscles are damaged in ways that spare the basal lamina sheaths of the muscle fibers, new myofibers develop within the sheaths and neuromuscular junctions form at the original synaptic sites on them. At the regenerated neuromuscular junctions, as at the original ones, the muscle fiber plasma membrane is characterized by infoldings and a high concentration of acetylcholine receptors (AChRs). The aim of this study was to determine whether or not the synaptic portion of the myofiber basal lamina sheath plays a direct role in the formation of the subsynaptic apparatus on regenerating myofibers, a question raised by the results of earlier experiments. The junctional region of the frog cutaneous pectoris muscle was crushed or frozen, which resulted in disintegration and phagocytosis of all cells at the synapse but left intact much of the myofiber basal lamina. Reinnervation was prevented. When new myofibers developed within the basal lamina sheaths, patches of AChRs and infoldings formed preferentially at sites where the myofiber membrane was apposed to the synaptic region of the sheaths. Processes from unidentified cells gradually came to lie on the presynaptic side of the basal lamina at a small fraction of the synaptic sites, but there was no discernible correlation between their presence and the effectiveness of synaptic sites in accumulating AChRs. We therefore conclude that molecules stably attached to the myofiber basal lamina at synaptic sites direct the formation of subsynaptic apparatus in regenerating myofibers. An analysis of the distribution of AChR clusters at synaptic sites indicated that they formed as a result of myofiber-basal lamina interactions that occurred at numerous places along the synaptic basal lamina, that their presence was not dependent on the formation of plasma membrane infoldings, and that the concentration of receptors within clusters could be as great as the AChR concentration at normal neuromuscular junctions.     

Acetylcholinesterase of the motor endplate and its response to muscle denervation

In innervated and denervated sternohyoid muscles of adult mice the AChE with a pH optimum at 7.2 was shown to occur in all three fiber types in two separate structural areals located: extrafibrillarly (synaptic cleft, postsynaptic folds, subsarcolemmal vesicles, T-tubules, interfibrillar space) and intrafibrillarly (perinuclear cisternae, SR including SR cisternae). There is not a stable connection between the two areas. The functional significance of the intrafibrillar AChE, in particular, is unknown. After muscle denervation, intrafibrillar AChE of the A and B fibers disappeares more quickly than that of C fibers. This phenomenon not only suggests a general, but possibly also a fiber-specific neurotrophic effect.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

PERK is required at the ER-mitochondrial contact sites to convey apoptosis after ROS-based ER stress

Endoplasmic reticulum stress is emerging as an important modulator of different pathologies and as a mechanism contributing to cancer cell death in response to therapeutic agents. In several instances, oxidative stress and the onset of endoplasmic reticulum (ER) stress occur together; yet, the molecular events linking reactive oxygen species (ROS) to ER stress-mediated apoptosis are currently unknown. Here, we show that PERK (RNA-dependent protein kinase (PKR)-like ER kinase), a key ER stress sensor of the unfolded protein response, is uniquely enriched at the mitochondria-associated ER membranes (MAMs). PERK^−/− cells display disturbed ER morphology and Ca²⁺ signaling as well as significantly weaker ER-mitochondria contact sites. Re-expression of a kinase-dead PERK mutant but not the cytoplasmic deletion mutant of PERK in PERK^−/− cells re-establishes ER-mitochondria juxtapositions and mitochondrial sensitization to ROS-mediated stress. In contrast to the canonical ER stressor thapsigargin, during ROS-mediated ER stress, PERK contributes to apoptosis twofold by sustaining the levels of pro-apoptotic C/EBP homologous protein (CHOP) and by facilitating the propagation of ROS signals between the ER and mitochondria through its tethering function. Hence, this study reveals an unprecedented role of PERK as a MAMs component required to maintain the ER-mitochondria juxtapositions and propel ROS-mediated mitochondrial apoptosis. Furthermore, it suggests that loss of PERK may cause defects in cell death sensitivity in pathological conditions linked to ROS-mediated ER stress.     

Interaction of nectin with afadin is necessary for its clustering at cell-cell contact sites but not for its cis dimerization or trans interaction

We have recently found a novel functional unit of cell-cell adhesion at cadherin-based adherens junctions, consisting of at least nectin, a homophilic cell adhesion molecule, and afadin, an actin filament-binding protein, which connects nectin to the actin cytoskeleton. Here we studied a mechanism of cell-cell adhesion of the nectin-afadin system by use of a cadherin-deficient L cell line stably expressing the intact form of mouse nectin-2alpha, a truncated form of nectin-2alpha incapable of interacting with afadin (nectin-2alpha-DeltaC), or a point-mutated form of nectin-2alpha capable of interacting with afadin and a cadherin-expressing EL cell line, which transiently expressed the point-mutated form of nectin-2alpha. We found that the interaction of nectin-2alpha with afadin was necessary for their clustering at cell-cell contact sites. However, nectin-2alpha-DeltaC showed cis dimerization and trans interaction, both of which did not require the interaction of nectin-2alpha with afadin. We have previously shown in EL cells that the interaction of nectin-1 with afadin is necessary for its recruitment to adherens junctions. We found that the trans interaction of nectin-2alpha was furthermore necessary for this recruitment. On the basis of these observations, we propose a model for the mechanism of cell-cell adhesion of nectin and roles of afadin in this mechanism.     

Scanning electron microscopy of nerve-muscle contacts in embryonic cell culture.

Scanning electron microscopy (SEM) of cell cultures of dissociated nerve and muscle from chick embryos has shown that developing muscle fibers can be contacted at many sites by one or more than one neuron, and that a single nerve can send branches to several myofibers. At these contact regions of nerve with muscle, the neurons send out terminal or lateral sprouts with fine tips which initially lack terminal swellings, but later acquire small “bouton”-like structures in contact with the sarcolemma, which resemble embryonic synapses. At these points, the sarcolemma does not appear to differ in ultrastructure from other surface regions of the myofiber. Transmission electron microscopy (TEM) has revealed the presence of both electron lucent and dense-cored vesicles at some nerve terminals. However, fluorescence histochemistry (Falck-Hillarp technique) failed to detect the presence of catecholamines in these cultures. The SEM pictures at substantially higher resolutions than the light microscope, and the enhanced three dimensional perspective of this technique, provide additional information about the developmental morphology of the nerve-muscle cell culture system. The results are correlated with previous findings by light microscopy, TEM and electrophysiology, and discussed in relationship to proposed innervation processes of skeletal muscle fibers in vivo.     

Modulation of mitochondrial contact sites formation in immature rat heart

Creatine phosphokinase-mediated transport of energy from the site of production to the site of consumption is a key process for meeting the energy-demands of reactions in cytosol. The mitochondrial creatine phosphokinase (mCPK) plays an important role in this process, with the enzyme activity localized particularly in the mitochondrial contact sites (MiCS). Earlier studies in adult animals have shown that the formation of MiCS varies in response to the energy demand and the physiological state of the heart, and it is stimulated by an increase in [Ca2+]i. However, there is little known about MiCS formation in juvenile hearts, characterized by metabolism different from adult hearts. In the present study we investigated the modulation of MiCS formation via Ca2+ in hearts of 14-day-old rats. The moderate response of MiCS to various stimuli (elevated extracellular Ca2+, diltiazem, cardiac arrest by Cd2+) may refer to a still increased intracellular Ca2+ concentration, the incomplete development of mitochondrial energy production as well as to persistingly high energy demand of the developing heart.     

Regulation of androgen receptor expression at the onset of functional overload in rat plantaris muscle

Skeletal muscle androgen receptor (AR) expression at the onset of functional overload (OV) has not been well described. It is also not known if overload and/or anabolic steroid differentially regulate AR expression. The purpose of this study was to examine AR gene expression at the onset of functional OV in rat plantaris muscle with and without nandrolone decanoate (ND) administration. The functional significance of AR protein induction was examined using skeletal alpha-actin promoter activity in transiently transfected CV-1 fibroblast cells. Male Sprague-Dawley rats ( approximately 125 g) were functionally overloaded for 1, 3, 7, or 21 days. A subset of animals was given an ND (6 mg/kg) injection at day 0 and then overloaded for 3 days. Control animals underwent sham surgeries. AR protein concentration increased 106 and 279% after 7 and 21 days of OV, respectively. AR mRNA increased 430% after 7 days of OV. AR protein expression in C2C12 murine myotubes subjected to 1% chronic radial stretch for 18 h was elevated 101% compared with control. ND treatment increased AR protein concentration 1,300% compared with controls, and there was no additional effect when ND and OV were combined. ND with 3 days of OV treatment increased AR mRNA expression 50% compared with control. AR overexpression in transiently transfected CV-1 fibroblast cells increased -424 bp skeletal alpha-actin promoter activity 80 to 1,800% in a dose-dependent fashion. Co-overexpression of either serum response factor (SRF) or active RhoA with AR overexpression induced a synergistic 36- and 28-fold induction of skeletal alpha-actin promoter. Cotransfection of AR, SRF, and active RhoA induced 180-fold increase in skeletal alpha-actin promoter activity. In conclusion, AR protein expression is increased after 7 days of functional OV, and this induction is regulated pretranslationally. AR induction in conjunction with SRF and RhoA signaling may be an important regulator of gene expression during overload-induced muscle growth.     

DLG1 is an anchor for the E3 ligase MARCH2 at sites of cell-cell contact

PDZ domain containing molecular scaffolds plays a central role in organizing synaptic junctions. Observations in Drosophila and mammalian cells have implicated that ubiquitination and endosomal trafficking, of molecular scaffolds are critical to the development and maintenance of cell-cell junctions and cell polarity. To elucidate if there is a connection between these pathways, we applied an integrative genomic strategy, which combined comparative genomics and proteomics with cell biological assays. Given the importance of ubiquitin in regulating endocytic processes, we first identified the subset of E3 ligases with conserved PDZ binding motifs. Among this subset, the MARCH family ubiquitin ligases account for the largest family and MARCH2 has been previously implicated in endosomal trafficking. Next, we tested in an unbiased fashion, if MARCH2 binds PDZ proteins in vivo using a modified tandem affinity purification strategy followed by mass spectrometry. Of note, DLG1 was co-purified from MARCH2, with subsequent confirmation that MARCH2 interacts with full-length DLG1 in a PDZ domain dependent manner. Furthermore, we demonstrated that MARCH2 co-localized with DLG1 at sites of cell-cell contact. In addition, loss of the MARCH2 PDZ binding motif led to loss of MARCH2 localization at cell-cell contact sites and MARCH2 appeared to localize away from cell-cell junctions. In in vivo ubiquitination assays we show that MARCH2 promotes DLG1 ubiquitination. Overall, these results suggest that PDZ ligands with E3 ligase activity may link PDZ domain containing tumor suppressors to endocytic pathways and cell polarity determination.     

α-Smooth muscle actin is transiently expressed in embryonic rat cardiac and skeletal muscles

Actin isoform expression may change during development, and in certain physiological, experimental and pathological situations. It is accepted that during sarcomeric (skeletal and cardiac) muscle development, the alpha-skeletal and alpha-cardiac isoforms of actin accumulate rapidly at the onset of muscle fibre formation, while there is a rapid fall in the expression of nonmuscle (beta and gamma) actin isoforms. Here we show that, before birth, both skeletal and myocardial cells express significant amounts of alpha-smooth muscle actin mRNA and protein. This expression is transient and disappears over the 1-7 days following birth. Our findings show that the program regulating actin isoform expression in sarcomeric muscle development is complex and that alpha-smooth muscle actin participates in this process.     

Immunoreactive sites and accumulation of somatomedin-C in rat Sertoli-spermatogenic cell co-cultures

Sertoli-spermatogenic cell co-cultures prepared from sexually immature rats (20-22 days old) and maintained in serum-free, hormone/growth factor-supplemented medium were used to determine the cell-specific localization of the growth factor somatomedin-C (SM-C). SM-C localization studies were carried out by indirect immunofluorescence using a monoclonal antibody (sm-1.2) to SM-C. In cultured rat hepatocytes, Sertoli and testicular peritubular cells, SM-C immunoreactivity was observed as a diffuse distribution of discrete immunofluorescent granules. Radio-immunoassay experiments using a rabbit antibody against human SM-C showed that testicular peritubular cells and Sertoli cells in primary culture accumulated SM-C in the medium. In spermatogenic cells co-cultured with subjacent Sertoli cells, immunoreactive SM-C was associated with pachytene spermatocytes but not with spermatogonia or early meiotic prophase spermatocytes (leptotene or zygotene). Both Sertoli cells and pachytene spermatocytes displayed binding sites for exogenously added SM-C. SM-C6 binding to spermatocytes reaching an advanced stage of meiotic prophase suggests a possible role of this growth factor in the meiotic process.     

Acetylcholinesterase activity and molecular forms during denervation and reinnervation in extensor digitorum longus muscle of the rat

The four principal molecular forms of acetylcholinesterase characteristic of the mammalian muscle (16.1 S., 12.5 S, 10.2 S, and 3.6. S) were identified by sucrose gradient sedimentation as the four activity peaks H, H₁, M and L.After denervation obtained by crushing the sciatic nerve five stages of the denervation-reinnervation process were examined. Days 7, 14, 22, 30, and 60 were chosen on the basis of previous electrophysiological and histochemical studies. The AChE activity showed an initial drop followed by recovery after nerve arrival at the muscle which was completed by day 60. Marked changes in the relative proportions of the four molecular forms were observed. The 16.1 S almost disappeared during the denervation period, reappeared after nerve arrival and was completely restored at day 60. Changes were also observed in the intermediate and lower forms and were tentatively related to processes of degradation, reaggregation and de novo synthesis.A comparison of the present data with those from parallel electrophysiological and histochemical studies suggests the presence and the functional role of molecular forms other than 16S in the neuromuscular junction.