Postsynaptic Neuromuscular Junction Research Articles

Differences in neuromuscular junctions of laryngeal and limb muscles in rats

Laryngeal muscles are specialized for fine control of voice, speech, and swallowing, and may differ from limb muscles in many aspects. Because muscles and their controlling motor neurons communicate via neuromuscular junctions (NMJs), we hypothesized that NMJs in laryngeal muscles have specialized characteristics different from limb muscles. In vivo study. Single muscle fibers from 12 Sprague-Dawley rats (six male, six female) were used to analyze the postsynaptic side of NMJs from laryngeal thyroarytenoid (TA), cricothyroid (CT), posterior cricoarytenoid (PCA), limb soleus (SOL), and extensor digitorum longus (EDL) muscles. NMJs were labeled with rhodamine-conjugated α-bungarotoxin. With confocal microscopy, we counted cluster fragments and measured the NMJ area, both absolute and normalized (corrected by muscle fiber diameter), for at least 10 single fibers from each muscle of each animal. Differences between genders were also compared. Cluster fragments of postsynaptic NMJs were more numerous in PCA and TA compared to CT, SOL, and EDL muscles (P < .01) in both male and female rats. NMJ cluster fragments were more numerous in female than in male rats only in the TA muscle (P < .01). The absolute area covered by the NMJs showed SOL > EDL > PCA > CT > TA (P < .01); however, with normalization the SOL = EDL = PCA > CT = TA. Differences found in NMJ surface and organization between laryngeal and limb muscle fibers may relate to specialized laryngeal muscle functions. Differences in NMJs between male and female rats were found only in the TA muscle, suggesting an underlying mechanism for some gender-specific laryngeal disorders related to abnormal TA muscle activity.

Passive and active immunization models of MuSK-Ab positive myasthenia: Electrophysiological evidence for pre and postsynaptic defects

Antibodies directed against the post-synaptic neuromuscular junction protein, muscle specific kinase (MuSK) are found in a small proportion of generalized myasthenia gravis (MuSK-MG) patients. MuSK is a receptor tyrosine kinase which is essential for clustering of the acetylcholine receptors (AChRs) at the neuromuscular junction, but the mechanisms by which MuSK antibodies (MuSK-Abs) affect neuromuscular transmission are not clear. Experimental models of MuSK-MG have been described but there have been no detailed electrophysiological studies and no comparisons between the MuSK-MG and the typical form with AChR-Abs (AChR-MG). Here we studied the electrophysiology of neuromuscular transmission after immunization against MuSK compared with immunization against AChR, and also after passive transfer of IgG from MuSK-MG or AChR-MG patients. Overt clinical weakness was observed in 6/10 MuSK-immunized and 3/9 AChR-immunized mice but not in those injected with patients' IgG. Miniature endplate potentials (MEPPS) were reduced in all weak mice consistent with the reduction in postsynaptic AChRs that was found. However, whereas there was an increase in the quantal release of acetylcholine (ACh) in the weak AChR-immunized mice, no such increase was found in the weak MuSK-immunized mice. Similar trends were found after the passive transfer of purified IgG antibodies from MuSK-MG or AChR-MG patients. Preliminary results showed that MuSK expression was considerably higher at the neuromuscular junctions of the masseter (facial) than in the gastrocnemius (leg) with no reduction in MuSK immunostaining at the neuromuscular junctions. Overall, these results suggest that MuSK antibodies act in at least two ways. Firstly by indirectly affecting MuSK's ability to maintain the high density of AChRs and secondly by interfering with a compensatory presynaptic mechanism that regulates quantal release and helps to preserve neuromuscular function. These results raise questions about how MuSK is involved in retrograde signaling, and the combination of post-synaptic defects with lack of presynaptic compensation may begin to explain the more severe disease in MuSK-MG patients.

Drosophila adducin regulates Dlg phosphorylation and targeting of Dlg to the synapse and epithelial membrane

Adducin is a cytoskeletal protein having regulatory roles that involve actin filaments, functions that are inhibited by phosphorylation of adducin by protein kinase C. Adducin is hyperphosphorylated in nervous system tissue in patients with the neurodegenerative disease amyotrophic lateral sclerosis, and mice lacking β-adducin have impaired synaptic plasticity and learning. We have found that Drosophila adducin, encoded by hu-li tai shao ( hts), is localized to the post-synaptic larval neuromuscular junction (NMJ) in a complex with the scaffolding protein Discs large (Dlg), a regulator of synaptic plasticity during growth of the NMJ. hts mutant NMJs are underdeveloped, whereas over-expression of Hts promotes Dlg phosphorylation, delocalizes Dlg away from the NMJ, and causes NMJ overgrowth. Dlg is a component of septate junctions at the lateral membrane of epithelial cells, and we show that Hts regulates Dlg localization in the amnioserosa, an embryonic epithelium, and that embryos doubly mutant for hts and dlg exhibit defects in epithelial morphogenesis. The phosphorylation of Dlg by the kinases PAR-1 and CaMKII has been shown to disrupt Dlg targeting to the NMJ and we present evidence that Hts regulates Dlg targeting to the NMJ in muscle and the lateral membrane of epithelial cells by controlling the protein levels of PAR-1 and CaMKII, and consequently the extent of Dlg phosphorylation.

CNS-targeted Viral Delivery of G-CSF in an Animal Model for ALS: Improved Efficacy and Preservation of the Neuromuscular Unit

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motoneurons. We have recently uncovered a new neurotrophic growth factor, granulocyte-colony stimulating factor (G-CSF), which protects α-motoneurons, improves functional outcome, and increases life expectancy of SOD-1 (G93A) mice when delivered subcutaneously. However, chronic systemic delivery of G-CSF is complicated by elevation of neutrophilic granulocytes. Here, we used adeno-associated virus (AAV) to directly target and confine G-CSF expression to the spinal cord. Whereas intramuscular injection of AAV failed to transduce motoneurons retrogradely, and caused a high systemic load of G-CSF, intraspinal delivery led to a highly specific enrichment of G-CSF in the spinal cord with moderate peripheral effects. Intraspinal delivery improved motor functions, delayed disease progression, and increased survival by 10%, longer than after systemic delivery. Mechanistically, we could show that G-CSF in addition to rescuing motoneurons improved neuromuscular junction (NMJ) integrity and enhanced motor axon regeneration after nerve crush injury. Collectively, our results show that intraspinal delivery improves efficacy of G-CSF treatment in an ALS mouse model while minimizing the systemic load of G-CSF, suggesting a new therapeutic option for ALS treatment.

Time Lapse in Vivo Visualization of Developmental Stabilization of Synaptic Receptors at Neuromuscular Junctions

The lifetime of nicotinic acetylcholine receptors (AChRs) in neuromuscular junctions (NMJs) is increased from <1 day to >1 week during early postnatal development. However, the exact timing of AChR stabilization is not known, and its correlation to the concurrent embryonic to adult AChR channel conversion, NMJ remodeling, and neuromuscular diseases is unclear. Using a novel time lapse in vivo imaging technology we show that replacement of the entire receptor population of an individual NMJ occurs end plate-specifically within hours. This makes it possible to follow directly in live animals changing stabilities of end plate receptors. In three different, genetically modified mouse models we demonstrate that the metabolic half-life values of synaptic AChRs increase from a few hours to several days after postnatal day 6. Developmental stabilization is independent of receptor subtype and apparently regulated by an intrinsic muscle-specific maturation program. Myosin Va, an F-actin-dependent motor protein, is also accumulated synaptically during postnatal development and thus could mediate the stabilization of end plate AChR.

The Nicotinic Acetylcholine Receptor and the Na,K-ATPase α2 Isoform Interact to Regulate Membrane Electrogenesis in Skeletal Muscle

The nicotinic acetylcholine receptor (nAChR) and the Na,K-ATPase functionally interact in skeletal muscle (Krivoi, I. I., Drabkina, T. M., Kravtsova, V. V., Vasiliev, A. N., Eaton, M. J., Skatchkov, S. N., and Mandel, F. (2006) Pflugers Arch. 452, 756-765; Krivoi, I., Vasiliev, A., Kravtsova, V., Dobretsov, M., and Mandel, F. (2003) Ann. N.Y. Acad. Sci. 986, 639-641). In this interaction, the specific binding of nanomolar concentrations of nicotinic agonists to the nAChR stimulates electrogenic transport by the Na,K-ATPase alpha2 isozyme, causing membrane hyperpolarization. This study examines the molecular nature and membrane localization of this interaction. Stimulation of Na,K-ATPase activity by the nAChR does not require ion flow through open nAChRs. It can be induced by nAChR desensitization alone, in the absence of nicotinic agonist, and saturates when the nAChR is fully desensitized. It is enhanced by noncompetitive blockers of the nAChR (proadifen, QX-222), which promote non-conducting or desensitized states; and retarded by tetracaine, which stabilizes the resting nAChR conformation. The interaction operates at the neuromuscular junction as well as on extrajunctional sarcolemma. The Na,K-ATPase alpha2 isozyme is enriched at the postsynaptic neuromuscular junction and co-localizes with nAChRs. The nAChR and Na,K-ATPase alpha subunits specifically coimmunoprecipitate with each other, phospholemman, and caveolin-3. In a purified membrane preparation from Torpedo californica enriched in nAChRs and the Na,K-ATPase, a ouabain-induced conformational change of the Na,K-ATPase enhances a conformational transition of the nAChR to a desensitized state. These results suggest a mechanism by which the nAChR in a desensitized state with high apparent affinity for agonist interacts with the Na,K-ATPase to stimulate active transport. The interaction utilizes a membrane-delimited complex involving protein-protein interactions, either directly or through additional protein partners. This interaction is expected to enhance neuromuscular transmission and muscle excitation.

Pre‐ and postsynaptic neuromuscular junction abnormalities in musk myasthenia

Autoantibodies to muscle-specific kinase (MuSK) can cause myasthenia gravis (MG). The pathophysiological mechanism remains unknown. We report in vitro electrophysiological and histological studies of the neuromuscular junction in a MuSK MG patient. Low levels of presynaptic acetylcholine release and small miniature endplate potentials were found. This combination of pre- and postsynaptic abnormalities was supported by histology, revealing partially denervated postsynaptic areas, and some degeneration of postsynaptic folds. Results suggest that anti-MuSK antibodies reduce the stability of muscle-nerve contact.

Drosophila twinfilin is required for cell migration and synaptic endocytosis

Precise actin regulation is essential for diverse cellular processes such as axonal growth, cell migration and endocytosis. twinfilin (twf) encodes a protein that sequesters actin monomers, but its in vivo functions are unclear. In this study, we characterized twf-null mutants in a metazoan for the first time and found that Drosophila twf negatively regulates F-actin formation in subcellular regions of rapid actin turnover in three different systems, namely postsynaptic neuromuscular junction (NMJ) synapses, migratory border cells and epithelial follicle cells. Loss of twf function results in defects in axonal growth in the brain and border cell migration in the ovary. Additionally, we found that the actin-dependent postsynaptic localization of glutamate receptor GluRIIA, but not GluRIIB, was specifically reduced in twf mutants. More importantly, we showed that twf mutations caused significantly reduced presynaptic endocytosis at NMJ synapses, as detected using the fluorescent dye FM1-43 uptake assay. Furthermore, electrophysiological analysis under high-frequency stimulation showed compromised neurotransmission in twf mutant synapses, confirming an insufficient replenishment of synaptic vesicles. Together, our results reveal that twinfilin promotes actin turnover in multiple cellular processes that are highly dependent on actin dynamics.

Remodeling of the neuromuscular junction precedes sarcopenia related alterations in myofibers

Several mechanisms contributing to the etiology of sarcopenia (age-related loss of muscle size) have been postulated. One of these attributes the loss of muscle mass to a preceding age-related denervation of myofibers. The aim of this study was to determine if signs of denervation were apparent at the neuromuscular junction (NMJ) before fiber atrophy, or fiber type conversion could be documented, and to reveal if a muscle’s activity level impacts its sensitivity to age-related denervation. Plantaris and soleus muscles were obtained from young adult (10 months) and early aged (21 months) rats. Pre- and post-synaptic NMJ morphology was quantified with cytofluorescent staining of nerve terminal branches and endplate regions, respectively. Myofiber profiles (fiber size and fiber type composition) were assessed with histochemical procedures. Results show that in the lightly recruited plantaris, significant ( P < 0.05) signs of denervation were noted in aged rats, while the same muscles displayed no change in myofiber profile. In the heavily recruited soleus, however, there was little evidence of denervation, and again no alterations in myofiber profile. These results indicate that age-related denervation occurs before myofiber atrophy, and that high amounts of neuromuscular activity may delay the onset of age-related denervation and sarcopenia.

Effect of locally delivered IGF‐1 on nerve regeneration during aging: An experimental study in rats

Age is an important predictor of neuromuscular recovery after peripheral nerve injury. Insulin-like growth factor 1 (IGF-1) is a potent neurotrophic factor that is known to decline with increasing age. The purpose of this study was to determine if locally delivered IGF-1 would improve nerve regeneration and neuromuscular recovery in aged animals. Young and aged rats underwent nerve transection and repair with either saline or IGF-1 continuously delivered to the site of the nerve repair. After 3 months, nerve regeneration and neuromuscular junction morphology were assessed. In both young and aged animals, IGF-1 significantly improved axon number, diameter, and density. IGF-1 also significantly increased myelination and Schwann cell activity and preserved the morphology of the postsynaptic neuromuscular junction (NMJ). These results show that aged regenerating nerve is sensitive to IGF-1 treatment.

Myasthenia gravis precipitated by trauma: Latent myasthenia and the concept of ‘threshold’

Myasthenia gravis is caused by antibodies to post-synaptic neuromuscular junction proteins, most commonly nicotinic acetylcholine receptors. Evidence indicates that acetylcholine receptor antibodies must be present in the circulation prior to the development of clinical or neurophysiological manifestations of myasthenia. We describe the onset of seropositive myasthenia within minutes of minor trauma to the chest and neck in a previously asymptomatic 68-year-old man, followed by protracted disease with persistent antibodies. Such rapid evolution of myasthenia following an identifiable stimulus has not been reported previously and suggests a humoral mechanism. We speculate that the remote effects of autoinflammation secondary to tissue microtrauma led to a sudden increase in muscle permeability and greater exposure of receptors to antibody, with resulting acute impairment of neuromuscular transmission. We also suggest that the trauma might have resulted in increased antibody production by remnant thymic tissue, leading to chronic isease.

Structural and Functional Diversity of Novel Coronin 1C (CRN2) Isoforms in Muscle

Coronin 1C (synonyms: coronin-3, CRN2), a WD40 repeat-containing protein involved in cellular actin dynamics, is ubiquitously expressed in human tissues. Here, we report on the identification and functional characterization of two novel coronin 1C isoforms, referred to as CRN2i2 and CRN2i3, which also associate with F-actin. Analyses of the coronin 1C gene disclosed a single promoter containing binding sites for myogenic regulatory factors and an alternative first exon 1b present in intron 1, which give rise to the novel isoforms. Chromatin immunoprecipitation studies demonstrate MyoD binding to a region of the CRN2 gene, which contains a highly conserved E-box element in exon 1a. Gel-filtration assays suggest that the largest isoform 3 exists as a monomer, in contrast to isoform 1 and isoform 2 appearing as trimers. CRN2i3, which can be induced by MyoD, is exclusively expressed in well-differentiated myoblasts as well as in mature skeletal muscle tissue. In human skeletal muscle, CRN2i3 is a novel component of postsynaptic neuromuscular junctions and thin filaments of myofibrils. Together, our findings postulate a role for CRN2 isoforms in the structural and functional organization of F-actin in highly ordered protein complexes.

How age impairs the response of the neuromuscular junction to nerve transection and repair: An experimental study in rats

Age is the most important predictor of clinical outcome after peripheral nerve injury. The stability of the neuromuscular junction (NMJ) after denervation is thought to be central to neuromuscular recovery. Stability is characterized by maintenance of the motor endplate and mRNA upregulation of the constituent nicotinic acetylcholinergic receptor (nAChR) subtypes and the muscle regulatory factors (MRFs). The purpose of this study was to determine the effect of age on the recovery and stability of the postsynaptic NMJ after peripheral nerve injury. Young and aged rats underwent transection and repair of the tibial nerve. At 1, 2, 4, 8, or 16 weeks following transection, the gastrocnemius was examined for electrical recovery, NMJ fragmentation and endplate area, mRNA, and protein levels of the MRFs and nAChR subtypes. After nerve injury, aged NMJ exhibited significant fragmentation and loss of motor endplate area while the young NMJ remained relatively stable. Concomitantly, age impaired peak upregulation of the MRFs and nAChRs. However, expression of gamma-nAChR and myogenin after nerve injury was not affected by age. These data support the claim that upregulation of the nAChRs and MRFs may play an important role in maintaining NMJ stability following nerve transection and repair. Furthermore, expression of gamma-nAChR and myogenin does not appear to prevent age-related NMJ fragmentation and loss of endplate area after nerve injury. These impairments of the aged NMJ response to injury may contribute to the poor neuromuscular recovery seen after nerve injury in this population.

Monozygous twins with neuromuscular transmission defects at opposite sides of the motor endplate

Disorders affecting the postsynaptic side of the neuromuscular junction include autoimmune myasthenia gravis (MG) as well as some of the congenital myasthenic syndromes (CMS). Lambert-Eaton myasthenic syndrome (LEMS) is an acquired autoimmune neuromuscular disorder in which autoantibodies are directed against the presynaptic calcium channels. Here we describe two monozygous twin brothers: case 1 was diagnosed with an indeterminate form of acquired postsynaptic neuromuscular junction defect at age 32 and case 2 with LEMS at age 47. Case 1 presented clinically with mild generalized myasthenic weakness, neurophysiological examination revealed disturbed neuromuscular transmission along with probable myositis and serum analysis regarding antibodies against the acetylcholine receptor and muscle-specific tyrosine kinase was negative. Case 2 presented with proximal muscle fatigue accompanied by areflexia at rest and antibodies against the P/Q-type voltage-gated calcium channels were present. Neurophysiologically, case 2 had reduced baseline compound motor action potential amplitudes on neurography, decrement on low-frequency repetitive nerve stimulation (RNS) and pathological increment on high frequency RNS. To our knowledge this is the first case report of its kind and adds an intriguing contrast to the more common diagnosis of CMS in monozygous twins.

LDL-receptor-related protein 4 is crucial for formation of the neuromuscular junction

Low-density lipoprotein receptor-related protein 4 (Lrp4) is a member of a family of structurally related, single-pass transmembrane proteins that carry out a variety of functions in development and physiology, including signal transduction and receptor-mediated endocytosis. Lrp4 is expressed in multiple tissues in the mouse, and is important for the proper development and morphogenesis of limbs, ectodermal organs, lungs and kidneys. We show that Lrp4 is also expressed in the post-synaptic endplate region of muscles and is required to form neuromuscular synapses. Lrp4-mutant mice die at birth with defects in both presynaptic and postsynaptic differentiation, including aberrant motor axon growth and branching, a lack of acetylcholine receptor and postsynaptic protein clustering, and a failure to express postsynaptic genes selectively by myofiber synaptic nuclei. Our data show that Lrp4 is required during the earliest events in postsynaptic neuromuscular junction (NMJ) formation and suggest that it acts in the early, nerveindependent steps of NMJ assembly. The identification of Lrp4 as a crucial factor for NMJ formation may have implications for human neuromuscular diseases such as myasthenia syndromes.

A Diagnostic and Management Dilemma

Neuromuscular junction disorders are usually categorized as either presynaptic or postsynaptic. The most frequently encountered disorder of the postsynaptic neuromuscular junction is acquired myasthenia gravis. Lambert-Eaton myasthenic syndrome is a well-known prototype of the presynaptic autoimmune disorders of neuromuscular transmission. These major disorders of neuromuscular transmission are relatively common and distinctly recognized, but co-occurrence of these disorders (overlap myasthenic syndrome) is rare and has so far attracted little attention. This report describes a patient with acquired myasthenia gravis and immunologic coexistence of Lambert-Eaton myasthenic syndrome (overlap myasthenic syndrome) in association with abdominal/uterine leiomyosarcoma. The patient presented with acute respiratory failure, making identification and management of her illness challenging. A general overview of the complexities associated with overlap between myasthenia gravis and Lambert-Eaton myasthenic syndrome is provided and this patient's complicated clinical course and response to therapy are discussed. To our knowledge, this is the first report of overlap myasthenic syndrome in conjunction with abdominal leiomyosarcoma. The immunologic coexistence of acquired myasthenia gravis and Lambert-Eaton myasthenic syndrome in a patient with a malignant smooth-muscle tumor is intriguing and suggests that a common paraneoplastic process targeting 2 different onconeural antigens was the underlying pathogenic mechanism in this patient.

Neuromuscular Recovery After Distraction Osteogenesis at Different Frequencies in a Rabbit Model

The muscle and nerve responses to stresses applied during distraction osteogenesis have not been clearly defined. This study hypothesized that distraction resulting in 30% lengthening decreases muscle force generation of the lengthened muscle and increasing the frequency of distraction attenuates the decrease of force generation accompanying lengthening. This study investigated the effects of different distraction frequencies on neuromuscular recovery in a rabbit model. Animals were assigned into group 1 (low-frequency distraction) and group 2 (high-frequency distraction). Distraction was continued until a 30% increase in the original tibial length was achieved. After consolidation of the osteotomy, knee and ankle range of motion, muscle force generation, and neuromuscular junction parameters were evaluated. Lengthening of 30% resulted in significantly decreased range of motion compared with the control leg (P < 0.05). Lengthening of 30% also substantially decreased force generation of the peroneus longus muscle. However, force generation of the peroneus longus muscle in the high-frequency group was 70.5% +/- 6.5% of the control side, significantly higher than that in the low-frequency distraction group (49.7% +/- 4.8% of the control side, P < 0.05). There was no statistical difference between the 2 groups in neuromuscular junction morphology, although an abnormal shape of the postsynaptic neuromuscular junction was observed after distraction. The use of a high-frequency distraction technique during limb lengthening may result in a reduction in impairment of knee and ankle range of motion and improved muscle function compared with that observed with the use of low-frequency distraction. Repeated microtrauma to the soft tissues associated with high-frequency distraction may facilitate the regenerative capacity of the soft tissues and result in an improved outcome of muscle and nerve function.

Self-reported Pain Affects Quality of Life in Myasthenia Gravis

Myasthenia gravis (MG) is an autoimmune-mediated disease of neuromuscular junction (NMJ) transmission that is well recognized and is commonly seen by practicing neurologists. The pathophysiology of the disease has been well defined, and is secondary to inhibition of postsynaptic NMJ transmission by

Treatment of autoimmune myasthenia gravis.

Autoimmune myasthenia gravis (MG) is associated with antibodies directed against the nicotinic acetylcholine receptor (AChR) in 85% of patients. Other postsynaptic neuromuscular junction antigens are implicated, e.g., muscle-specific receptor tyrosine kinase (MuSK), in a number of the remaining 15% of patients, so-called seronegative MG. The autoimmune attack generally leads to decreased concentrations of the AChR and damage to the structure of the endplate itself. This information has guided the empiric treatment of patients with MG and has suggested new treatment strategies. Whereas the outcome of patients with MG has improved because of more effective symptomatic treatment, including advances in critical care medicine and the use of cholinesterase inhibitors, the greatest advances have come from therapies that directly reduce the autoimmune attack or modify its effects on the AChR and the surrounding endplate. Immune-directed treatment of patients with MG, which is guided by this information and by data from the management of other autoimmune disease, is aimed at inducing an immunologic remission and then maintaining that remission. Remission induction is usually accomplished through the use of high-dose corticosteroids, frequently in conjunction with IV immunoglobulin or plasmapheresis. Maintenance of the remission is usually accomplished by slow tapering of the corticosteroids along with the use of "steroid-sparing" agents, which include azathioprine, thymectomy, and possibly mycophenolate. Therapy usually begins with cholinesterase inhibitors. If necessary, immune-directed treatment is added, beginning with either thymectomy or high-dose corticosteroids. The short-term therapies, i.e., IV immunoglobulin or plasmapheresis, may be effective in the early stages of treatment or later during an exacerbation. Steroid-sparing medications are usually added to facilitate the tapering phase.

Abl-ity to Form Synapses

Synaptogenesis, the process whereby the specialized junction between a neuron and its target cell develops, depends on an intricate interplay of signals between the presynaptic and postsynaptic cells. At the neuromuscular junction (NMJ), agrin, which is secreted by the presynaptic neuron, acts through muscle-specific receptor tyrosine kinase (MuSK) to stimulate clustering of muscle acetylcholine receptors (AChRs). AChR clustering involves activation of an unknown downstream tyrosine kinase and reorganization of the actin cytoskeleton. Finn et al. identified the Abl nonreceptor tyrosine kinases 1 and 2 (Abls 1 and 2), which have actin-bundling ability and have been implicated in the formation of cell-to-cell junctions (as well as in the pathogenesis of some leukemias), as critical components of the signaling cascade downstream of agrin and MuSK. The authors used Western analysis to identify Abls 1 and 2 in mouse muscle and immunohistochemistry to localize them to the postsynaptic NMJ. Pharmacologic analysis in C2C12, a murine myoblast cell line, as well as expression of Abl mutants, indicated that Abl activity was involved in both MuSK phosphorylation and in AChR clustering downstream of agrin and MuSK. Coexpression experiments in HEK cells indicated that MuSK and Abl1 phosphorylated each other. Western analysis of immunoprecipitates indicated that Abl formed a complex with MuSK; agrin treatment promoted formation of this complex. The authors propose a model in which Abl kinases promote AChR clustering through both phosphorylation and induction of a postsynaptic actin scaffold.A. I. Finn, G. Feng, A. M. Pendergast, Postsynaptic requirement for Abl kinases in assembly of the neuromuscular junction. Nat. Neurosci. 6, 717-723 (2003). [Online Journal]