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Muscle Specific Kinase (MuSK) is a transmembrane tyrosine kinase vital for forming and maintaining the mammalian neuromuscular junction (NMJ: the synapse between motor nerve and skeletal muscle). MuSK expression switches on during skeletal muscle differentiation. MuSK then becomes restricted to the postsynaptic membrane of the NMJ, where it functions to cluster acetylcholine receptors (AChRs). The expression, activation and turnover of MuSK are each regulated by signals from the motor nerve terminal. MuSK forms the core of an emerging signalling complex that can be acutely activated by neural agrin (N-agrin), a heparin sulfate proteoglycan secreted from the nerve terminal. MuSK activation initiates complex intracellular signalling events that coordinate the local synthesis and assembly of synaptic proteins. The importance of MuSK as a synapse organiser is highlighted by cases of autoimmune myasthenia gravis in which MuSK autoantibodies can deplete MuSK from the postsynaptic membrane, leading to complete disassembly of the adult NMJ., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Rapsyn is a protein that interacts with the cytoplasmic face of the nicotinic acetylcholine receptors (AChR) to cluster them within postsynaptic membrane of muscle. Here we show that intracellular AChRs are also affected by rapsyn. When rapsyn was co-transfected with AChR into QT-6 fibroblasts, (125)I-alpha-bungarotoxin binding indicated a reduction in the fraction of AChRs expressed on the cell surface, compared to cells expressing AChRs alone. Double fluorescent labeling showed that intracellular AChRs accumulated in patches at the cell periphery, beneath rapsyn-associated cell surface AChR clusters. These patches were observed even when cells were grown in medium containing excess unlabelled alpha-bungarotoxin to mask internalized AChRs, suggesting that they arose from hindered trafficking of newly formed AChRs to the cell surface. Similarly, in the muscle cell line, C2, overexpression of rapsyn resulted in the co-localization of aggregates of intracellular alpha-bungarotoxin binding sites with rapsyn beneath cell surface AChR microaggregates. The results indicate that rapsyn can modify the trafficking of AChRs within the cell and suggest a role in selectively targeting newly synthesized intracellular AChRs to the postsynaptic membrane., (Copyright 2000 Wiley-Liss, Inc.)

Recombinant acetylcholine receptors (AChRs) expressed on the surface of cultured fibroblasts become organized into discrete membrane domains when the 43-kD postsynaptic protein (43k) is co-expressed in the same cells (Froehner, S.C., C. W. Luetje, P. B. Scotland, and J. Patrick, 1990. Neuron. 5:403-410; Phillips, W. D., M. C. Kopta, P. Blount, P. D. Gardner, J. H. Steinbach, and J. P. Merlie. 1991. Science (Wash. DC). 251:568-570). Here we show that AChRs present on the fibroblast cell surface prior to transfection of 43k are recruited into 43k-rich membrane domains. Aggregated AChRs show increased resistance to extraction with Triton X-100, suggesting a 43k-dependent linkage to the cytoskeleton. Myotubes of the mouse cell line C2 spontaneously display occasional AChR/43k-rich membrane domains that ranged in diameter up to 15 microns, but expressed many more when 43k was overexpressed following transfection of 43k cDNA. However, the membrane domains induced by recombinant 43k were predominantly small (< or = 2 microns). We were then interested in whether the cytoskeletal component, dystrophin related protein (DRP; Tinsley, J. M., D. J. Blake, A. Roche, U. Fairbrother, J. Riss, B. C. Byth, A. E. Knight, J. Kendrick-Jones, G. K. Suthers, D. R. Love, Y. H. Edwards, and K. E. Davis, 1992. Nature (Lond.). 360:591-593) contributed to the development of AChR clusters. Immunofluorescent anti-DRP staining was present at the earliest stages of AChR clustering at the neuromuscular synapse in mouse embryos and was also concentrated at the large AChR-rich domains on nontransfected C2 myotubes. Surprisingly, anti-DRP staining was concentrated mainly at the large, but not the small AChR clusters on C2 myotubes suggesting that DRP may be principally involved in permitting the growth of AChR clusters.

Many avian muscles contain a characteristic topographical distribution of fibre types. In order to study the role of nerves in the establishment and distribution of these fibre types, monoclonal antibodies (McAb) to the heavy chain subunit of myosin (MHC) were produced. The anti-fast McAb (2B12) bound to adult fast MHC and cross-reacted with the embryonic isoform of MHC. The anti-slow McAb (3D1) bound specifically to the heavy chain of slow myosin 2. By indirect immunofluorescence, anti-fast (2B12) stained all myotubes in the anterior latissimus dorsi and triceps and biceps muscles at stage 37 (11 days embryonic), whilst anti-slow (3D1) staining was largely restricted to the future slow fibres of these muscles. Brachial levels of the neural tube were surgically removed at stage 12 (2 days embryonic) so that muscles developed aneurally. Muscles at aneural stage 37 were smaller than normal, but the distribution of myotube types was not altered; all myotubes present still stained with anti-fast antibody while anti-slow staining was restricted to the anterior latissimus dorsi and myotubes in the deep parts of the triceps brachii pars scapularis, triceps brachii par humeralis and biceps brachii muscles (the future slow fibres of normal muscles). The results suggest that despite an overall reduction in MHC in aneural muscles, specialized fast and slow primary myotubes arise independently of the nerve in appropriate regions of the muscle.

The embryonic precursors of the avian slow (type I and III) and fast (type II) fibers can be distinguished from each other early in muscle formation (stage 28, V. Hamburger and H. L. Hamilton, J. Morphol, 88, 49-92, 1951) on the basis of the differential sensitivity of their myosin ATPases. To test the neural dependence of fiber type differentiation, the source of motor innervation was eliminated by excision of the brachial neural tube at stages 16-18 before muscles are innervated. Removal of the brachial neural tube did not affect the number of primary myotubes in a sample muscle of the forelimb (ulnimetacarpalis dorsalis, UMD) up until stage 36. Myosin ATPase staining at a variety of pHs revealed the typical patterns of fiber types in muscles of neural-tube free embryos in stages 35-37. These muscles included the anterior latissimus dorsi, brachialis, and UMD which showed presumptive type III staining (type IIIEMB), the pronator superficialis and flexor carpi ulnaris which showed embryonic type II staining (type IIEMB), and the triceps brachii muscles which showed characteristic arrangements of both type IEMB and type IIEMB fibers. The normal patterns of type IEMB and type IIEMB myotubes were also seen in muscles containing a heterogeneous mixture of fiber types such as the biceps brachii, extensor metacarpi radialis, and adductor indicis muscles, although the intensity of acid-stable ATPase staining of the type IEMB myotubes in these muscles was lower than in innervated muscles. It is concluded that the earliest differentiation of muscle fiber types is independent of the nervous system.

The size and distribution of acetylcholine receptor clusters (AChR-C) on normal and aneural developing muscle fibres of the chick wing were studied by labelling AChR with fluorescent conjugates of alpha-bungarotoxin (alpha-BGT). AChR-C of a size typical of initial synaptic contacts (5 micron long) were present at 7 days incubation, shortly after the appearance of nerves, and were grouped in bands corresponding to muscle nerve branches. A regular distribution of large (approximately equal to 5 micron) AChR-C separated by 100-200 micron had developed by 10-14 days in the slow-tonic anterior latissimus dorsi and ulnimetacarpalis dorsalis muscles. The role of motor innervation in the formation of AChR-C was assessed by removing the brachial neural tube at 2 days incubation in order to prevent nerves entering the wing. Neural-tube removal prevented the appearance of the large AChR-C normally associated with the early synaptic contacts. Small AChR-C (less than 2 micron long) appeared in aneural muscles, but these were not grouped into bands characteristic of the large AChR-C in normal muscles. The results suggest that the formation of junctional AChR-C is dependent on nerves.

The spatial distribution of intracellular acetylcholine receptors along the length of fibres from the avian posterior latissimus dorsi muscle has been investigated during embryonic development, when distributed synaptic sites are eliminated from the muscle fibres. Cell surface AChR were irreversibly blocked with unlabelled alpha-bungarotoxin (alpha-BGT). Muscles were then fixed and ultrasonically dissociated into fibre fragments, treated with 0.5% saponin and stained with 125I-alpha-BGT. This revealed an intracellular pool of curare sensitive binding sites equivalent to about 10% of total cell AChR. The spatial distribution of this pool was studied by autoradiography. Large (longer than 2 microns) AChR-clusters (AChR-C) characteristic of neuromuscular contacts were localized on the same fibres by immunofluorescence with an anti-AChR antibody. At E11, relatively high levels of intracellular AChR were observed throughout the length of fibres. Between E11 and E18 intracellular AChR declined (19 fold) in extrajunctional parts of fibres but remained high in segments of fibre corresponding to AChR-clusters. Treatment of E14 embryos with an inhibitor of protein synthesis (cycloheximide) reduced intracellular AChR to 22 +/- 6% (mean +/- SE) of control levels, suggesting that most of the intracellular binding represented newly-synthesized AChR. Between E11 and E18 cell nuclei were found to accumulate beneath AChR-C. The mean density of nuclei in segments of fibre corresponding to AChR-C increased 5 fold between E11 and E18, but remained unchanged in extrajunctional segments. It is suggested that the elimination of excess distributed AChR-C may be due to the preferential accumulation of nuclei at a single AChR-C on each fibre accompanied by the down regulation of AChR synthesis associated with nuclei at the remaining AChR-C.

Changes in the distribution of large acetylcholine receptor clusters (AChR-Cs) on developing fast-twitch fibres of the chicken posterior latissimus dorsi (PLD) muscle have been studied during the period of loss of polyneuronal innervation using fluorescein-conjugated alpha-bungarotoxin. Embryonic muscles were ultrasonically dissociated into single fibre fragments and presumptive fast-twitch fibres were distinguished from the minority of slow-type fibres in the PLD by immunofluorescence using an antibody against slow-type myosin. Whereas mature PLD muscle fibres are focally innervated, at embryonic day 11 (E11) many fibre fragments from the PLD displayed two or more large (longer than 2 micron) AChR-Cs. Double labelling with anti-neurofilament antibody suggested that most of these AChR-Cs (82 +/- 2%) were associated with neuromuscular contacts. There was a progressive decline in the number of large (synaptic) AChR-Cs per 1000 micron of fibre, from 3.2 +/- 0.5 at E11 to 0.4 +/- 0.1 at E18. No further decline occurred between E18 and one week post-hatch. Primary generation muscle cells identified at E11 and E16 by tritiated thymidine labelling showed a decline in the number of large AChR-Cs per 1000 micron proportional to that seen in the fibre population as a whole, suggesting that distributed synaptic AChR-Cs are eliminated from individual fibres as they mature. When embryos were treated with d-tubocurarine starting at E6 the loss of distributed AChR-Cs from fast-type PLD fibres between E11 and E14 did not occur, suggesting that neuromuscular activity may play an important role in establishing the focal synaptic site AChR-C.

The development of the focal localization of large acetylcholine receptor clusters (AChR-Cs) on avian fast muscle fibres has been investigated in the triceps brachii pars humeralis (TH) muscle of the chick embryo. The mature TH muscle consists of both fast fibres, which usually receive a focal innervation at single synaptic sites, and slow fibres which receive a distributed innervation at multiple synaptic sites. Single fibre fragments dissociated from the embryonic muscle were typed using anti-myosin antibodies; fluorescently labelled alpha-bungarotoxin was used to identify large AChR-Cs which serve as synaptic markers. In contrast to the mature focal innervation, at embryonic day 11 (E11), many fast-type fibres in the TH muscle displayed large, distributed AChR-Cs (3.7 +/- 0.7 per 1000 microns fibre length; n = 6 embryos) like neighbouring slow-type fibres. By E16 distributed AChR-Cs were rare on fast type fibres (0.9 +/- 0.2 per 1000 microns fibre length). As it was possible that the frequency of fast fibres with distributed AChR-Cs declined simply as a consequence of the increase in number of secondary generation fibres, tritiated thymidine was injected at E7 in order to identify the primary generation fibres at E14. The great majority of fast fibres that were heavily labelled with thymidine at E14 appeared to possess a focal AChR-C. The results suggest that at E11 fast-type primary fibres in the TH muscle receive a distributed innervation very similar to neighbouring slow-type fibres; this subsequently evolves into the mature focal innervation following the elimination of synaptic sites between E11 and E14.