Aggregates of acetylcholine receptors are associated with plaques of a basal lamina heparan sulfate proteoglycan on the surface of skeletal muscle fibers.

Abstract

Hybridoma techniques have been used to generate monoclonal antibodies to an antigen concentrated in the basal lamina at the Xenopus laevis neuromuscular junction. The antibodies selectively precipitate a high molecular weight heparan sulfate proteoglycan from conditioned medium of muscle cultures grown in the presence of [3~S]methionine or [35S]-sulfate. Electron microscope autoradiography of adult X. laevis muscle fibers exposed to 12Sl-labeled antibody confirms that the antigen is localized within the basal lamina of skeletal muscle fibers and is concentrated at least fivefold within the specialized basal lamina at the neuromuscular junction. Fluorescence immunocytochemical experiments suggest that a similar proteoglycan is also present in other basement membranes, including those associated with blood vessels, myelinated axons, nerve sheath, and notochord.During development in culture, the surface of embryonic muscle cells displays a conspicuously non-uniform distribution of this basal lamina proteoglycan, consisting of large areas with a low antigen site-density and a variety of discrete plaques and fibrils. Clusters of acetylcholine receptors that form on muscle cells cultured without nerve are invariably associated with adjacent, congruent plaques containing basal lamina proteoglycan. This is also true for clusters of junctional receptors formed during synaptogenesis in vitro. This correlation indicates that the spatial organization of receptor and proteoglycan is coordinately regulated, and suggests that interactions between these two species may contribute to the localization of acetylcholine receptors at the neuromuscular junction.The vertebrate neuromuscular junction is a region of elaborate morphological specialization for both the skeletal muscle fiber and motor neuron. This morphological complexity reflects a corresponding chemical specialization. It is well established, for example, that the postsynaptic membrane of the muscle fiber contains a high concentration of acetylcholine receptors (AChR), ~ and that the packing density of this integral membrane protein drops at least 50-fold within a few micrometers of the nerve terminal (1). It is also known that acetylcholinesterase (ACHE) is concentrated within the synaptic cleft (2, J Abbreviations used in this paper: ACHE, acetylcholinesterase; AChR, acetylcholine receptor; aBGT, a-bungarotoxin; FITC, fluorescein isothiocyanate; and TRITC, tetramethylrhodamine isothiocyanate.3), where it appears to be associated with the basal lamina (4,5). Immunocytochemical experiments indicate the existence of further chemical specialization in the muscle cytoskeleton (6-8), basal lamina (9), and the nerve terminal (10, 11). Undoubtedly the list of components concentrated at the neuromuscular junction will continue to increase as improved immunological techniques permit the identification and study of new molecular entities.In contrast to such evidence for chemical specialization, little is known about the cellular mechanisms responsible for the elaboration...

Distribution of synaptic specializations along isolated motor units formed in Xenopus nerve-muscle cultures

“…4). Besides AChRs, several muscle components have been implicated in the generation of NARPs, including components of the extracellular matrix (Burden et al, 1979;Anderson and Fambrough, 1983;Vogel et al, 1983;McMahan and Slater, 1984;Fallon et al, 1985) and the cytoskeletal system (Bloch, 1986;Froehner, 1986;Peng and Poo, 1986). The supply of any of these might limit the capacity of muscle cells to generate NARPs.…”

“…Initially, these 2 synaptic specializations may develop independently of each other, but with time the incidence of spatial alignment increases (Bixby and Reichardt, 1985). Components of the synaptic basal lamina have been implicated in the development of both these specializations (Sanes et al, 1978;Burden et al, 1979;Anderson and Fambrough, 1983;Vogel et al, 1983;McMahan and Slater, 1984;Fallon et al, 1985;Anderson, 1986).…”

Propriétés des fibres musculaires squelettiques. 1. Influence de l'innervation motrice

“…La stimulation du muscle squelettique adulte provoque une augmentation très importante de la concentration en GMPc musculaire (Nestler et al, 1978). De même, l'addition de GMPc à des cultures de muscle mime les effets de l'activité sur l'AChE synaptique (Rubin et al, 1980 Fambrough, 1983 ;Sanes et Chiu, 1983 (1983,1984), Nitkin etal. (1983Nitkin etal.…”

Extracellular matrix organization in developing muscle: correlation with acetylcholine receptor aggregates.

“…These enzymes selectively degrade the different classes of glycosaminoglycan side chains that characterize each species of proteoglycan. Control experiments demonstrated that whereas commercial preparations of hyaluronidase and keratanase exhibited detectable levels of protease contamination, the chondroitinase ABC and two separate heparinase preparations did not (2). In experiments where [35S]sulfate-labeled immunoprecipitates were treated with the various enzymes before electrophoretic analysis (Fig.…”