Agrin-induced Reorganization of Extracellular Matrix Components on Cultured Myotubes: Relationship to AChR Aggregation

  • Ralph M. Ni
  • Published 1990

Abstract

Agrin, an extracellular matrix-associated protein extracted from synapse-rich tissues, induces the accumulation of acetylcholine receptors (AChRs) and other synaptic components into discrete patches on cultured myotubes. The appearance of agrin-like molecules at neuromuscular junctions suggests that it may direct synaptic organization in vivo. In the present study we examined the role of extracellular matrix components in agrin-induced differentiation. We used immunohistochemical techniques to visualize the spatial and temporal distribution of laminin, a heparan sulfate proteoglycan (HSPG), fibronectin, and type IV collagen on cultured chick myotubes during agrininduced aggregation of AChRs. Myotubes displayed significant amounts of laminin and HSPG, lesser amounts of type IV collagen, and little, if any, fibronectin. Agrin treatment caused cell surface laminin and HSPG to patch, while collagen and fibronectin distributions were generally unaffected. Many of the agrin-induced laminin and HSPG patches colocalized with AChR patches, raising the possibility of a causal relationship between matrix patching and AChR accumulations. However, patching of AChRs (complete within a few hours) preceded that of laminm or HSPG (not complete until 15-20 h), making it unlikely that matrix accumulations initiate AChR patching at agrin-induced sites. Conversely, when AChR patching was blocked by treatment with anti-AChR antibody mAb 35, agrin was still able to effect patching of laminin and HSPG. Taken together, these findings suggest that agrin-induced accumulations of AChR and laminin/HSPG are not mechanistically linked. F UNCTIONAL transmission across the neuromuscular junction requires the precise localization of cell surface, cytoskeletal, and extracellular components at the point of nerve-muscle contact (reviewed by Peng, 1987; Schuetze and Role, 1987; Steinbach and Bloch, 1986; Rubin and Barald, 1983; Dennis, 1981; Fambrough, 1979). It is quite remarkable that these components accumulate in such a discrete region, which represents only ~,0.1% of the myofiber surface. Acetylcholine receptors (AChRs) ~, for example, are packed into the synaptic membrane at densities approaching 15-20,000 per square micron, hut are virtually absent elsewhere on the myofiber surface (reviewed in Salpeter and Loring, 1985). To understand the mechanisms involved in the formation and maintenance of synaptic structure, we have treated cultured myotubes with agrin, a synaptic-organizing molecule that appears to be related to factors that function at synapses in vivo. Studies by McMahan and colleagues demonstrated that in vivo matrix-associated factors can play a significant role in the regeneration of neuromuscular structure (Sanes et al., Dr. Nitkin's present address is NICHD-MRDDB, Bldg. EPN, Rm. 631, 6130 Executive Blvd., Bethesda, MD 20892. Address reprint requests to him. 1. Abbreviations used in this paper: AChR, acetyicholine receptor; HSPG, heparan sulfate proteoglycan. 1978; Burden et al., 1979; McMahan and Slater, 1984; Anglister and McMahan, 1985; see also Bader, 1981). This led to the screening of matrix-enriched fractions of Torpedo electric organ for factors that affect AChR distribution (Rubin and McMahan, 1982; Nitldn et al., 1983;~ Godfrey et al., 1984). In this manner a proteinaceous factor, termed agrin, was identified and characterized (Nitldn et al., 1987). Agrin induces on cultured myotubes dense accumulations of AChRs, which are also associated with acetylcholinesterase (Wallace et al., 1985; Wallace, 1986) and other synaptic components (Wallace, 1989). mAbs against the active component recognize a related series of polypeptides with molecular masses of 70, 95, 135, and 150 kD (Nitldn et al., 1987; see also Godfrey et al., 1988a). These mAbs were also used to demonstrate that agrin-like molecules are concentrated at neuromuscular junctions in vivo (Fallon et al., 1985; Reist et al., 1987; Godfrey et al., 1988a). Therefore, not only is agrin a useful means of inducing "synaptic" differentiation on cultured myotubes, but it appears to be related to factors that direct synaptic development in vivo. Several observations suggest that agrin-induced AChR patching occurs through a specific, physiological cellular mechanism. Agrin effects myotubes in a dose-dependent manner, although at higher agrin levels the number of AChR patches per myotube plateaus off (Godfrey et al., 1984; Wallace, 1989), suggesting that AChR patching may be limited © The Rockefeller University Press, 0021-9525/90/09/1161/10 $2.00 The Journal of Cell Biology, Volume 11 I, September 199

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Cite this paper

@inproceedings{Ni1990AgrininducedRO, title={Agrin-induced Reorganization of Extracellular Matrix Components on Cultured Myotubes: Relationship to AChR Aggregation}, author={Ralph M. Ni}, year={1990} }