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Formation of the synaptic basal lamina at vertebrate neuromuscular junction involves the accumulation of numerous specialized extracellular matrix molecules including a specific form of acetylcholinesterase (AChE), the collagenic-tailed form. The mechanisms responsible for its localization at sites of nerve- muscle contact are not well understood. To understand synaptic AChE localization, we synthesized a fluorescent conjugate of fasciculin 2, a snake alpha-neurotoxin that tightly binds to the catalytic subunit. Prelabeling AChE on the surface of Xenopus muscle cells revealed that preexisting AChE molecules could be recruited to form clusters that colocalize with acetylcholine receptors at sites of nerve-muscle contact. Likewise, purified avian AChE with collagen-like tail, when transplanted to Xenopus muscle cells before the addition of nerves, also accumulated at sites of nerve-muscle contact. Using exogenous avian AChE as a marker, we show that the collagenic-tailed form of the enzyme binds to the heparan-sulfate proteoglycan perlecan, which in turn binds to the dystroglycan complex through alpha-dystroglycan. Therefore, the dystroglycan-perlecan complex serves as a cell surface acceptor for AChE, enabling it to be clustered at the synapse by lateral migration within the plane of the membrane. A similar mechanism may underlie the initial formation of all specialized basal lamina interposed between other cell types.
Figure 2. AChE clustering in cultured Xenopus muscle cells. (A and B) A spontaneously formed hot spot of AChE and AChR visualized by R-fasciculin 2 and OG-BTX labeling. (C–E) An AChE cluster induced by a HB-GAM–coated bead. The culture was prelabeled with R-fasciculin 2 and OG-BTX before bead application. Thus, this cluster was formed from preexistent AChE and AChR. (F–H) Clustering of preexistent AChE at the NMJ. The muscle culture was innervated with spinal cord neurons after prelabeling with fluorescent toxins. Both AChE and AChR become clustered at sites of nerve–muscle contact (indicated by arrows in H) formed along the length of this neurite.
Figure 3. Transplantation of quail AChE onto Xenopus muscle cells. Cultured Xenopus muscle cells were incubated with collagenic-tailed quail A12 AChE and its binding to the cell surface detected with avian AChE-specific mAb 1A2. Transplanted AChE colocalized with perlecan on the cell surface in both the clustered (A and B) and diffuse states (C and D). Arrows point to the precise correspondence between the AChE and perlecan labeling.
Figure 4. Transplanted collagenic-tailed AChE is enriched at AChR clusters. (A and B) Attachment of A12 AChE to sites of AChR accumulation on Xenopus muscle cells. (C and D) Clustering of transplanted A12 AChE at the NMJ. After the muscle culture was treated with exogenous AChE, it was innervated by spinal neurons to induce NMJ formation. This AChE, detected by mAb 1A2, becomes clustered at the developing NMJ.
Figure 5. Nonspecific binding of globular G2/G4 forms of quail AChE to Xenopus muscle cells. The AChE was detected with mAb 1A2 (A) and the cell was double-labeled with anti-perlecan antibody (B). In comparison to A12 AChE, these forms showed only low level, non-specific binding to the cell surface without any correlation with the perlecan labeling.
Figure 6. Binding of AChE to perlecan. (A) Binding of AChE to perlecan-conjugated Sepharose beads. Perlecan secreted by quail myotubes was captured on mAb #33-conjugated Sepharose beads which were then used to determine the binding of AChE to this HSPG. The AChE bound to the beads was quantified by radiometric assay. Only the collagenic-tailed A12 form of AChE exhibited strong binding to perlecan. The binding of the globular G2/G4 forms showed only background activity. (B) Binding assayed with BIAcore. Perlecan was covalently conjugated to a sensor chip and this surface was used to assess the binding of AChE. Samples were injected into the flow cell over the chip and the net change in RU at the termination of the sample injection and buffer wash was plotted. Using buffer injection as the baseline, G2/G4 AChE showed nearly no binding to perlecan. In contrast, A12 AChE showed strong binding, which was only reversed by high salt. The samples were injected sequentially as shown in the abscissa (from left to right). For each sample, the mean of 20 data points and the standard deviation are given at the bottom of the trace.
Figure 7. Clustering of preexistent perlecan induced by HB-GAM–coated beads. The muscle cell was prelabeled with anti-perlecan antibodies, treated with HB-GAM–coated beads, and then labeled with fluorescent secondary antibody 24 h later (A and B). (A) Distribution of perlecan at site of bead contact; (B) localization of HB-GAM–coated bead. All sites of perlecan accumulation at sites of bead contact (C) also showed accumulation of DG (D).
Figure 8. Colocalization of AChE and DG in culture. Endogenous AChE was labeled with R-Fasciculin 2 and DG was labeled with anti–β-DG antibody followed by FITC-conjugated second antibody. (A and B) Colocalization of AChE and DG at a NMJ. (C and D) Colocalization of AChE and DG at a bead–muscle contact.
Figure 9. Colocalization of AChE, perlecan and DG on myotomal muscle fibers in vivo. (A and B) Perlecan and AChE colocalization at the ends of myotomal muscle fibers. Arrows point to the membrane invaginations of the myotendinous junction. (C and D) Clustering of DG at the NMJ, marked by AChR clusters (D) labeled with fluorescent α-bungarotoxin, and at the myotendinous junction (arrows). (E and F) Colocalization of DG and AChE at intersomitic junctions (low magnification). (G–H) Colocalization of DG and AChE at sarcolemma specializations shown at higher magnification. Arrows point to invaginations of the myotendinous junction.
Figure 10. Model illustrating the involvement of the dystroglycan-perlecan complex in synaptic localization of the collagenic-tailed AChE form. (A) Before innervation, the COOH terminus of perlecan can interact with the transmembrane DG complex (50). The A12 collagenic-tailed AChE in turn binds to the heparan-sulfate chains of perlecan via its collagen-like tail. This association with a transmembrane protein complex enables the AChE to undergo lateral movement (arrows) on the cell surface in the similar manner as AChRs. (B) In response to innervation, the AChE-perlecan-DG complex is clustered by a mechanism similar to the lateral migration-mediated localization of AChRs. Other postsynaptic proteins, such as dystrophin, utrophin, syntrophin, and the sarcoglycan complex, as well as F-actin, may be involved in the formation and/or the stabilization of the cluster.
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