XB-ART-10483J Neurobiol September 15, 2000; 44 (4): 436-45.
Agrin fragments differentially induce ectopic aggregation of acetylcholine receptors in myotomal muscles of Xenopus embryos.
Agrin is an extracellular synaptic protein that organizes the postsynaptic apparatus, including acetylcholine receptors (AChRs), of the neuromuscular junction. The COOH-terminal portion of agrin has full AChR-aggregating activity in culture, and includes three globular domains, G1, G2, and G3. Portions of the agrin protein containing these domains bind to different cell surface proteins of muscle cells, including alpha-dystroglycan (G1-G2) and heparan sulfate proteoglycans (G2), whereas the G3 domain is sufficient to aggregate AChRs. We sought to determine whether the G1 and G2 domains of agrin potentiate agrin activity in vivo, as they do in culture. Fragments from the COOH-terminal of a neuronal agrin isoform (4,8) containing G3, both G2 and G3, or all three G domains were overexpressed in Xenopus embryos during neuromuscular synapse formation in myotomal muscles. RNA encoding these fragments of rat agrin was injected into one-cell embryos. All three fragments increased the ectopic aggregation of AChRs in noninnervated regions near the center of myotomes. Surprisingly, ectopic aggregation was more pronounced after overexpression of the smallest fragment, which lacks the heparin- and alpha-dystroglycan-binding domains. Synaptic AChR aggregation was decreased in embryos overexpressing the fragments, suggesting a competition between endogenous agrin secreted by nerve terminals and exogenous agrin fragments secreted by muscle cells. These results suggest that binding of the larger agrin fragments to alpha-dystroglycan and/or heparan sulfate proteoglycans may sequester the fragments and inhibit their activity in embryonic muscle. These intermolecular interactions may regulate agrin activity and differentiation of the neuromuscular junction in vivo.
PubMed ID: 10945898
Article link: J Neurobiol
Species referenced: Xenopus laevis
Genes referenced: dag1 tbxt.2
Article Images: [+] show captions
|Figure 1 Diagram of agrin fragments overexpressed in the embryo. RNAs encoding COOH-terminal fragments of rat agrin were synthesized from cDNA templates encoding one (T3), two (T2), or three (T1) G-domains (see Materials and Methods). All fragments used were from a neuronal agrin isoform containing both y4 and z8 inserts (Ferns et al., 1993). Epitope tags encoding both FLAG and polyhistidine sequences (“T”) were present on the N-terminus of the fragments, along with a signal sequence (“S”) to ensure secretion of the proteins. Predicted molecular mass of the fragments shown (amino acids only) is about 104.8, 61.3, and 26.4 kDa for T1-–T3, compared to 101.9 kDa for “C-agrin” (Ferns et al., 1993), which is similar to T1 except that it lacks epitope tags. Location of the minimal fragment required for AChR aggregating activity and the heparin and dystroglycan-binding domains are indicated by bold lines at bottom.|
|Figure 2 Ectopic acetylcholine receptor (AChR) aggregation induced by overexpression of COOH-terminal agrin fragments. Images of AChR aggregates labeled with rhodamine–a-bungarotoxin (bright areas) were obtained by confocal microscopy. Projected series of images are shown. Horizontal stripe of AChR aggregates at the top of each panel consisted of synaptic aggregates at the ends of myotomes. Ectopic aggregates were concentrated in a band near the center of myotomes (arrows). (A) Control muscle from embryo injected with 2.8 fmol GFP RNA. (B,C,D) Muscle from embryos injected with T1, T2, or T3 agrin fragment RNA (8.7 fmol). Bar 5 20 mm.|
|Figure 3 Immunofluorescence for overexpressed agrin fragments is found in nonsynaptic regions in myotomal muscles of embryos. Rat agrin fragments were labeled with antibody to the FLAG epitope (C,E) or with a mixture of antibodies against FLAG and rat agrin (A). Immunofluorescence was most prominent within ectoderm (not shown) and in the central portion of myotomal muscles (A,C,E), where ectopic AChR aggregates (B,D,F) were induced, but was also seen in the synaptic region of muscles. Control embryos (injected with GFP RNA; not shown) lacked immunofluorescence. (A,B) Injected with T1 agrin RNA; (C,D) T2 RNA; (E,F) T3 RNA (all 17.4 fmol). Bar 5 20 mm. A semiquantitative estimate of amount of the overexpressed agrin fragments in the ectopic region, made by integrating area of immunofluorescent deposits with intensity, gave values of 2.9, 1.2, and 2.9 3 105 for the microscopic fields shown in (A), (T1); (C), (T2); and (E), (T3), respectively.|
|Figure 4 Axonal pattern is unaffected by expression of agrin fragments. Ectopic AChR aggregation was induced by the T3 fragment containing the COOH-terminal G3 domain of agrin. Axons were labeled with HNK-1 antibody (green), and AChR aggregates with rhodamine–a-bungarotoxin (red). Axons were present in the intermyotomal septal (synaptic) regions (top and bottom of both panels) in control embryos injected with GFP RNA (A) and in embryos expressing the T3 agrin fragment (B). Ectopic AChR aggregates were focused in a band near the center of the myotome (arrows, B) and were not contacted by axons detectable with this antibody. Bar 5 20 mm.|
|Figure 5 Ectopic AChR aggregation is induced by similar amounts of injected RNA encoding different agrin fragments. Agrin fragment RNA (T1, l; T2, n; T3, ) was injected into one-cell stage Xenopus embryos. AChR aggregation was measured as described in Experimental Methods. Ectopic aggregation (A) refers to total AChR aggregate area outside of the innervated (synaptic) region at the ends of myotomes. Values shown are ratios of ectopic aggregation in experimental and control embryos (6SEM). Synaptic AChR aggregation (B) was measured in the same experiment within the usually innervated region adjacent to the intermyotomal septum and is presented as a percentage (6SEM) of control (GFP RNA-injected) synaptic AChR aggregate area. Synaptic AChR aggregation decreased as ectopic AChR aggregation increased.|