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. 2011 Jan 21;30(4):706–718. doi: 10.1038/emboj.2010.355

Figure 3.

Figure 3

OIG-4 is secreted and clustered at neuromuscular junctions (NMJs) while the mutated protein GFP-OIG-4(G84R) is retained intracellularly. (A) The GFP-OIG-4 translational fusion but not the mutated GFP-OIG-4(G84R) rescues the oig-4 mutant phenotypes. Graph represents the percentage of dead animals after overnight exposure to levamisole 0.6 mM (mean±s.e.m., n=3 independent experiments of 3–5 independent lines, total number of animals tested: 100–300). Ex: extrachromosomal transgene; Is: genome integrated transgene; expression was achieved using the oig-4 promoter (Poig-4) or the muscle-specific promoter Pmyo-3 (muscle). (B) GFP-OIG-4 is secreted. The GFP-OIG-4 translational fusion expressed by the muscle promoter Pmyo-3 in transgenic oig-4 mutants is detected in coelomocytes. Coelomocytes (dotted circle) are visualized by Nomarski (i, iii) and epifluorescence microscopy (ii, iv) in wild-type (i, ii) and transgenic animals (iii, iv). Asterisk indicates intestine autofluorescence; scale bar=10 μm. (C) GFP-OIG-4 forms clusters at NMJs GFP-OIG-4 were detected in the nerve ring (nr), and in both the dorsal and ventral nerve cords (dc, vc) using anti-GFP immunofluorescence staining (i). GFP-OIG-4 clusters are juxtaposed to the pre-synaptic cholinergic boutons visualized by immunostaining of the vesicular acetylcholine transporter (VAChT) UNC-17 (ii–iv) and colocalize with L-AChR clusters stained by antibodies against the UNC-38 subunit (v–vii). Scale bar=10 μm. (D) GFP-OIG-4(G84R) is retained in the muscle cell bodies (i, ii) and does not reach NMJs visualized by anti-UNC-17 immunostaining (iii). Arrows indicate muscle cells. Scale bar=10 μm.