Fig. 5: The P3 domain of UNC-40 binds the FERM-FA domains of FRM-3.
a Schematic representation of UNC-40 predicted domains: IG: Ig-like; FNIII: fibronectin type-III; TM: transmembrane region; P1, P2 and P3: conserved intracellular motifs. The putative truncated UNC-40 protein resulting from the e1430 mutation is indicated. A hemagglutinin (HA) epitope tag was inserted to replace the last 33 amino acids containing the P3 region. b Transmitted-light images of wild-type, unc-40 null (e1430) and unc-40 ∆P3 (kr457) young adult animals crawling on plates (12 h after L4 stage). Scale bar = 100 μm. c Morphology of muscle cells assessed using a Phim-4::yfp reporter combined with a pan-neuronal Prgef-1::dsRed marker. Arrows, neurites. Stars, muscle arms. d Number of muscle arms per cell in the wild-type and unc-40 mutants. Box plot as in Fig. 1g. One-way ANOVA followed by Tukey’s multiple comparison test of each group compared with the wild type. ***p < 0.001, **p < 0.01. e, f Axonal morphology of the AVM touch neuron expressing a Pmec-7::yfp reporter. Scoring of guidance defects was repeated three times (n = 35 worms per genotype per individual experiment). Percentages of abnormal guidance events compared by one-way ANOVA followed by Tukey’s multiple comparison test of each group. Box plot as in Fig. 1g. ***p < 0.001, **p < 0.01. g Actin organization visualized by COR-1-BFP expressed under the control of the Phim-4 promoter in the outer rows of muscle quadrants. Stars: muscle arms; arrowheads: nerve cord. A SNB-1-GFP marker was expressed in GABAergic motoneurons to visualize synaptic boutons. Arrows: neuronal cell bodies. COR-1-BFP also labels sarcomeres (above the blue line, on the top of each picture). h–j Confocal detection of RFP-labeled GABAARs expressed from the unc-49::rfp knock-in locus and frm-3 FERM-FA-GFP expressed under the control of the muscle-specific promoter Pmyo-3. The overall fluorescence levels of GABAAR (i) and of FERM + FA (j) were normalized to the wild type. Box plots are as in Fig. 1e. One-way ANOVA followed by Tukey’s multiple comparison tests of each group compared with the wild type. ***p < 0.001. *p < 0.05. Scale bars = 10 μm except Fig. 5b where it is 100 μm.
