Fig. 4: The FERM-FA tandem of FRM-3 acts cell-autonomously to support GABAAR clustering. | Nature Communications

Fig. 4: The FERM-FA tandem of FRM-3 acts cell-autonomously to support GABAAR clustering.

From: The netrin receptor UNC-40/DCC assembles a postsynaptic scaffold and sets the synaptic content of GABAA receptors

Fig. 4

a Confocal detection of RFP-labeled GABAAR expressed from the unc-49::rfp knock-in locus kr296 in frm-3(0) mutants alone and upon muscle-specific expression of full-length and truncated versions of FRM-3-GFP (domain schematic as in Fig. 1a). Fluorescence intensity of RFP-UNC-49 was normalized to the wild type. Box plot 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; n.s: not significant. b Analysis of FRM-3-GFP distribution in frm-3(0) mutants expressing full-length and truncated versions of FRM-3-GFP. The proportion of animals with synaptic or diffuse GFP distribution was scored for each chimera. c GST pull-down analysis of FERM-FA domain dimerization. GST-LIN-2B was used as a positive control. The samples were analyzed by immunoblotting using an anti-HA antibody. The same membrane was stained with Ponceau S red to show GST expression. Molecular weights (Mw) are shown on the right. d, e In vivo co-immunoprecipitation experiments between UNC-40 and FRM-3 full length or FERM-FA domain. UNC-40-RFP was precipitated from whole worm extracts by RFP-trap-A bead. GFP fused components were detected by western blot. Precipitation efficiency was tested by Western Blot using an anti-RFP antibody. Specificity of co-IP detection was shown in e. f Alignment and predicted secondary structure of human DCC and UNC-40 P3 region, based on PROMALS3D analysis. Consensus_ss (secondary structure): “h” shows residues involved in an alpha-helix structure; the corresponding amino acid are marked in red. Consensus_aa: aliphatic: l; aromatic: @; hydrophobic: h; polar residues: p; tiny: t; small: s; negatively charged: -; charged: c. g GST pull-down analysis of the FERM-FA domain interaction with UNC-40ICD and UNC-40ICDΔP3. GST fused to LIN-2B was used as positive control. Samples were analyzed by immunoblotting using an anti-HA antibody. The same membrane was stained by Ponceau S red to shown GST expression. Molecular weights (Mw) of corresponding proteins are shown on the right. Scale bars = 10 μm.

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