SDN-1 recruits LIN-2/CASK at cholinergic synapses to cluster α7-like N-AChRs.(A) Coimmunoprecipitation analysis of the interaction between LIN-2B and ACR-16. The worm lysates of animals expressing ACR-16-wrmScarlet with or without coexpression of GFP-LIN-2B in muscle were precipitated by wrmScarlet nanobody beads and detected by anti-GFP or anti-wrmScarlet antibodies. (B) Confocal detection and quantification of ACR-16-wrmScarlet in control, lin-2(0), and lin-2(0) animals expressing GFP-LIN-2A or GFP-LIN-2B driven by the muscle-specific Pmyo-3 promoter. (C and D) Confocal detection and quantification of ACR-16-wrmScarlet (A) or UNC-29-RFP (B) fluorescence levels in control and sdn-1(ΔEYFA) animals that carry a mutation deleting the SDN-1 C-terminal PDZ-binding motif. (E) GST pull-down analysis of LIN-2B interaction with the entire intracellular part of SDN-1 (SDN-1ICD) or after deleting the PDZ-binding motif (SDN-1ICD ΔEFYA). Samples were analyzed 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. (F) Confocal detection and fluorescence profiles of RFP-LIN-2A and NLG-1-GFP in control and sdn-1(0) animals expressing rfp-lin-2a and nlg-1-gfp driven by the muscle-specific promoter Pmyo-3. (G) Quantification of the fluorescence levels of RFP-LIN-2A at the dorsal nerve cord. (H) Pearson’s correlation coefficient between RFP-LIN-2A and NLG-1-GFP fluorescence. (I) Fluorescence levels of RFP-LIN-2A at GABA synapses. RFP-LIN-2A fluorescence level was quantified in GABA synaptic regions as defined by the presence of the NLG-1-GFP marker (see Material and methods for details). (J and K) Confocal detection (J) and quantification (K) of mNG-SDN-1. (L) Confocal detection and fluorescence profiles of RFP-LIN-2A and mNG-SDN-1 full length (control) or lacking the PDZ-binding motif (sdn-1(ΔEYFA)) in animals expressing rfp-lin-2a driven by the muscle-specific promoter Pmyo-3. (M) Pearson’s correlation coefficient between RFP-LIN-2A and mNG-SDN-1 full length or mNG-SDN-1 (ΔEYFA). Individual values are shown as dots. (N) Confocal detection and quantification of LIN-2A-GFP. Scale bars, 10 µm.
Figure 6.

SDN-1 recruits LIN-2/CASK at cholinergic synapses to cluster α7-like N-AChRs. (A) Coimmunoprecipitation analysis of the interaction between LIN-2B and ACR-16. The worm lysates of animals expressing ACR-16-wrmScarlet with or without coexpression of GFP-LIN-2B in muscle were precipitated by wrmScarlet nanobody beads and detected by anti-GFP or anti-wrmScarlet antibodies. (B) Confocal detection and quantification of ACR-16-wrmScarlet in control, lin-2(0), and lin-2(0) animals expressing GFP-LIN-2A or GFP-LIN-2B driven by the muscle-specific Pmyo-3 promoter. (C and D) Confocal detection and quantification of ACR-16-wrmScarlet (A) or UNC-29-RFP (B) fluorescence levels in control and sdn-1(ΔEYFA) animals that carry a mutation deleting the SDN-1 C-terminal PDZ-binding motif. (E) GST pull-down analysis of LIN-2B interaction with the entire intracellular part of SDN-1 (SDN-1ICD) or after deleting the PDZ-binding motif (SDN-1ICD ΔEFYA). Samples were analyzed 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. (F) Confocal detection and fluorescence profiles of RFP-LIN-2A and NLG-1-GFP in control and sdn-1(0) animals expressing rfp-lin-2a and nlg-1-gfp driven by the muscle-specific promoter Pmyo-3. (G) Quantification of the fluorescence levels of RFP-LIN-2A at the dorsal nerve cord. (H) Pearson’s correlation coefficient between RFP-LIN-2A and NLG-1-GFP fluorescence. (I) Fluorescence levels of RFP-LIN-2A at GABA synapses. RFP-LIN-2A fluorescence level was quantified in GABA synaptic regions as defined by the presence of the NLG-1-GFP marker (see Material and methods for details). (J and K) Confocal detection (J) and quantification (K) of mNG-SDN-1. (L) Confocal detection and fluorescence profiles of RFP-LIN-2A and mNG-SDN-1 full length (control) or lacking the PDZ-binding motif (sdn-1(ΔEYFA)) in animals expressing rfp-lin-2a driven by the muscle-specific promoter Pmyo-3. (M) Pearson’s correlation coefficient between RFP-LIN-2A and mNG-SDN-1 full length or mNG-SDN-1 (ΔEYFA). Individual values are shown as dots. (N) Confocal detection and quantification of LIN-2A-GFP. Scale bars, 10 µm.

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