Martin, Ashley et al. (2015) International Worm Meeting "Regulation of the nicotinic acetylcholine receptor ACR-16."

Sancar, Feyza, Martin, Ashley, Richmond, Janet

[International Worm Meeting, 2015]

At the C. elegans body wall neuromuscular junctions (NMJs) there are two cholinergic ionotropic receptor types, one that is heteromeric and activated by levamisole (LAChR) and one that is homomeric, alpha-7-like, and activated by nicotine (NAChR). LEV-9, LEV-10, and OIG-4 have been implicated in the clustering of LAChRs, but the expression of the colocalized NAChR appears completely normal when these genes are perturbed. The only receptor subunit known to be required for the C. elegans NAChR is ACR-16, which can form functional homo-pentameric receptors. Rapsyn is a possible regulator of the alpha-7 receptor, but we find the levels of ACR-16::GFP and evoked responses of rpy-1 mutants to be the same as wild type. This suggests that other, unidentified proteins play a role in the regulation of the ACR-16 receptor.A forward genetic screen was performed to isolate candidate genes involved in ACR-16 regulation. The screen utilized a single-copy integrant of ACR-16::GFP to isolate mutants that decrease the level of ACR-16::GFP expression. From this screen 3 mutants were identified. Electrophysiological recordings demonstrated a reduction in the evoked NMJ responses in these mutants. Further characterization suggested that LAChRs are unaffected as there was no change in response to pressure ejected-levamisole in the mutants and the fluorescence level of RFP-tagged LAChRs was also normal. Behavioral assays performed on the EMS mutant lines in an unc-63 mutant background revealed a more severe uncoordinated phenotype when compared to unc-63 alone, similar to an unc-63;acr-16 double mutant. These mutant phenotypes do not appear to be due to a synaptogenesis defect, as immunostaining for the cholinergic vesicular marker, UNC-17, appeared to be wild type in the majority of the EMS mutant backgrounds. Responses to pressure-ejected nicotine revealed a reduction in amplitude for two of the mutants, while the amplitude remained wild type in the third. This may relate to different roles in the regulation of ACR-16 in these mutants. Whole genome sequencing, using the Variant Density Mapping approach, has revealed 3 candidate genes for these mutations. Reference mutant strains for all 3 genes phenocopy the ACR-16 expression defects, and two strains fail to complement their respective EMS mutants; a complementation assay for the third mutant is underway. Further characterization of these genes is ongoing and should reveal mechanisms regulating the ACR-16 receptor.

Martin, Ashley A. et al. (2011) International Worm Meeting "Screening for ACR-16 clustering mutants."

Richmond, Janet E., Sancar, Feyza, Martin, Ashley A.

[International Worm Meeting, 2011]

At the C. elegans body wall neuromuscular junctions (NMJs) there are two cholinergic ionotropic receptor types, one that is heteromeric and activated by the anthelmintic levamisole (LAChR) and one that is homomeric and activated by nicotine (NAChR). Screens designed to isolate levamisole-resistant mutants in C. elegans have identified subunits of the LAChR as well as additional genes implicated in receptor function. These latter genes affect LAChR assembly and trafficking as well as receptor clustering at the NMJ. Specifically LEV-9, LEV-10, and OIG-4 have been implicated in the clustering of LAChRs, but remarkably the expression of the colocalized NAChR appears completely normal when these gene products are perturbed. The only receptor subunit known to be required for the C. elegans NAChR is ACR-16, which can form functional homo-pentameric receptors in a heterologous expression system. This receptor has been shown to contribute to the quickly desensitizing component of the NMJ evoked response that accounts for ~80% of the maximum evoked cholinergic response amplitude. At vertebrate skeletal NMJs the nAChR clustering mechanism is known to involve the proteins Agrin, MuSK, and Rapsyn. Similarly, the C. elegans ACR-16 receptor is regulated by the receptor tyrosine kinase, CAM-1, which is homologous to MuSK. However, the function of ACR-16 appears to be unaffected in C. elegans rapsyn mutants (rpy-1)(unpublished data JR), suggesting that CAM-1 may interact with other tethering factors to target ACR-16 to the NMJ. Forward and reverse genetic screens were therefore performed to isolate candidate genes involved in the clustering mechanism of ACR-16. The screens utilized a single-copy insertion of ACR-16::GFP in acr-16 and unc-63;acr-16 mutant backgrounds to isolate mutants that specifically impacted ACR-16::GFP function and expression pattern. From these screens, one gene candidate was identified by RNAi and 25 mutants were isolated following EMS mutagenesis. Mutants exhibited either a reduction in ACR-16::GFP expression at NMJs or ectopic ACR-16::GFP puncta. Preliminary electrophysiological recordings demonstrated a reduction in the evoked NMJ response in four of the EMS mutants as well as the RNAi construct. In all five cases this reduction corresponded to a similar reduction in ACR-16::GFP expression levels. Further characterization of these mutants will require an examination of synaptogenesis, muscle morphology, as well as an evaluation of the post-synaptic response to exogenous ACh. Those mutants that have normal synaptogenesis and muscle morphology will be SNP-mapped and genes of interest will be further characterized.

Martin, Ashley A. et al. (2013) International Worm Meeting "Regulation of the nicotinic acetylcholine receptor ACR-16."

Martin, Ashley A., Richmond, Janet E., Sancar, Feyza

[International Worm Meeting, 2013]

At the C. elegans body wall neuromuscular junctions (NMJs) there are two cholinergic ionotropic receptor types, one that is heteromeric and activated by levamisole (LAChR) and one that is homomeric and activated by nicotine (NAChR). Screens designed to isolate levamisole-resistant mutants in C. elegans have identified subunits of the LAChR and genes that affect LAChR assembly, trafficking, and receptor clustering at the NMJ. Specifically LEV-9, LEV-10, and OIG-4 have been implicated in the clustering of LAChRs, but the expression of the colocalized NAChR appears completely normal when these genes are perturbed. The only receptor subunit known to be required for the C. elegans NAChR is ACR-16, which can form functional homo-pentameric receptors. Published data implicates CAM-1 as well as LIN-17, CWN-2, and DSH-1 as working in a pathway to help localize ACR-16 to the synapse. However, it is possible that other proteins also play a role in the regulation of this receptor. A forward genetic screen was performed to isolate candidate genes involved in the regulation of ACR-16. The screen utilized a single-copy insertion of ACR-16::GFP in an unc-63;acr-16 mutant background to isolate mutants that specifically impact the ACR-16::GFP expression pattern. From this screen, 3 mutants were identified. All mutants exhibited a significant reduction in ACR-16::GFP expression at NMJs. Electrophysiological recordings also demonstrated a reduction in the evoked NMJ responses in the EMS mutants. Further characterization of these mutants demonstrated that the LAChRs may be unaffected as there was no change in response to pressure ejected-levamisole in the mutants and the fluorescence level of RFP-tagged LAChRs was also normal. Immunostaining for the cholinergic vesicular marker, UNC-17 appeared to be wild type, eliminating synaptogenesis defects. Responses to pressure-ejected nicotine revealed a reduction in amplitude for two of the mutants, while the amplitude remained wild type in the third. This may relate to different defects of ACR-16 in these mutants. Whole genome sequencing of these mutations and continued characterizations are underway.