Janet Richmond (2005) WormBook "Synaptic function."

Janet Richmond

[WormBook, 2005]

C. elegans has emerged as a powerful genetic model organism in which to study synaptic function. Most synaptic proteins in the C. elegans genome are highly conserved and mutants can be readily generated by forward and reverse genetics. Most C. elegans synaptic protein mutants are viable affording an opportunity to study the functional consequences in vivo. Recent advances in electrophysiological approaches permit functional analysis of mutant synapses in situ. This has contributed to an already powerful arsenal of techniques available to study synaptic function in C. elegans. This review highlights C. elegans mutants affecting specific stages of the synaptic vesicle cycle, with emphasis on studies conducted at the neuromuscular junction.

Janet Richmond et al. (2002) West Coast Worm Meeting "Identifying the role of endophilin in synaptic vesicle endocytosis"

Warren Davis, Alexander M van der Bleik, Dawn Signor Matthies, Janet Richmond, Steffen Runz, Erik M Jorgensen, Daniel A Rube, Kim Schuske

[West Coast Worm Meeting, 2002]

Endophilin is an SH3 domain-containing protein which binds the proline-rich domains of synaptojanin and dynamin. Both dynamin and synaptojanin play a major role in clathrin-mediated endocytosis. Recent studies on the Drosophila endophilin mutant, D-endoA/endo, demonstrated that mutant animals have a depletion of synaptic vesicles in nerve terminals, indicating a defect in vesicle recycling (1,2). Endophilin is encoded by the unc-57 gene. We are characterizing unc-57 mutants to determine 1) if endophilin is required for synaptic vesicle endocytosis in C. elegans, 2). if the role of endophilin is to bind and localize synaptojanin and/or dynamin to sites of endocytosis, and 3). if the lipid modifying activity of endophilin is required for its function. Consistent with a role in endocytosis,unc-57 is expressed in many, if not all neurons and in the spermatheca. In addition, GFP-tagged UNC-57 protein is present at synapses. Physiological recordings demonstrate that unc-57 mutants have reduced evoked and endogenous activity. This is consistent with a defect in either exocytosis or endocytosis. However, synapses fatigue more rapdily in mutants than wild-type animals. These data are consistent with endophilin playing a role in synaptic vesicle recycling. Moreover, proteins normally endocytosed into vesicles are diffusely distributed on neuronal processes. Specifically, a GFP-tagged synaptic vesicle protein, synaptobrevin (VAMP), is diffusely localized in the unc-57(ok310) mutant background rather than punctate as is seen in wild-type worms. This result is similar to the phenotype of other endocytosis mutants such as unc-26, dyn-1, unc-11, and snt-1. Finally, proof of an endocytosis defect will require an ultrastructural analysis; these experiments are in progress. It has been suggested that endophilin is required to bind and localize dynamin and synaptojanin to sites of endocytosis. To test this we are looking at the localization of DYN-1::GFP and UNC-26::GFP in an unc-57 mutant background. Our data suggests that at a gross level, DYN-1:GFP is correctly localized in unc-57 mutants. We do not yet know if UNC-26 is localized. Schmidt et al. recently showed that endophilin has a lipid modifying function - specifically, lysophosphatidic acid acyl transferase activity (LPAAT) (3). This lipid modifying activity converts lysophopatidic acid to phosphatidic acid. It was proposed that a specific lipid composition may be required for plasma membrane invagination during synaptic vesicle endocytosis. We are considering a variety of approaches to determine if C. elegans endophilin has LPAAT activity and if that activity is required for its role in synaptic vesicle endocytosis.

Janet E. Richmond (2006) WormBook "Electrophysiological recordings from the neuromuscular junction of C. elegans."

Janet E. Richmond

[WormBook, 2006]

Electrophysiology provides a quantifiable measure of synaptic activity useful in the functional analysis of synaptic proteins. Recent advances in the application of this technique to C. elegans provides a means of coupling genetics to electrophysiological analysis, providing new insights into the molecular mechanisms regulating neurotransmission. Here we describe a dissection technique that exposes the neuromuscular junctions of C. elegans for electrophysiological analysis. This technique can be adapted to record from virtually any excitable cell in the worm.

Janet E Richmond (2008) C.elegans Neuronal Development Meeting "Molecular Bases for Synaptic Vesicle Docking and Priming at C. Elegans Neuromuscular Junctions"

Janet E Richmond

[C.elegans Neuronal Development Meeting, 2008]

In order to release neurotransmitters, synaptic vesicles must first become fusion competent. Several proteins including UNC-13 and the SNARE complex components, syntaxin, SNAP-25 and synaptobrevin are considered to play essential roles in rendering synaptic vesicles fusion competent in a multi-step process termed priming. Once primed, vesicles juxtaposed to the membrane of presynaptic release sites are poised to release their contents into the synaptic cleft in response to a calcium signal. On the basis of ultrastructural data derived from chemically fixed synapses, the plasma membrane juxtaposition of vesicles has long been thought to persist in the absence of priming, suggesting that an independent vesicle-docking step precedes priming. Several proteins including RAB-3 and UNC-18 have been implicated in this upstream docking process. However, recent ultrastructural analyses of priming defective mutants following high-pressure freeze fixation have revealed dramatic reductions in the number of docked vesicles, implying that docking is actually a morphological correlate of priming. What then are the functional roles of proteins previously assigned a docking function? In order to address this question we have re-examined the ultrastructural phenotypes of rab-3 and unc-18 mutants using high-pressure freeze fixation. The results of this analysis will be presented.

Janet E Richmond et al. (2001) International C. elegans Meeting "Evidence that UNC-13 acts via syntaxin to promote vesicle priming"

Robby M Weimer, Erik M Jorgensen, Janet E Richmond

[International C. elegans Meeting, 2001]

Release of neurotransmitters from synaptic vesicles requires the fusion of the synaptic vesicle membrane with the plasma membrane. Membrane fusion at nerve terminals is extremely rapid and requires the vesicle to be in a readied or primed state. Priming is thought to require the formation of the SNARE complex, comprised of the vesicle protein synaptobrevin and the plasma membrane proteins SNAP-25 and syntaxin. In solution syntaxin adopts a default-closed conformation that is incompatible with SNARE complex assembly. In this closed state, the amino terminus of syntaxin occludes its SNARE motif preventing interactions with the other SNARE proteins. UNC-13 is another protein that is required for vesicle priming. UNC-13 binds the amino terminus of syntaxin in a region not involved in interactions with the other SNARE proteins. One possible model for UNC-13 function is that it promotes the open configuration of syntaxin by binding to the syntaxin amino terminus and thus facilitates interactions with the other SNARE proteins. If the function of UNC-13 is to open syntaxin then one would predict that a constitutively open form of syntaxin should bypass the requirement for UNC-13 in vesicle priming. Dulubova et al. ( EMBO J. 1999 Aug 16;18(16):4372-82.) identified two amino acid substitutions which cause syntaxin to constitutively adopt the open state. We engineered mutations into C. elegans syntaxin at these conserved residues (UNC-64 L166A/E167A). Expression of the open form of syntaxin in unc-13(s69) mutants significantly improved the behavioral deficits of unc-13(s69) animals . Furthermore, primed vesicles and evoked release were restored to wild-type levels in unc-13(s69) mutants expressing the open form of syntaxin. In contrast, overexpression of wild-type syntaxin failed to rescue the behavioral or priming defects of unc-13(s69 ) mutants. The ability of open syntaxin to rescue evoked release was specific to unc-13 mutants, since open syntaxin failed to rescue evoked release in the synaptobrevin null mutant snb-1(js124) or the neuronal calcium channel mutant unc-2(e55) . These data support a model in which UNC-13 primes synaptic vesicles for fusion by promoting the open configuration of syntaxin.

Han H-P et al. (1992) Worm Breeder's Gazette "Expression and Localization of the cha-1 and unc-17 Gene Products"

Rand JB, Duerr JS, Han H-P

[Worm Breeder's Gazette, 1992]

EXPRESSION AND LOCALIZATION OF THE cha-l AND unc-17 GENE PRODUCTS He-ping Han, Janet Duerr, and Jim Rand, Oklahoma Medlcal Research Foundation, Oklahoma Clty, OK 73104

Martin, Ashley A. et al. (2017) International Worm Meeting "The C. elegans sarcoplasmic reticulum calcium-ATPase regulates nicotinic acetylcholine receptor ACR-16 expression."

Martin, Ashley A., Richmond, Janet E.

[International Worm Meeting, 2017]

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). Conserved components of a novel synaptic cleft scaffold have been implicated in the clustering of LAChRs, but the expression of the colocalized NAChR appears completely normal when these proteins are absent. This suggests that other, unidentified proteins regulate NAChR expression. The only receptor subunit known to be required for the C. elegans NAChR is ACR-16, which can form functional homo-pentameric receptors. A forward genetic screen was performed to isolate candidate genes involved in ACR-16 expression. The screen utilized a single-copy integrant of ACR-16::GFP to isolate mutants that decrease the synaptic level of ACR-16::GFP. From this screen a mutant was identified, and whole genome sequencing, using the Variant Density Mapping approach, revealed the sarcoplasmic reticulum calcium-ATPase sca-1 as a likely candidate gene for this mutation. A sca-1 reference mutant strain phenocopies the ACR-16::GFP expression defects. The expression and localization of LAChRs and inhibitory GABA receptors seem unaffected in a sca-1 mutant background based on wild-type levels of LAChR::RFP and GABAR::GFP fluorescence. Behavioral assays show a more severe uncoordinated phenotype in a sca-1;LAChR mutant background, consistent with an affect on ACR-16 receptors. The affects of sca-1 on ACR-16 receptor function are not caused by defects in NMJ patterning, as there is no change in the number of synapses and muscle structure is wild type in these mutants. Electrophysiological recordings show a significant reduction in the evoked response of sca-1 mutants suggesting a functional affect on the receptors. Responses to pressure-ejected nicotine in sca-1 mutants are wild type implying that receptors are present on the muscle membrane but mislocalized. The reintroduction of SCA-1 driven by a muscle specific promoter is able to rescue the reduction in levels of ACR-16::GFP and the increased uncoordination seen in sca-1;LAChR double mutants. Due to the role of SCA-1 in calcium level maintenance, calcium imaging was performed. In sca-1 mutants there is a significant increase in baseline levels of calcium, however, stimulated calcium levels are significantly reduced as compared to the control. Preliminary calcium imaging in acr-16 mutants suggests there is a similar phenotype to what is seen in sca-1 mutants. This suggests a role for calcium homeostasis mediated through ACR-16 receptors. Further evaluation of this mechanism is on going.

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.

Rapti, Georgia et al. (2009) International Worm Meeting "A single immunoglobulin domain protein required for the localization of acetylcholine receptors at the C.elegans neuromuscular junction."

Rapti, Georgia, Richmond, Janet, Bessereau, Jean-Louis

[International Worm Meeting, 2009]

Acetylcholine receptors sensitive to the nematode-specific cholinergic agonist levamisole (L-AChRs) form clusters at the C. elegans neuromuscular junctions (NMJs) and their proper localization is critical for efficient excitatory neurotransmission. Here we demonstrate that oig-4 (one Ig domain-4) is a new gene required for L-AChR synaptic clustering. We have previously shown that L-AChR clustering requires two proteins, LEV-9 and LEV-10, which form an extracellular synaptic scaffold. LEV-9, a CCP-domain rich secreted protein, physically interacts with the ectodomain of LEV 10, a CUB-domain rich type 1 transmembrane protein. Interestingly, none of the proteins of the L-AChR/LEV-9/LEV-10 complex forms clusters at the synapse by itself, indicating that additional components are necessary for synaptic localization of this complex. lev-10 and lev-9 mutants show partial resistance to levamisole: they sense levamisole and hypercontract but do not die on drug concentrations lethal for wild-type animals. To identify new components of the L-AChR clustering machinery we performed a screen for mutants with partial resistance to levamisole after EMS mutagenesis and we isolated a new mutant allele, kr39. Using classical genetic mapping we identified a mis-sense point mutation in the predicted gene oig-4. We isolated a second oig-4 mutant allele in a non-complementation screen. In both mutants, levamisole-resistance is rescued by providing an oig-4 genomic fragment. Immunostaining of L-AChR subunits and in vivo imaging of knock-in strains with fluorescent L-AChR subunits demonstrate that L-AChRs do not localize properly at the NMJs of oig-4 mutants. Moreover, the LEV-10 protein is no longer detected at synapses of these mutants. However, presynaptic boutons are properly formed. OIG-4 is specifically required for the L-AChR clustering as the UNC-49 GABA receptor and the ACR-16 N-AChRs are clustered normally at NMJs of oig-4 mutants. oig-4 is predicted to encode a secreted protein with a single immunoglobulin (Ig) domain. We demonstrated that OIG-4 is expressed predominantly in the body wall muscle. Its muscle expression is sufficient to rescue the partial levamisole-resistance phenotype. A functional OIG-4-GFP protein is secreted when expressed by the muscle and it forms clusters at NMJs. Altogether, these results suggest that we have identified a new extracellular synaptic protein that might be part of the L-AChR/LEV-9/LEV-10 scaffold and may link this complex to local synaptic determinants.

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.