Zachary T Eggers et al. (2003) International Worm Meeting "Determining the role of Y18D10A.17, a putative member of the clathrin endocytotic pathway, during cytokinesis in embryos"

Jayne M Squirrell, Zachary T Eggers, John G White

[International Worm Meeting, 2003]

Cytokinesis, the division of one cell into two daughter cells, is of fundamental importance for the growth and development of all living organisms. Recently, systematic screens of genes in the C. elegans genome have been made using RNAi. These screens have identified many genes that are required for early embryonic development. When the function of the Y18D10A.17 gene is blocked using RNAi, defects in cytokinesis are observed in the developing embryo (Zipperlen et al., 2001 EMBO J 20:3984). Y18D10A.17 codes for a protein that has similarity to a yeast suppressor of clathrin deficiency gene (SCD6). Mutations in SCD6 do not affect viability. As SCD6 was identified in a screen for suppression of a clathrin deficiency, it might be that SCD6 is a novel member of the clathrin vesicle budding pathway or, alternatively, a component of an as yet unidentified by-pass pathway. Using multiphoton microscopy of laser-permeabilized embryos that had been RNAi suppressed for Y18D10A.17 expression in the presence of a membrane probe, we found the cytokinesis fails after cleavage furrows fully ingress, i.e, at scission when the persisting intercellular canal is broken and the membranes re-seal. We found that that the late accumulation of membrane to the furrow tip that is seen in the wild type did not occur in the Y18D10A.17 suppressed embryos. Observations of the tubulin organization in these embryos revealed that they undergo exaggerated spindle rocking at the P0 stage and have no visible microtubules in the spindle midzone at anaphase. Furthermore, ZEN-4, a component of the midzone, is less abundant and appears to be mislocalized in Y18D10A.17-depleted embryos. These observations strongly suggest that Y18D10A.17 has an essential role in the process of scission. Additionally, this gene appears to be conserved yet novel, with a possible involvement with clathrin-mediated endocytosis. We therefore are very interested in determining the specific role of Y18D10A.17 in the final stages of cytokinesis.

Christopher Hopkins et al. (2007) International Worm Meeting "UNC-18 transports syntaxin to the synapse and promotes SNARE-mediated vesicle fusion."

Shigeki Wantanabe, Janet Richmond, Erik Jorgensen, Christopher Hopkins

[International Worm Meeting, 2007]

In C. elegans neurons, UNC-18 is necessary for evoked synaptic vesicle release, but its mechanism is unknown. Prior experiments suggest that UNC-18 acts either before or after vesicle docking. We propose UNC-18 has both pre- and post-docking functions: 1) it is a molecular chaperone that traffics syntaxin to the synapse in a non-active (closed) conformation and 2) a facilitator of vesicular release independent of closed syntaxin binding. Furthermore, we propose phosphorylation of UNC-18 provides the molecular switch converting UNC-18 to a form that can bind assembled SNARE complexes. Molecular chaperone: unc-18 null mutants have a severe locomotion defect. One model suggests that UNC-18 acts via the SNARE protein syntaxin. UNC-18 exhibits high affinity for syntaxin in the closed state. In immunoprecipitation pull-down assays, anti-GFP beads recover high levels of both UNC-18::GFP and syntaxin, indicating the two proteins physically interact. The mutant protein UNC-18(R39C) does not bind syntaxin with high affinity. unc-18 mutants exhibit abnormal localization of syntaxin to neuronal cell bodies. These defects are completely rescued by an UNC-18::GFP expression construct, which suggest UNC-18 is required for normal syntaxin localization. In a UNC-18(R39C)::GFP point mutant construct, pull-down recovery of syntaxin is nearly abolished (400 x decrease). Additionally, the R39C construct partially retains a cell body staining defect of syntaxin localization. These results indicate the tight-binding of UNC-18 to syntaxin facilitates syntaxin transport to the synapse. Release facilitator: Electron microscopy of unc-18 null shows a 2.5 fold decrease in docked synaptic vesicles and electrophysiology reveals the remaining docked vesicles are incompetent for evoked release. UNC-18(R39C) rescues the Unc phenotype yet a docking defect remains. These results indicate UNC-18 has a post-docking role that promotes vesicle release.

Robby M Weimer et al. (2001) International C. elegans Meeting "Dual roles of UNC-18 in synaptic transmission."

Erik M Jorgensen, Robby M Weimer, Janet E Richmond, Warren S Davis

[International C. elegans Meeting, 2001]

Neurotransmitter is released from presynaptic nerve terminals by the fusion of synaptic vesicles with the plasma membrane. Synaptic vesicle exocytosis requires the formation of SNARE complexes, comprised of synaptobrevin, SNAP-25 and syntaxin. In solution, syntaxin adopts a closed configuration that is incompatible with SNARE complex formation. UNC-18, a neuronal protein implicated in vesicle exocytosis, binds to syntaxin in the closed configuration. Loss-of-function mutations in unc-18 nearly eliminate vesicle exocytosis. A model based on these data proposed that UNC-18 bound closed syntaxin, promoted the opening of syntaxin, and thereby promoted the formation of the SNARE complex and hence exocytosis. A prediction of this model is that if one could eliminate the syntaxin - UNC-18 interaction then neurotransmission would be blocked. To test this model we engineered mutations into syntaxin that reduce UNC-18 binding, and mutations into UNC-18 that reduce syntaxin binding. We have tested the consequence of these mutations on synaptic vesicle exocytosis. Scanning alanine replacement experiments of the mammalian syntaxin homolog identified two residues, L165/E166, that when mutated to alanine render syntaxin incapable of binding UNC-18. We made the corresponding mutations into C. elegans syntaxin, L166A/E167A. Pulldown experiments demonstrate that this mutant form of syntaxin does not bind UNC-18 from worm lysates. We expressed this mutant form of syntaxin in a null background, unc-64(js115) . Mutant syntaxin that does not bind UNC-18 is capable of rescuing the lethality and locomotory phenotypes associated with the syntaxin null, suggesting that the mutant protein functions in vivo and that UNC-18 function is not via syntaxin. In fact, such worms are hypersensitive to Aldicarb, suggesting that UNC-18 interactions with syntaxin inhibit release. Studies of the UNC-18 homolog in Drosophila , ROP, identified a mutation, R50C, that decreases the affinity of ROP for syntaxin without affecting other known UNC-18 interactions. We made the corresponding mutation in UNC-18, R39C, and expressed the protein in unc-18(e234) animals. Expression of R39C UNC-18 rescues the paralysis associated with e234 suggesting the mutant protein functions in vivo and that the interaction of UNC-18 with syntaxin is not relevant to the UNC-18 role in facilitation of exocytosis. However, animals expressing the mutant UNC-18 are hypersensitive to the effects of Aldicarb, suggesting that more acetylcholine is released in these animals. Thus, UNC-18 binding to syntaxin appears to inhibit rather than facilitate vesicle exocytosis. Finally, if UNC-18 is required to promote the open configuration of syntaxin for SNARE complex formation then a constitutively open form of syntaxin should bypass the requirement for UNC-18. The L165/E166 mutations which reduce UNC-18 binding also renders syntaxin in a constitutively open configuration. To test if the open form of syntaxin can bypass the requirement of UNC-18 we made doubles with unc-18(e234) . Expression of open syntaxin in the unc-18 mutant fails to suppress the Unc-18 phenotype. This suggests that the facilitory role of UNC-18 is not to promote the open configuration of syntaxin for SNARE complex formation. Consistent with this result our preliminary ultrastructural analysis of unc-18 mutant synapses suggests that the facilitory role of UNC-18 is in synaptic vesicle docking, which precedes SNARE complex formation. Together, these data suggest that UNC-18 has dual roles in synaptic transmission: a facilitory role in vesicle docking that precedes SNARE complex formation, and an inhibitory role via the SNARE protein syntaxin. We are currently testing in vivo whether the inhibitory role of UNC-18 is via inhibition of SNARE complex formation.

Yin Li et al. (2008) C.elegans Neuronal Development Meeting "FLP-18 Neuropeptide Signaling Functions to Increase C. elegans Body Curvature"

Niels Ringstad, Yin Li, Bob Horvitz

[C.elegans Neuronal Development Meeting, 2008]

The C. elegans genome contains a large family of flp genes, which encode FMRFamide-related peptides. However, the functions of these neuropeptides are poorly understood. We studied the neuropeptide gene flp-18 by using its overexpression phenotype as an in vivo assay for peptide activity. Overexpression of flp-18 shortened body length, increased body curvature, decreased velocity and caused uncoordinated reverse locomotion. We sought suppressors of the behavioral defects caused by flp-18 overexpression by testing candidate genes and by mutant screens. We found that the G protein-coupled receptor gene Y58G8A.4 is required for the body length and posture defects of flp-18 overexpressors, suggesting that Y58G8A.4 mediates the control of body shape by FLP-18 signaling. Overexpression of Y58G8A.4 caused the same behavioral defects as overexpression of flp-18. The receptor overexpression phenotype was suppressed by deletion of flp-18, indicating that Y58G8A.4 receptor activity is regulated by FLP-18 peptides in vivo. Because flp-18 deletion mutants were grossly wild-type we tested whether FLP-18 peptides function redundantly with small-molecule transmitters. Specifically, we examined animals multiply mutant for flp-18 and genes required for the synthesis or transmission of acetylcholine, GABA, tyramine and octopamine or serotonin. We found that deletion of flp-18 partially suppressed the increased-curvature defect of cha-1 and unc-29 mutants, which have decreased cholinergic signaling, suggesting that one function of FLP-18 is to increase curvature. To identify cellular circuits that use FLP-18 peptides to control posture, we generated a functional flp-18::gfp reporter transgene. We observed expression of flp-18::gfp in head neurons, approximately a dozen neurons in the ventral nerve cord, the dorsal nerve cord and one tail neuron. flp-18::gfp was coexpressed with a cholinergic marker in some ventral nerve cord motor neurons, but not in head or tail neurons. A functional Y58G8A.4::gfp reporter gene was expressed primarily in body-wall muscles. These expression patterns suggest that FLP-18 peptides regulate body curvature by acting directly on body-wall muscles and might be released from cholinergic ventral cord motor neurons.

Deng, Jichao et al. (2021) International Worm Meeting "DAF-18/PTEN functions in the muscles and proximal somatic gonad to couple to promote oocyte arrest in the absence of sperm"

Deng, Jichao, Gagne, Olivier, Narbonne, Patrick

[International Worm Meeting, 2021]

DAF-18/PTEN is an important tumour suppressor, and loss of PTEN activity is associated with various types of cancers. In C. elegans hermaphrodites that lack sperm, such as fog-1 mutants, DAF-18 is required for oocytes to arrest and accumulate in the proximal gonad. As such, fog-1; daf-18 double mutants lay unfertilised oocytes, and fail to downregulate germline stem cell (GSC) proliferation. However, the tissue(s) in which DAF-18 acts to permit oocyte arrest and accumulation in the absence of sperm, and to eventually suppress GSC proliferation, is still unknown. Here, we transgenically rescued daf-18, specifically in the gut, neurons, hypodermis, muscles, proximal somatic gonad, and germline, of fog-1; daf-18 null mutants. Interestingly, we found that daf-18 expression in the muscles, or in the proximal somatic gonad, was sufficient to allow unfertilized oocytes to arrest and accumulate in the proximal gonad of fog-1;daf-18 double mutants. DAF-18 is also required to suppress the proliferation of germ and somatic gonadal cells during dauer formation, wherein DAF-18 acts in the somatic gonadal cells. Using our germline-specific DAF-18 transgene, we further show that maternally-provided DAF-18 is sufficient to rescue the dauer phenotype of daf-18(-) mutants, but not the arrest of unfertilized oocytes in spermless adults. Overall, our results demonstrate that DAF-18 can act from at least two separate tissues to non-autonomously mediate the arrest of oocytes in the absence of sperm, and allow for their accumulation in the proximal gonad. These results may provide new insights into the how PTEN prevents tumour formation.

Ingrid Masse et al. (2005) International Worm Meeting "Lifespan and dauer regulation by tissue-specific activities of Caenorhabditis elegans DAF-18."

Laurent Molin, Florence Solari, Marc Billaud, INGRID MASSE

[International Worm Meeting, 2005]

In Caenorhabditis elegans, the insulin/IGF1 DAF-2 receptor controls entry into dauer and longevity. DAF-2 signalling cascade includes the PI3 Kinase homolog AGE-1 and the FOXO transcription factor DAF-16. The DAF-2 pathway is downregulated by DAF-18 which is encoded by the ortholog of the human tumor suppressor gene PTEN. We have previously shown that, like PTEN, DAF-18 antagonizes the activity of PI3 kinase/AGE-1. To further explore the role of DAF-18 in the regulation of the insulin pathway, we investigated which tissue(s) DAF-18 functions in to regulate dauer formation and lifespan. Our data show that complete dauer formation requires daf-18 expression in several tissues and that the remodelling of dauer tissues depends on both cell autonomous and cell nonautonomous daf-18 function(s). Conversely, daf-18 expression increases adult lifespan in all individual tissues tested. Furthermore, we show that the role of DAF-18 in dauer and lifespan control depends on DAF-16 activation, which is regulated by both cell autonomous and cell nonautonomous DAF-18 function(s) and in a tissue specific manner. Overall our data strongly suggest that several tissues act as signalling centres to mediate DAF-18 function and that DAF-18 could act outside the canonical DAF-2/DAF-16 pathway to regulate dauer and lifespan.

Elena Gracheva et al. (2008) C.elegans Neuronal Development Meeting "Interactions Between TOM-1, UNC-64 and UNC-18 in Synaptic Transmission"

Martine Berthelot-Grosjean, Edward Maryon, Janet Richmond, Elena GRACHEVA

[C.elegans Neuronal Development Meeting, 2008]

The UNC-18/Munc18 protein family is implicated in several stages of exocytosis. Here we present evidence for two UNC-18-dependent synaptic vesicle (SV) targeting steps termed tethering (near the plasma membrane) and docking (contact with the plasma membrane). Docked SVs arise primarily from UNC-13-dependent priming, aprocess leading to SNARE complex assembly and SV fusion competence. The loss of docked SVs in unc-18 mutants, therefore, supports a growing view that UNC-18 functions in this process. Although docked SVs are depleted in unc-13 mutants, tethered SVs accumulate, suggesting UNC-18-dependent tethering occurs in the absence of SV priming. UNC-18 is thought to associate with the plasma membrane via syntaxin interactions. Consistent with this notion, membrane bound UNC-18 is abolished in syntaxin (unc-64) mutant synapses. We, therefore, asked whether SV targeting is impacted by loss of syntaxin. In unc-64 mutant synapses, tethered and docked SVs are eliminated, indicating both SV steps require UNC-18 and syntaxin. Since Munc18-1 is known to interact with closed and open syntaxin conformations, we next examined synapses in which syntaxin was rendered constitutively open. Open syntaxin reduced SV tethering but docking was enhanced, indicating that UNC-18/closed syntaxin binding is required for SV tethering,where as UNC-18/open syntaxin is required for SV docking. We have previously shown that C. elegans tomosyn (TOM-1) negatively regulates SV priming.Vertebrate tomosyn is known to compete with Munc18-1 for syntax inbinding. Similarly, we found increased membrane-associated TOM-1 in unc-18 mutants and conversely, increased membrane-bound UNC-18 in tom-1 mutants. We therefore, predicted that loss of tomosyn would enhance UNC-18/syntaxin interactions at C. elegans synapses, resulting in increased SV tethering and docking. Analysis of tom-1 mutants confirmed this prediction, indicating that TOM-1 negatively regulates both UNC-18-dependent SV-targeting steps. We next demonstrated that in tom-1;unc-18 double mutants, SV docking but not tethering was partially restored resulting in an increased primed SV pool. Together, these data provide evidence for two molecularly distinct UNC-18-dependent SV-targeting mechanisms; tethering, requiring UNC-18/closed syntaxin and docking, in which UNC-18/open syntaxin promotes SV priming.TOM-1 negatively regulates both UNC-18 dependent steps, thereby limiting the number of fusion-competent SVs.

Christopher Hopkins et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Probing UNC-18 Interactions to SNARE Proteins: Pull-downs, Phosphorylation Dependence, and Affinity Measurements"

Christopher Hopkins, Erik Jorgensen

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Synaptic transmission requires the SNARE complex proteins syntaxin, SNAP-25, and synaptobrevin. UNC-18 is also required for neurotransmitter release but its role remains controversial. Affinity experiments with recombinant proteins demonstrate UNC-18 retains a high affinity interaction to syntaxin but does not interact with SNAP-25 or synaptobrevin. UNC-18 may be a negative regulator of fusion by binding syntaxin and inhibiting SNARE complex formation. Alternatively, in yeast, the UNC-18 homolog (Sec1p) appears to be a facilitator of SNARE complex formation because interaction with its cognate syntaxin homolog (SSO1p) occurs only when a SNARE complex is formed. To determine if UNC-18 functions before or after SNARE complex formation, UNC-18 protein complexes were purified from UNC-18::GFP rescued C. elegans via anti-GFP affinity chromatography. As expected, western blots demonstrate that syntaxin co-immunopurifies with the UNC-18::GFP. To demonstrate specificity, a point mutation (R39C) of UNC-18 was found not to co-purify syntaxin. Probing with anti-SNAP-25 or anti-synaptobrevin failed to yield a strong signal, thus the isolated UNC-18 complex is predominantly an interaction to syntaxin and not to a SNARE complex. Unc-18 is a phosphoprotein with strong phosphorylation consensus sequences for phosphorylation by PKC, cdk5, and CAMKII. The inclusion of phosphatase inhibitors in lysis conditions leads to a dramatic reduction in syntaxin co-purification, which suggest phosphorylation mediates interaction of UNC-18 and syntaxin. With phosphatase inhibitors, an increase in SNAP-25 co-purification was observed (synaptobrevin not tested). Therefore phosphorylation of UNC-18 and its binding partners appears to be critical to their interaction affinity. To directly measure UNC-18 affinity to its binding partners a chip-based TIRF method is being developed (in collaboration with Joel Harris at the U of Utah) to directly measure on rate and off rates at the single molecule level.

Ogawa H et al. (1997) International C. elegans Meeting "UNC-18 MEDIATES BINDING OF SYNTAXIN TO THE SYNAPTIC VESICLES"

Ogawa H, Harada S-i, Rand JB, Yamamoto H, Sassa T, Hosono R

[International C. elegans Meeting, 1997]

Genetic studies have revealed that the essential function of the unc-18 gene is the synaptic transmission. n-Sec-1, the mammalian UNC-18 homolog is believed to be associated with a synaptic plasma membrane protein prior to synaptic vesicle docking. If this were the case for the C. elegans UNC-18, the gene mutations would result in constitutive fusions of synaptic vesicles. However, the gene mutations result in the accumulation of synaptic vesicles and neurotransmitters. To examine the conflicting observations, we analyzed the binding of wild-type and mutant UNC-18 to syntaxin. UNC-18 is a hydrophilic, globular, and neutral protein (pI 6.95). In the Sf21 cells the protein is predominantly localized in the cytoplasm, indicating its intrinsic solublility. To investigate further the intracellular distribution of UNC-18, we isolated synaptic vesicles by the Percoll gradient fractionation. UNC-18 was detected on synaptic vesicles by Western blotting. We cloned C. elegans cDNAs encoding the mammalian syntaxin1A homolog (Ce syntaxin). We tested the binding of UNC-18 to GST-Ce syntaxin fusion protein. UNC-18 binds to the recombinant syntaxin in vitro with high affinity. These findings indicate that the protein is periplasmic associating with both synaptic vesicles and plasma membrane, although intrinsically soluble. In vesicle traffic UNC-18 may be a regulator with the plasma membrane through syntaxin. UNC-18 may cause a conformational change of syntaxin and allowing it to bind to synaptic vesicles. To test the hypothesis, we sequenced cDNAs of 15 unc-18 mutants. We found eight missense mutations. We are examing the ability of the mutant UNC-18 proteins to bind to Ce syntaxin and to associate with synaptic vesicles.

Sarah E. Brisbin et al. (2007) International Worm Meeting "Analysis of DAF-18/PTEN in VAB-1 Eph RTK Signaling."

Ian D. Chin-Sang, Sarah E. Brisbin

[International Worm Meeting, 2007]

The elucidation of signaling pathways involved in cell movements is critical for understanding complex developmental processes. Accordingly, consideration must be given to the function of the individual components of such signaling cascades. DAF-18, a C. elegans homolog of the PTEN human tumor suppressor, is a component of the DAF-2 Insulin Receptor-like signaling pathway involved in dauer formation, longevity and metabolism. Our lab has shown that DAF-18 can physically bind VAB-1, an Eph Receptor Tyrosine Kinase with roles in axon guidance and embryonic morphogenesis. To corroborate these varied functions as well as explore additional roles of DAF-18 in the worm, affinity purified DAF-18 antibodies were used for in situ detection of DAF-18 as well as Western blotting. Results of Western blotting confirm that DAF-18 is present in N2 as well as our over-expressing lines at the expected molecular weight of 110 kDa but is absent in daf-18 (ok480), suggesting this represents a molecular null allele. Here we show that DAF-18 is expressed in amphid neurons, ventral nerve cord, nerve ring and the germline precursor cells, Z2 and Z3. The presence of DAF-18 in neuronal tissues is consistent with the known expression pattern of VAB-1, further substantiating an in vivo interaction. Additionally, we have shown endogenous VAB-1 is also expressed in the Z2 and Z3 germline precursors. Previous literature implicates VAB-1 in regulating oocyte maturation, suggesting expression in the germline. DAF-18 has recently been implicated in regulating germline proliferation in L1 arrest, and our results are consistent with DAF-18 functioning in the germline. While the exact mechanism of the DAF-18/VAB-1 interaction requires further investigation, preliminary molecular evidence proposes VAB-1 may be a negative regulator of DAF-18. DAF-18 has been shown to exhibit PIP3 lipid phosphatase activity and we are currently testing whether DAF-18 has protein phosphatase activity on VAB-1. Taken together these results support the in vivo interaction of DAF-18 and VAB-1 which we will be exploring further with 4D analysis to ascertain the role of daf-18 in embryonic morphogenesis, double mutation analysis, aging and dauer studies.