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[
West Coast Worm Meeting,
2002]
We have found that the Hawaiian race CB4856 exhibits higher male fertility than N2 Bristol. This difference is due (at least in part) to increased length of fertility in CB4856 compared to N2: Adult CB4856 males are typically fertile for six or more days, while adult N2 males are sterile after four. We are working to determine the physiological cause of this difference (sperm production, aging, etc.) as well as its genetic and molecular basis.
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[
West Coast Worm Meeting,
2004]
Synaptic vesicles undergo three steps during exocytosis: docking, priming and fusion. Fusion is thought to be mediated by the SNARE complex. SNARE complexes are composed of a helical bundle of the synaptic vesicle protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP-25. SNARE complex formation is regulated by UNC-18 and UNC-13. Our previous data indicated that UNC-18 mediates docking by binding tightly to a closed form of syntaxin. UNC-13 provides a priming function by promoting the formation of an open form of syntaxin. Presumably, this allows syntaxin to form a complex with SNAP-25, which in turn can bind synaptobrevin and allow SNARE complex formation. Genetic data from other organisms contradict this simple model. Specifically, it is not clear whether UNC-13 functions before or after UNC-18. There are three possible scenarios: UNC-18 acts during docking independent of syntaxin, UNC-18 acts during priming before UNC-13 function, UNC-18 acts in fusion after UNC-13 function. These models make different predictions about the association of UNC-18 and synaptobrevin with syntaxin in wild-type vs
unc-13 mutants. A biochemical approach will be used to resolve the steps at which UNC-18 and UNC-13 facilitates vesicle exocytosis. A tandem affinity purification (TAP) tag of protein A and the calmodulin-binding-domain was fused to C-terminus of syntaxin. Rescue of syntaxin-null worms was achieved with extra-chromosomal expression of the construct. Syntaxin purification resulted in co-purification of UNC-18 in the wild type. To demonstrate if UNC-18 is present in SNARE complexes, two types of fusion constructs are being made. A myc-tagged synaptobrevin construct and a flag-tagged UNC-18 will be independently transformed into worms with chromosomally-integrated TAP-tagged syntaxin. Complexes isolated by TAP tag purification will be further purified by affinity to either an anti-myc or an anti-flag antibody affinity chromatography. Western blots and mass spectrometry will then be used to resolve which model prevails.
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[
Neuronal Development, Synaptic Function, and Behavior Meeting,
2006]
Synaptic vesicle exocytosis is thought to occur in three ordered steps: docking, priming and fusion. Priming is defined as the engagement of SNARE proteins between the vesicle and plasma membranes, and fusion is also a SNARE-dependent process. However, the mechanism of docking, in which vesicles become closely associated with the plasma membrane, is not known. Previously, the docking step was believed to be independent of the SNARE proteins. We now find that the SNARE protein syntaxin is required for virtually all synaptic vesicle docking. Further, the syntaxin-binding proteins UNC-13 and UNC-18 are required for docking of specific vesicle populations. Docking defects in UNC-13 and UNC-18 are rescued by a constitutively open syntaxin mutant, demonstrating that docking proceeds through the open conformation of syntaxin. Our results suggest that docking may be indistinguishable from priming, and may involve SNARE interactions between syntaxin and the vesicular SNARE protein synaptobrevin. Alternatively, syntaxin may dock vesicles via a SNARE-independent mechanism, possibly involving syntaxin?s Habc domain. We are currently performing experiments to distinguish between these models.
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[
Neuronal Development, Synaptic Function, and Behavior Meeting,
2006]
Synaptic vesicles exocytosis is thought to occur through three ordered steps: docking, priming, and fusion. SNARE (soluble N-ethylmaleimide-sensitive fusion attachment protein receptors) proteins are involved in all membrane-bound trafficking within eukaryotes including synaptic vesicle exocytosis. The SNAREs are known to function in the priming and fusion step of exocytosis. We have now demonstrated that docking is virtually eliminated in mutants of the SNARE protein syntaxin. In addition, we find that mutants in two syntaxin binding proteins, UNC-13 and UNC-18, also have docking defects. The docking defect in
unc-13 and
unc-18 can be fully rescued by the open form of syntaxin, in which an inhibitory domain (Habc) is hindered from interacting with the SNARE motif. We conclude that docking is mediated by open syntaxin with UNC-13 and UNC-18 acting upstream.
But how does open syntaxin mediate docking? In the open form, both the Habc domain and the SNARE motif are available for interactions with additional proteins. Specifically the open form is available to interact with the other SNARE proteins synaptobrevin and SNAP-25 leading to the formation of the core complex. The formation of the core complex is thought to be the molecular equivalent to the functional step of priming. Two models can be proposed. In the first, the formation of the core complex corresponds to docking in addition to functioning in priming. We are testing this by looking for docking defects in synaptobrevin mutants. A docking defect in synaptobrevin would implicate the formation of the core complex in docking. We have shown that synaptobrevin mutants do not eliminate fusion, but do result in a significant reduction in fusion events. The remaining fusion could be due to redundancy with other synaptobrevin homologs. EM studies for docking defects are underway. Normal docking in synaptobrevin mutants would suggest the alterative model: that the Habc domain binds to a docking factor on the synaptic vesicles. This model is also being tested by expressing the SNARE motif or the Habc motif alone and looking for rescue of docking defects in syntaxin null animals.
*these authors contributed equally
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[
International Worm Meeting,
2005]
The SNAREs are conserved molecules used in all membrane trafficking events. Yet, despite the SNARE molecules' central importance in trafficking, their exact function in the process remains less well understood. The prevailing model for the molecular mechanism of membrane fusion suggests that a vesicular SNARE protein contacts SNARE proteins on the target membrane. The individual SNARE proteins then wind around each other pulling the vesicle to the target membrane. When reconstituted into lipid bilayers the SNARE proteins are able to fuse membranes in vitro. However, in vivo the elimination of individual SNARE molecules does not always eliminate fusion. Unfortunately, in all organisms studied the elimination of SNAREs results in early lethality, making the few in vivo studies of SNARE function extremely difficult. We have generated a system in C. elegans to circumvent the lethality and have begun to use this system to conduct in vivo experiments aimed at both a basic characterization of SNARE protein function in fusion and finally structure function experiments with one of the SNARE proteins, syntaxin. Neurotransmission at a chemical synapse is a specialized form of trafficking and also uses members of the SNARE family. In C. elegans the SNAREs used in neurotransmission are encoded by
unc-64,
snb-1, and
ric-4 (M. Nonet, personal communication) which correspond to syntaxin, synaptobrevin, and SNAP-25 respectively. Like other organisms studied, null mutants in each of these genes are lethal. In order to get around the lethality, we have generated mosaic animals expressing the SNAREs, under the control of the
unc-17 promoter, specifically in the cholinergic neurons in a null background. These animals are rescued to adulthood and importantly the synapses in these animals not using acetylcholine should lack the respective SNARE protein. The neuromuscular junction in C. elegans is accessible to electrophysiological studies and is innervated by both an excitatory acetylcholine component and an inhibitory GABA component, which can readily be isolated with the application of the cholinergic receptor blocker, d-tubocurare. Using a combination of electron microscopy and electrophysiology, we can now assess the morphological and functional consequences of removing SNAREs. In addition, we can now put back altered forms of the SNARE proteins into these mosaic animals without having to rely on the altered forms rescuing the animal. Currently, we have focused our studies on the
snb-1 and
unc-64 genes. Experiments on these animals will be presented.
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[
International Worm Meeting,
2005]
Single nucleotide polymorphism (SNP) mapping against CB4856 has quickly become an indispensable technique in the worm field since its introduction by Stephen Wicks et al. in 2001 (Nat Genet 2001 28: 160-164). Because SNPs are present every few thousand base pairs, SNP mapping allows mapping to extremely narrow intervals. Because mapping is done in an essentially wild-type background, very subtle or complex phenotypes can be mapped that could not be mapped using visible mutations. Typically, SNPs that alter a restriction enzyme recognition site (snip-SNPs) have been detected by running digested PCR products on agarose gels. This technique is reliable and readily accessible to any molecular biology lab; however, it requires substantial hands-on time for each reaction, as each SNP typically requires different PCR cycling and restriction digestion conditions. A number of methods have been published that provide automated analysis of SNP genotypes, including florescence polarization (Swan et al. 2002 Genome Res 12: 1100-1105) or capillary electrophoresis (Zipperlen et al. 2005 Genome Biol 6: R19). Although these methods have the advantage of reducing hands-on time for determining SNP genotypes, they require investment in expensive equipment that is not common in many molecular biology labs. In order to provide a more efficient detection protocol for snip-SNPs, we have developed a set of 48 primer pairs that detect SNPs evenly spaced across the C. elegans genome, and that work under identical PCR and restriction digestion conditions. We present a scheme using these reagents to quickly and reliably map mutations using high-throughput equipment and techniques. We have used our SNP mapping techniques to map many phenotypically subtle or genetically complex mutants, including a subtle behavioral defect resulting from three independent mutations. In fact our techniques are simple and relatively inexpensive enough to have been used successfully by undergraduates in a teaching laboratory setting.
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[
International Worm Meeting,
2005]
The neuronal SNARE protein syntaxin (UNC-64) contains an autoinhibitory domain (the Habc domain) that binds syntaxins SNARE domain and prevents SNARE complex formation (closed syntaxin). A mutant syntaxin in which the autoinhibitory domain does not bind the SNARE domain (open syntaxin) can rescue the L1 lethal phenotype of null syntaxin mutants. However, we found that a mutant syntaxin with the autoinhibitory domain deleted (naked syntaxin) cannot rescue the syntaxin null. Naked syntaxin might fail to rescue because it does not support release, or because it supports constitutive release at lethal levels. To determine the effect on release of naked syntaxin in neurons that lack wild-type syntaxin, we rescued the L1 lethal phenotype of the syntaxin null by expressing syntaxin in a limited number of neurons. In neurons that lacked wild-type syntaxin, the presence of naked syntaxin had no effect on morphology or on synaptic vesicle localization and distribution. Constitutive release is likely to result in a reduction in synaptic vesicles. Thus, these data suggest that naked syntaxin is unable to support exocytosis. However, in the wild-type background, naked syntaxin increased Aldicarb sensitivity, showing that it can promote release in combination with full-length syntaxin. These data suggest that the Habc domain may promote release. To test the effect of the Habc domain on release, we used two mutant syntaxins: one that contained only the Habc domain (free Habc), and one that contained the Habc domain tethered to syntaxins trans-membrane domain by a bacterial alpha-helix (tethered Habc). Free Habc had no effect under any condition tested. However, tethered Habc could rescue the syntaxin null when co-expressed with naked syntaxin, but not when expressed alone. To assess synaptic function in rescued animals, we measured miniature post-synaptic potentials (minis) at the neuromuscular junction. We found that rescued animals have high levels of neurotransmitter release. Together, these results demonstrate that interactions between the Habc domain and the SNARE domain have an essential function in promoting syntaxin-mediated vesicle release. Further, these interactions can function in trans, but depend on membrane localization. We are currently testing whether the Habc domain functions during syntaxin transport and localization or at the synapse itself.
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[
MicroPubl Biol,
2019]
Infections by helminths are one of the major health problems in developing countries. Fast adaptation of parasitic organisms has conferred resistance to drug therapies used for decades (Burns et al., 2015), causing a threat to control worm parasites and generating an urgent need for new emergent molecules with therapeutic potential, in particular for nematodes. The nematode Caenorhabditis elegans is phylogenetically-related to parasitic species of helminths in which high throughput assays are challenging; thus, this species is an excellent model to study and evaluate novel anthelmintic drugs (Gilleard, 2004; Yoon et al., 2006). One interesting molecular target for drug development are the GABA receptors of parasitic helminths which are evolutionarily preserved in invertebrates and vertebrates and are present in C. elegans (Castillo et al., 1967; del Castillo et al., 1964; Jorgensen, 2005). GABA receptors from C. elegans are ionotropic channels that belong to the Cys-loop superfamily of ligand gated ion channels widely spread in multiple species, including humans (Olsen and Sieghart, 2008).
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[
Methods Cell Biol,
1995]
The number of easily distinguishable mutant phenotypes in Caenorhabditis elegans is relatively small, and this constrains the number of factors that can be followed in standard genetic crosses. Consequently, a new mutation is mapped, first to a chromosome using two-factor data from one or more crosses, and then to a chromosomal subregion by successive three-factor crosses. Mapping would be more efficient if it were possible to score a large number of well-distributed markers in a single cross. The advent of the polymerase chain reaction makes this approach feasible by allowing polymorphic genomic regions to serve as genetic markers that are easily scored in DNA released from individual animals. The only "phenotype" is a band on a gel, so the segregation of many of these markers can be followed in a single cross. Following the terminology proposed by Olsen et al. (1989), we refer to polymorphisms that can be scored by appropriately designed polymerase chain reaction (PCR) assays as polymorphic seqeunce-tagged sites (STSs)...
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[
Mech Ageing Dev,
2007]
An explanation is offered for the increased lifespan of Caenorhabditis elegans when mRNA translation is inhibited due to loss of the initiation factor IFE-2 [Hansen, M., Taubert, T., Crawford, D., Libina, N., Lee, S.-J., Kenyon, C., 2007. Lifespan extension by conditions that inhibit translation in Caenorhabditis elegans. Ageing Cell 6, 95-110; Pan, K.Z., Palter, J.E., Rogers, A.N., Olsen, A., Chen, D., Lithgow, G.J., Kapahi, P., 2007. Inhibition of mRNA translation extends lifespan in Caenorhabditis elegans. Ageing Cell 6, 111-119; Syntichaki, P., Troulinaki, K., Tavernarakis, N., 2007. eIF4E function in somatic cells modulates ageing in Caenorhabditis elegans. Nature 445, 922-926]. It is suggested that the general reduction of protein synthesis, due to the decreased frequency of mRNA translation, also lowers the cellular load of erroneously synthesized polypeptides which the constitutive protein homeostatic apparatus (proteases and chaperones proteins) normally eliminates. This situation results in "spare" proteolytic and chaperone function which can then deal with those proteins modified post-synthetically, e.g. by oxidation and/or glycation, which are thought to contribute to the senescent phenotype. This increased availability of proteolytic and chaperone functions may thereby contribute to the observed increase in organism stress resistance and lifespan.