Differential expression and function of synaptotagmin 1 isoforms in Caenorhabditis elegans

Mathews, Eleanor; Mullen, Gregory P.; Crowell, John A.; Duerr, Janet S.; McManus, John; Duke, Angie; Gaskin, Jennifer; Rand, James B.

doi:10.1016/j.mcn.2007.01.009

Cited by 7 publications

(11 citation statements)

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“…We therefore examined UNC-41 localization in snt-1(md290) mutants; md290 is a null allele of snt-1 [33]. We found that endogenous UNC-41, as assayed by immunostaining, was localized to synapses (Figure 5).…”

Section: Resultsmentioning

confidence: 99%

UNC-41/Stonin Functions with AP2 to Recycle Synaptic Vesicles in Caenorhabditis elegans

Mullen

Grundahl

et al. 2012

PLoS ONE

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The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (Drosophila Stoned B, mammalian stonin 2) have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the unc-41 gene, which encodes the stonin ortholog in the nematode Caenorhabditis elegans. Transgenic expression of Drosophila stonedB rescues unc-41 mutant phenotypes, demonstrating that UNC-41 is a bona fide member of the stonin family. In unc-41 mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of snt-1 unc-41 double mutants is stronger than snt-1 mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that unc-41 mutants have defects in synaptic vesicle membrane endocytosis, including a ∼50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in apm-2 mutants, which lack the µ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41 acts in the same endocytic pathway as µ2 adaptin.

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Section: Resultsmentioning

confidence: 99%

UNC-41/Stonin Functions with AP2 to Recycle Synaptic Vesicles in Caenorhabditis elegans

Mullen

Grundahl

et al. 2012

PLoS ONE

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“…To test this hypothesis, we replaced the signal peptide and N-terminal domain of SUP-1 with the analogous region from SNT-1/synaptotagmin (Nonet et al 1993;Mathews et al 2007), an unrelated synaptic vesicle protein with the same (type I) membrane topology. We found that this chimeric protein suppressed the unc-17(e245) mutant phenotype and restored BiFC fluorescence (Figure 8).…”

Section: The Transmembrane Domain Of Sup-1 Mediates Interaction With mentioning

confidence: 99%

“…In contrast, deletion of the lumenal region between the signal sequence and the TMD eliminated suppression and most of the BiFC fluorescence. The little remaining fluorescence was restricted to the cell bodies, presumably due to improper trafficking of the SUP-1 protein.To test this hypothesis, we replaced the signal peptide and N-terminal domain of SUP-1 with the analogous region from SNT-1/synaptotagmin (Nonet et al 1993;Mathews et al 2007), an unrelated synaptic vesicle protein with the same (type I) membrane topology. We found that this chimeric protein suppressed the unc-17(e245) mutant phenotype and restored BiFC fluorescence (Figure 8).…”

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confidence: 99%

Genetic Interactions Between UNC-17/VAChT and a Novel Transmembrane Protein in Caenorhabditis elegans

et al. 2012

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The unc-17 gene encodes the vesicular acetylcholine transporter (VAChT) in Caenorhabditis elegans. unc-17 reduction-offunction mutants are small, slow growing, and uncoordinated. Several independent unc-17 alleles are associated with a glycine-toarginine substitution (G347R), which introduces a positive charge in the ninth transmembrane domain (TMD) of UNC-17. To identify proteins that interact with UNC-17/VAChT, we screened for mutations that suppress the uncoordinated phenotype of UNC-17(G347R) mutants. We identified several dominant allele-specific suppressors, including mutations in the sup-1 locus. The sup-1 gene encodes a single-pass transmembrane protein that is expressed in a subset of neurons and in body muscles. Two independent suppressor alleles of sup-1 are associated with a glycine-to-glutamic acid substitution (G84E), resulting in a negative charge in the SUP-1 TMD. A sup-1 null mutant has no obvious deficits in cholinergic neurotransmission and does not suppress unc-17 mutant phenotypes. Bimolecular fluorescence complementation (BiFC) analysis demonstrated close association of SUP-1 and UNC-17 in synapse-rich regions of the cholinergic nervous system, including the nerve ring and dorsal nerve cords. These observations suggest that UNC-17 and SUP-1 are in close proximity at synapses. We propose that electrostatic interactions between the UNC-17(G347R) and SUP-1(G84E) TMDs alter the conformation of the mutant UNC-17 protein, thereby restoring UNC-17 function; this is similar to the interaction between UNC-17/ VAChT and synaptobrevin. C OORDINATED muscle contraction in both vertebrates and nematodes requires release of the neurotransmitter acetylcholine (ACh) from motor neurons. ACh is synthesized in motor neurons by the enzyme choline acetyltransferase (ChAT) and transported into synaptic vesicles by the vesicular ACh transporter (VAChT). Following transmitter release, cholinergic signaling is terminated primarily through the action of acetylcholinesterase (AChE), which hydrolyses the released ACh into choline and acetyl-CoA. In the nematode Caenorhabditis elegans, the ChAT and VAChT proteins are encoded by the cha-1 and unc-17 genes, respectively (Alfonso et al. , 1994b. Mutations that functionally eliminate either protein are lethal , while reduction-of-function mutations result in animals that are small, slow growing, uncoordinated, and resistant to AChE inhibitors such as aldicarb .The unc-17 missense mutation e245 replaces a glycine with an arginine (G347R) in the ninth transmembrane domain (TMD9) of the VAChT protein . Genetic screens designed to identify mutations that improve the locomotion of e245 homozygotes have identified several loci that suppress the deleterious effects of the VAChT G347R mutation. Sandoval et al. (2006) found that the sup-8 locus corresponds to the previously characterized snb-1 gene, which encodes the v-SNARE synaptobrevin. The single sup-8 allele is associated with a missense mutation that results in a charge alteration (I97D) in the TMD of synaptobrevin. T...

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“…Interestingly, sytI is also a target of A-to-I RNA editing in some insects and squid ( Reenan 2005 ; Yang et al 2008 ). In addition, sytI also undergoes exon duplication and alternative splicing or other functional wobble splicing in C. elegans and A. californica ( Nakhost et al 2004 ; Mathews et al 2007 ). These events provide another means to create subtle changes at posttranscriptional stages.…”

Section: Discussionmentioning

confidence: 99%

Molecular Determinants and Evolutionary Dynamics of Wobble Splicing

Yang

Yin

et al. 2009

Molecular Biology and Evolution

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Alternative splicing at tandem splice sites (wobble splicing) is widespread in many species, but the mechanisms specifying the tandem sites remain poorly understood. Here, we used synaptotagmin I as a model to analyze the phylogeny of wobble splicing spanning more than 300 My of insect evolution. Phylogenetic analysis indicated that the occurrence of species-specific wobble splicing was related to synonymous variation at tandem splice sites. Further mutagenesis experiments demonstrated that wobble splicing could be lost by artificially induced synonymous point mutations due to destruction of splice acceptor sites. In contrast, wobble splicing could not be correctly restored through mimicking an ancestral tandem acceptor by artificial synonymous mutation in in vivo splicing assays, which suggests that artificial tandem splice sites might be incompatible with normal wobble splicing. Moreover, combining comparative genomics with hybrid minigene analysis revealed that alternative splicing has evolved from the 3′ tandem donor to the 5′ tandem acceptor in Culex pipiens, as a result of an evolutionary shift of cis element sequences from 3′ to 5′ splice sites. These data collectively suggest that the selection of tandem splice sites might not simply be an accident of history but rather in large part the result of coevolution between splice site and cis element sequences as a basis for wobble splicing. An evolutionary model of wobble splicing is proposed.

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Differential expression and function of synaptotagmin 1 isoforms in Caenorhabditis elegans

Cited by 7 publications

References 58 publications

UNC-41/Stonin Functions with AP2 to Recycle Synaptic Vesicles in Caenorhabditis elegans

UNC-41/Stonin Functions with AP2 to Recycle Synaptic Vesicles in Caenorhabditis elegans

Genetic Interactions Between UNC-17/VAChT and a Novel Transmembrane Protein in Caenorhabditis elegans

Molecular Determinants and Evolutionary Dynamics of Wobble Splicing

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