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Differential roles for snapin and synaptotagmin in the synaptic vesicle cycle.

Yu SC, Klosterman SM, Martin AA, Gracheva EO, Richmond JE - PLoS ONE (2013)

Bottom Line: The kinetics of synaptic transmission were unaffected at snpn-1 mutant neuromuscular junctions (NMJs), but the number of docked, fusion competent vesicles was significantly reduced.However, analyses of snt-1 and snt-1;snpn-1 double mutants suggest that the docking role of SNPN-1 is independent of Synaptotagmin.Based on these results we propose that the primary role of Snapin in C. elegans is to promote vesicle priming, consistent with the stabilization of SNARE complex formation through established interactions with SNAP-25 upstream of the actions of Synaptotagmin in calcium-sensing and endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, United States of America.

ABSTRACT
Evoked synaptic transmission is dependent on interactions between the calcium sensor Synaptotagmin I and the SNARE complex, comprised of Syntaxin, SNAP-25, and Synaptobrevin. Recent evidence suggests that Snapin may be an important intermediate in this process, through simultaneous interactions of Snapin dimers with SNAP-25 and Synaptotagmin. In support of this model, cultured neurons derived from embryonically lethal Snapin mutant mice exhibit desynchronized release and a reduced readily releasable vesicle pool. Based on evidence that a dimerization-defective Snapin mutation specifically disrupts priming, Snapin is hypothesized to stabilize primed vesicles by structurally coupling Synaptotagmin and SNAP-25. To explore this model in vivo we examined synaptic transmission in viable, adult C. elegans Snapin (snpn-1) mutants. The kinetics of synaptic transmission were unaffected at snpn-1 mutant neuromuscular junctions (NMJs), but the number of docked, fusion competent vesicles was significantly reduced. However, analyses of snt-1 and snt-1;snpn-1 double mutants suggest that the docking role of SNPN-1 is independent of Synaptotagmin. Based on these results we propose that the primary role of Snapin in C. elegans is to promote vesicle priming, consistent with the stabilization of SNARE complex formation through established interactions with SNAP-25 upstream of the actions of Synaptotagmin in calcium-sensing and endocytosis.

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Neuronally expressed C. elegans Snapin and Synaptotagmin regulate locomotory behavior.(A) The Snapin (snpn-1) gene structure and location of the snpn-1(tm1892) deletion. (B) The mean ± SEM number of body bend reversals triggered by a single head tap were significantly reduced in snpn-1(tm1892), snt-1(md290) and the double mutants. (C) The mean ± SEM values for thrashing responses of snpn-1 mutants placed in solution show a modest decrease compared to snt-1 and snt-1;snpn-1 doubles mutants. It should be noted that subtle differences between snt-1 single and double mutants would be difficult to discern in this assay, given the already very low thrashing rates observed in snt-1 mutants. Significance values for all mutants are ≤0.0001 relative to wild-type. (D) Expression of GFP::SNPN-1 under the snpn-1 promoter co-localized with mCherry driven by the pannueronal pRab-3 promoter throughout the C. elegans nervous system.
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pone-0057842-g001: Neuronally expressed C. elegans Snapin and Synaptotagmin regulate locomotory behavior.(A) The Snapin (snpn-1) gene structure and location of the snpn-1(tm1892) deletion. (B) The mean ± SEM number of body bend reversals triggered by a single head tap were significantly reduced in snpn-1(tm1892), snt-1(md290) and the double mutants. (C) The mean ± SEM values for thrashing responses of snpn-1 mutants placed in solution show a modest decrease compared to snt-1 and snt-1;snpn-1 doubles mutants. It should be noted that subtle differences between snt-1 single and double mutants would be difficult to discern in this assay, given the already very low thrashing rates observed in snt-1 mutants. Significance values for all mutants are ≤0.0001 relative to wild-type. (D) Expression of GFP::SNPN-1 under the snpn-1 promoter co-localized with mCherry driven by the pannueronal pRab-3 promoter throughout the C. elegans nervous system.

Mentions: The single C. elegans Snapin homolog (snpn-1) encodes a 122 amino acid protein that is 29% identical (59% similar) to mouse Snapin. We obtained a deletion mutant, snpn-1(tm1892), which eliminates 520 genomic base pairs, spanning the upstream regulatory sequence, the start codon, the first exon and intron, and half of the second exon of the three exon snpn-1 coding region (Fig. 1A). Based on the extent of the snpn-1 deletion and the complete absence of a detectable transcript following qRT-PCR (data not shown), this mutant is thought to be a molecular [33]. Unlike mouse Snapin mutants which die shortly after birth, C. elegans snpn-1(tm1892) mutants are viable and fertile, allowing us to assess the behavioral consequences in freely moving adult snpn-1 s. A gentle tap to the head produces a reliable backing response in wild-type worms that can be scored as number of body bend reversals. As shown in Figure 1B, snpn-1(tm1892) mutants produced significantly fewer body bend reversals in response to a head tap when compared to wild-type worms (p = 0.0008). Similarly, the thrashing response of snpn-1(tm1892) mutants elicited by placing worms in M9 medium, was significantly depressed when compared to the wild type after 3 minutes (p<0.0001) (Fig. 1C). Given that the interaction between Snapin and Synaptotagmin suggests a functional link between these two proteins, we next assessed the behavioral consequences of eliminating SNPN-1 on the behavior of C. elegans snt-1 mutants. Where as the head tap response and thrashing measurements of snt-1(md290) mutants were more severely impacted in comparison to snpn-1 mutants (Fig. 1B,C), the snt-1;snpn-1 double mutants showed no further reduction, with the exception of the third minute of thrashing (P = 0.016) suggesting that any additive functions of SNT-1 and SNPN-1 are marginal and require prolonged activity.


Differential roles for snapin and synaptotagmin in the synaptic vesicle cycle.

Yu SC, Klosterman SM, Martin AA, Gracheva EO, Richmond JE - PLoS ONE (2013)

Neuronally expressed C. elegans Snapin and Synaptotagmin regulate locomotory behavior.(A) The Snapin (snpn-1) gene structure and location of the snpn-1(tm1892) deletion. (B) The mean ± SEM number of body bend reversals triggered by a single head tap were significantly reduced in snpn-1(tm1892), snt-1(md290) and the double mutants. (C) The mean ± SEM values for thrashing responses of snpn-1 mutants placed in solution show a modest decrease compared to snt-1 and snt-1;snpn-1 doubles mutants. It should be noted that subtle differences between snt-1 single and double mutants would be difficult to discern in this assay, given the already very low thrashing rates observed in snt-1 mutants. Significance values for all mutants are ≤0.0001 relative to wild-type. (D) Expression of GFP::SNPN-1 under the snpn-1 promoter co-localized with mCherry driven by the pannueronal pRab-3 promoter throughout the C. elegans nervous system.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585204&req=5

pone-0057842-g001: Neuronally expressed C. elegans Snapin and Synaptotagmin regulate locomotory behavior.(A) The Snapin (snpn-1) gene structure and location of the snpn-1(tm1892) deletion. (B) The mean ± SEM number of body bend reversals triggered by a single head tap were significantly reduced in snpn-1(tm1892), snt-1(md290) and the double mutants. (C) The mean ± SEM values for thrashing responses of snpn-1 mutants placed in solution show a modest decrease compared to snt-1 and snt-1;snpn-1 doubles mutants. It should be noted that subtle differences between snt-1 single and double mutants would be difficult to discern in this assay, given the already very low thrashing rates observed in snt-1 mutants. Significance values for all mutants are ≤0.0001 relative to wild-type. (D) Expression of GFP::SNPN-1 under the snpn-1 promoter co-localized with mCherry driven by the pannueronal pRab-3 promoter throughout the C. elegans nervous system.
Mentions: The single C. elegans Snapin homolog (snpn-1) encodes a 122 amino acid protein that is 29% identical (59% similar) to mouse Snapin. We obtained a deletion mutant, snpn-1(tm1892), which eliminates 520 genomic base pairs, spanning the upstream regulatory sequence, the start codon, the first exon and intron, and half of the second exon of the three exon snpn-1 coding region (Fig. 1A). Based on the extent of the snpn-1 deletion and the complete absence of a detectable transcript following qRT-PCR (data not shown), this mutant is thought to be a molecular [33]. Unlike mouse Snapin mutants which die shortly after birth, C. elegans snpn-1(tm1892) mutants are viable and fertile, allowing us to assess the behavioral consequences in freely moving adult snpn-1 s. A gentle tap to the head produces a reliable backing response in wild-type worms that can be scored as number of body bend reversals. As shown in Figure 1B, snpn-1(tm1892) mutants produced significantly fewer body bend reversals in response to a head tap when compared to wild-type worms (p = 0.0008). Similarly, the thrashing response of snpn-1(tm1892) mutants elicited by placing worms in M9 medium, was significantly depressed when compared to the wild type after 3 minutes (p<0.0001) (Fig. 1C). Given that the interaction between Snapin and Synaptotagmin suggests a functional link between these two proteins, we next assessed the behavioral consequences of eliminating SNPN-1 on the behavior of C. elegans snt-1 mutants. Where as the head tap response and thrashing measurements of snt-1(md290) mutants were more severely impacted in comparison to snpn-1 mutants (Fig. 1B,C), the snt-1;snpn-1 double mutants showed no further reduction, with the exception of the third minute of thrashing (P = 0.016) suggesting that any additive functions of SNT-1 and SNPN-1 are marginal and require prolonged activity.

Bottom Line: The kinetics of synaptic transmission were unaffected at snpn-1 mutant neuromuscular junctions (NMJs), but the number of docked, fusion competent vesicles was significantly reduced.However, analyses of snt-1 and snt-1;snpn-1 double mutants suggest that the docking role of SNPN-1 is independent of Synaptotagmin.Based on these results we propose that the primary role of Snapin in C. elegans is to promote vesicle priming, consistent with the stabilization of SNARE complex formation through established interactions with SNAP-25 upstream of the actions of Synaptotagmin in calcium-sensing and endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, United States of America.

ABSTRACT
Evoked synaptic transmission is dependent on interactions between the calcium sensor Synaptotagmin I and the SNARE complex, comprised of Syntaxin, SNAP-25, and Synaptobrevin. Recent evidence suggests that Snapin may be an important intermediate in this process, through simultaneous interactions of Snapin dimers with SNAP-25 and Synaptotagmin. In support of this model, cultured neurons derived from embryonically lethal Snapin mutant mice exhibit desynchronized release and a reduced readily releasable vesicle pool. Based on evidence that a dimerization-defective Snapin mutation specifically disrupts priming, Snapin is hypothesized to stabilize primed vesicles by structurally coupling Synaptotagmin and SNAP-25. To explore this model in vivo we examined synaptic transmission in viable, adult C. elegans Snapin (snpn-1) mutants. The kinetics of synaptic transmission were unaffected at snpn-1 mutant neuromuscular junctions (NMJs), but the number of docked, fusion competent vesicles was significantly reduced. However, analyses of snt-1 and snt-1;snpn-1 double mutants suggest that the docking role of SNPN-1 is independent of Synaptotagmin. Based on these results we propose that the primary role of Snapin in C. elegans is to promote vesicle priming, consistent with the stabilization of SNARE complex formation through established interactions with SNAP-25 upstream of the actions of Synaptotagmin in calcium-sensing and endocytosis.

Show MeSH
Related in: MedlinePlus