In Vivo Analysis of Conserved C. elegans Tomosyn Domains

Neurosecretion is critically dependent on the assembly of a macromolecular complex between the SNARE proteins syntaxin, SNAP-25 and synaptobrevin. Evidence indicates that the binding of tomosyn to syntaxin and SNAP-25 interferes with this assembly, thereby negatively regulating both synaptic transmission and peptide release. Tomosyn has two conserved domains: an N-terminal encompassing multiple WD40 repeats predicted to form two β-propeller structures and a C-terminal SNARE-binding motif. To assess the function of each domain, we performed an in vivo analysis of the N- and C- terminal domains of C. elegans tomosyn (TOM-1) in a tom-1 mutant background. We verified that both truncated TOM-1 constructs were transcribed at levels comparable to rescuing full-length TOM-1, were of the predicted size, and localized to synapses. Unlike full-length TOM-1, expression of the N- or C-terminal domains alone was unable to restore inhibitory control of synaptic transmission in tom-1 mutants. Similarly, co-expression of both domains failed to restore TOM-1 function. In addition, neither the N- nor C-terminal domain inhibited release when expressed in a wild-type background. Based on these results, we conclude that the ability of tomosyn to regulate neurotransmitter release in vivo depends on the physical integrity of the protein, indicating that both N- and C-terminal domains are necessary but not sufficient for effective inhibition of release in vivo

Control of Synaptic Transmission Through SNARE Complex Regulation

Synaptic transmission requires the assembly of a highly conserved complex composed of the SNARE proteins Syntaxin, SNAP-25, and Synaptobrevin. The Syntaxin binding protein Tomosyn has been implicated in the regulation of this secretory process through interactions with the SNARE complex. Here we used truncated Tomosyn constructs in C. elegans to demonstrate that both the N and C terminal domains contribute to the inhibitory function of this protein. Based on this observation, we isolated a novel Tomosyn N –terminal interacting protein, VPS-39. Our analysis suggests that VPS-39 does regulate Tomosyn levels, but the major synaptic phenotype is consistent with a permissive role in exocytosis. The characterization of the vps-39 mutant phenotype indicates a role upstream of UNC-13 function. Based on known biochemical interactions and our own experimental data we postulate that VPS-39 promotes the transition of Syntaxin from its default closed to open configuration required for priming. We also explored the role of a second SNARE binding protein, Snapin. Our analysis indicates that Snapin is involved in synaptic vesicle docking consistent with its known interaction with SNAP-25. Importantly, our genetic analysis suggests that this function is independent and upstream of the calcium sensor, Synaptotagmin. Together these observations further our understanding of the mechanisms regulating SNARE complex assembly, a critical event underlying normal synaptic transmission

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

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 null 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

Over-expression of TOM1-A SNARE or ΔSNARE constructs do not inhibit synaptic release in the wild-type background.

<p><b>A.</b> Representative evoked traces for full-length TOM-1A (SY1237), SNARE (SY1234) and ΔSNARE (SY1235) expressing transgenes in the wild-type background. (<b>B</b>) Average evoked amplitude and (<b>C</b>) Average charge integral were only significantly reduced by full-length TOM-1A relative to wild type (***, p = 0.0005, and p = 0.0007 for B and C, respectively). All data are expressed as mean ± SEM, the sample size (n) is indicated as a number in each bar, significance values obtained with the Mann Whitney T-test.</p

SNARE and ΔSNARE domains of TOM-1A fail to rescue <i>tom-1(nu468)</i> mutants.

<p><b>A.</b> Schematic showing full-length TOM-1A (SY1242) and the SNARE (SY1230) and ΔSNARE (SY1231) truncated constructs used to generate the integrated transgenics. The position of the early stop at amino acid W212 for <i>tom-1(nu46</i>8) is indicated by the arrow <b>B.</b> Representative traces of evoked post-synaptic responses and plots of evoked amplitude (***, p = 0.006) (<b>C</b>), evoked charge integral (**,p = 0.0014, ***, p = 0.007) (<b>D</b>) and evoked half-time decay ((**,p = 0.001, ***, p<0.0001) (<b>E</b>). All data are expressed as mean ± SEM. The Mann Whitney T-test was used to determine significance values relative to <i>tom-1(nu468)</i>. The sample size (n) is indicated as a number in each bar.</p

Flag-tagged TOM-1A SNARE and ΔSNARE transgenics phenocopy untagged lines.

<p><b>A.</b> Representative evoked response traces for SNARE::FLAG (SY1232) and ΔSNARE::FLAG (SY1233) expressing lines. <b>B.</b> Plots of average evoked amplitude and (<b>C</b>) evoked charge integral. All data are expressed as mean ± SEM, the sample size (n) is indicated as a number in each bar. Mann Whitney T-tests showed values were not significantly different.</p

Both TOM1-A SNARE and ΔSNARE are stably expressed and localized at synapses.

<p><b>A.</b> The FLAG tagged SNARE and ΔSNARE constructs are of the predicted size on Westerns. <b>B.</b> Representative confocal images of SNARE::FLAG and ΔSNARE::FLAG expression in the ventral nerve cord (VNC) anterior to the vulva, the region used for electrophysiological recording. Staining in the lateral nerve cord (LNC) was also observed. Scale bar is 50 µm.</p

ΔΔCt-values for TOM-1A transgenic lines.

<p>Transgene mRNA levels were determined by qRT-PCR using primers specific for TOM-1A N-terminal (starting at bp1840) and the SNARE domain in the <i>tom-1(nu468)</i> mutant background. ΔΔC(t) values were normalized to <i>tom-1(nu468)</i> using <i>act-1</i> transcript levels as a calibrator.</p

Inverse-relationship between predicted full-length TOM-1A expression levels and synaptic function.

<p><b>A.</b> Representative evoked post-synaptic responses from the NMJ of <i>tom-1(nu468)</i>, wild type and two TOM-1A integrated lines, SY1229 and SY1242, expressed in the <i>tom-1(nu468)</i> mutant background respectively. <b>B.</b> Average charge integral for evoked responses of <i>tom-1(nu468)</i> (n = 20), wild type (n = 73) and <i>tom-1(nu468)</i> over-expressing TOM-1A integrated lines SY1242 (∼6 fold mRNA levels) (n = 7) and SY1229 (∼12 fold mRNA levels) (n = 7) plotted against predicted TOM-1A expression levels based on quantitative real-time RT-PCR (qRT-PCR) normalized to <i>C. elegans</i> actin (<i>act-1</i>) transcript levels. Data plotted as mean ± SEM (significance values relative to <i>tom-1(nu468)</i>, *** p≤0.0001, Mann Whitney T-test). Representative evoked NMJ traces are displayed above each strain.</p

Co-expression of SNARE and ΔSNARE constructs failed to reconstitute TOM-1A function.

<p><b>A.</b> Representative evoked response traces for <i>tom-1(nu468)</i>, and with full-length TOM-1A over-expression (SY1242) or co-expression of SNARE and ΔSNARE (SY1239), (<b>B</b>) average evoked amplitudes (***, p = 0.0006) and (<b>C</b>) average evoked charge integrals (**, p = 0.0021). (<b>D</b>) Representative evoked response traces for wild type alone, and with TOM-1A over-expression (SY1237) or co-expression of SNARE and ΔSNARE (SY1240), (<b>E</b>) average evoked amplitudes (**, p = 0.0033) and (<b>F</b>) evoked charge integrals (**, p = 0.0037). All data are expressed as mean ± SEM, the sample size (n) is indicated as a number in each bar, significance values obtained with the Mann Whitney T-test.</p