InteractiveFly: GeneBrief

Tomosyn: Biological Overview | References

Gene name - Tomosyn

Synonyms -

Cytological map position - 11B6-11B7

Function - signaling

Keywords - neuromuscular junction - SNARE binding protein - potently inhibits exocytosis by sequestering SNARE proteins in nonfusogenic complexes - a decoy snare - enables tonic release in Ib motoneurons by reducing SNARE complex formation and suppressing probability of release to generate decreased levels of synaptic vesicle fusion and enhanced resistance to synaptic fatigue - involved in a specific component of late associative memory

Symbol - Tomosyn

FlyBase ID: FBgn0030412

Genetic map position -

NCBI classification - R-SNARE_STXBP5_6: SNARE domain of STXBP5, STXBP6 and related proteins

Cellular location - cytoplasmic

NCBI links: EntrezGene, Nucleotide, Protein

GENE orthologs: Biolitmine

Synaptic vesicle release probability (P(r)) is a key presynaptic determinant of synaptic strength established by cell intrinsic properties and further refined by plasticity. To characterize mechanisms that generate P(r) heterogeneity between distinct neuronal populations, this study examined glutamatergic tonic (Ib) and phasic (Is) motoneurons in Drosophila with stereotyped differences in P(r) and synaptic plasticity. The decoy SNARE Tomosyn is differentially expressed between these motoneuron subclasses and contributes to intrinsic differences in their synaptic output. Tomosyn expression enables tonic release in Ib motoneurons by reducing SNARE complex formation and suppressing P(r) to generate decreased levels of synaptic vesicle fusion and enhanced resistance to synaptic fatigue. In contrast, phasic release dominates when Tomosyn expression is low, enabling high intrinsic P(r) at Is terminals at the expense of sustained release and robust presynaptic potentiation. In addition, loss of Tomosyn disrupts the ability of tonic synapses to undergo presynaptic homeostatic potentiation (PHP) (Sauvola, 2021).

Ca2+-dependent fusion of synaptic vesicles (SVs) is the primary mechanism for neurotransmission and is mediated by the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) family. Following an action potential, SNARE proteins located on the SV and plasma membrane zipper into an energetically favorable coiled-coil bundle to induce SV fusion. Neurotransmitter release results in a postsynaptic response that varies in size depending on the strength of the synapse, which can be regulated from both pre-and post-synaptic compartments. The postsynaptic cell controls sensitivity to neurotransmitters by governing receptor field composition, while the presynaptic neuron establishes the probability (Pr) of SV fusion. Highly stereotyped differences in Pr exist across neurons, with many neuronal populations broadly classified as tonic or phasic depending on their spiking patterns, Pr and short-term plasticity characteristics. How cell-intrinsic properties establish differences in presynaptic Pr between neuronal classes, and how release strength is further refined via plasticity, remain incompletely understood (Sauvola, 2021).

The Drosophila melanogaster larval neuromuscular junction (NMJ) provides a robust genetic system for characterizing mechanisms mediating synaptic communication and tonic versus phasic release properties. Larval body wall muscles are co-innervated by two glutamatergic motoneuron populations that drive locomotion, including the tonic-like Ib and phasic-like Is subtypes. Tonic Ib terminals display lower initial Pr and sustained release during stimulation, whereas phasic Is terminals show higher intrinsic Pr and rapid depression. The Drosophila NMJ also undergoes robust presynaptic homeostatic potentiation (PHP) that rapidly increases Pr to compensate for disruptions to postsynaptic glutamate receptor (GluR) function. In addition to intrinsic release differences, the Ib and Is subtypes display distinct capacity for PHP. How tonic and phasic neurons differentially regulate Pr during normal synaptic communication and plasticity is largely unknown (Sauvola, 2021).

The highly conserved SNARE regulatory protein Tomosyn negatively controls SV release and has been proposed to participate in synaptic plasticity. Tomosyn has an N-terminal WD40 repeat domain and a C-terminal SNARE motif with homology to the SV v-SNARE Synaptobrevin 2 (Syb2). Tomosyn inhibits presynaptic release by binding the t-SNAREs Syntaxin1 (Syx1) and SNAP-25 to prevent Syb2 incorporation into the SNARE complex fusion machinery (Sauvola, 2021).

To further examine the role of Tomosyn in synaptic transmission and plasticity, CRISPR was used to generate mutations in the sole Drosophila tomosyn gene. Structure-function analysis revealed the SNARE domain is critical for release inhibition, while the scaffold region promotes enrichment of Tomosyn to SV-rich sites. Despite enhanced evoked release, tomosyn mutants fail to maintain high levels of SV output during sustained stimulation due to rapid depletion of the immediately releasable SV pool. Tomosyn is highly enriched at Ib synapses and generates tonic neurotransmission properties characterized by low Pr and sustained release in this population of motoneurons. Indeed, optogenetic stimulation and optical quantal analysis demonstrate an exclusive role for Tomosyn in regulating intrinsic release strength in tonic motoneurons. PHP expression primarily occurs at tonic synapses and is abolished in tomosyn mutants, suggesting Tomosyn is also essential for acute PHP expression. Together, these data indicate Tomosyn mediates the tonic properties of Ib motoneurons by suppressing Pr to slow the rate of SV usage, while decreasing Tomosyn suppression enables Pr enhancement during PHP. Conversely, the absence of Tomosyn in Is motoneurons facilitates phasic release properties by enabling an intrinsically high Pr that quickly depletes the releasable SV pool, resulting in rapid synaptic depression and reduced capacity for PHP (Sauvola, 2021).

The findings reported in this study indicate the conserved presynaptic release suppressor Tomosyn functions in setting presynaptic output and plasticity differences for a tonic/phasic pair of motoneurons that co-innervate Drosophila larval muscles. CRISPR-generated mutations in Drosophila tomosyn revealed synchronous, asynchronous and spontaneous SV release are all elevated in the absence of the protein. While single evoked responses were enhanced, rapid depression of release was observed during train stimulation, suggesting loss of Tomosyn biases synapses toward a more phasic pattern of SV release. To directly test whether Tomosyn plays a unique role in tonic synapses, Ib and Is motoneurons were separately stimulated using optogenetics to measure their isolated contributions. These experiments revealed a 4-fold increase in output from Ib neurons with no change to Is release. Optical quantal analysis confirmed the Ib specific effect of Tomosyn and demonstrated enhanced evoked responses in tomosyn is due to higher intrinsic Pr across the entire AZ population. Endogenously-tagged Tomosyn was more abundant at Ib synapses than Is, consistent with Tomosyn's role in regulating Ib release. Together, these data indicate the intrinsically high Pr and rapid depression normally found in Is motoneurons is due in part to a lack of Tomosyn inhibition of SV usage at phasic synapses. High-frequency stimulation experiments demonstrate Tomosyn does not regulate the size of the immediately releasable SV pool (IRP) but rather regulates IRP usage to ensure sustained availability of SVs during prolonged stimulation, as the IRP is strongly biased towards early release in tomosyn mutants. A model is proposed where Drosophila synapses are more phasic in release character by default, with tonic release requiring higher levels of Tomosyn to generate a fusion bottleneck that enables extended periods of stable release by slowing the rate of SV usage (Sauvola, 2021).

How Tomosyn normally suppresses SV release has been unclear. The most widely hypothesized mechanism is that Tomosyn competes with Syb2 for binding t-SNAREs. By forming fusion-incompetent SNARE complexes that must be disassembled by NSF, a pool of t-SNAREs is kept in reserve and can be mobilized by alleviating Tomosyn inhibition. Indeed, enhanced SNARE complex formation was found in Drosophila tomosyn mutants, consistent with the model that Tomosyn's SNARE domain acts as a decoy SNARE to inhibit productive SNARE complex assembly. Expression of the Tomosyn scaffold alone failed to rescue the null phenotype, while overexpression of the scaffold had no effect on evoked release. As such, these data indicate that while the scaffold is required for full Tomosyn function, it does not directly inhibit fusion. These observations are consistent with the mechanism proposed in C. elegans, but differ from studies in cultured mammalian cells suggesting the scaffold acts as an independent release suppressor by inhibiting Syt1. Characterization of Drosophila tomosyn/syt1 double mutants demonstrated Tomosyn suppresses release independent of Syt1, arguing the scaffold must serve a function that enhances the inhibitory activity of the SNARE domain independent of Syt1. Indeed, this study found the Tomosyn SNARE motif was mislocalized without the WD40 scaffold, arguing this region indirectly supports Tomosyn's inhibitory activity by ensuring proper localization so the SNARE domain can compete for t-SNARE binding. Similar to studies in C. elegans and mammals, this study found Drosophila Tomosyn co-localized with other SV proteins. Human Tomosyn transgenes also rescued elevated evoked and spontaneous release in tomosyn mutants, indicating functional conservation of its inhibitory properties. Overexpression of either Drosophila or human Tomosyn in a wildtype background also decreased release, demonstrating presynaptic output can be bi-directionally controlled by varying Tomosyn expression levels (Sauvola, 2021).

In addition to intrinsic release differences between tonic and phasic motoneurons, this study found Tomosyn also controls presynaptic homeostatic potentiation (PHP). This form of synaptic plasticity occurs when presynaptic motoneurons upregulate Pr and quantal content to compensate for decreased GluR function and smaller quantal size. Inducing PHP with the allosteric GluR inhibitor Gyki revealed Tomosyn is required for expression of this form of acute PHP at Ib terminals. Removing Tomosyn inhibition at Ib synapses generates a ~ 4 fold enhancement in evoked release, more than sufficient to compensate for a twofold reduction in evoked response size from two equally contributing motoneurons. Indeed, AZ Pr mapping revealed Ib synapses potentiate in the presence of Gyki while Is terminals showed no change, indicating enhanced release from Ib is sufficient to homeostatically compensate for Gyki-induced decreases in quantal size. Although future studies will be required to determine the molecular cascade through which Tomosyn mediates PHP expression, prior work indicates PKA phosphorylation of Tomosyn reduces its SNARE binding properties and decreases its inhibition of SV release. Given Gyki-induced PHP expression requires presynaptic PKD (Nair, 2020), an attractive hypothesis is that PKD phosphorylates Tomosyn and reduces its ability to inhibit SNARE complex formation. Similar to tomosyn mutants, this could promote SV availability by generating a larger pool of free t-SNAREs to support enhanced docking of SVs at AZs. Increased docking would elevate single AZ Pr by increasing the number of fusion-ready SVs upon Ca2+ influx, similar to the effect observed with quantal imaging (Sauvola, 2021).

Despite the importance of Tomosyn in regulating release character between tonic and phasic motoneurons, tomosyn null mutants are viable into adulthood. As such, the entire range of Tomosyn expression can be used by distinct neuronal populations in vivo to set presynaptic output. Tonic Ib terminals shift towards phasic release with no effect on Is output in tomosyn null mutants, resulting in a collapse of presynaptic release diversity between these two neuronal subgroups. Like tomosyn, null mutants in syt7 are viable and show dramatically enhanced evoked release. Tomosyn/syt7 double mutants show even greater increases in release output, arguing multiple non-essential presynaptic proteins can independently fine tune synaptic strength within the presynaptic terminal. Together, these experiments demonstrate Tomosyn is a highly conserved release inhibitor that varies in expression between distinct neuronal subtypes to regulate intrinsic Pr and plasticity, providing a robust mechanism to generate presynaptic diversity across the nervous system (Sauvola, 2021).

Tomosyn-dependent regulation of synaptic transmission is required for a late phase of associative odor memory

Synaptic vesicle secretion requires the assembly of fusogenic SNARE complexes. Consequently proteins that regulate SNARE complex formation can significantly impact synaptic strength. The SNARE binding protein tomosyn has been shown to potently inhibit exocytosis by sequestering SNARE proteins in nonfusogenic complexes. The tomosyn-SNARE interaction is regulated by protein kinase A (PKA), an enzyme implicated in learning and memory, suggesting tomosyn could be an important effector in PKA-dependent synaptic plasticity. This hypothesis was tested in Drosophila, in which the role of the PKA pathway in associative learning has been well established. It was first determined that panneuronal tomosyn knockdown by RNAi enhanced synaptic strength at the Drosophila larval neuromuscular junction, by increasing the evoked response duration. Next memory performance was assayed 3 min (early memory) and 3 h (late memory) after aversive olfactory learning. Whereas early memory was unaffected by tomosyn knockdown, late memory was reduced by 50%. Late memory is a composite of stable and labile components. Further analysis determined that tomosyn was specifically required for the anesthesia-sensitive, labile component, previously shown to require cAMP signaling via PKA in mushroom bodies. Together these data indicate that Tomosyn has a conserved role in the regulation of synaptic transmission and provide behavioral evidence that Tomosyn is involved in a specific component of late associative memory (Chen, 2011).

Synaptic transmission is dependent on the formation of SNARE complexes between the vesicle SNARE synaptobrevin and the plasma membrane SNAREs syntaxin and SNAP-25. SNARE complex assembly produces fusion-competent (primed) vesicles, docked at the plasma membrane. Originally identified as a syntaxin-binding protein, tomosyn has emerged as a negative regulator of secretion by directly competing with synaptobrevin to form nonfusogenic tomosyn SNARE complexes (Fujita, 1998; Hatsuzawa, 2003; Pobbati, 2004). The N terminus of tomosyn also promotes SNARE complex oligomerization, sequestering SNARE monomers required for priming, and impedes the function of the calcium-sensor synaptotagmin. By these means tomosyn is involved in regulating SNARE complex assembly and controlling the size of the readily releasable pool of synaptic vesicles. Evidence suggests that the interaction between tomosyn and the SNARE machinery can be modulated by cAMP-dependent protein kinase A (PKA) phosphorylation of tomosyn, a second messenger cascade previously implicated in several forms of behavioral and synaptic plasticity. This study characterized the synaptic function of Drosophila tomosyn and probed the functional relevance of tomosyn in the regulation of behavioral plasticity (Chen, 2011).

The results demonstrate that tomosyn suppresses synaptic function and is necessary in mushroom body intrinsic neurons specifically for a long-term cAMP-dependent component of associative olfactory learning in Drosophila. The prominent biophysical change observed at the fly NMJ after tomosyn RNAi is a prolonged EJC resulting in an increased total charge transfer, similar to that observed at C. elegans tomosyn mutant synapses. Although the underlying cause of the altered EJC duration in either C. elegans or Drosophila has yet to be determined, one potential explanation is the occurrence of late fusion events possibly resulting from ectopically primed vesicles distal to release sites. This hypothesis is supported by ultrastructural data from C. elegans tomosyn mutants, in which a twofold increase in the number of morphologically docked vesicles was observed, the additional vesicles positioned further from the presynaptic density (Gracheva, 2006). Analyses of priming defective unc-13 and syntaxin C. elegans mutants, in which docked vesicles were found to be greatly reduced, suggest that vesicle docking is a morphological correlate of priming. On the basis of these data, it has been proposed that distally primed vesicles in C. elegans tomosyn mutants exhibit delayed release relative to those close to the presynaptic density, the presumed site of calcium entry, leading to a broadened evoked response (Chen, 2011).

The observed increase in synaptic release at the fly NMJ after tomosyn RNAi adds to a growing body of evidence that tomosyn suppresses synaptic strength. For example, the twofold increase in docked vesicles observed at C. elegans tomosyn mutant synapses correlates with a doubling of the readily releasable pool assessed by applying hyperosmotic saline and corresponds to the enhanced evoked EJC charge integral. The increase in quantal content and reduction in PPD at the fly NMJ after tomosyn RNAi is also consistent with an enhanced primed vesicle pool. Similar conclusions were reached for changes in paired-pulse facilitation at central synapses of mouse tomosyn mutants (Chen, 2011).

The observation that tomosyn knockdown at the fly NMJ results in the addition of mEJCs with slower decay kinetics could also be a manifestation of ectopic vesicle priming. Alternatively, this change in fly mEJCs could reflect a change in fusion pore dynamics in the absence of tomosyn, a possibility given previously observed genetic and/or physical interactions between tomosyn and several key components of the exocytic machinery, including the SNARE proteins synaptotagmin, Munc-13, and Munc-18. Because the presynaptic density is responsible for localizing elements of the vesicle fusion machinery such as UNC-13 and calcium channels, spatial misregulation of vesicle docking in the absence of tomosyn may be related to these changes in fusion properties. However, definitive evidence for ectopically primed vesicles at the fly NMJ after tomosyn RNAi is not yet available, and therefore the cause of the altered evoked response kinetics remains speculative (Chen, 2011).

Evidence indicates that vertebrate tomosyn is a PKA target. Although it cannot yet be definitively establish that tomosyn function is down-regulated by PKA phosphorylation at fly synapses pending the availability of a tomosyn null mutant, the fact that tomosyn RNAi phenocopies the NMJ response to cAMP activation and that these two treatments show nonadditivity supports this notion (Chen, 2011).

On the basis of this analysis of NMJ function, it is predicted that, as in mouse tomosyn mutants, synapses within the fly CNS will experience similar increases in synaptic strength after tomosyn RNAi or cAMP activation. This prediction is supported by the specific ASM aversive odor learning deficit observed in the fly olfactory CNS after tomosyn knockdown in mushroom body Kenyon cells, the site of cAMP-mediated associative memory formation (Chen, 2011).

Drosophila aversive odor learning has long been used to investigate the molecular and cellular mechanisms underlying associative memory formation. In the Drosophila mushroom bodies, neuronal signals representing odor cues and electric shock converge onto type I adenylyl cyclase encoded by rutabaga (rut-AC I) to initiate cAMP signals necessary and sufficient to form engrams underlying associative odor memory. These instructive cAMP signals localize to the Kenyon cells in which presynaptic changes are thought to represent a particular odor memory. Stabilization of aversive odor memory over time requires signals from dorsal-paired medial (DPM) neurons, the putative release sites of amnesiac peptide — the fly homolog to mammalian pituitary adenylate cyclase-activating polypeptide (PACAP) — onto mushroom body Kenyon cells. Amnesiac neuropeptides are known to stimulate cAMP production, whereas the major fly PKA is required from 30 min to 3 h after acquisition to sustain late odor memory. The two distinct components of late odor memory, ASM and ARM, differ in both temporal dynamics and molecular mechanisms. Whereas ARM requires the active zone protein Bruchpilot, ASM is not only tomosyn-dependent, according to the current observations, but also requires synapsin, a conserved PKA phosphorylation target associated with synaptic vesicles. Adult synapsin mutant flies exhibit impaired ASM in aversive odor learning. Furthermore, PKA-dependent phosphorylation of synapsin within Kenyon cells is necessary to support larval appetitive odor learning. Thus, both tomosyn and synapsin are required for the cAMP-dependent ASM phase of associate learning (Chen, 2011).

How might synapsin and tomosyn function together in ASM? Mechanistically, PKA phosphorylation of synapsin is implicated in the mobilization and supply of synaptic vesicles from the reserve pool to the active zone, whereas PKA phosphorylation of tomosyn promotes SNARE complex assembly (Baba, 2005). This suggests that enhanced vesicle delivery and increased priming capacity through PKA regulation of synapsin and tomosyn, respectively, may act in concert to maintain synaptic strength in support of ASM. Because knockdown of either protein disrupts ASM, it seems that both vesicle mobilization and enhanced priming capacity are required for this phase of synaptic plasticity (Chen, 2011).

Several lines of evidence suggest that PKA-dependent modulation of tomosyn function provides a possible molecular mechanism for the transduction of cAMP signaling into synaptic plasticity within the olfactory system underlying ASM. First, at the level of the NMJ, it was shown that tomosyn regulates synaptic strength and that this regulation occludes cAMP-dependent synaptic enhancement. Second, within the olfactory neuronal network, it was demonstrated that tomosyn function is likely required within Kenyon cells to support ASM: the cells that receive instructive signals to initiate cAMP-dependent synaptic changes underlying appropriate behavioral plasticity. Third, synaptic output from Kenyon cells is necessary to support both early and late odor memories. Fourth, DPM signaling onto Kenyon cells is required for late aversive odor memory, and DPM neurons stain positive for the amnesiac peptide, which is known to stimulate cAMP production. Fifth, enhanced synaptic transmission in cultured neurons induced by the vertebrate amnesiac homolog PACAP requires PKA-dependent tomosyn phosphorylation (Baba, 2005). Sixth, postacquisitional PKA activity is necessary for late aversive odor memory and is likely mediated by an A kinase-anchoring protein (AKAP)-bound pool of PKA holoenzymes within Kenyon cells. Finally, biochemical evidence demonstrating that PKA-dependent phosphorylation of tomosyn reduces its affinity for the SNARE machinery suggests a potential mechanistic link between cAMP signaling within Kenyon cells and the up-regulation of synaptic strength (Chen, 2011).

On the basis of this evidence, it is postulated that loss of tomosyn inhibitory function leads to a generalized up-regulation of synaptic strength at Drosophila synapses. In Kenyon cells enhanced synaptic transmission resulting from loss of tomosyn likely occludes cAMP-dependent plasticity. This speculation is supported by the observed occlusion of forskolin-dependent synaptic enhancement at the NMJ after tomosyn RNAi. Similarly, PACAP-induced synaptic plasticity in cultured neurons is occluded when tomosyn is no longer phosphorylatable by PKA (Baba, 2005). It is further speculated that within Kenyon cells the phosphorylation of Drosophila tomosyn is due to postacquisitional, Amnesiac-induced PKA activity. This hypothesis would fit with the observed requirement of AKAP-bound PKA for late but not early ASM. It is thus tempting to speculate that tomosyn phosphorylation is dependent on localized signaling via AKAPs at specific subdomains of Kenyon cells that occur after early ASM is established (Chen, 2011).

Functions of Tomosyn orthologs in other species

The ubiquitin-proteasome system functionally links neuronal Tomosyn-1 to dendritic morphology

Altering the expression of Tomosyn-1 (Tomo-1), a soluble, R-SNARE domain-containing protein, significantly affects behavior in mice, Drosophila, and Caenorhabditis elegans. Yet, the mechanisms that modulate Tomo-1 expression and its regulatory activity remain poorly defined. This study found that Tomo-1 expression levels influence postsynaptic spine density. Tomo-1 overexpression increased dendritic spine density, whereas Tomo-1 knockdown (KD) decreased spine density. These findings identified a novel action of Tomo-1 on dendritic spines, which is unique because it occurs independently of Tomo-1's C-terminal R-SNARE domain. This study also demonstrated that the ubiquitin-proteasome system (UPS), which is known to influence synaptic strength, dynamically regulates Tomo-1 protein levels. Immunoprecipitated and affinity-purified Tomo-1 from cultured rat hippocampal neurons was ubiquitinated, and the levels of ubiquitinated Tomo-1 dramatically increased upon pharmacological proteasome blockade. Moreover, Tomo-1 ubiquitination appeared to be mediated through an interaction with the E3 ubiquitin ligase HRD1, as immunoprecipitation of Tomo-1 from neurons co-precipitated HRD1, and this interaction increases upon proteasome inhibition. Further, in vitro reactions indicated direct, HRD1 concentration-dependent Tomo-1 ubiquitination. It is also noted that the UPS regulates both Tomo-1 expression and functional output, as HRD1 KD in hippocampal neurons increased Tomo-1 protein level and dendritic spine density. Notably, the effect of HRD1 KD on spine density was mitigated by additional KD of Tomo-1, indicating a direct HRD1/Tomo-1 effector relationship. In summary, these results indicate that the UPS is likely to participate in tuning synaptic efficacy and spine dynamics by precise regulation of neuronal Tomo-1 levels (Saldate, 2018).

UNC-13L, UNC-13S, and Tomosyn form a protein code for fast and slow neurotransmitter release in Caenorhabditis elegans

Synaptic transmission consists of fast and slow components of neurotransmitter release. This study shows that these components are mediated by distinct exocytic proteins. The Caenorhabditis elegans unc-13 gene is required for SV exocytosis, and encodes long and short isoforms (UNC-13L and S). Fast release was mediated by UNC-13L, whereas slow release required both UNC-13 proteins and was inhibited by Tomosyn. The spatial location of each protein correlated with its effect. Proteins adjacent to the dense projection mediated fast release, while those controlling slow release were more distal or diffuse. Two UNC-13L domains accelerated release. C2A, which binds RIM (a protein associated with calcium channels), anchored UNC-13 at active zones and shortened the latency of release. A calmodulin binding site accelerated release but had little effect on UNC-13's spatial localization. These results suggest that UNC-13L, UNC-13S, and Tomosyn form a molecular code that dictates the timing of neurotransmitter release (Hu, 2013).

Tomosyn inhibits synaptic vesicle priming in Caenorhabditis elegans

Caenorhabditis elegans TOM-1 is orthologous to vertebrate tomosyn, a cytosolic syntaxin-binding protein implicated in the modulation of both constitutive and regulated exocytosis. To investigate how TOM-1 regulates exocytosis of synaptic vesicles in vivo, this study analyzed C. elegans tom-1 mutants. Electrophysiological analysis indicates that evoked postsynaptic responses at tom-1 mutant synapses are prolonged leading to a two-fold increase in total charge transfer. The enhanced response in tom-1 mutants is not associated with any detectable changes in postsynaptic response kinetics, neuronal outgrowth, or synaptogenesis. However, at the ultrastructural level, a concomitant increase was observed in the number of plasma membrane-contacting vesicles in tom-1 mutant synapses, a phenotype reversed by neuronal expression of TOM-1. Priming defective unc-13 mutants show a dramatic reduction in plasma membrane-contacting vesicles, suggesting these vesicles largely represent the primed vesicle pool at the C. elegans neuromuscular junction. Consistent with this conclusion, hyperosmotic responses in tom-1 mutants are enhanced, indicating the primed vesicle pool is enhanced. Furthermore, the synaptic defects of unc-13 mutants are partially suppressed in tom-1 unc-13 double mutants. These data indicate that in the intact nervous system, TOM-1 negatively regulates synaptic vesicle priming (Gracheva, 2006).

PKA-catalyzed phosphorylation of tomosyn and its implication in Ca2+-dependent exocytosis of neurotransmitter

Neurotransmitter is released from nerve terminals by Ca2+-dependent exocytosis through many steps. SNARE proteins are key components at the priming and fusion steps, and the priming step is modulated by cAMP-dependent protein kinase (PKA), which causes synaptic plasticity. This study shows that the SNARE regulatory protein tomosyn is directly phosphorylated by PKA, which reduces its interaction with syntaxin-1 (a component of SNAREs) and enhances the formation of the SNARE complex. Electrophysiological studies using cultured superior cervical ganglion (SCG) neurons revealed that this enhanced formation of the SNARE complex by the PKA-catalyzed phosphorylation of tomosyn increased the fusion-competent readily releasable pool of synaptic vesicles and, thereby, enhanced neurotransmitter release. This mechanism was indeed involved in the facilitation of neurotransmitter release that was induced by a potent biological mediator, the pituitary adenylate cyclase-activating polypeptide, in SCG neurons. The roles and modes of action of PKA and tomosyn in Ca2+-dependent neurotransmitter release are described (Baba, 2005).

Two distinct genes drive expression of seven tomosyn isoforms in the mammalian brain, sharing a conserved structure with a unique variable domain

Tomosyn was previously identified as a syntaxin-binding protein that inhibits soluble NSF (n-ethylmaleimide-sensitive fusion protein) attachment protein receptor (SNARE)-mediated secretion. This study set out to investigate the distribution of tomosyn mRNA in the mammalian brain and found evidence for the presence of two paralogous genes designated tomosyn-1 and -2. In a collection of tomosyn-2 cDNA clones, four splice variants (named xb-, b-, m- and s-tomosyn-2) were observed derived from the skipping of exons 19 and 21. This feature is conserved with tomosyn-1 that encodes three splice variants. To compare the expression pattern of tomosyn-1 and -2, in situ hybridization experiments were performed with gene-specific probes. Both genes were expressed in the nervous system, clearly following distinct spatial and developmental expression patterns. Real-time quantitative PCR experiments indicated that tomosyn-1 expression was up-regulated less than threefold between developmental stages E10 and P12, whereas tomosyn-2 expression increased 31-fold. Not only the transcription level, but also the splice composition of tomosyn-2 mRNA shifted during development. It is concluded that two distinct genes drive expression of seven tomosyn isoforms. Their expression patterns support a role in regulating neuronal secretion. All isoforms share conserved WD40 and SNARE domains separated by a hypervariable module, the function of which remains to be clarified (Groffen, 2005).

Structural basis for the inhibitory role of tomosyn in exocytosis

Upon Ca2+ influx synaptic vesicles fuse with the plasma membrane and release their neurotransmitter cargo into the synaptic cleft. Key players during this process are the Q-SNAREs syntaxin 1a and SNAP-25 and the R-SNARE synaptobrevin 2. It is thought that these membrane proteins gradually assemble into a tight trans-SNARE complex between vesicular and plasma membrane, ultimately leading to membrane fusion. Tomosyn is a soluble protein of 130 kDa that contains a COOH-terminal R-SNARE motif but lacks a transmembrane anchor. Its R-SNARE motif forms a stable core SNARE complex with syntaxin 1a and SNAP-25. This study presents the crystal structure of this core tomosyn SNARE complex at 2.0-Å resolution. It consists of a four-helical bundle very similar to that of the SNARE complex containing synaptobrevin. Most differences are found on the surface, where they prevented tight binding of complexin. Both complexes form with similar rates as assessed by CD spectroscopy. In addition, synaptobrevin cannot displace the tomosyn helix from the tight complex and vice versa, indicating that both SNARE complexes represent end products. Moreover, data bank searches revealed that the R-SNARE motif of tomosyn is highly conserved throughout all eukaryotic kingdoms. This suggests that the formation of a tight SNARE complex is important for the function of tomosyn (Pobbati, 2004).

The R-SNARE motif of tomosyn forms SNARE core complexes with syntaxin 1 and SNAP-25 and down-regulates exocytosis

Tomosyn is a 130-kDa syntaxin-binding protein that contains a large N-terminal domain with WD40 repeats and a C-terminal domain homologous to R-SNAREs. This study shows that tomosyn forms genuine SNARE core complexes with the SNAREs syntaxin 1 and SNAP-25. In vitro studies with recombinant proteins revealed that complex formation proceeds from unstructured monomers to a stable four-helical bundle. The assembled complex displayed features typical for SNARE core complexes, including a profound hysteresis upon unfolding-refolding transitions. No stable complexes were formed between the SNARE motif of tomosyn and either syntaxin or SNAP-25 alone. Furthermore, both native tomosyn and its isolated C-terminal domain competed with synaptobrevin for binding to endogenous syntaxin and SNAP-25 on inside-out sheets of plasma membranes. Tomosyn-SNARE complexes were effectively disassembled by the ATPase N-ethylmaleimide-sensitive factor together with its cofactor alpha-SNAP. Moreover, the C-terminal domain of tomosyn was as effective as the cytoplasmic portion of synaptobrevin in inhibiting evoked exocytosis in a cell-free preparation derived from PC12 cells. Similarly, overexpression of tomosyn in PC12 cells resulted in a massive reduction of exocytosis, but the release parameters of individual exocytotic events remained unchanged. It is concluded that tomosyn is a soluble SNARE that directly competes with synaptobrevin in the formation of SNARE complexes and thus may function in down-regulating exocytosis (Hatsuzawa, 2003).

Yeast homologues of tomosyn and lethal giant larvae function in exocytosis and are associated with the plasma membrane SNARE, Sec9

Two related yeast proteins, Sro7p and Sro77p, have been identified based on their ability to bind to the plasma membrane SNARE (SNARE) protein, Sec9p. These proteins show significant similarity to the Drosophila tumor suppressor, Lethal (2) giant larvae and to the neuronal syntaxin-binding protein, tomosyn. SRO7 and SRO77 have redundant functions since loss of both gene products leads to a severe cold-sensitive growth defect that correlates with a severe defect in exocytosis. Similar to Sec9, Sro7/77 functions in the docking and fusion of post-Golgi vesicles with the plasma membrane. In contrast to a previous report, no defect is seen in actin polarity under conditions where a dramatic effect is seen on secretion. This demonstrates that the primary function of Sro7/77 is in exocytosis rather than in regulating the actin cytoskeleton. Analysis of the association of Sro7p and Sec9p demonstrates that Sro7p directly interacts with Sec9p both in the cytosol and in the plasma membrane and can associate with Sec9p in the context of a SNAP receptor complex. Genetic analysis suggests that Sro7 and Sec9 function together in a pathway downstream of the Rho3 GTPase. Taken together, these studies suggest that members of the lethal giant larvae/tomosyn/Sro7 family play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane (Lehman, 1999).

Three splicing variants of tomosyn and identification of their syntaxin-binding region

A neural tissue-specific syntaxin-1-binding protein, tomosyn, is capable of dissociating Munc18/n-Sec1/rbSec1 from syntaxin-1 to form a 10S tomosyn complex, an intermediate complex converted to the 7S SNARE complex. Two splicing variants of tomosyn have been isolated: one has 36 amino acids (aa) insertion and another has 17 aa deletion. The original one has been named m-tomosyn, the big one b-tomosyn, and the small one s-tomosyn. s-Tomosyn as well as m-tomosyn is mainly expressed in brain whereas b-tomosyn is ubiquitously expressed. All the isoforms bind to syntaxin-1, but not to syntaxin-2, -3, or -4, and have a region highly homologous to VAMP, another syntaxin-binding protein. This region is necessary but not sufficient for high-affinity binding of tomosyn to syntaxin-1 (Yokoyama, 1999).

Tomosyn: a syntaxin-1-binding protein that forms a novel complex in the neurotransmitter release process

Syntaxin-1 is a component of the synaptic vesicle docking and/or membrane fusion soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex (7S and 20S complexes) in nerve terminals. Syntaxin-1 also forms a heterodimer with Munc18/n-Sec1/rbSec1 in a complex that is distinct from the 7S and 20S complexes. A novel syntaxin-1-binding protein, tomosyn, has been identified that is capable of dissociating Munc18 from syntaxin-1 and forming a novel 10S complex with syntaxin-1, soluble N-etyhlmaleimide-sensitive factor attachment (SNAP) 25, and synaptotagmin. The 130 kDa isoform of tomosyn is specifically expressed in brain, where its distribution partly overlaps with that of syntaxin-1 in nerve terminals. High level expression of either syntaxin-1 or tomosyn results in a specific reduction in Ca2+-dependent exocytosis from PC12 cells. These results suggest that tomosyn is an important component in the neurotransmitter release process where it may stimulate SNARE complex formation (Fujita, 1998).


Search PubMed for articles about Drosophila Tomosyn

Baba, T., Sakisaka, T., Mochida, S. and Takai, Y. (2005). PKA-catalyzed phosphorylation of tomosyn and its implication in Ca2+-dependent exocytosis of neurotransmitter. J Cell Biol 170(7): 1113-1125. PubMed ID: 16186257

Chen, K., Richlitzki, A., Featherstone, D. E., Schwarzel, M. and Richmond, J. E. (2011). Tomosyn-dependent regulation of synaptic transmission is required for a late phase of associative odor memory. Proc Natl Acad Sci U S A 108: 18482-18487. PubMed ID: 22042858

Fujita, Y., et al. (1998). Tomosyn: a syntaxin-1-binding protein that forms a novel complex in the neurotransmitter release process. Neuron 20(5): 905-15. PubMed Citation: 9620695

Gracheva, E. O., Burdina, A. O., Holgado, A. M., Berthelot-Grosjean, M., Ackley, B. D., Hadwiger, G., Nonet, M. L., Weimer, R. M. and Richmond, J. E. (2006). Tomosyn inhibits synaptic vesicle priming in Caenorhabditis elegans. PLoS Biol 4(8): e261. PubMed ID: 16895441

Groffen, A. J., Jacobsen, L., Schut, D. and Verhage, M. (2005). Two distinct genes drive expression of seven tomosyn isoforms in the mammalian brain, sharing a conserved structure with a unique variable domain. J Neurochem 92(3): 554-568. PubMed ID: 15659226

Hatsuzawa, K., Lang, T., Fasshauer, D., Bruns, D. and Jahn, R. (2003). The R-SNARE motif of tomosyn forms SNARE core complexes with syntaxin 1 and SNAP-25 and down-regulates exocytosis. J Biol Chem 278(33): 31159-31166. PubMed ID: 12782620

Hu, Z., Tong, X. J. and Kaplan, J. M. (2013). UNC-13L, UNC-13S, and Tomosyn form a protein code for fast and slow neurotransmitter release in Caenorhabditis elegans. Elife 2: e00967. PubMed ID: 23951547

Lehman, K., et al. (1999). Yeast homologues of tomosyn and lethal giant larvae function in exocytosis and are associated with the plasma membrane SNARE, Sec9. J. Cell Biol. 146(1): 125-40. PubMed Citation: 10402465

Lu, Z., Chouhan, A. K., Borycz, J. A., Lu, Z., Rossano, A. J., Brain, K. L., Zhou, Y., Meinertzhagen, I. A. and Macleod, G. T. (2016). High-Probability Neurotransmitter Release Sites Represent an Energy-Efficient Design. Curr Biol 26(19): 2562-2571. PubMed ID: 27593375

Nair, A. G., Muttathukunnel, P. and Muller, M. (2021). Distinct molecular pathways govern presynaptic homeostatic plasticity. Cell Rep 37(11): 110105. PubMed ID: 34910905

Newman, Z. L., Hoagland, A., Aghi, K., Worden, K., Levy, S. L., Son, J. H., Lee, L. P. and Isacoff, E. Y. (2017). Input-Specific Plasticity and Homeostasis at the Drosophila Larval Neuromuscular Junction. Neuron 93(6): 1388-1404 e1310. PubMed ID: 28285823

Pobbati, A. V., Razeto, A., Boddener, M., Becker, S. and Fasshauer, D. (2004). Structural basis for the inhibitory role of tomosyn in exocytosis. J Biol Chem 279(45): 47192-47200. PubMed ID: 15316007

Saldate, J. J., Shiau, J., Cazares, V. A. and Stuenkel, E. L. (2018). The ubiquitin-proteasome system functionally links neuronal Tomosyn-1 to dendritic morphology. J Biol Chem 293(7): 2232-2246. PubMed ID: 29269412

Sauvola, C. W., Akbergenova, Y., Cunningham, K. L., Aponte-Santiago, N. A. and Littleton, J. T. (2021). The decoy SNARE Tomosyn sets tonic versus phasic release properties and is required for homeostatic synaptic plasticity. Elife 10. PubMed ID: 34713802

Yokoyama, S., Shirataki, H., Sakisaka, T. and Takai, Y. (1999). Three splicing variants of tomosyn and identification of their syntaxin-binding region. Biochem Biophys Res Commun 256(1): 218-222. PubMed ID: 10066450

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date revised: 12 November 2022

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