Anna Burdina et al. (2008) C.elegans Neuronal Development Meeting "Structure-Function analysis of C. elegans TOM-1"

Anna Burdina, Elena GRACHEVA, Janet Richmond

[C.elegans Neuronal Development Meeting, 2008]

Synaptic vesicle priming is a prerequisite for fusion requiring the assembly of SNARE complexes between syntaxin, SNAP-25 and synaptobrevin. We have recently shown that C. elegans tomosyn (TOM-1) negatively regulates this priming process; tom-1 mutants exhibiting aldicarb-hypersensitivity and enhanced evoked release due to increased vesicle priming. Overexpression of full-length TOM-1A reversed the tom-1 phenotype producing aldicarb-resistance and a deficit in evoked release due to fewer primed vesicles. While the function of TOM-1 is now established, the molecular mechanism by which TOM-1 regulates priming remains to be ascertained. TOM-1 is a large cytosolic protein composed of 14 N-terminal WD-40 repeats linked to a C-terminal SNARE binding domain. Similar to vertebrate tomosyn, the C-terminal SNARE domain of TOM-1 forms an SDS-sensitive complex with syntaxin and SNAP-25 proposed to inhibit SNARE complex assembly, thereby limiting vesicle priming. However, it is unknown whether the TOM-1 SNARE domain, is necessary or sufficient to inhibit priming. To address this question, we generated integrated lines expressing either the TOM-1 SNARE domain alone or TOM-1A lacking the SNARE domain (delta SNARE) in a tom-1 mutant background. Both constructs produced mRNA levels similar to the rescuing full-length TOM-1A construct. However, in sharp contrast to full-length TOM-1A, neither the TOM-1 SNARE domain or delta-SNARE integrants appreciably rescued the aldicarb-hypersensitive phenotype of tom-1 mutants. Consistent with these results, the synaptically evoked responses of neither truncated TOM-1A constructs rescued the enhanced release of tom-1 mutants or the accumulation of primed vesicles. To examine whether these truncated TOM-1A constructs were stably expressed, we generated flag-tagged integrants of each construct in tom-1 mutants. The flag-tagged versions of truncated TOM-1A also failed to rescue tom-1 mutants. We confirmed that both constructs were stably expressed by Western blot analysis of lysed worms and further demonstrated by whole-mount immunohistochemistry, that both constructs were expressed at the neuromuscular synapses from which our synaptic recordings were obtained. Together these results suggest that the TOM-1 SNARE domain is necessary but not sufficient to negatively-regulate synaptic vesicle priming at C. elegans synapses and imply that regions upstream of the SNARE domain contribute to the function of this regulatory protein.

Gracheva EO et al. (2006) PLoS Biol "Tomosyn inhibits synaptic vesicle priming in Caenorhabditis elegans."

Burdina AO, Hadwiger G, Berthelot-Grosjean M, Richmond JE, Ackley BD, Gracheva EO, Weimer RM, Nonet ML, Holgado AM

[PLoS Biol, 2006]

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, we analyzed C. elegans tom-1 mutants. Our 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, we observe a concomitant increase 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.

Meenakshi Balaraman et al. (2007) International Worm Meeting "Characterization of TOM-1- vesicle interactions."

Elena O. Gracheva, Meenakshi Balaraman, Dennis Touroutine, Janet E. Richmond, Anna O. Burdina

[International Worm Meeting, 2007]

Tomosyn is a highly conserved protein identified on the basis of its interaction with the SNARE protein syntaxin, an essential component of the synaptic vesicle exocytotic machinery.C.elegans tomosyn (TOM-1) has recently been shown to negatively regulate neurotransmission; tom-1 mutants exhibiting aldicarb hypersensitivity attributed to increased synaptic vesicle priming leading to enhanced synaptic transmission.Whole-mount immunohistochemistry indicates that endogenous TOM-1 is highly enriched at C.elegans synapses, consistent with a previously published TOM-1::GFP expression pattern. This synaptic localization requires the kinesin motor protein, UNC-104 that is known to transport synaptic vesicles to terminals. Recent molecular analysis of vertebrate synaptic vesicles confirms that tomosyn is associated with synaptic vesicles and our TOM-1 immunoEM analysis suggests that TOM-1 also associates with dense core vesicles. Surprisingly however, the sub-synaptic distribution of TOM-1 based on immunoEM suggests that TOM-1 is selectively excluded from primed vesicles that are destined for release within the active zone. We would therefore like to identify the region of TOM-1 that is required for its vesicle association and establish how TOM-1 interacts with vesicles. tom-1 encodes several isoforms that share a common C-terminal SNARE binding motif, a hypervariable linker and except for the short TOM-1B isoform, possess an N-terminal WD-40 repeat domain. GFP-tagged TOM-1 lacking the SNARE domain appears to be normally targeted to the synapse, suggesting this domain is not required for vesicle association during transport to the synapse. Similarly, we have shown that a tom-1 allele with an in-frame deletion excluding most of the WD40 repeat domain is also targeted to synapses. This suggests that TOM-1 vesicle association requires the remaining regions of the WD40 not excised in this mutant or the linker domain. To narrow down and define the region of TOM-1 required for vesicle association, we will examine the localization of truncated TOM-1 constructs lacking either the entire WD-40 domain, or the SNARE domain. Once we have identified the TOM-1 domain required for synaptic localization we will take several approaches to determine the nature of the interaction between TOM-1 and secretory vesicles including; 1) a yeast two-hybrid screen for possible vesicle-associated interacting proteins, 2) perform a clonal screen using a GFP-tagged version of the minimal vesicle-association domain to identify mutants in which the GFP signal is mislocalized and 3) screen for additional aldicarb-hypersensitive mutants that may disrupt genes required for the vesicle localization of TOM-1.

Gracheva EO et al. (2007) J Neurosci "Tomosyn negatively regulates CAPS-dependent peptide release at Caenorhabditis elegans synapses."

Richmond JE, Berthelot-Grosjean M, Touroutine D, Gracheva EO, Parekh H, Burdina AO

[J Neurosci, 2007]

The syntaxin-interacting protein tomosyn is thought to be a key regulator of exocytosis, although its precise mechanism of action has yet to be elucidated. Here we examined the role of tomosyn in peptide secretion in Caenorhabditis elegans tomosyn (tom-1) mutants. Ultrastructural analysis of tom-1 mutants revealed a 50% reduction in presynaptic dense-core vesicles (DCVs) corresponding to enhanced neuropeptide release. Conversely, overexpression of TOM-1 led to an accumulation of DCVs. Together, these data provide the first in vivo evidence that TOM-1 negatively regulates DCV exocytosis. In C. elegans, neuropeptide release is promoted by the calcium-dependent activator protein for secretion (CAPS) homolog UNC-31. To test for a genetic interaction between tomosyn and CAPS, we generated tom-1;unc-31 double mutants. Loss of TOM-1 suppressed the behavioral, electrophysiological, and DCV ultrastructural phenotypes of unc-31 mutants, indicating that TOM-1 antagonizes UNC-31-dependent DCV release. Because unc-31 mutants exhibit synaptic transmission defects, we postulated that loss of DCV release in these mutants and the subsequent suppression by tom-1 mutants could simply reflect alterations in synaptic activity, rather than direct regulation of DCV release. To distinguish between these two possibilities, we analyzed C. elegans Rim mutants (unc-10), which have a comparable reduction in synaptic transmission to unc-31 mutants, specifically attributed to defects in synaptic vesicle (SV) exocytosis. Based on this analysis, we conclude that the changes in DCV release in tom-1 and unc-31 mutants reflect direct effects of TOM-1 and UNC-31 on DCV exocytosis, rather than altered SV release.

Anna Burdina et al. (2007) International Worm Meeting "Dissecting the Function of C. elegans Tomosyn Domains."

Anna Burdina, Elena GRACHEVA, Janet Richmond

[International Worm Meeting, 2007]

Calcium-dependent release of neurotransmitters requires the prior assembly of SNARE complexes between the vesicle SNARE, synaptobrevin and the plasma membrane SNAREs, syntaxin and SNAP-25 during the priming stage of the vesicle cycle. We have recently shown that C. elegans tomosyn (TOM-1) negatively regulates this priming step. TOM-1 has two conserved domains; an N-terminal WD40 repeat domain and a C-terminal SNARE domain. The ability of the TOM-1 SNARE domain to form non-fusogenic complexes with syntaxin and SNAP-25 in competition with synaptobrevin, is proposed to mediate the inhibitory action of TOM-1 in exocytosis. To directly test the role of the SNARE motif and establish the functional significance of the WD40 repeats we have taken the following approaches. We have generated transgenes encoding full-length or truncated TOM-1A in which either the WD40 or SNARE domains are eliminated or in which only the WD40 or SNARE domains are expressed. Neuronal expression of the full-length TOM-1A transcript reverses the exuberant release observed in tom-1 mutants. We are now examining the functional consequences of expressing each of the truncated tom-1 constructs in both tom-1 mutants and wild-type worms. Two pieces of evidence suggest that the WD40 domain of tomosyn may be important for its function: 1) In yeast tomosyn homologues the WD40 domain is conserved whereas the SNARE-binding domain is not. 2) We have examined a tom-1 allele, ok188, in which an in-frame deletion removes the majority of the WD40 repeats, but the protein is stably expressed at synapses based on TOM-1 antibody staining. tom-1(ok188) animals phenocopy synaptic defects of other tomosyn mutants, i.e. they are aldicarb-hypersensitive and have an increased evoked response. Therefore, the WD40 domain appears to be necessary for the inhibitory function of TOM-1 in exocytosis. Based on this evidence we undertook a yeast two-hybrid screen, using the WD40 domain as a bait to identify candidate binding partners of tomosyn. One of the proteins identified in this screen shares homology with a yeast trafficking protein; 3 independent hits representing overlapping regions of this protein, suggest a minimal binding domain responsible for the interaction with tomosyn. Loss-of-function mutants in this protein are viable but exhibit maternal sterility. Homozygous mutant progeny from a balanced strain exhibit increased aldicarb sensitivity and mild locomotory defects similar to tom-1 mutants. Further characterization of this mutant is underway to test the possible functional relationship between this and TOM-1 in the regulation of exocytosis.

Ling Chen et al. (2004) West Coast Worm Meeting "Functional Characterization of a Putative Apoptotic Suppressor, tfg-1, in C. elegans"

Joel H Rothman, Ling Chen, Tom McCloskey

[West Coast Worm Meeting, 2004]

Among the genes identified in genome-wide RNAi screens for novel apoptotic regulators (see abstract by McCloskey and Rothman) is tfg-1 , the C. elegans homologue of human TFG (TRK-fused gene). TFG was originally identified on the basis of its rearrangement in chromosomal translocations present in papillary thyroid carcinomas and anaplastic large cell lymphomas. These rearrangements create in-frame fusions of TFG coding sequences with the receptor tyrosine kinase, ALK (anaplastic lymphoma kinase). TFG transcription is also up-regulated in response to activation of the TNF receptor family member TALL-1, which promotes proliferation of B lymphocytes and autoimmunity. We have found that that loss of tfg-1 activity results in a slight increase in CED-3-independent cell death. While this effect is mild, we also observed that RNAi of tfg-1 results in small, uncoordinated, and sterile phenotypes, all three of which are suppressed by a ced-4 , but not a ced-3 mutation. Thus, the Sma Unc Ste phenotypes might be the result of CED-4-dependent apoptosis, as is seen in icd-1 and icd-2 mutants (see abstract by Bloss and Rothman). While the function of human TFG in tumorigenesis is unknown, these results suggest that tfg-1 might act to suppress apoptosis, leading to the hypothesis that TFG might contribute to tumor formation by inappropriately repressing apoptosis, in analogy to the action of the anti-apoptotic factor, Bcl-2, in promoting lymphomas. Such an anti-apoptotic function could also explain a role for TFG in autoimmunity. Current studies are directed at illuminating the action of tfg-1 in repression of apoptosis.

Barnes TM (1994) Worm Breeder's Gazette "Genome Parametrics, or Look, I Can Use Query and Tablemaker!"

Barnes TM

[Worm Breeder's Gazette, 1994]

Genome Parametrics, or Look, I can use Query and Tablemaker! Tom Barnes Deptartment of Biology, McGill University, Montreal PQ H3AlB1 Canad

Barnes TM (1994) Worm Breeder's Gazette "The Structure of the C. elegans Genome and Implications For Function"

Barnes TM

[Worm Breeder's Gazette, 1994]

The Structure of the C. elegans Genome and Implications for Function. Tom Barnes, Department of Biology, McGill University, Montreal, PQ H3A lBl Canada

Mahadik, Snehal et al. (2021) International Worm Meeting "TOM-1/Tomosyn is an inhibitor of growth cone protrusion and works with the UNC-6/Netrin receptor UNC-5"

Mahadik, Snehal, Lundquist, Erik

[International Worm Meeting, 2021]

Previous work from the Lundquist lab showed that the UNC-6/Netrin receptors UNC-40 and UNC-5 regulate growth cone protrusion. UNC-40 stimulates protrusion whereas UNC-5 inhibits protrusion, and asymmetric distribution of protrusive activity across the growth cone results in directed growth cone migration away from UNC-6/Netrin (the Polarity/Protrusion model). unc-5 mutant VD growth cones display unpolarized and excessive protrusion. UNC-5 inhibits protrusion using the FMO flavin monoxygenases, likely via actin inhibition, and by restricting growth cone microtubule entry via UNC-33/CRMP, which might in turn regulate vesicle entry which drives protrusion. To explore the role of vesicle fusion in growth cone protrusion, we analyzed tom-1/tomosyn mutants. Tomosyn normally occludes formation of the SNARE complex by interacting with and inhibiting syntaxin-1. VD growth cones of tom-1 null mutants were similar to wild-type. However, tom-1 null mutants suppressed the effects of constitutively-activated MYR::UNC-5, which alone causes small growth cones with little protrusion. This suggests that TOM-1 is normally required for the inhibitory effects of MYR::UNC-5 on growth cone protrusion. Mutations specifically affecting tom-1 long isoforms showed small and non-protrusive growth cones, and did not suppress MYR::UNC-5. This suggests that TOM-1 short and long isoforms might have opposing roles, with TOM-1 short normally inhibiting protrusion, and TOM-1 long stimulating protrusion. Short isoform specific mutations suppressed MYR::UNC-5, and long isoform specific mutations suppressed excess protrusion in unc-5 loss-of-function mutants, consistent with this idea. Finally, transgenic expression of full-length tom-1(+) resulted in small and non-protrusive growth cones in both wild-type and unc-5 mutant backgrounds, consistent with a role of TOM-1 in inhibiting protrusion downstream of UNC-5. In the polarity/protrusion model of growth cone outgrowth, UNC-6/Netrin inhibits growth cone protrusion via the UNC-5 receptor. Previous studies showed that UNC-5 inhibits protrusion via the FMOs and possible actin destabilization, and by preventing MT entry via UNC-33/CRMP. These results suggest that UNC-5 inhibits protrusion via a third pathway, employing TOM-1/tomosyn to prevent vesicle fusion and growth cone protrusion.

Burdina AO et al. (2011) PLoS One "In vivo analysis of conserved C. elegans tomosyn domains."

Shtessel L, Burdina AO, Klosterman SM, Ahmed S, Richmond JE

[PLoS One, 2011]

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 -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.