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SYMPOSIUM REPORT |
1 Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL, USA
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Abstract |
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(Received 8 June 2007;
accepted after revision 6 July 2007;
first published online 12 July 2007)
Corresponding author J. E. Richmond: Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL, USA. Email: jer{at}uic.edu
Synaptic transmission is a highly orchestrated process in which calcium influx triggers the release of neurotransmitters from fusion-competent secretory vesicles within microseconds. Exocytosis requires the prior assembly of tertiary SNARE complexes between the vesicle SNARE (soluble NSF-attachment protein receptor), synaptobrevin and the plasma membrane-associated SNAREs, syntaxin and SNAP-25 (Sollner et al. 1993; Hanson et al. 1997; Lonart & Sudhof, 2000; Chen & Scheller, 2001) (Fig. 1D ). The central importance of SNARE complex assembly in neurotransmission is illustrated by the severe release defects that result from exposure to SNARE cleaving clostridial toxins or by genetic ablation of SNARE proteins (Broadie et al. 1995; Schoch et al. 2001; Washbourne et al. 2001). The regulation of SNARE complex assembly is thus a potentially important mechanism by which proteins can adjust synaptic strength.
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C. elegans is an excellent model organism in which to study the molecular basis of synaptic transmission for the following reasons (Nonet, 1999). First, the nervous system of this small soil nematode is relatively simple, with a total of 302 invariant neurons. Second, examination of the annotated C. elegans genome indicates that synaptic proteins are highly conserved, thus functional information obtained through the analysis of worms is applicable to vertebrate homologues. Third, it is relatively easy to isolate synaptic mutants by forward and reverse screens, and many of these mutants are viable. Fourth, there is a rich repertoire of tools and techniques available to characterize the synaptic phenotypes of acquired mutants, including the recent development of an electrophysiological preparation that allows us to record from the neuromuscular junctions (NMJs) of these worms (Richmond & Jorgensen, 1999) and high-pressure freeze fixation techniques that allow us to image synaptic ultrastructure and immunolabel synaptic proteins (Rostaing et al. 2004).
C. elegans encodes a single tomosyn gene, tom-1, for which we have obtained a number of loss-of-function mutants (Dybbs et al. 2005). tom-1 mutants have only mild behavioural defects such as reduced brood size, decreased thrashing rates in solution and aberrant head-tap responses, indicating that TOM-1 is functionally important but is not an essential synaptic protein. Pharmacological assays on freely moving animals suggest that acetylcholine transmission is enhanced in tom-1 mutant worms. To elucidate the physiological basis of this phenotype we directly measured release at cholinergic NMJs of dissected tom-1 mutants. Evoked synaptic responses in these mutants were significantly broader than wild-type, resulting in a twofold increase in the evoked charge integral (WT 18 ± 1 pC, n = 49, versus 38 ± 3 pC, n = 30, for tom-1(ok285)) (Gracheva et al. 2006; McEwen et al. 2006) (Fig. 1A). We attributed the tom-1 mutant phenotype to a change in presynaptic ACh release for the following reasons. First, neither the amplitude nor the kinetics of the miniature postsynaptic responses was altered, suggesting that postsynaptic reception was normal. Second, TOM-1 was found to be enriched in presynaptic terminals based on immunohistochemical staining. Third, we were able to reverse the tom-1 mutant synaptic phenotype, by expressing TOM-1 in the presynaptic cholinergic motor neurons. How might tomosyn regulate presynaptic release? We confirmed that like its mammalian orthologues, the C-terminal SNARE domain of C. elegans TOM-1 is capable of forming a complex with syntaxin and SNAP-25. Since this interaction is predicted to inhibit synaptic vesicle priming we examined the synaptic responses of tom-1 mutants to hyperosmotic saline, to measure the size of the fusion-competent, primed vesicle pool. The twofold increase in hyperosmotic responses of tom-1 mutant synapses indicates that vesicle priming is enhanced in the absence of TOM-1 (Gracheva et al. 2006; McEwen et al. 2006). We have recently described a morphological correlate of primed vesicles, based on ultrastructural comparisons of wild-type and priming defective unc-13 mutant C. elegans synapses prepared by high-pressure freeze fixation (Weimer et al. 2006) (Fig. 1A). This analysis suggests that vesicle contact with the plasma membrane is primarily a reflection of vesicle priming. When we examined tom-1 mutant synaptic profiles, we observed a twofold increase in the number of morphologically contacting vesicles (WT 8.5 ± 0.3% of vesicles contacting the plasma membrane/synaptic profile, n = 250 profiles versus 15.6 ± 0.75%, n = 74 profiles for tom-1(ok285)) consistent with the enhanced release observed electrophysiologically (Gracheva et al. 2006) (Fig. 1A). Conversely, overexpression of TOM-1 at cholinergic synapses caused a reduction in both evoked responses and number of morphologically docked vesicles when compared with the wild type. Based on these data we propose that tomosyn acts as a negative regulator of synaptic vesicle priming, possibly by competing with vesicle-associated synaptobrevin for complex formation with syntaxin and SNAP-25, thereby restricting vesicle fusion-competence.
We have previously shown that UNC-13 is required to prime synaptic vesicles, possibly by promoting the availability of the syntaxin SNARE domain required for SNARE complex assembly (Richmond et al. 1999, 2001). Given that TOM-1 can negatively regulate priming, possibly by occupying the syntaxin SNARE domain, we asked whether genetically ablating TOM-1 could rescue the unc-13 mutant priming defect. We found partial restoration of evoked and hyperosmotic synaptic responses in tom-1–unc-13 double mutants (Fig. 1B and C), accompanied by partial restoration of the plasma membrane contacting vesicle pool. Therefore, these data suggest that TOM-1 antagonizes UNC-13-dependent priming.
We next asked whether TOM-1 similarly regulates the priming of peptide-containing dense core vesicles (DCVs). The priming and fusion-competence of DCVs is thought to require the cytosolic protein, CAPS (calcium-dependent activator protein for secretion) (Walent et al. 1992; Ann et al. 1997; Berwin et al. 1998) encoded by unc-31 in C. elegans. unc-31 mutants exhibit an uncoordinated, semi-paralysed phenotype, which can be rescued by expressing UNC-31 in cholinergic neurons (Charlie et al. 2006). Furthermore, immunohistochemical analysis reveals localized expression of UNC-31 protein at cholinergic synapses, which we show in this study by immuno-EM is the result of CAPS association with DCVs. To determine the physiological basis for the behavioural defects in unc-31 mutants we recorded NMJ-evoked responses. The unc-31 mutant synapses exhibited a 50% reduction in evoked release amplitude. This reduction in synaptic vesicle release is thought to be the result of impaired peptidergic signalling primarily through G protein-coupled receptors that are known to be upstream regulators of synaptic transmission in C. elegans. To assess whether peptide release was impaired in unc-31 mutants, we examined the ultrastructure of unc-31 synapses and observed a threefold accumulation of synaptic DCVs, consistent with the proposed function of UNC-31 in promoting DCV release. Conversely, when we examined the number of DCVs in tom-1 mutant synapses we observed 50% fewer DCVs, whereas over expression of TOM-1 caused DCV accumulations similar to those of unc-31 mutants. The loss of DCVs in tom-1 mutants could be a reflection of increased peptide release, the replenishment of DCVs failing to keep pace with the enhanced release caused by loss of TOM-1. Alternatively the depletion of DCVs in tom-1 mutants may reflect reduced DCV biogenesis or trafficking to the synapse. To distinguish between these possibilities we used a recently developed peptide secretion assay, in which the levels of fluorescently tagged peptides secreted by cholinergic neurons can be measured (Sieburth et al. 2006). This assay takes advantage of the fact that worms sequester secreted peptides in scavenger cells called coelomocytes, therefore the level of fluorescence accumulating in coelomocytes provides a read-out of fluorescently tagged peptide secretion. Using this approach we were able to show that tom-1 mutants have enhanced peptide release. In contrast, we confirmed previous reports showing that unc-31 mutants have reduced peptide release (Sieburth et al. 2006), suggesting that tomosyn and CAPS have opposing functions in the regulation of peptidergic transmission.
To examine whether there is a genetic interaction between unc-31 and tom-1 we generated double mutants. The behavioural defects of tom-1; unc-31 mutants were less pronounced than unc-31 alone. Consistent with this observation, NMJ-evoked responses improved in the double mutants although did not reach wild-type levels. The accumulation of DCVs observed in unc-31 mutants was also partially suppressed in tom-1; unc-31 mutants. These data indicate that tom-1 negatively regulates CAPS-dependent DCV release.
Since we have shown that both unc-31 and tom-1 also affect synaptic vesicle release we wondered whether the observed effects on DCV release were secondary consequences of altered synaptic activity. To test this, we examined mutants in unc-10, a gene encoding Rim, which is an active zone protein implicated in the targeting and priming of synaptic vesicles (Koushika et al. 2001; Weimer et al. 2006). We chose this mutant because the evoked release in unc-10 mutants is similar to that of both unc-31 mutants and TOM-1-overexpressing animals (Koushika et al. 2001; Gracheva et al. 2006). Since we found significantly fewer DCVs in unc-10 mutants, relative to unc-31 and TOM-1 overexpression, we concluded that DCV accumulation is due to loss of a direct action of UNC-31 in DCV release and to inhibition of DCV release in the case of TOM-1 overexpression. To address whether the loss of DCVs in tom-1 mutants is a direct consequence of losing TOM-1 or an indirect result of enhanced synaptic activity we next generated tom-1; unc-10 mutants. The evoked release of these double mutants was partially improved relative to unc-10 single mutants, but remained much lower than wild-type and dramatically lower than tom-1 mutants. However, when we examined DCV numbers in tom-1; unc-10 double mutants we observed the same decrease as in tom-1 single mutants, indicating that tom-1 mutants cause enhanced DCV exocytosis even under conditions where synaptic activity is impaired. These data suggest that TOM-1 directly inhibits CAPS-dependent DCV release.
In summary we have demonstrated that both synaptic vesicle priming and DCV release are negatively regulated by tomosyn. We have also shown that tom-1 genetically interacts with genes encoding both the synaptic vesicle priming factor UNC-13 and UNC-31 which promotes DCV fusion. On the basis of these results we propose that tomosyn through its interactions with the SNARE protein syntaxin, restricts the priming processes of both secretory organelles (Fig. 1D).
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Footnotes |
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References |
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TopAbstractReferences |
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Berwin B, Floor E & Martin TF (1998). CAPS (mammalian UNC-31) protein localizes to membranes involved in dense-core vesicle exocytosis. Neuron 21, 137–145.[CrossRef][Medline]
Broadie K, Prokop A, Bellen HJ, O'Kane CJ, Schulze KL & Sweeney ST (1995). Syntaxin and synaptobrevin function downstream of vesicle docking in Drosophila. Neuron 15, 663–673.[CrossRef][Medline]
Charlie NK, Schade MA, Thomure AM & Miller KG (2006). Presynaptic UNC-31 (CAPS) is required to activate the G
s pathway of the Caenorhabditis elegans synaptic signaling network. Genetics 172, 943–961.
Chen YA & Scheller RH (2001). SNARE-mediated membrane fusion. Nat Rev Mol Cell Biol 2, 98–106.[CrossRef][Medline]
Dybbs M, Ngai J & Kaplan JM (2005). Using microarrays to facilitate positional cloning: identification of tomosyn as an inhibitor of neurosecretion. PLoS Genet 1, 6–16.[Medline]
Fujita Y, Shirataki H, Sakisaka T, Asakura T, Ohya T, Kotani H, Yokoyama S, Nishioka H, Matsuura Y, Mizoguchi A, Scheller RH & Takai Y (1998). Tomosyn: a syntaxin-1-binding protein that forms a novel complex in the neurotransmitter release process. Neuron 20, 905–915.[CrossRef][Medline]
Gracheva EO, Burdina AO, Holgado AM, Berthelot-Grosjean M, Ackley BD, Hadwiger G, Nonet ML, Weimer RM & Richmond JE (2006). Tomosyn inhibits synaptic vesicle priming in Caenorhabditis elegans. PLoS Biol 4, e261.[CrossRef][Medline]
Hanson PI, Heuser JE & Jahn R (1997). Neurotransmitter release – four years of SNARE complexes. Curr Opin Neurobiol 7, 310–315.[CrossRef][Medline]
Hatsuzawa K, Lang T, Fasshauer D, Bruns D & 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, 31159–31166.
Hattendorf DA, Andreeva A, Gangar A, Brennwald PJ & Weis WI (2007). Structure of the yeast polarity protein Sro7 reveals a SNARE regulatory mechanism. Nature 446, 567–571.
Koushika SP, Richmond JE, Hadwiger G, Weimer RM, Jorgensen EM & Nonet ML (2001). A post-docking role for active zone protein Rim. Nat Neurosci 4, 997–1005.[CrossRef][Medline]
Lonart G & Sudhof TC (2000). Assembly of SNARE core complexes prior to neurotransmitter release sets the readily releasable pool of synaptic vesicles. J Biol Chem 275, 27703–27707.
McEwen JM, Madison JM, Dybbs M & Kaplan JM (2006). Antagonistic regulation of synaptic vesicle priming by Tomosyn and UNC-13. Neuron 51, 303–315.[CrossRef][Medline]
Nonet ML (1999). Studying Mutants that Affect Neurotransmitter Release in C. elegans. Oxford University Press, Oxford.
Pobbati A, Razeto A, Boddener M, Becker S & Fasshauer D (2004). Structural basis for the inhibitory role of tomosyn in exocytosis. J Biol Chem 279, 47192–47200.
Richmond JE, Davis WS & Jorgensen EM (1999). UNC-13 is required for synaptic vesicle fusion in C. elegans. Nat Neurosci 2, 959–964.[CrossRef][Medline]
Richmond JE & Jorgensen EM (1999). One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction. Nat Neurosci 2, 791–797.[CrossRef][Medline]
Richmond JE, Weimer RM & Jorgensen EM (2001). An open form of syntaxin bypasses the requirement for UNC-13 in vesicle priming. Nature 412, 338–341.[CrossRef][Medline]
Rostaing P, Weimer RM, Jorgensen EM, Triller A & Bessereau JL (2004). Preservation of immunoreactivity and fine structure of adult C. elegans tissues using high-pressure freezing. J Histochem Cytochem 52, 1–12.
Schoch S, Deak F, Konigstorfer A, Mozhayeva M, Sara Y, Sudhof TC & Kavalali ET (2001). SNARE function analyzed in synaptobrevin/VAMP knockout mice. Science 294, 1117–1122.
Sieburth D, Madison JM & Kaplan JM (2006). PKC-1 regulates secretion of neuropeptides. Nat Neurosci 10, 49–57.[CrossRef][Medline]
Sollner T, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S, Tempst P & Rothman JE (1993). SNAP receptors implicated in vesicle targeting and fusion. Nature 362, 318–324.
Walent JH, Porter BW & Martin TF (1992). A novel 145 kd brain cytosolic protein reconstitutes Ca2+-regulated secretion in permeable neuroendocrine cells. Cell 70, 765–775.[CrossRef][Medline]
Washbourne P, Cansino V, Mathews JR, Graham M, Burgoyne RD & Wilson MC (2001). Cysteine residues of SNAP-25 are required for SNARE disassembly and exocytosis, but not for membrane targeting. Biochem J 357, 625–634.[CrossRef][Medline]
Weimer RM, Gracheva EO, Meyrignac O, Miller KG, Richmond JE & Bessereau JL (2006). UNC-13 and UNC-10/rim localize synaptic vesicles to specific membrane domains. J Neurosci 26, 8040–8047.
Yizhar O, Matti U, Melamed R, Hagalili Y, Bruns D, Rettig J & Ashery U (2004). Tomosyn inhibits priming of large densecore vesicles in a calcium-dependent manner. Proc Natl Acad Sci U S A 101, 2578–2583.
Yokoyama S, Shirataki H, Sakisaka T & Takai Y (1999). Three splicing variants of tomosyn and identification of their syntaxin-binding region. Biochem Biophys Res Commun 256, 218–222.[CrossRef][Medline]
Zhang W, Lilja L, Mandic SA, Gromada J, Smidt K, Janson J, Takai Y, Bark C, Berggren PO & Meister B (2006). Tomosyn is expressed in β-cells and negatively regulates insulin exocytosis. Diabetes 55, 574–581.
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Acknowledgements |
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