Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Dec 29.
Published in final edited form as: Nat Neurosci. 2003 Sep 14;6(10):10.1038/nn1118. doi: 10.1038/nn1118

Defects in synaptic vesicle docking in unc-18 mutants

Robby M Weimer 1,4,5, Janet E Richmond 2,5, Warren S Davis 1, Gayla Hadwiger 3, Michael L Nonet 3, Erik M Jorgensen 1
PMCID: PMC3874415  NIHMSID: NIHMS77227  PMID: 12973353

Abstract

Sec1-related proteins function in most, if not all, membrane trafficking pathways in eukaryotic cells. The Sec1-related protein required in neurons for synaptic vesicle exocytosis is UNC-18. Several models for UNC-18 function during vesicle exocytosis are under consideration. We have tested these models by characterizing unc-18 mutants of the nematode Caenorhabditis elegans. In the absence of UNC-18, the size of the readily releasable pool is severely reduced. Our results show that the near absence of fusion-competent vesicles is not caused by a reduction in syntaxin levels, by a mislocalization of syntaxin, by a defect in fusion or by a failure to open syntaxin during priming. Rather, we found a reduction of docked vesicles at the active zone in unc-18 mutants, suggesting that UNC-18 functions, directly or indirectly, as a facilitator of vesicle docking.

Synaptic vesicles must be tethered to sites of release and then become competent for fusion with the plasma membrane. These processes are called docking and priming, respectively1. The molecular processes of docking are not well understood. Priming appears to involve the formation of trans-SNARE complexes, in which the synaptic vesicle protein synaptobrevin (also called VAMP) forms a helical bundle with the plasma membrane proteins syntaxin and SNAP-25 (ref. 2). The formation of SNARE complexes is sufficient for membrane fusion in vitro3, but may require other components in vivo4,5.

Another protein that is involved in synaptic vesicle exocytosis is UNC-18 (ref. 6). unc-18 mutants were first isolated in C. elegans on the basis of their severe locomotion defects7. The UNC-18 protein is similar to the yeast SEC1 protein8, and orthologs of UNC-18 function at the synapses of fruit flies (Rop)9 and vertebrates (Muncl8-1; also known as Stxbpl, N-secl, Rb-secl)10. Genetic studies of UNC-18 in various organisms suggest that UNC-18 is required for synaptic vesicle exocytosis. For example, unc-18 mutants in C. elegans are severely paralyzed and are resistant to inhibitors of cholinesterase but sensitive to acetylcholine (ACh) receptor agonists11, implicating UNC-18 in the exocytosis of ACh. Four potential functions for UNC-18 in exocytosis have been proposed12: a trafficking factor for syntaxin13, a facilitator of fusion14, a facilitator of priming15 or a facilitator of docking16.

The first model proposes that UNC-18 has an indirect role in vesicle exocytosis by trafficking syntaxin to the plasma membrane. In cell culture, syntaxin is not transported to the plasma membrane unless Muncl8-1 is coexpressed with the SNARE protein13. In yeast, the syntaxin homolog TLG2 is degraded by the proteasome unless the cognate UNC-18 homolog is present to protect it17. Consistent with this idea is the fact that in Muncl8-1 knockout mice, syntaxin levels are reduced by half16.

A second model suggests that UNC-18 may function in the final step of exocytosis, that is, in the fusion of the synaptic and plasma membranes. Specifically, overexpression of a mutant form of Muncl8-1 in adrenal chromaffin cells alters the dynamics of the fusion pore in single-granule exocytosis14, suggesting that the positive role for UNC-18 occurs at the fusion step after the formation of the SNARE complex. A role in fusion is consistent with the absence of synaptic vesicle fusion events at central synapses in mice lacking Muncl8-1 (ref. 10). Moreover, these data are consistent with studies in yeast in which an UNC-18 homolog, Seclp, has been shown to function after the formation of the SNARE complex18. However, fusion of dense core vesicles still occurs in Muncl8-1 knockout mice16.

A third model proposes that UNC-18 facilitates the priming step of synaptic vesicle exocytosis through its interaction with the SNARE protein syntaxin15. Syntaxin can adopt two configurations19. In the open configuration, the SNARE domain of syntaxin can interact with SNAP-25 and synaptobrevin. In the closed configuration, the amino terminus of syntaxin folds over the SNARE domain and occludes interactions with the other SNARE proteins19. Thus, syntaxin must be ‘opened’ for the SNARE complex to form. UNC-18 proteins from C. elegans and vertebrates bind syntaxin with nanomolar affinity6. Furthermore, syntaxin rendered constitutively open fails to bind UNC-18 in pulldown assays19. One possible model for UNC-18 is that binding to the closed state of syntaxin is a prerequisite for the opening of syntaxin; once syntaxin is in the open state, UNC-18 is released, which allows the formation of the SNARE complex15,19. Consistent with this model, expression of a truncated form of the yeast syntaxin homolog, Tlg2p, partially suppresses mutants of the UNC-18 homolog Vps45p17.

A fourth model proposes that UNC-18 functions before vesicle priming and fusion to promote synaptic vesicle docking. Dense-core vesicles in Muncl8-1 knockout mice fail to associate with the plasma membrane in chromaffin cells16. Given that dense-core vesicle exocytosis uses many of the same proteins as synaptic vesicle exocytosis20, UNC-18 may function to promote synaptic vesicle docking as well. However, no docking defect was observed for synaptic vesicles at synapses in the hippocampus of Muncl8-1 mutant mice10, suggesting that UNC-18 function may be different for synaptic vesicle exocytosis and dense core vesicle exocytosis.

In addition to promoting release, UNC-18 has been implicated as an inhibitor of vesicle exocytosis21. Specifically, overexpression of UNC-18 inhibits exocytosis via its interactions with syntaxin22. Conversely, point mutations in UNC-18 or syntaxin that destabilize binding result in increased release of neurotransmitter22–24. This role for UNC-18 in exocytosis is not addressed by this study, but rather we confine our analysis to the facilitatory function of UNC-18 in exocytosis.

We tested models for UNC-18 function, and here we report that although mutations in unc-18 result in severe defects in neurotransmission (i) UNC-18 is not required for the stabilization or trafficking of syntaxin (ii) UNC-18 is not absolutely required for fusion (iii) UNC-18 is not required to open syntaxin to facilitate priming and (iv) UNC-18 facilitates the docking of synaptic vesicles to the plasma membrane. We conclude that UNC-18 promotes, either directly or indirectly, the docking of synaptic vesicles to the plasma membrane.

RESULTS

UNC-18 is not required for neuronal development or maintenance

The unc-18 locus was identified in screens for uncoordinated animals in the nematode C. elegans7. unc-18 mutants are severely paralyzed. This paralysis could be caused by defects in the development of the nervous system. In fact, Muncl8-1 knockout mice show widespread neurodegeneration after development10. To test whether the uncoordinated phenotype of unc-18 mutants is caused by defects in the development or maintenance of the nervous system, we characterized the architecture of the motor neurons and the neuromuscular junctions in adult unc-18(e234), unc-18(e81) and unc-18(md299) null mutant animals. unc-18(e234) and unc-18(e81) alleles are nonsense mutations25, and unc-18(md299) is a multigenic deletion that removes the promoter and open reading frame of the unc-18 gene (data not shown).

To assess neuronal outgrowth and maintenance, we analyzed the structure of the GABA nervous system using a green fluorescent protein (GFP) tag. The GABA nervous system in unc-18(e234), unc-18(e81) and unc-18(md299) adult animals was indistinguishable from that of the wild type (Fig. 1a). Axons extended along both the ventral and dorsal nerve cords and were stably maintained in adults.

Figure 1.

Figure 1

Open in a new tab

Normal morphology of the GABA nervous system in unc-18 null mutants. (a) GABA motor neuron anatomy is normal in unc-18 null mutants. GABA motor neurons were visualized using a GFP construct (oxls12; ref. 50) in wild-type, unc-18(e234), unc-18(e81) and unc-18(md299) adult worms. In all images, dorsal is up, anterior is right, and the scale bar is 100 µm. (b) The density and distribution of GABA synaptic varicosities is normal in unc-18 mutants. Clusters of synaptic vesicles were visualized using a construct (nls52) in which the vesicle protein synaptobrevin is fused to GFP and is expressed specifically in the GABA neurons26. All images are of the ventral nerve cord near the VD6 cell body; scale bar is 10 µm. (c) The distribution of postsynaptic GABA receptor clusters is normal in unc-18 mutants. GABA receptor clusters in the muscles were visualized using an integrated array (oxls22) expressing GFP-tagged GABA receptors in the muscle27. All pictures are of the dorsal nerve cord near the posterior reflex of the gonad in adult worms; the scale bar is 10 µm. (d) Both GABA and ACh receptors are functional in unc-18 mutant animals. GABA or ACh (10−4 M) was pressure-ejected for 100 ms (arrows) onto body wall muscles of dissected wild-type and unc-18 adults. The mean induced currents (± s.e.m.) elicited by either GABA or ACh were indistinguishable in wild-type and unc-18 mutant worms. GABA currents are inward in our solutions at the holding potential of −60 mV.

Similarly, presynaptic nerve terminals develop and are maintained in the absence of UNC-18 (Fig. 1b). Briefly, synaptic vesicles were visualized by the expression of GFP-tagged synaptobrevin in GABA motor neurons26. The distribution of synapses as visualized by GFP-labeled clusters of synaptic vesicles in unc-18 mutant animals was indistinguishable from that of the wild type (mean distance between synaptic vesicle clusters along the ventral nerve cord: wild type, 2.82 ± 0.20 µm, n = 4 animals; unc-18(e234), 2.92 ± 0.14 µm, n = 5; unc-18(e81), 2.77 ± 0.15 µm, n = 8; unc-18(md299), 3.09 ± 0.23 µm, n = 3).

Furthermore, postsynaptic receptor function was normal in the absence of UNC-18. The distribution of GFP-tagged GABA receptors27 in unc-18 mutants was indistinguishable from wild type (Fig. 1c). Exogenous application of either GABA or ACh elicited robust currents from the muscles of unc-18 null mutants, suggesting that the neurotransmitter receptors are functional in the absence of UNC-18 (Fig. 1d; GABA: wild type, 2403 ± 333 pA, n = 5; unc-18(e234), 2317 ± 402 pA, n = 5; unc-18(e81), 2181 ± 185 pA, n = 5; unc-18(md299), 2620 ± 217 pA, n = 3; P > 0.5; ACh: wild type, 1761 ± 190 pA, n = 8; unc-18(e234), 1410 + 220 pA, n = 6; unc-18(e81), 1327 ± 167 pA, n = 6; unc-18(md299), 1385 ± 250 pA, n = 4; P > 0.1). Therefore, the primary defect associated with the loss of UNC-18 function is not due to a defect in neuronal outgrowth, synapse development or maintenance of the nervous system. These data suggest that UNC-18 is required for synaptic function.

UNC-18 is not essential to synapse function

To directly test whether UNC-18 is required for synapse function, we quantified whole-cell voltage-clamped currents from C. elegans neuromuscular junctions28 (Fig. 2a). Stimulation of the ventral nerve cord of a wild-type animal resulted in robust postsynaptic currents (2,042 ± 82 pA, n = 32). However, in the unc-18 null mutants, evoked responses were reduced to approximately 20% of the wild-type responses (unc-18(e234), 491 ± 107 pA, n = 12; unc-18(e81), 307 ± 50 pA, n = 16; unc-18(md299), 485 ± 55 pA, n = 28; P < 0.0001). These data suggest that UNC-18 promotes the release of neurotransmitter from presynaptic nerve terminals.

Figure 2.

Figure 2

Open in a new tab

Neurotransmitter release is markedly reduced in unc-18 mutant animals. (a) Evoked responses are reduced in unc-18 mutants. Average evoked amplitude recorded in 5 mM Ca2+ plotted as mean ±s.e.m. (b) Endogenous release rates were reduced in unc-18 mutants. However, synaptic vesicle fusion still occurred in the absence of UNC-18. Miniature postsynaptic currents were measured in 5 mM Ca2+, and the frequency of synaptic vesicle fusions are plotted as mean ±s.e.m. Throughout this study, miniature postsynaptic currents represent release of both ACh- and GABA-containing vesicles from excitatory and inhibitory motor neurons. (c) The calcium-dependence of release was not altered in unc-18 mutants. The average evoked release in unc-18(md299) mutants was reduced compared to the wild type (WT) at all calcium concentrations tested, but when normalized to the maximum current, the calcium-dependence of evoked release did not differ between unc-18(md299) and the wild type. (d) Spontaneous vesicle fusion events were also reduced in unc-18 mutants. Miniature postsynaptic currents were measured in 0 mM Ca2+, and the frequency of synaptic vesicle fusions is plotted as mean ±s.e.m. (e) The size of the readily releasable pool was reduced in unc-18 mutants as assayed by application of hyperosmotic saline. The total number of miniature postsynaptic currents were measured during a 2-s application of hyperosmotic saline in 5 mM Ca2+, and the total quanta released are plotted as mean ± s.e.m.

The evoked currents in the unc-18 mutants were quite small; the decreased response could have been caused by defects in the number of synaptic vesicles released or a decrease in the quantal size. Individual synaptic vesicle fusion events can be observed in C. elegans by recording endogenous miniature postsynaptic currents that are caused by the release of neurotransmitter from one or a few vesicles. Endogenous release events were frequent in wild-type animals (48.3 ± 3.9 fusions/s, n = 46; Fig. 2b). In unc-18 mutants, the frequency of vesicle fusion was decreased to about 14% of the wild-type frequency (e234, 5.5 ± 0.9 fusions/s, n = 32; e81, 6.3 ± 0.8 fusions/s, n = 38; P < 0.0001), but even in the complete absence of the unc-18 locus, single fusion events still occurred (md299, 8.5 ± 0.8 fusions/s, n = 28, P < 0.0001). Moreover, quantal currents in unc-18(e234) (32.3 ± 1.9 pA, n = 30), in unc-18(e81) (37.6 ± 2.7 pA, n = 30) and in unc-18(md299) (40.5 ± 2.6 pA, n = 40) were not smaller but were in fact slightly larger than the wild-type quantal currents (26.6 ± 1.0 pA, n = 34). These results suggest that UNC-18 is required for normal levels of secretion but is not absolutely required for synaptic vesicle fusion.

The readily releasable pool is reduced in unc-18 mutants

The decrease in release observed in unc-18 mutants could be due to a defect in calcium sensing, in the number of vesicles primed for release, or in the probability of release of a primed vesicle. To determine whether calcium sensing was defective, we measured evoked responses over a range of external calcium concentrations. The amplitude of evoked release in unc-18(md299) was roughly 16% compared to that of wild type at each calcium concentration (n > 3; Fig. 2c, left). However, normalized responses were identical in the wild type and in the deletion allele unc-18(md299) (Fig. 2c, right). Thus, the calcium-dependence of release does not differ between unc-18(md299) and the wild type (calculated Hill coefficients were 2.8 forwildtype and2.9 for unc-18(md299)).

A comparative measure of vesicle priming can be determined by measuring the frequency of calcium-independent spontaneous vesicle fusions. In the wild type in 0-mM external calcium, vesicle fusion events occurred at a frequency of 6.9 ±1.9 fusions per second (n = 8; Fig. 2d). In the unc-18 mutants, fusion event frequency in 0-mM calcium was 11% that of the wild type (e234, 0.66 ± 0.2 fusions/s, n = 4; e81, 0.81 ± 0.1 fusions/s, n = 3; md299, 0.89 ± 0.2 fusions/s, n = 4; P <0.01).

A decrease in vesicle fusion events could be caused by a decrease in the probability of fusion of a primed vesicle or by a decrease in the number of vesicles primed for release. The number of primed vesicles can be determined by application of hypertonic solution to the neuromuscular junction during recording. Hypertonic solution causes vesicles close to the plasma membrane to spontaneously fuse29. In the wild type, an average of 122.7 ± 13.1 (n = 10) vesicles were released from the plasma membrane during a 2-s application of hypertonic solution in the presence of 5mm calcium (Fig. 2e). In unc-18 mutants, however, the number of quanta released by hypertonic solution was 33% of that in the wild type (unc-18(e234), 23.3 ± 6.3 quanta, n = 13; unc-18(e81), 57.5 ± 8.1, n = 14; unc-18(md299), 40.3 ± 5.1, n = 6; P < 0.0004). To confirm that hypertonic release is not calcium-dependent, we assayed release in the absence of external calcium and observed that release in unc-18(md299) was 20% that of the wild type (Supplementary Fig. 1). Since the pool of vesicles released by hypertonic saline is thought to represent the number of primed vesicles30, these data suggest that there is a reduction in the number of primed vesicles in unc-18 mutants.

UNC-18 is not required for syntaxin trafficking

Synaptic vesicle priming requires the SNARE protein syntaxin31. Therefore, a defect in synaptic transmission in unc-18 mutants could be due to a requirement for UNC-18 to traffic syntaxin to the synapse or to stabilize syntaxin levels. This model predicts that syntaxin should be mislocalized or absent in an unc-18 mutant animal.

To test these predictions, we compared the localization of syntaxin in wild-type and unc-18 mutant animals. In situ immunohistochemi-cal staining showed that syntaxin is present throughout neuronal processes of wild-type animals, and that syntaxin distribution in unc-18 mutant animals is indistinguishable from that in wild type (Fig. 3a). Moreover, immunogold labeling of syntaxin on ultra-thin sections showed that syntaxin is associated with axonal plasma membranes in both wild-type and unc-18 mutant unc-18(e234) (Supplementary Fig. 2). Therefore, UNC-18 is not required for trafficking of syntaxin to the plasma membrane in neurons.

Figure 3.

Figure 3

Open in a new tab

Syntaxin is localized but unstable in unc-18 mutant animals. (a) Syntaxin localization was unaffected in unc-18 mutant animals. Syntaxin was visualized in wild-type, unc-18(e234), unc-18(e81) and unc-18(md299) animals using an anti-syntaxin antibody48. Syntaxin localization in unc-18 mutant animals was indistinguishable from the wild type. Syntaxin was localized throughout the nervous system, including in the ventral nerve cord (VNC) and commissural axons (arrows). (b) Syntaxin levels were reduced in unc-18 mutant animals. Equivalent concentrations of whole-worm protein extracts from wild type, unc-18(e234), unc-18(e81), unc-18(md299), the syntaxin splicing mutant unc-64(mdl30), the synaptobrevin hypomorph snb-l(md247), SNAP-25 null mutant ric-4(ox45), unc-18(md299) syx(+)—a strain overexpressing syntaxin from the integrated array oxls33 in an unc-18 syntaxin double null mutant—and syntaxin heterozygotes unc-64(js1 15)/+were analyzed by immunoblotting with anti-UNC-18, anti-syntaxin, anti-synaptobrevin and anti-SNAP-25 antibodies. Syntaxin levels were normalized to the wild type based on the ratio of syntaxin to synaptobrevin in each genotype, except for snb-1(md247), where syntaxin levels were normalized based on the ratio of syntaxin to SNAP-25.

To determine whether syntaxin levels are reduced in the absence of UNC-18, we quantified syntaxin levels in wild-type and unc-18 animals. Quantitative western analysis demonstrated that syntaxin levels are reduced roughly by half in unc-18 mutants compared to the wild type (Fig. 3b). Similarly, the density of anti-syntaxin gold beads was reduced in unc-18 mutants compared to the wild type (Supplementary Fig. 2). Note that mutations in other synaptic components, such as synaptobrevin and SNAP-25 also led to a 50% reduction in syntaxin levels. These data raise the possibility that the Unc-18 phenotype could be caused by a reduction in syntaxin levels.

If the Unc-18 phenotype is caused by a 50% reduction in syntaxin, then (i) animals expressing half as much syntaxin should phe-nocopy unc-18 mutants, and (ii) overexpres-sion of syntaxin should rescue the Unc-18 phenotype. Animals heterozygous for the C. elegans syntaxin null mutation unc-64(js115) express reduced levels of syntaxin (Fig. 3b). However, these animals do not phe-nocopy unc-18 mutants. Specifically, animals heterozygous for the syntaxin allele unc-64(jsll5) do not show an uncoordinated phenotype. Moreover, syntaxin heterozygotes had normal levels of evoked release (wild-type, 2,042 ± 82 pA, n = 32; unc-64(js115)/+, 2,030 ± 334 pA, n = 3; P > 0.95) and endogenous activity (wild-type, 48.3 ± 3.9 fusions/s, n = 46; unc-64(js115)/+, 40.1 ± 11.1 fusions/s, n = 3; P > 0.6), whereas unc-18 mutants showed severe defects in exocytosis (Fig. 4a,b). Furthermore, overexpression of wild-type syntaxin did not rescue unc-18 physiological defects. Specifically, overexpression of wild-type syntaxin (Fig. 3b, unc-18(md299) syx(+)) from a transgene32 failed to rescue either evoked release (unc-18(md299), 485 ± 55 pA, n = 28; unc-18(md299) syntaxin(+), 550.3 ± 76 pA, n = 10; P < 0.0001) or endogenous activity (unc-18(md299), 8.58 ± 0.81 fusions/s, n = 28; unc-18(md299) syntaxin(+), 11.7 ± 2.3 fusions/s, n = 10; P < 0.0001) to wild-type levels (Fig. 4a,b, WT versus unc-18 syx(+)). Thus, the 50% reduction in syntaxin levels in unc-18 mutants cannot be responsible for the observed decrease in the number of primed synaptic vesicles.

Figure 4.

Figure 4

Open in a new tab

Overexpression of either wild-type or open syntaxin does not bypass the requirement for UNC-18 in synaptic transmission. (a) Evoked responses were not restored to unc-18 mutants overexpressing wild-type syntaxin or open syntaxin. Average evoked amplitude recorded in 5 mM Ca2+ plotted as mean ± s.e.m. (b) Endogenous activity was not restored to unc-18 mutants overexpressing wild-type or open syntaxin. Miniature postsynaptic currents were recorded in 5 mM Ca2+, and the frequency of vesicle fusions plotted as the mean ± s.e.m. Syntaxin is encoded by the unc-64 gene. In all syntaxin overexpressing strains, the endogenous locus is unc-64(js115), the syntaxin null allele. ‘syntaxin(+)’ and ‘syx(+)’ signify the integrated array overexpressing wild-type syntaxin (oxls33) and ‘syntaxin(open)’ and ‘syx(open)’ signify the integrated array overexpressing the open form of syntaxin (oxls34).

UNC-18 is not required to open syntaxin

Formation of the SNARE complex, and thus priming, requires syntaxin to adopt an open configuration to expose its SNARE binding domain19. UNC-18 binding to syntaxin may be required for the conversion of syntaxin from the closed state to the open state19, which then promotes the formation of the SNARE complex. This model predicts that expression of a constitutively open form of syntaxin should bypass the requirement of UNC-18 for neurotransmitter release.

To test this prediction, we engineered a form of syntaxin that is constitutively in the open state. Two residues in syntaxin (L165,E166) have been identified that, when mutated to alanines, render syntaxin constitutively open and unable to bind UNC-18 in pulldown assays19. When the corresponding mutations (L166A,E167A) are engineered in C. elegans syntaxin, the mutant protein also no longer binds UNC-18 in pulldown experiments32. Moreover, the mutant protein is able to function in place of wild-type syntaxin in vivo32. Specifically, syntaxin-null animals, expressing the open form of syntaxin, release neurotransmitter from nerve terminals at wild-type levels32. Neurotransmitter release via open syntaxin requires the normal presynaptic machinery, such as other SNARE proteins and calcium, but open syntaxin bypasses the requirement of proteins that function in vesicle priming, such as UNC-13 and Rim32,33.

To test whether the open form of syntaxin could bypass the requirement for UNC-18, we expressed the open form of syntaxin in unc-18(md299) mutants. Overexpression of open syntaxin (3.0-fold) failed to rescue the severe paralysis associated with the unc-18 null mutant. Consistent with the locomotion phenotype, neurotransmitter release was not restored to unc-18 null animals expressing open syntaxin (Fig. 4a,b). Specifically, expression of open syntaxin in unc-18 null mutants failed to rescue either evoked release (unc-18(md299), 485 ± 55 pA, n = 28; unc-18(md299) syntaxin (open), 333 ± 105 pA, n = 7; P < 0.0001) or endogenous activity (unc-18(md299), 8.58 ± 1.81 fusions/s, n = 28; unc-18(md299) syntaxin (open), 7.0 ± 1.5 fusions/s, n = 6; P < 0.0001) to wild-type levels. In fact, open syntaxin slightly exacerbated the phenotype of unc-18. Similar results were obtained using other unc-18 mutations (e234 and e81) and using another array expressing open syntaxin (oxEx314) (data not shown). These data suggest that the main function of UNC-18 is not to open syntaxin during synaptic vesicle priming.

UNC-18 facilitates synaptic vesicle docking

The physiological data indicate that there is a decrease in the size of the readily releasable pool of synaptic vesicles in unc-18 mutants. Docking of synaptic vesicles with the plasma membrane is a prerequisite for vesicle priming, and thus contributes to the size of the readily releasable pool. To determine whether the distribution of synaptic vesicles is normal in unc-18 mutants, we characterized the ultrastructure of neuromuscular junctions in unc-18 mutants. Synaptic vesicles were abundant at presynaptic nerve terminals in unc-18(e234), unc-18(e81) and unc-18(md299) synapses (Fig. 5a), indicating that there is no defect in the generation of vesicles. Moreover, synaptic vesicles accumulated at abnormally high levels at the synapses of unc-18 mutants (wild-type, 394 ± 65, n = 14; unc-18(e234), 606 ± 64, n = 14; unc-18(e81), 500 ± 57, n = 18; wild-type, 263 ± 37, n = 24; unc-18(md299), 434 ± 41 n = 23) (Fig. 5b), consistent with a defect in exocytosis.

Figure 5.

Figure 5

Open in a new tab

Fewer synaptic vesicles associate with the plasma membrane in the absence of UNC-18. (a) Electron micrographs of neuromuscular junctions from wild-type, unc-18(e234), unc-18(e81), unc-18(md299), unc-13(s69) and unc-13 animals. Values for the wild type vary because wild-type control animals were fixed in parallel for each mutant. Arrows indicate presynaptic specializations, and triangles indicate docked synaptic vesicles. Scale bar represents 0.5 µm. (b) The average number of synaptic vesicles per neuromuscular junction plotted as the mean ± s.e.m. (c) The proportion of docked synaptic vesicles plotted as mean ± s.e.m.

To determine whether UNC-18 is involved in docking, we quantified the number of docked vesicles at unc-18 synapses. The number of vesicles docked at synapses in most organisms investigated seems to be a fixed fraction of the reserve pool. Thus, as the number of vesicles in the reserve pool increases, the number of docked vesicles also increases. This increase in the number of docked vesicles can be observed in synapses of larger size34,35 or in mutants that change the size of the reserve pool36–40. For this reason, the size of the docked pool is calculated as the fraction of vesicles at a synapse that are associated with the membrane. For this study, docked vesicles were defined as those in contact with the plasma membrane in sections containing a presynaptic specialization (Fig. 5a, triangles). For the deletion allele md299, wild-type and mutant sections were shuffled and scored blind. In the wild type, the proportion of vesicles in contact with the plasma membrane at a synapse is around 5–6% (Fig. 5c). In other mutants that accumulate synaptic vesicles to the same degree as unc-18, the proportion of docked vesicles remains fixed. For example, at unc-13 mutant synapses, synaptic vesicles accumulate (wild-type, 297 ± 30, n = 19; unc-13(s69), 741 ± 131, n = 13; Fig. 5a,b); however, the proportion of those vesicles contacting the plasma membrane was 6.8 ± 0.6% (n = 69 synaptic profiles)— approximately the same as the proportion in the wild type in matched studies (5.6 ± 0.5%, n = 46 synaptic profiles; Fig. 5c)28,40. In the absence of UNC-18, the number of synaptic vesicles associated with the plasma membrane was only 36% of the wild type (wild type, 5.8 ± 0.5%, n = 59; unc-18(e234), 1.5 ± 0.2%, n = 65; unc-18(e81), 2.1 ± 0.2%, n = 75; wild-type, 4.9 + 0.6%, n = 24; unc-18(md299), 2.4 ± 0.2%, n = 24; P< 0.0001). The reduction in the number of docked vesicles observed in unc-18 mutants is not as severe as the physiological defect; clearly, not all docked vesicles are in the readily releasable pool. These data suggest that docking and priming proceed through multiple steps, one of which is affected by UNC-18.

DISCUSSION

UNC-18 is the Sec1-related protein required for synaptic vesicle exocytosis. In this study, we have explored defects in unc-18 mutants in the nematode C. elegans. These studies are possible in adults because UNC-18 is not required for nervous system development in C. elegans. In mice, loss of UNC-18 does not cause defects in neurogenesis or in synaptic development, but the protein is required to prevent neuronal apoptosis10. This effect is likely to be indirect, such that loss of neurosecretion leads to programmed cell death of neurons. By contrast, in C. elegans unc-18 mutants, the nervous system develops normally and is intact even in adult animals. Receptor clustering and function in the postsynaptic muscles also appears normal. Thus, studies of exocytosis in unc-18 adult neuromuscular junctions have allowed us to test various models that have been proposed for UNC-18 function. Our data refute several of these models, and partially conflict with observations in other organisms.

In the first model, UNC-18 is proposed to be required for trafficking of syntaxin. In yeast, the UNC-18 homolog Vps45p is required to prevent the nearly complete degradation of the cognate t-SNARE Tlg2p17. In unc-18 mutants, syntaxin levels are reduced by 50%, but this reduction cannot account for the Unc-18 phenotype. Animals heterozygous for the syntaxin null allele unc-64(js115) express half as much syntaxin, but they do not show defects in vesicle exocytosis. In addition, overexpression of syntaxin in an unc-18 mutant animal does not ameliorate the UNC-18 electrophysiological phenotype. In tissue culture cells, UNC-18 is required for the trafficking of syntaxin from the Golgi apparatus to the plasma membrane13. However, in unc-18 mutants, syntaxin is distributed normally along axons and at presynaptic sites. Thus, it seems unlikely that the severe defects observed in C. elegans unc-18 mutants are due to alterations in syntaxin trafficking.

Second, some experiments indicate that UNC-18 might function at the fusion step of exocytosis. For example, no synaptic vesicle fusions were observed in CA1 hippocampal neurons from Muncl8-1 mutants10. Moreover, overexpression of a mutant form of Muncl8-1 in adrenal chromaffin cells altered the dynamics of the fusion pore in granule exocytosis14. In contrast to Muncl8-1 mutant mice, our data show that synaptic vesicle fusion still occurs in the absence of UNC-18. Furthermore, the observed reduction in vesicle exocytosis in unc-18 mutants can be attributed to a decreased pool of primed vesicles. The persistence of fusion events in C. elegans unc-18 mutants parallels the observations from chromaffin cells of Muncl8-1 null mice in which dense-core granule fusion events still occur16.

A third model proposes that UNC-18 facilitates the priming step of synaptic vesicle exocytosis by promoting the open configuration of syntaxin and thus allowing the formation of the SNARE complex15,19. We tested this model by determining whether a constitutively open form of syntaxin could bypass the requirement for UNC-18. However, open syntaxin did not suppress the unc-18 mutant phenotype, indicating that UNC-18 is not required to stabilize the open configuration of syntaxin during priming.

A fourth model proposes that UNC-18 functions before vesicle priming and fusion to promote synaptic vesicle docking. Dense-core vesicles in Muncl8-1 knockout mice fail to associate with the plasma membrane in chromaffin cells16. Given that dense-core vesicle exocytosis uses many of the same proteins as synaptic vesicle exocytosis20, UNC-18 may function to promote synaptic vesicle docking as well. However, no docking defect was observed for synaptic vesicles at synapses in the hippocampus of Muncl8-1 mutant mice10, suggesting that UNC-18 function may be different for synaptic vesicle exocytosis and dense-core vesicle exocytosis. Our data conflict with this observation: the fraction of synaptic vesicles docked at the plasma membrane was reduced in unc-18 mutants. To resolve this discrepancy between the mouse and C. elegans synaptic vesicle docking data, we will require a better understanding of the mechanisms of docking in these two organisms. We conclude that UNC-18 promotes, either directly or indirectly, the docking of synaptic vesicles to the plasma membrane.

Models for UNC-18 function

Docking has been largely defined by morphological criteria; that is, docked vesicles are identified as those that are adjacent to the plasma membrane at the active zone in electron micrographs. There is strong physiological evidence for the priming and fusion steps in exocytosis, but the docking step has depended on this relatively weak ultrastructural definition. In concrete terms, it has not been clear whether docked vesicles in electron micrographs are a pool of vesicles engaged in distinct molecular interactions or simply vesicles forced against the membrane by the high density of vesicles in the reserve pool. Reduced docking in unc-18 mutants indicates that docked vesicles represent a pool of vesicles that can be influenced by loss of particular proteins, specifically UNC-18. However, our data do not address whether the role of UNC-18 in docking is direct or indirect.

In the simplest model, UNC-18 is directly involved in docking, by interaction with other tethering proteins. Since UNC-18 is a cytosolic protein, these tethering proteins must be localized on both the vesicular and plasma membranes. An obvious interacting protein on the plasma membrane is syntaxin, and data from yeast indicate that UNC-18 could act in SNARE preassembly41. However, data from Drosophila melanogaster and squid indicate that syntaxin is not required for docking31,42,43. Furthermore, additional factors at the plasma membrane would be required, since UNC-18 and syntaxin are not restricted to active zones8,44,45. On the synaptic vesicle, potential targets include the Rab proteins, which are implicated in vesicle docking and exhibit genetic interactions with the Sec1-related proteins46. In C. elegans, however rab-3 mutants do not phenocopy unc-18 mutants47, suggesting either that Rab proteins are redundant or that UNC-18 is required for Rab3-independent processes at the synapse. Although synaptotagmin has not been shown to physically interact with UNC-18, it has been implicated in docking39 and may eventually be shown to act in concert with UNC-18. Finally, UNC-18 physically interacts with a number of other proteins including Mint, Doc2 and granuphilin6.

The data presented here could also support an alternative model in which UNC-18 has an indirect role in docking. One can imagine many scenarios; for example, UNC-18 could be involved at a post-fusion stage by aiding in the disassembly of SNARE complexes. Specifically, UNC-18 could bind and protect syntaxin after N-ethyl-maleimide–sensitive factor (NSF)-mediated disassembly of the cis-SNARE complex. This binding could prevent the reformation of the cis-SNARE complex once these proteins are separated but while they are still in the plasma membrane. In unc-18 mutants, functional syntaxin would be depleted because it is tied up in cis-SNARE complexes. This model is intellectually appealing because it takes into account the tight physical interaction between UNC-18 and syntaxin. In this model, the lack of free SNARE proteins either on the vesicle or on the plasma membrane would interfere with the ability of vesicles to dock with the plasma membrane.

METHODS

Genetics

Strains were maintained using standard methods as described previously7. The Bristol N2 strain was used as the wild type. The unc-18 alleles e234 and e81 contain point mutations that result in premature stop codons at amino acid positions 141 and 528, respectively25. The viable unc-18 deficiency allele md299 contains a large (>6 kb) deletion. This deletion does not remove the 3’ end of the gene, but does delete the unc-18 open reading frame and promoter. In addition, it removes the promoter of the syntrophin gene, which lies just upstream of unc-18 (data not shown). The strains NM464 (genotype unc-64(md130)111), NM204 (genotype snt-1(md290)11), NM467 (genotype snb-1(md247)V) and NM979 (genotype unc-64(js115)/bli-5(e518)111) have been previously described (for details, see Supplementary Methods online). The ric-4(ox45) strain is EG45, and the ox45 mutation disrupts the gene encoding the C. elegans SNAP-25 homolog (I. Lee, Y. Lee, I. Rand, M.L. Nonet & B.J. Myer, unpublished data). The syntaxin(+) strain is EG1986 oxh33[unc-64(+); Punc-122:GFP] I; unc-64(js115) III and the syntaxin(open) strain is EG1985 oxIs34 [unc-64(L166A,E167A); Pmyo-2:GFP] unc-64(js115) III described previously32. The unc-18(md299) syntaxin(+) strain is EG2768 oxIs33 I; unc-64(js115) III; unc-18(md299) X. The unc-18(md299) syntaxin(open) strain is EG2767 oxIs34 unc-64(js115) III; unc-18(md299) X.

Electrophysiology and worm dissection

Electrophysiological methods were performed as previously described28,40 (for details, see Supplementary Methods online). The hyperosmotic saline, provided by addition of sucrose to a final osmolarity of 800 mosm, was applied by pressure-ejection. In this assay, individual quanta can be distinguished; thus, the size of the readily releasable pool was calculated as the total number of discrete miniature postsynaptic currents measured during a 2-s application of hyperosmotic saline in 5 mM Ca2+.

Syntaxin in situ immunohistochemistry

Immunohistochemistry was performed on dissected wild-type and unc-18 mutant worms fixed in 4% paraformaldehyde made in phosphate buffered saline (PBS). Fixed animals were stained with a syntaxin antibody48 diluted 1:8,000 followed by a goat-anti rabbit Alexa Fluor 488 secondary antibody. The preparations were imaged on a Biorad confocal microscope.

Electron microscopy

Adult nematodes were prepared for transmission electron microscopy as described previously40. Ribbons of ultrathin sections (35 nm) were imaged using a Hitachi H-7100 electron microscope. Images were quantified using NIH Image software. A synapse was defined as a set of serial sections containing a presynaptic specialization and each flanking section containing more than one synaptic vesicle. Docked synaptic vesicles were defined as vesicles contacting the plasma membrane on the side of the axon with a presynaptic specialization. In all cases mutants and wild-type controls were fixed in parallel. For unc-18(md299), micrographs were shuffled with micrographs from the wild-type control and scored blind.

Western blots

For western analysis, protein lysates were collected from mixed stage populations, separated by SDS-PAGE, transferred to nitrocellulose and probed with specific antibodies (for detailed protocols, see Supplementary Methods online). Blots were blocked for 1 h at room temperature, incubated in primary antibody overnight at 4 °C, and antibodies were detected using ECL. SNAP-25 was detected with affinity-purified rabbit 6470 antibodies (G. Hadwiger & M.L.N., unpublished data), synaptobrevin with affinity-purified rabbit 1092 antibodies49, syntaxin with affinity-purified rabbit 939 antibodies48 and UNC-18 with rabbit 601 antisera at 1:7,00045 (a kind gift of J. Duerr & J. Rand). Intensities were quantified using NIH image.

Supplementary Material

Figure S1
Figure S2
Supplementary methods

ACKNOWLEDGMENTS

We thank J. Rand for providing unc-18(md299) and antisera to UNC-18, M. Bastiani for providing the confocal microscope, the Caenorhabditis Genetics Center for providing unc-18(e234) and unc-18(e81), E. Maryon for providing the Pt09a9.10:gfp reporter construct, and J.-L. Bessereau, P. Rostaing and A. Triller for assistance with developing immuno-electron microscopy techniques. This work was supported by National Institutes of Health grants to J.E.R. and E.M J., a US Public Health Service grant to M.L.N., and an INSERM Poste Vert grant to R.MW.

Footnotes

Note: Supplementary information is available on the Nature Neuroscience website.

COMPETING INTERESTS STATEMENT

The authors declare that they have no competing financial interests.

References

  • 1.Robinson LJ, Martin TF. Docking and fusion in neurosecretion. Curr. Opin. Cell Biol. 1998;10:483–492. doi: 10.1016/s0955-0674(98)80063-x. [DOI] [PubMed] [Google Scholar]
  • 2.Rizo J, Sudhof TC. SNAREs and Muncl8 in synaptic vesicle fusion. Nat. Rev. Neurosci. 2002;3:641–653. doi: 10.1038/nrn898. [DOI] [PubMed] [Google Scholar]
  • 3.Weber T, et al. SNAREpins: minimal machinery for membrane fusion. Cell. 1998;92:759–772. doi: 10.1016/s0092-8674(00)81404-x. [DOI] [PubMed] [Google Scholar]
  • 4.Ungermann C, Sato K, Wickner W. Defining the functions of trans-SNARE pairs. Nature. 1998;396:543–548. doi: 10.1038/25069. [DOI] [PubMed] [Google Scholar]
  • 5.Peters C, et al. Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion. Nature. 2001;409:581–588. doi: 10.1038/35054500. [DOI] [PubMed] [Google Scholar]
  • 6.Toonen RF, Verhage M. Vesicle trafficking: pleasure and pain from SM genes. Trends Cell Biol. 2003;13:177–186. doi: 10.1016/s0962-8924(03)00031-x. [DOI] [PubMed] [Google Scholar]
  • 7.Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77:71–94. doi: 10.1093/genetics/77.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Gengyo-Ando K, et al. The C. elegans unc-18 gene encodes a protein expressed in motor neurons. Neuron. 1993;11:703–711. doi: 10.1016/0896-6273(93)90080-b. [DOI] [PubMed] [Google Scholar]
  • 9.Harrison SD, Broadie K, van de Goor J, Rubin GM. Mutations in the Drosophila Rop gene suggest a function in general secretion and synaptic transmission. Neuron. 1994;13:555–566. doi: 10.1016/0896-6273(94)90025-6. [DOI] [PubMed] [Google Scholar]
  • 10.Verhage M, et al. Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science. 2000;287:864–869. doi: 10.1126/science.287.5454.864. [DOI] [PubMed] [Google Scholar]
  • 11.Miller KG, et al. A genetic selection for Caenorhabditis elegans synaptic transmission mutants. Proc. Natl. Acad. Sci. USA. 1996;93:12593–12598. doi: 10.1073/pnas.93.22.12593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Weimer RM, Jorgensen EM. Controversies in synaptic vesicle exocytosis. J. Cell Sci. 2003;116:3661–3666. doi: 10.1242/jcs.00687. [DOI] [PubMed] [Google Scholar]
  • 13.Rowe J, Calegari F, Taverna E, Longhi R, Rosa P. Syntaxin 1A is delivered to the apical and basolateral domains of epithelial cells: the role of munc-18 proteins. J. Cell Sci. 2001;114:3323–3332. doi: 10.1242/jcs.114.18.3323. [DOI] [PubMed] [Google Scholar]
  • 14.Fisher RJ, Pevsner J, Burgoyne RD. Control of fusion pore dynamics during exocytosis by Muncl8. Science. 2001;291:875–878. doi: 10.1126/science.291.5505.875. [DOI] [PubMed] [Google Scholar]
  • 15.Misura KM, Scheller RH, Weis WI. Three-dimensional structure of the neuronal-Secl-syntaxin la complex. Nature. 2000;404:355–362. doi: 10.1038/35006120. [DOI] [PubMed] [Google Scholar]
  • 16.Voets T, et al. Muncl8-1 promotes large dense-core vesicle docking. Neuron. 2001;31:581–591. doi: 10.1016/s0896-6273(01)00391-9. [DOI] [PubMed] [Google Scholar]
  • 17.Bryant NJ, James DE. Vps45p stabilizes the syntaxin homologue Tlg2p and positively regulates SNARE complex formation. EMBO J. 2001;20:3380–3388. doi: 10.1093/emboj/20.13.3380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Grote E, Carr CM, Novick PJ. Ordering the final events in yeast exocytosis. J. Cell Biol. 2000;151:439–452. doi: 10.1083/jcb.151.2.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Dulubova I, et al. A conformational switch in syntaxin during exocytosis: role of muncl8. EMBO J. 1999;18:4372–4382. doi: 10.1093/emboj/18.16.4372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Zucker RS. Exocytosis: a molecular and physiological perspective. Neuron. 1996;17:1049–1055. doi: 10.1016/s0896-6273(00)80238-x. [DOI] [PubMed] [Google Scholar]
  • 21.Schulze KL, et al. rop, a Drosophila homolog of yeast Seel and vertebrate n- Secl/Munc-18 proteins, is a negative regulator of neurotransmitter release in vivo. Neuron. 1994;13:1099–1108. doi: 10.1016/0896-6273(94)90048-5. [DOI] [PubMed] [Google Scholar]
  • 22.Wu MN, Littleton JT, Bhat MA, Prokop A, Bellen HJ. ROP, the Drosophila Seel homolog, interacts with syntaxin and regulates neurotransmitter release in a dosage-dependent manner. EMBO J. 1998;17:127–139. doi: 10.1093/emboj/17.1.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Wu MN, et al. Syntaxin 1A interacts with multiple exocytic proteins to regulate neurotransmitter release in vivo. Neuron. 1999;23:593–605. doi: 10.1016/s0896-6273(00)80811-9. [DOI] [PubMed] [Google Scholar]
  • 24.Wu MN, Schulze KL, Lloyd TE, Bellen HJ. The ROP-syntaxin interaction inhibits neurotransmitter release. Eur. J. Cell Biol. 2001;80:196–199. doi: 10.1078/0171-9335-00143. [DOI] [PubMed] [Google Scholar]
  • 25.Sassa T, et al. Regulation of the UNC-l8-Caenorhabditis elegans syntaxin complex by UNC-13. J. Neurosci. 1999;19:4772–4777. doi: 10.1523/JNEUROSCI.19-12-04772.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Jorgensen EM, et al. Defective recycling of synaptic vesicles in synaptotagmin mutants of Caenorhabditis elegans. Nature. 1995;378:196–199. doi: 10.1038/378196a0. [DOI] [PubMed] [Google Scholar]
  • 27.Bamber BA, Beg AA, Twyman RE, Jorgensen EM. The Caenorhabditis elegans unc-49 locus encodes multiple subunits of a heteromultimeric GABA receptor. J. Neurosci. 1999;19:5348–5359. doi: 10.1523/JNEUROSCI.19-13-05348.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Richmond JE, Jorgensen EM. One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction. Nat. Neurosci. 1999;2:791–797. doi: 10.1038/12160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Bekkers JM, Stevens CF. Osmotic stimulation of presynaptic terminals. In: Kettenmann H, Grantyn R, editors. Practical Electrophysiological Methods. John Wiley & Sons; 1992. pp. 150–154. [Google Scholar]
  • 30.Lonart G, Sudhof TC. Assembly of SNARE core complexes prior to neurotransmitter release sets the readily releasable pool of synaptic vesicles. J. Biol. Chem. 2000;275:27703–27707. [PubMed] [Google Scholar]
  • 31.Broadie K, et al. Syntaxin and synaptobrevin function downstream of vesicle docking in Drosophila. Neuron. 1995;15:663–673. doi: 10.1016/0896-6273(95)90154-x. [DOI] [PubMed] [Google Scholar]
  • 32.Richmond JE, Weimer RM, Jorgensen EM. An open form of syntaxin bypasses the requirement for UNC-13 in vesicle priming. Nature. 2001;412:338–341. doi: 10.1038/35085583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Koushika SP, et al. A post-docking role for active zone protein Rim. Nat. Neurosci. 2001;4:997–1005. doi: 10.1038/nn732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Harris KM, Sultan P. Variation in the number, location and size of synaptic vesicles provides an anatomical basis for the nonuniform probability of release at hip-pocampal CA1 synapses. Neuropharmacology. 1995;34:1387–1395. doi: 10.1016/0028-3908(95)00142-s. [DOI] [PubMed] [Google Scholar]
  • 35.Murthy VN, Schikorski T, Stevens CF, Zhu Y. Inactivity produces increases in neurotransmitter release and synapse size. Neuron. 2001;32:673–682. doi: 10.1016/s0896-6273(01)00500-1. [DOI] [PubMed] [Google Scholar]
  • 36.Hunt JM, et al. A post-docking role for synaptobrevin in synaptic vesicle fusion. Neuron. 1994;12:1269–1279. doi: 10.1016/0896-6273(94)90443-x. [DOI] [PubMed] [Google Scholar]
  • 37.Koenig JH, Kosaka T, Ikeda K. The relationship between the number of synaptic vesicles and the amount of transmitter released. J. Neurosci. 1989;9:1937–1942. doi: 10.1523/JNEUROSCI.09-06-01937.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Li J, Schwarz TL. Genetic evidence for an equilibrium between docked and undocked vesicles. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1999;354:299–306. doi: 10.1098/rstb.1999.0381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Reist NE, et al. Morphologically docked synaptic vesicles are reduced in synaptotagmin mutants of Drosophila. J. Neurosci. 1998;18:7662–7673. doi: 10.1523/JNEUROSCI.18-19-07662.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Richmond JE, Davis WS, Jorgensen EM. UNC-13 is required for synaptic vesicle fusion in C. elegans. Nat. Neurosci. 1999;2:959–964. doi: 10.1038/14755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Peng R, Gallwitz D. Sly1 protein bound to Golgi syntaxin Sed5p allows assembly and contributes to specificity of SNARE fusion complexes. J. Ceil Biol. 2002;157:645–655. doi: 10.1083/jcb.200202006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Marsal J, et al. Block of transmitter release by botulinum C1 action on syntaxin at the squid giant synapse. Proc. Natl. Acad. Sci. USA. 1997;94:14871–14876. doi: 10.1073/pnas.94.26.14871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.O’Connor V, et al. Disruption of syntaxin-mediated protein interactions blocks neurotransmitter secretion. Proc. Natl. Acad. Sci. USA. 1997;94:12186–12191. doi: 10.1073/pnas.94.22.12186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Ogawa H, Harada S, Sassa T, Yamamoto H, Hosono R. Functional properties of the unc-64 gene encoding a Caenorhabditis elegans syntaxin. J. Biol. Chem. 1998;273:2192–2198. doi: 10.1074/jbc.273.4.2192. [DOI] [PubMed] [Google Scholar]
  • 45.Lickteig KM, et al. Regulation of neurotransmitter vesicles by the homeodomain protein UNC-4 and its transcriptional corepressor UNC-37/groucho in Caenorhabditis elegans cholinergic motor neurons. J. Neurosci. 2001;21:2001–2014. doi: 10.1523/JNEUROSCI.21-06-02001.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Ossig R, Dascher C, Trepte HH, Schmitt HD, Gallwitz D. The yeast SLY gene products, suppressors of defects in the essential GTP-binding Yptl protein, may act in endoplasmic reticulum-to-Golgi transport. Mol. Cell. Biol. 1991;11:2980–2993. doi: 10.1128/mcb.11.6.2980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Nonet ML, et al. Caenorhabditis elegans rab-3 mutant synapses exhibit impaired function and are partially depleted of vesicles. J. Neurosci. 1997;17:8061–8073. doi: 10.1523/JNEUROSCI.17-21-08061.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Saifee O, Wei L, Nonet ML. The Caenorhabditis elegans unc-64 locus encodes a syntaxin that interacts genetically with synaptobrevin. Mol. Biol. Cell. 1998;9:1235–1252. doi: 10.1091/mbc.9.6.1235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Nonet ML, Saifee O, Zhao H, Rand JB, Wei L. Synaptic transmission deficits in Caenorhabditis elegans synaptobrevin mutants. J. Neurosci. 1998;18:70–80. doi: 10.1523/JNEUROSCI.18-01-00070.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Mclntire SL, Reimer RJ, Schuske K, Edwards RH, Jorgensen EM. Identification and characterization of the vesicular GABA transporter. Nature. 1997;389:870–876. doi: 10.1038/39908. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1
NIHMS77227-supplement-Figure_S1.pdf (82.8KB, pdf)
Figure S2
NIHMS77227-supplement-Figure_S2.webloc (286B, webloc)
Supplementary methods
NIHMS77227-supplement-Supplementary_methods.webloc (286B, webloc)

RESOURCES