Previously, syntaxin was not thought to be required for docking. By contrast, our results demonstrate that syntaxin is required for docking synaptic vesicles at the C. elegans neuromuscular junction. Vesicles are docked in two pools: the active zone pool and the perisynaptic pool. We also find that UNC-13 is required for synaptic vesicle docking. However, while both pools of docked vesicles depend absolutely on syntaxin, UNC-13 only plays a role at the active zone. Finally, the docking function of UNC-13 is completely bypassed by open syntaxin.
The observed docking defects in the syntaxin and unc-13 mutant synapses are likely to be caused by a direct role of these proteins in the docking pathway rather than by an indirect effect on neuronal health. First, syntaxin acts cell autonomously: expressing syntaxin in the acetylcholine neurons rescues docking in these cells but not in downstream neurons in the motor circuit. Second, chronic lack of syntaxin does not lead to developmental abnormalities in the cell. Synaptic vesicles and synaptic vesicle components such as synaptobrevin are transported to the synapse, vesicles are clustered, dense projections and adherens junctions appear normal at the ultrastructural level, the postsynaptic receptors cluster appropriately, and the receptors are functional: the synapses appear to be intact. Third, syntaxin appears to play a late role in docking. The syntaxin-binding protein UNC-13 is required for docking as well, and open syntaxin can rescue the docking defect in unc-13 mutants, suggesting that syntaxin acts downstream of UNC-13 during docking. These data are most consistent with a direct role for syntaxin in the docking of synaptic vesicles.
A role for syntaxin in docking conflicts with previous studies [30
]. It is unlikely that syntaxin function is not conserved among organisms; it is more likely that the conflicting results arise from the difficulties in studying docking. The different conclusions might be attributed to two causes: definitions for docking and the potential for residual syntaxin. First, different definitions for docking were used in these various studies. In the present study only vesicles contacting the membrane were considered docked (). This definition was used in studies of vertebrate synapses comparing the docked and readily releasable pools [26
]. By contrast, previous syntaxin studies, as well as our previous UNC-13 studies, defined docked vesicles as those near the plasma membrane (less than 30, 40, or 50 nm, [30
]). If we analyze our current data using the 30 nm definition, we also do not detect decreases in docking (for example, vesicles within 30 nm per profile, matched wild-type GABA 5.6 ± 0.2; syntaxin(−) GABA from EG3817 5.4 ± 0.3; p
= 0.49). It was not possible to reanalyze our previous data with our current definition of docking, because the glutaraldehyde fixation used in the previous experiments did not preserve membranes well enough to distinguish between docked and undocked vesicles. Tethering proteins span larger distances than the SNARE proteins and thus are thought to function in those vesicles that are close to but not contacting the plasma membrane [78
]. Our data thus suggest that syntaxin is not required to tether synaptic vesicles to the membrane. In contrast to synaptic vesicles, secretory vesicles require syntaxin for tethering [35
The second possible explanation for the discrepancy is that residual syntaxin could have mediated docking in previous experiments. In the studies on squid and cultured hippocampal cells, syntaxin was acutely disrupted by protease digestion; nevertheless, about 10% of synaptic vesicle fusions remained, suggesting that some syntaxin was still present [31
]. Further, syntaxin may itself be redundant, in agreement with the almost complete lack of a phenotype in syntaxin knockout mice [81
]. Studies in Drosophila
used mutation rather than protease cleavage to disrupt syntaxin. In fly syntaxin mutants, vesicle fusions were 5% the wild-type rate [30
]; much greater than the fusion rate observed in the syntaxin mosaics in C. elegans
(less than 0.2% of the wild-type rate). In Drosophila
, there is a significant maternal contribution of syntaxin [61
], and it has been suggested that syntaxin might perdure until late embryogenesis [30
]. In our own data, although syntaxin is not detectable by antibody staining, we do observe a few docked vesicles and a few spontaneous fusions ( and ). These rare events are likely due to residual syntaxin, either as a result of read-through of the stop codon in unc-64(js115)
or as a result of misexpression from our rescuing array. Thus, syntaxin is likely to be essential for all synaptic vesicle docking.
In addition to syntaxin, docking in the active zone also relies on UNC-13. The docking defect in unc-13
mutants is completely bypassed by open syntaxin but not by closed syntaxin. This observation suggests that UNC-13′s function in docking is to promote open syntaxin. However, open syntaxin does not completely restore exocytosis in unc-13
mutant animals. Specifically, in unc-13
mutants expressing open syntaxin evoked response is 38% of the wild type. Further, we find that the presence of open syntaxin only slightly improves locomotion in unc-13
mutants (unpublished data). The simplest explanation is that UNC-13 has a second function after docking to increase the probability of fusion [82
]. Alternatively, levels of open syntaxin might not be sufficient to support normal exocytosis in the absence of UNC-13. It is worth noting that this strain has changed with time; previously the strain was more active and evoked responses were more robust [77
]. By contrast, some recently derived strains have no evoked response [84
]. It is possible that expression levels have declined in these strains. We propose that only a few molecules of open syntaxin suffice for docking a vesicle, but that multiple molecules of open syntaxin are required to mediate normal exocytosis. Thus, very high expression levels of open syntaxin might be required to bypass the function of UNC-13. In a wild-type synapse, UNC-13 is specifically localized to active zones [54
], where it can locally generate the high levels of open syntaxin that are required for release.
How does open syntaxin interact with synaptic vesicles during docking? There are two regions of syntaxin that could be involved: the Habc domain and the SNARE motif. In the open state of syntaxin both of these regions are free to interact with vesicle proteins. It is possible that the Habc domain mediates docking independently of SNARE function. In this model, the other SNARE proteins would not be required for docking. In support of this idea, previous data suggest that genetic and toxin disruption of synaptobrevin and SNAP-25 does not disrupt docking [30
]. However, these studies used differing definitions of vesicle docking, perhaps obscuring specific docking defects. Further, it has been suggested that redundant SNARE proteins compensate for the loss of the synaptic SNAREs in these experiments [81
]. If the SNARE motif of syntaxin mediates docking then it is likely that the SNARE proteins synaptobrevin and SNAP-25, which interact with the SNARE motif of syntaxin, will also be required for docking. In this case, formation of the SNARE complex would mediate docking, as originally predicted in the SNARE hypothesis [32
], and the distinction between morphological docking and priming would not exist.