Genetic analysis has demonstrated that Syt 1 is essential for calcium-dependent synchronous release, underlying the fourth order cooperativity of synaptic vesicle fusion, but does not abolish asynchronous calcium-dependent release (Geppert et al., 1994
; Yoshihara and Littleton, 2002
; Stevens and Sullivan, 2003
). These observations are consistent with the current two calcium sensor model for synaptic transmission (Yamada and Zucker, 1992
), with Syt 1 functioning as the calcium sensor regulating the fast synchronous component of release and an unidentified calcium sensor mediating the slow asynchronous component. Other synaptotagmin isoforms are obvious candidates for the asynchronous calcium sensor. In addition, synaptotagmins have unique calcium-binding properties (Sugita et al., 2002
) and undergo heterooligomerization in vitro (Littleton et al., 1999
; Desai et al., 2000
). Several plasticity models have been proposed, suggesting differential expression of synaptotagmin isoforms on synaptic vesicles might regulate presynaptic release probability (Littleton et al., 1999
; Wang et al., 2001
) or transitions from full fusion to kiss-and-run (Wang et al., 2003
). These hypotheses require that synaptotagmins have a similar expression pattern to Syt 1 and localize presynaptically at synaptic terminals. We have addressed these hypotheses in vivo by performing an extensive expression and localization study of the entire synaptotagmin family in D. melanogaster
. Our localization data argue against the possibility that other synaptotagmin isoforms function with Syt 1 to regulate neurotransmitter release. Instead, the remaining synaptotagmin isoforms likely regulate distinct membrane trafficking steps in vivo.
Syt 4 was found in the postsynaptic compartment, suggesting it regulates a postsynaptic membrane trafficking pathway. We cannot rule out that a small fraction of Syt 4 may also be present in some presynaptic compartments, though it does not localize to Syt 1–positive synaptic vesicles. The detection of the Syt 4 protein by Western analysis and immunocytochemistry with our new antisera is abolished in syt 4
null mutants, confirming the antisera accurately reflects the subcellular localization of Syt 4. These results indicate that previous detection of Syt 4 on synaptic vesicles (Littleton et al., 1999
) reflected cross-reactivity of the old antisera with Syt 1. Given that Syt 4 does not colocalize on Syt 1–positive synaptic vesicles, the reduction of neurotransmitter release by Syt 4 up-regulation observed in Drosophila
(Littleton et al., 1999
) is unlikely to be due to heteromultimerization of the two proteins on vesicles and may instead reflect competitive binding to Syt 1 effectors or altered presynaptic calcium buffering.
In terms of Syt 4's postsynaptic localization, there is evidence in several experimental systems for a regulated form of postsynaptic vesicular trafficking (Ludwig et al., 2002
). Studies in hippocampal culture neurons indicate that long-term labeling with FM1–43 loads dendritic organelles that undergo rapid calcium-triggered exocytosis that is blocked by tetanus toxin (Maletic-Savatic and Malinow, 1998
). In addition, pharmacological blockage of postsynaptic membrane fusion reduces LTP (Lledo et al., 1998
), suggesting postsynaptic vesicle trafficking contributes to synaptic plasticity. Mammalian Syt 4 has been localized within dendrites and soma (Ibata et al., 2002
), suggesting Syt 4 and the related homologue Syt 11 may also function postsynaptically. Although the exact role for regulated postsynaptic fusion remains unclear, possibilities include the release of retrograde signals, trafficking of postsynaptic receptors, and/or trafficking of synaptic cell adhesion proteins.
The remaining synaptotagmins were not ubiquitously localized to synapses. Unlike Syt 1 or Syt 4, we could not detect Syt 7 at synapses, but found it was expressed in both neuronal and nonneuronal tissues. Mammalian Syt 7 has been found in secretory lysosomes (Martinez et al., 2000
) and in synaptic active zones where it has been postulated to function as a plasma membrane calcium sensor (Sugita et al., 2001
). Genetic studies of Syt 7 will be required to determine if it also functions at Drosophila
active zones. Peripheral Syt β staining was restricted to muscle fiber 8 synapses that are known to release the neuropeptide leukokinin (Cantera and Nassel, 1992
). In the CNS, Syt β was observed in a pair of bilateral neurons that may be the DPM neurosecretory neurons known to secrete the amnesiac neuropeptide. The only staining outside the nervous system is detected at tracheal branch points, where a group of myomodulin-releasing neurosecretory cells are located (O'Brien and Taghert, 1998
). These localization studies suggest Syt β is a candidate calcium sensor for mediating dense core vesicle fusion and release of neuropeptides. Similar to Syt β, Syt α showed specific expression in another set of putative CNS neuropeptide-releasing neurons, as well as within the mushroom bodies. In the periphery, staining was restricted to the LBD neurosecretory neuron, which is consistent with a role in neuropeptide release. In addition, the localization of Syt α in mushroom bodies and the possible localization of Syt β in DPM neurons makes these isoforms attractive candidates for potential roles in vesicular trafficking pathways contributing to neuronal plasticity. We were unable to localize the two remaining synaptotagmins, Syt 12 and Syt 14. It is likely that the proteins are below the detection level of our antisera, which is consistent with the microarray and in situ experiments, indicating that these isoforms are expressed at low levels in embryos and adults. Unlike the other synaptotagmins, these two isoforms lack most of the calcium coordination residues in C2A and C2B in both vertebrates and flies, indicating that they may function in trafficking pathways not regulated by calcium.
In summary, Drosophila synaptotagmin isoforms identify unique membrane-trafficking compartments. A summary of the expression of both the mRNA and protein for each synaptotagmin family member is shown in . Our data indicate that only the Syt 1 isoform is found on synaptic vesicles and so argue against heterooligomerization models. In addition, we find that Syt 4 and Syt 7 cannot rescue the behavioral or physiological defects in syt 1 mutants, suggesting that synaptotagmins define unique membrane trafficking pathways within neurons. It is possible synaptotagmins function in an analogous manner to control vesicle fusion, but do so in distinct compartments. Given that Syt 4 localizes to the postsynaptic compartment, our findings indicate that calcium-dependent membrane trafficking occurs on both sides of the synapse.
Figure 9. Summary of the expression pattern of the Drosophila synaptotagmin family. The results from embryonic in situ experiments are shown in the left panel, whereas the two right panels highlight protein expression in the third instar larval CNS and periphery. (more ...)