In order to identify genes which are required for synapse formation, we carried out a genetic screen using a synapse-specific GFP marker: Punc-25-VAMP-GFP(1). VAMP-GFP fusion protein is specifically localized to the presynaptic zones of neurons due to the association of VAMP with synaptic vesicles(2).
unc-25 promoter activates the GFP expression in a group of GABAergic neurons namely Ds. We isolated 12 new genes (named syd for synapse defective) which affect the shape and localization of the VAMP-GFP marker when mutated(3). These syd genes do not alter the axonal morphology, suggesting that they may affect synapse formation. Here we report the molecular and functional analysis of two of the genes,
syd-2 and
syd-3. In wild-type worms, the VAMP-GFP marker is expressed as uniformly- sized punctate clusters representing individual synaptic zones of D neurons. In
syd-2 null mutant the GFP clusters are diffused, suggesting that the presynaptic specializations are disrupted. We mapped
syd-2 onto the X chromosome, and showed that
syd-2 encodes a member of the LIP (LAR-interacting protein) protein family. LAR is a member of the receptor-tyrosine phosphatase (RPTPase) family, and LIP proteins were previously demonstrated to interact with the second phosphatase domain of the LAR-type RPTPase by the yeast two-hybrid system(4).
syd-2 is expressed in both neurons and muscles in C. elegans. In neurons SYD-2 is specifically localized at the presynaptic zones. Such a subcellular localization is not affected by mutations in
unc-104, a gene required for the transport of synaptic vesicles to the presynaptic zones, suggesting that SYD-2 protein is not associated with the synaptic vesicles. Both mosaic analysis and molecular rescuing experiments show that the expression of SYD-2 at the presynaptic zones is necessary and sufficient to rescue the VAMP-GFP marker expression defect in
syd-2 mutant. We are currently analyzing the ultrastructural defects of
syd-2 mutant by electron microscopy. We propose that
syd-2 is required for the establishment of presynaptic zone structure through regulating the activity and/or localization of receptor tyrosine phosphorylation signaling cascade in the nerve termini. In
syd-3 mutants the VAMP- GFP clusters are diffused and unevenly distributed along the ventral and dorsal nerve cord. We mapped
syd-3 onto chromosome V.
syd-3 encodes a protein containing a RCC1 type guanine exchange factor (GEF) domain. Similar to SYD-2, SYD-3 protein is also localized at the presynaptic zones in neurons. This suggests that
syd-3 may also play roles in establishing the presynaptic specialization. We are currently testing if the GEF domain is required for
syd-3 function and analyzing the ultrastructure defects of
syd-3 mutants. Reference: 1) Jorgensen, EM et al. Nature 378:196-199, 1995 2) Nonet, ML et al. J. of Neuroscience 18:70-80, 1998 3) Zhen, M and Jin, Y WBG14(5):40, 1997 4) Serra-Pages, C. et al., EMBO J.14(12):2827-2838, 1995