We performed a forward genetic screen for active zone defective mutants using a GFP active zone marker hpIs3 (Punc-25-SYD-2::GFP). One recovered mutant,
hp102, displays defects in SYD-2::GFP expression and localization. In addition,
hp102 animals display a loopy coiler Unc behaviour. Genetic analysis revealed that
hp102 is allelic to
unc-77(
e625) and
nca-1(
gk9) . We confirmed this by identifying molecular lesions to
nca-1 in both
hp102 and
unc-77(
e625). Also, genomic fragments containing only the
nca-1 gene fully rescue the Unc behaviour of
hp102.
nca-1(
gk9) is a deletion mutant predicted to be a null protein, as it retains only the first two pore domains of the alpha subunit. However,
nca-1(
gk9) does not display any obvious Unc behaviour as seen in
hp102 or
e625.
hp102/nDf41 worms are similar to
hp102/hp102 in both behaviour and hpIs3 phenotype, suggesting that
hp102 is a genetic null.
hp102/gk9 displays a severe Unc phenotype, suggesting that
gk9 may be hypomorphic. Transgenic animals carrying a genomic fragment encoding the first two pore domains of NCA-1 can partially rescue the behaviour of
hp102. These data suggest that NCA-1 may not be functioning as a traditional calcium channel alpha subunit. The defects seen with SYD-2::GFP correlate with defects seen with other synaptic markers such as juIs1 (Punc-25-SNB-1::GFP) and oxIs22 (UNC-49B::GFP), suggesting that
nca-1 functions to regulate synaptic differentiation. We also observe defects in hpIs1 (Punc-25-SAD-1::GFP) expression and localization. Mutations in
sad-1, which encodes a Ser/Thr kinase implicated in synapse formation and axon termination, suppress the coiler behaviour of
nca-1(
hp102). In addition,
nca-1;
sad-1 double mutants display synaptic defects seen with juIs1 and hpIs3 similar to
sad-1 mutants, suggesting that
sad-1 is epistatic to
nca-1. Furthermore,
sad-1 mutations suppress another mutant recovered from our screen,
hp121.
hp121 is an allele of
unc-7 and displays less SYD-2::GFP puncta.
nca-1;
unc-7 double mutants display enhanced behavioural, synapse and nervous system defects. These data suggest that
nca-1 and
unc-7 function in parallel pathways that converge onto the
sad-1 pathway. We are investigating the nature of NCA-1 involvement in synaptogenesis and within the SAD-1 pathway. Current progress will be presented.