Synaptic connections rely upon specialized machinery to ensure proper morphology and neuronal transmission. We describe the identification of a putative calcium signaling cascade and its role in regulating synaptic morphology. This pathway is comprised of the voltage gated calcium channel (VGCC) subunits
unc-2 and
unc-36 along with a previously uncharacterized gene, F30A10.1, predicted to encode an EF-hand containing protein that we are naming the homolog of calmyrin-1 (
calm-1).
unc-2,
unc-36 and
calm-1 were discovered to regulate synapse morphology by suppressing the defects observed in nidogen mutants. Nidogen is a conserved ECM molecule, and when absent results in irregularly shaped, elongated synaptic puncta. Removing
unc-2,
unc-36 or
calm-1 in a
nid-1 background returns synapses to wild type size. These results indicate the abnormal synaptic areas of
nid-1 mutants require functional calcium signaling.
unc-2,
unc-36 and
calm-1 single mutants display enlarged, regularly shaped synaptic areas and an overall reduction in synaptic puncta. Double mutant analysis between VGCC;
calm-1 reveals these genes work in a linear pathway. Synaptic areas in a gain of function allele of
unc-2 resemble the diffuse, elongated nidogen synapses, and
calm-1, through epistatic analysis, was found to operate downstream of the VGCCs. This suggests CALM-1 is responsive to calcium influx from activated calcium channels and is subsequently activated to regulate synaptic morphology.If this is true, CALM-1 would be expected to interact with any number of target proteins after calcium binding and activation. We found that CALM-1 binds RACK-1 in a calcium dependent manner. RACK-1 is an intracellular scaffold protein that interacts with signaling and adhesion molecules.
rack-1 mutants phenocopy nidogen synaptic defects, and these defects can be suppressed by
calm-1.
Synapses can be added dynamically during organismal growth and based on our synaptic phenotype data, we believe to have uncovered one possible mechanism. Nidogen may serve as an adhesive force providing synaptic stability as indicated by mutant synaptic spreading. Conversely, calcium signal component (
unc-2,
unc-36 and
calm-1) mutations result in slightly enlarged, punctate synapses and suppress the spreading of nidogen mutants. A proposed mechanism may be a calcium signaling pathway that, in response to growth signals, mediates synaptic adhesion through the nidogen complex. Modulation of synaptic adhesiveness may allow for addition of new synapses during development.