Calcium signaling components and their effect on synaptic morphology during neuronal development

Abstract

Along with regulating synaptic transmission, voltage-gated calcium channel (VGCC) function is responsible for a myriad of cellular outputs, ranging from gene expression to shaping synaptic morphology. Despite the morphological role of VGCCs, the proteins working downstream of VGCCs to regulate synaptic morphology remain mostly unknown, and their identification would provide insight into the shaping of synapses through calcium signaling. Chapter I introduces the Caenorhabditis elegans VGCC subunits unc-2 and unc-36 as regulators of D-type GABAergic neuromuscular junction morphology. In addition to synaptic defects found in single mutants, loss-of-function mutations in VGCC subunits, independent of neurotransmission, suppressed the enlarged synaptic areas resulting from mutations in the extracellular matrix protein nidogen (nid-1). Furthermore, time-lapse microscopy revealed UNC-2 function was required for proper synaptic dynamics that occurred during the L4 larval stage of organismal growth. Specifically, the dynamics observed in wild-type animals were slowed or absent in unc-2 mutants. Since wild-type synapses undergo enlargement - increasing to areas similar to nid-1 - and subsequently divide into two smaller puncta, we conclude UNC-2 is responsible for a temporal switch between synaptic stability and growth to add synapses during development. In chapter III, I detail the characterization of the calm-1 (ortholog of calmyrin), a gene encoding an EF hand protein that was identified in a nid-1 suppressor screen. Double mutant analysis between calm-1 and VGCC subunits suggests calm-1 acts downstream of VGCCs to regulate synaptic morphology. This result is the first to find a calmyrin protein that affects synapse morphology downstream of VGCCs. A calcium-dependent CALM-1 pulldown identified the intracellular scaffolding protein RACK-1 as a protein that may be targeted via VGCC/CALM-1 signaling. Similar to nid-1, rack-1 mutants displayed abnormally shaped synaptic areas that were suppressed by calm-1 mutants. In conclusion, my genetic analyses suggest synaptic growth through VGCCs is normally inhibited by NID-1. But this inhibition is relieved during development to allow synaptogenesis to occur, commensurate with increases in organismal size, from the expansion and budding of existing synaptic connections. Thus, the cycling between synaptic adhesion and growth allows for a rapid and localized mechanism to add new synapses.