Identification of genes involved in synaptogenesis using a fluorescent active zone marker in Caenorhabditis elegans.
Active zones are presynaptic regions where synaptic vesicles fuse with plasma membrane to release neurotransmitters. Active zones are highly organized structurally and are functionally conserved among different species. Synapse defective-2 (SYD-2) family proteins regulate active zone morphology in Caenorhabditis elegans and Drosophila. Here, we demonstrate by immunoelectron microscopy that at C. elegans synapses, SYD-2 localizes strictly at active zones and can be used as an active zone marker when fused to green fluorescent protein (GFP). By driving expression of SYD-2::GFP fusion protein in GABAergic neurons, we are able to visualize discrete fluorescent puncta corresponding to active zones in living C. elegans. During development, the number of GABAergic synapses made by specific motoneurons increases only slightly from larvae to adult stages. In contrast, the number of SYD-2::GFP puncta doubles, suggesting that individual synapses accommodate the increasing size of their synaptic targets mainly by incorporating more active zone materials. Furthermore, we used this marker to perform a genetic screen to identify genes involved in the development of active zones. We recovered 16 mutants with altered SYD-2::GFP expression, including alleles of five genes that have been implicated previously in synapse formation or nervous-system development. Mapping of 11 additional mutants suggests that they may represent novel genes involved in active zone formation.
Yeh
Edward
E
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5, Canada.
Kawano
Taizo
T
Weimer
Robby M
RM
Bessereau
Jean-Louis
JL
Zhen
Mei
M
D000818:Animals; D015415:Biomarkers; D017173:Caenorhabditis elegans / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000502:physiology*; D002352:Carrier Proteins / Q000502:physiology; D002462:Cell Membrane; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D036341:Intercellular Signaling Peptides and Proteins; D016253:Microscopy, Immunoelectron / Q000379:methods; D008969:Molecular Sequence Data; D038081:Organogenesis / Q000502:physiology*; D010750:Phosphoproteins / Q000502:physiology; D017729:Presynaptic Terminals / Q000378:metabolism / Q000648:ultrastructure; D013569:Synapses / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D013572:Synaptic Vesicles / Q000378:metabolism / Q000648:ultrastructure; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D015415:Biomarkers; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D036341:Intercellular Signaling Peptides and Proteins; D010750:Phosphoproteins; C490598:SYD-2 protein, C elegans; C083141:phorbol ester binding protein; C437625:unc-10 protein, C elegans; D049452:Green Fluorescent Proteins; D005680:gamma-Aminobutyric Acid