PKA activation bypasses the requirement for UNC-31 in the docking of dense core vesicles from C. elegans neurons.
The nematode C. elegans provides a powerful model system for exploring the molecular basis of synaptogenesis and neurotransmission. However, the lack of direct functional assays of release processes has largely prevented an in depth understanding of the mechanism of vesicular exocytosis and endocytosis in C. elegans. We address this technical limitation by developing direct electrophysiological assays, including membrane capacitance and amperometry measurements, in primary cultured C. elegans neurons. In addition, we have succeeded in monitoring the docking and fusion of single dense core vesicles (DCVs) employing total internal reflection fluorescence microscopy. With these approaches and mutant perturbation analysis, we provide direct evidence that UNC-31 is required for the docking of DCVs at the plasma membrane. Interestingly, the defect in DCV docking caused by UNC-31 mutation can be fully rescued by PKA activation. We also demonstrate that UNC-31 is required for UNC-13-mediated augmentation of DCV exocytosis.
Zhou
Ke-Ming
KM
Key Laboratory of Molecular Biophysics, Ministry of Education, and Joint Laboratory of Institute of Biophysics, Huazhong University of Science and Technology, Wuhan 430074, China.
Dong
Yong-Ming
YM
Ge
Qian
Q
Zhu
Dan
D
Zhou
Wei
W
Lin
Xian-Guang
XG
Liang
Tao
T
Wu
Zheng-Xing
ZX
Xu
Tao
T
D000818:Animals; D017173:Caenorhabditis elegans / Q000378:metabolism* / Q000648:ultrastructure; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002352:Carrier Proteins; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D005089:Exocytosis / Q000502:physiology; D049452:Green Fluorescent Proteins; D007425:Intracellular Membranes / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology; D009420:Nervous System / Q000378:metabolism* / Q000648:ultrastructure; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D009489:Neurosecretion / Q000502:physiology; D018377:Neurotransmitter Agents / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D012701:Serotonin / Q000378:metabolism; D013570:Synaptic Membranes / Q000378:metabolism; D009435:Synaptic Transmission / Q000502:physiology
D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018377:Neurotransmitter Agents; C107581:UNC-31 protein, C elegans; C083141:phorbol ester binding protein; D049452:Green Fluorescent Proteins; D012701:Serotonin; D017868:Cyclic AMP-Dependent Protein Kinases