Characterization of docking and fusion of synaptic-like microvesicles in PC12 cells using TIRFM

Neurotransmitters are released by the fusion of synaptic vesicles with presynaptic membrane,which has been extensively studied. The analysis of single vesicle fusion kinetics reveals that there exist fusion modes of "kiss and run" and "kiss and stay" which may be favored by neurons especially during strong firing beside full fusion. Pre-fusion steps of translocation,docking and priming along the exo-cytotic pathway play important roles in neurotransmitter release and its regulation. In the present report,we used dual-color imaging of VAMP2-pHluorin and VAChT-TDimer2 under total internal reflection fluorescence microscope(TIRFM) to monitor the docking and fusion of synaptic-like microvesicles(SLMVs) in PC12 cells stimulated by high K+. Our results show that "kiss and run" is a dominative fu-sion mode in PC12 cells under high K+-challenge,and the dwell time of SLMVs is prolonged by the high K+ stimulation that suggests an enhancement of vesicle priming.

Neurotransmitters are released by the fusion of synapUc vesicles with presynaptic membrane, which has been extensively studied. The analysis of single vesicle fusion kinetics reveals that there exist fusion modes of ＂kiss and run＂ and ＂kiss and stay＂ which may be favored by neurons especially during strong firing beside full fusion. Pre-fusion steps of translocation, docking and priming along the exocytotic pathway play important roles in neurotransmitter release and its regulation. In the present report, we used dual-color imaging of VAMP2-pHluorin and VAChT-TDimer2 under total internal reflection fluorescence microscope （TIRFM） to monitor the docking and fusion of synaptic-like mlcrovesicles （SLMVs） in PC12 cells stimulated by high K^＋. Our results show that ＂kiss and run＂ is a dominative fusion mode in PC12 cells under high K^＋-challenge, and the dwell time of SLMVs is prolonged by the high K^＋ stimulation that suggests an enhancement of vesicle priming.