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Neurotransmitters, Gliotransmitters, and Modulators

開催日 2014/9/12
時間 11:00 - 12:00
会場 Poster / Exhibition(Event Hall B)

Dual imaging of SVs and DCVs exocytosis

  • P2-017
  • 中島 柚依 / Yui Nakajima:1 篠田 陽 / Yo Shinoda:1 古市 貞一 / Teiichi Furuichi:1,2 
  • 1:東京理科大院理工応用生物科学 / Dept. of Appl. Biol. Sci., Fac. of Sci. and Technol.,Tokyo Univ. of Sci., Chiba, Japan 2:理研BSI / RIKEN Brain Science Institute, Saitama, Japan 

Exocytosis of synaptic vesicles (SVs) mediates the release neurotransmitters into synaptic cleft through the sequential process of vesicle docking, priming and fusion, when the action potential arrives at the presynaptic terminals. In neuronal cells, there is another secretory vesicle, called dense-core vesicles (DCVs) that play a major role in the release of neuropeptides and peptide hormones. Very little, however, is known about the regulatory process of DCV exocytosis. To clarify the distinction between SV and DCV exocytosis regarding the underlying mechanisms including subcellular release patterns and stimulus-dependent release kinetics, simultaneous cell imaging of both exocytosis events must be informative. We constructed a pH-sensitive red fluorescent protein mOrange2 fused with the DCV luminal protein chromogranin A (ChgA), "ChgA-mOrange2" as a fluorescent probe for DCV exocytosis. We also used a pH-sensitive green fluorescent protein pHluorin (pH) fused with the SV membrane protein synaptophysin (SYP), "SYP-pH" as a fluorescent probe for SV exocytosis. Two probe constructs were co-transfected into rat primary cultured cortical neurons and were subjected to live-cell time-lapse imaging. At present, we successfully detected exocytosis events indicated by ChgA-mOrange2 and SYP-pH, most of which likely showed different subcellular sites and different mode. We will further repeat dual imaging and will present the data on their comparative exocytosis mechanisms.

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