Observing dynamic interactions of store-operated calcium channel proteins by fluorescence lifetime imaging microscopy

• Main Authors: Ping-Chun Huang, 黃柄鈞
• Other Authors: De Ming Yang
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/8vmdqv

碩士 === 國立陽明大學 === 生醫光電工程研究所 === 97 === The Ca2+ influx is essential for initiating messange to regulate several vital processes. The store-operated Ca2+ channels (SOCs) constructed by Orai1 with ER membrane peotein STIM1 were recently found to be a unique Ca2+ entry pathway which is the major Ca2+ signaling cascade for non-excitable cells. The SOCs also functional exist in various types of cells such as excitable neuroendocrines like rat pheochromocytoma PC12 cells where voltage-gated Ca2+ channels are the major entry route. Althogh Orai1 together with STIM1 are thought to be interact with each other thorugh biochemical evidence, it is still unrevealed to define the detail mechanism about their dynamic interactions during store-operated conditions in situ. To solve this problem in this thesis, the green fluorescence protein (EGFP) targeted Orai1 (to either N- or C-terminal; wild type or double deletion type or Orai1, as donor) and mOrange flagged STIM1 (as acceptor) were used as a fluorescence resonance energy transfer (FRET) pair within living PC12 cells under a new constructed one-photon excitation fluorescence lifetime imaging microscopy (FLIM) adapting the strategy of time-correlated single photon counting (TCSPC). The activity of SOCs, namely the store-operated Ca2+ entry (SOCE) was measured using fura-2 imaging method. The ER Ca2+ ATPase inhibitor thapsigargin (TG) was used to activate the SOCE. The lifetime map and histogram/distribution of each single cells were acquired under FLIM. Both the color coded liftime map (plasma membrane) and the distribution (100 ps left shift) of EGFP tagged Orai1 changed signficantly after 20-minute administering of TG. The efficiency xi of FRET from each experimental sets was also calculatecd and compared. In summary, we further show the dynamic interactions between the essential SOC protein Orai1 and STIm1 with the novel FLIM-FRET technique within living PC12 cells.