| Summary: | 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.
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