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研究生:黃柄鈞
研究生(外文):Ping-Chun Huang
論文名稱:以螢光生命週期顯微術觀測鈣庫調節鈣離子通之道調控蛋白質間的動態交互作用
論文名稱(外文):Observing dynamic interactions of store-operated calcium channel proteins by fluorescence lifetime imaging microscopy
指導教授:楊德明楊德明引用關係
指導教授(外文):De Ming Yang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生醫光電工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:101
中文關鍵詞:螢光共振能量轉移螢光生命期顯微術鈣庫調控鈣離子通道蛋白質交互作用
外文關鍵詞:FRETFLIMSOCsProtein-protein interactionsOrai1STIM1
相關次數:
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本論文以螢光生命週期顯微術,搭配雷射共軛焦顯微鏡以及全波段
光譜儀來探討活體細胞內鈣庫調控鈣離子通道的調控蛋白質Orai1 以
及STIM1 之間的動態交互作用。我們以EGFP 搭配mOrange 做為一
組FRET pair,以螢光轉染的方式在大鼠腎上腺髓質嗜鉻細胞株PC12
上表現具有螢光的Orai1 以及STIM1,以內質網鈣離子幫浦阻斷劑
(Thapsigargin,TG) 排空內質網內的鈣離子,達到鈣庫調控鈣離子通
道啟動的要件,利用螢光生命週期顯微術量測EGFP 以及mOrange
的螢光共振能量轉移效應,來表現在通道啟動時Orai1 以及STIM1
這兩種蛋白質的動態表現。在TG 刺激20 分鐘後,成功的觀測到這
兩個蛋白質發生交互作用的位置是位於細胞膜表面。並藉由螢光生命
週期的分析,我們可以知道這兩種蛋白質在作用時是以哪一種形式結
合。我們也發現以兩個指數方程式去計算螢光分子的生命週期時,第
二個基底(component)的變化與FRET 的發生效率有正向的關連性,在
螢光生命週期的量化分析上,提供另一種計算的方式。
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.
目錄
頁碼
圖表目次 i
英文縮寫表 vii
中文摘要 ix
英文摘要 x
第一章 緒論 1
第二章 鈣庫調控鈣離子通道 4
一、 SOCs 的相關調控蛋白質 6
二、 STIM1 6
三、 Orai1 7
第三章 螢光能量共振轉移 9
一、 FRET 的量測 12
二、 螢光生命週期顯微術 14
第四章 實驗方法與實驗設計 18
第五章 樣品製備與實驗儀器 20
一、 實驗材料與試劑 20
二、 細胞培養 20
三、 螢光蛋白轉染 21
四、 Ca2+ imaging 21
五、 全波段光譜掃描儀 22
六、 雷射共焦掃描顯微鏡 22
七、 螢光生命週期顯微鏡 22
第六章 實驗結果 25
一、 EGFP 與mOrange 可做為FRET pair 25
二、 蛋白質活性檢驗 27
三、 雷射共焦掃描顯微鏡影像 29
四、 螢光生命週期顯微鏡影像 34
五、 螢光生命週期分佈曲線 41
第七章 結果討論 45
一、 以兩個components 來計算螢光蛋白質的lifetime 45
二、 Orai1 與STIM1 間的交互作用 47
三、 Orai1 與Orai1 間的交互作用 53
四、 從FRET 效率推測螢光分子之間的距離 56
第八章 結論與未來展望 58
第九章 參考文獻 60
第十章 附錄 64
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