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研究生:林坤漢
研究生(外文):Kun-Han Lin
論文名稱:藉由表現Rab3A與其突變蛋白研究Rab3A在分泌過程所扮演之角色
論文名稱(外文):Role of Rab3A in neurotransmitter release:A study by overexpression of Rab3A and its mutants
指導教授:高閬仙高閬仙引用關係
指導教授(外文):Lung-Sen Kao
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:84
中文關鍵詞:囊泡胞吐反應小G蛋白Rab3A突變蛋白全反射螢光顯微鏡
外文關鍵詞:synptic vesiclesexocytosisRab3ARab3A MutantsTIRFM
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Rab3A是一種小G蛋白,其功能是調控胞吐作用。在過去研究中認為它是屬於抑制性調控胞吐反應,而且過程會與下游作用蛋白如rabphilin3A, calmodulin, RIM 等一起作用。然而缺乏直接證據可證實Rab3A是於何時、何處與如何參與囊泡分泌,因此在本研究中,我將不同的Rab3A突變蛋白與EGFP, ECFP,及EYFP三種螢光蛋白融合成螢光突變蛋白,並且在PC12細胞內大量表現。搭配一套可即時偵測細胞內螢光融合蛋白的全反射螢光顯微鏡系統,以直接觀察Rab3A及其突變蛋白在細胞內螢光變化。
EGFP-Rab3A在細胞是成點狀分佈。然而在C端區域缺失(Rab3A- Tc)及維持與GDP結合狀態(Rab3A-T36N)的兩種突變蛋白,是均勻分佈在細胞質,但卻不會影響囊泡標誌蛋白NPY的分佈。有趣的是,維持與GTP結合狀態(Rab3A-Q81L)的突變蛋白在細胞內雖成點狀分佈,但細胞膜附近的數量較Rab3A多,而且也會影響囊泡標誌蛋白NPY的在細胞表面分佈數量。這些結果表示GTP結合狀態的Rab3A藉由C端與囊泡作用。
在刺激後Rab3A螢光在下降前會先迅速提升,但是Rab3A-T36N卻沒有螢光先上升再下降的情形;整體螢光下降程度也不及Rab3A。Rab3A-Tc也有類似情形。而在Rab3A-F59S(與rabphilin3A間作用的突變)及Rab3A-Q81L雖然在刺激後其螢光在下降前也會先上升,但上升的速度上不及Rab3A,其中Rab3-Q81L的速度最為緩慢,而且下降程度也不及Rab3A。由上述的結果推測Rab3A可帶領囊泡前往座落(docking)於細胞膜附近並調控胞吐作用的最後步驟。
Rab3A is a small GTP-binding protein and is implicated in regulated exocytosis. It is thought that Rab3A is a negative regulator of exocytosis and exerts its action by interacting with many effectors including rabphilin3A, calmodulin, RIM. However, it is not known where, when, and how Rab3A regulates exocytosis. In my study, the fusion proteins of Rab3A and its mutants with various fluorescent proteins were constructed and over-expressed in PC12 cells. Then, an on-line total internal reflection fluorescence microscopic system was used to examine the subcellular localization and monitor the movement of these fusion proteins during exocytosis.
The fluorescence of EGFP-wild type Rab3A show punctuate patterns as expected because that Rab3A is known to associate with the secretory vesicles. The C-terminal truncated Rab3A and GDP form Rab3A mutant (Rab3A T36N) show diffuse patterns in the cytosol but the distribution of secretory vesicles appears to be normal, as shown by the distribution of the marker of secretory vesicles, neuropeptide Y. Interestingly, although the GTP form Rab3A mutant (Rab3A Q81L) shows punctuate pattern the number of vesicle near cell surface was more than that of the control cells. The results suggest that C-terminal is responsible for the interaction of Rab3A and vesicles and that vesicle interacts with GTP form Rab3A.
Changes in the fluorescence intensity of Rab3A and its mutants in individual PC12 cells upon stimulation were then monitored and analyzed. In general, the fluorescence of Rab3A decreased upon stimulation. The decrease is less in cells overexpressed GTP form, GDP form and C-terminal truncated Rab3A. The fluorescence of some cells show a transient increase before the decrease. This transient increase has never been observed in the GDP from Rab3A-expressed cells and was delayed in the cells that expressed GTP form Rab3A. For the mutant that lacks the interaction with rabphilin3A (Rab3A F59S), the transient increase was also delayed but to a less extent than that expressed the GTP from Rab3A. These results indicated that Rab3A is involved in the movement of vesicles toward cell membrane, and the final steps of exocytosis.
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