| Abstract: | Rab3A被認為是一種參與調控式胞吐的小G蛋白。根據目前的研究認為Rab3A的作用是在囊泡的導引與膜融合，但是沒有直接證據證明其作用的位置與實際參與的生化反應。為了解決這個問題，首先一定要了解Rab3A在活體細胞中的位置與其在胞吐作用時的分布變化。本計畫將Rab3A以螢光蛋白(Enhanced cyan fluorescent protein, ECFP)結合，在PC12嗜鉻性瘤細胞中表現，以螢光顯微鏡來觀察Rab3A在活體細胞中的位置與其在胞吐作用時的分布變化。本年度成功地完成計畫所需的基本實驗配備，實驗材料與實驗條件，包括(1)分泌囊泡標記蛋白之螢光融合蛋白(NPY-GFP、Chg-DsRed2、VAMP2-YFP)與Rab3A結合蛋白之螢光融合蛋白(YFP-Rabphilin3A)的真核表達載體的構築)；(2)建立分泌與蛋白質分布同步觀察的螢光顯微鏡系統；(3)建立觀察螢光蛋白質動態分布的顯微影像分析系統。根據這些基礎工作所得的實驗條件，進行觀察GFP-Rab3A在PC12細胞中的分布。由免疫螢光顯微影像發現融合蛋白可以被Rab3A的抗體辨認。此外，本結果與過去免疫螢光染色結果相似，EGFP-Rab3A在細胞呈點狀分布，且集中在核周圍與細胞膜附近。當ECFP-Rab3A與分泌囊泡標記之螢光融合蛋白(NPY-GFP)共同表現時，發現ECFP-Rab3A與標記融合蛋白有類似的分布，顯示ECFP-Rab3A位於分泌囊泡上。此兩項實驗結果證明螢光融合蛋白並不會影響Rab3A的正確位置。當以高鉀溶液刺激同時表現NPY-GFP與ECFP-Rab3A的PC12細胞，雙光螢光顯微鏡紀錄，發現ECFP-Rab3A螢光隨著NPY-GFP螢光下降而下降，此一結果顯示ECFP-Rab3A分布在胞吐時有轉移的現象。為了更精密地了解此種移動是移出聚焦面還是離開分泌囊泡擴散至細胞質，以內全反射螢光顯微鏡(Total internal reflection fluorescence microscopy, TIRFM)觀察ECFP-Rab3A在PC12細胞中的動態分布。在此之前，先觀察分泌囊泡標記蛋白之螢光融合蛋白(NPY-GFP)，以了解我們的TIRFM系統是否可以觀察單一囊泡的分泌時的各步驟，結果發顯此TIRFM系統可以觀察分泌囊泡的移動、定位、膜融合。確定此TIRFM的表現之後，以此TIRFM分析EYFP-Rab3A在PC12細胞中的動態分布，並以PowerPoint進行初步分析。光點的大小與形狀，發現其大小約0.3微米到1微米之間，小光點其形狀近似圓形，大多分布在細胞膜附近呈靜止狀態，此顯示小光點可能為準備分泌的囊泡。有一部分囊泡從細胞內部往細胞膜附近移動，顯示Rab3A可能作用於囊泡的導引與膜融合反應。部分光點有互相融合，有部分光點會從細胞膜向細胞中心移動，此種現象類似內胞食體(Endosome)的行為，此顯示出Rab3A亦有可能參與突觸囊泡的回收與生成。當以高鉀刺激細胞時，發現EYFP-Rab3A的螢光會增強而後模糊減弱，似乎證明EYFP-Rab3A會導引分泌囊泡而後從囊泡離開。然而此一現象亦有可能是細胞底部因加藥而貼近玻璃所致，需要進一步以反射干涉顯微鏡進一步檢測。
Rab3A is a small GTP-binding protein thought to regulate regulated exocytosis. Recent investigations indicate Rab3A plays a role in docking and fusion steps of exocytosis, but there is no direct evidence to prove where Rab3A acts. To solve this problem, tracking Rab3A localization during exocytosis in vivo need to be established. In this study, we have setup a simple system to detect Rab3A translocalization in vivo. In this study, we have accomplished basic reagents, facility and experimental conditions; including construct of expressing vectors of fluorescent protein-tagged organelle markers (NPY-EGFP, Chg-DsRed2, VAMP2-EYFP) and Rab3A-binding protein (EYFP-Rabphilin3A), simultaneous detection system for secretion and protein translocalization, and detection and analysis system for dynamic protein localization. Based on these accomplishments, we detect protein localization of Rab3A in PC12 cells during exocytosis. Cells expressing EGFP-Rab3A are analyzed by immuohistochemistry, EGFP-Rab3A can be recognized by anti-Rab3A antibody. Besides, subcellular localization Rab3A is similar to previous investigations that Rab3A-associated compartments are punctuated and concentrate in cell membrane and perinuclear region. When ECFP-Rab3A and NPY-EGFP, one of secretory vesicle markers, are co-expressed in-PC 12 cells, ECFP-Rab3A and NPY-EGFP are co-localized. These two results indicate that fluorescent protein doesn't interfere normal subcellular localization of Rab3A. When cells are stimulated with high potassium, fluorescent intensity of NPY-EGFP decrease due to release of NPY-EGFP in to cytosol. Interestingly, ECFP-Rab3A decreases, too. This indicates that Rab3A translocalize during exocytosis. To verify whether translocalization of ECFP-Rab3A is moving out of the focal plane or dissociating from secretory vesicles and diffusing into cytosol, TIRFM (Total internal reflection fluorescence microscopy) is used for detection of dynamic localization of ECFP-Rab3A during secretion. Before this study, we use NPY-EGFP, soluble content of secretory vesicles, to confirm our TIRFM capable of detecting all steps of single exocytosis, and find that our system can detect movement, docking and fusion of vesicles during secretion. After this, we use TIRFM to detect behavior of EYFP-Rab3A in living PC12 cells and analyze dynamic protein localization by PowerPoint. Sizes of fluorescent dots are ranging from 0.3micron to 1 micron. Fluorescent dots are static near plasma membrane, and these may be docked vesicles. Some fluorescent dots move from center of the cell to plasma membrane, and this indicates that Rab3A involves in docking of vesicles. Some of vesicles move oppositely and fuse each other, and these phenomena are similar to those of endosomes. Therefore, Rab3A seems to also play a role in recycle of vesicles. When high potassium stimulates cells expressing EYFP-Rab3A, fluorescence of EYFP-Rab3A increases and becomes blurring. This may be due to Rab3A moving toward plasma membrane and dissociating from secretory vesicles. But this observation may be also due to increasing membrane associated with coverslip when high potassium is applied. And we need to use reflection interference contrast microscopy to rule out this possibility. |
