鈣和ROS通路關(guān)鍵信號(hào)蛋白的可視化分析研究報(bào)告

林金星 李瑞麗

摘 要：我們完成了對(duì)多種生物活性分子探針的化學(xué)修飾，為下一階段的應(yīng)用研究奠定了探針基礎(chǔ)。首先，在細(xì)胞膜通透性方面，發(fā)現(xiàn)我們的核酸分子探針，由于其高度的化學(xué)修飾和較短的鏈長，已經(jīng)實(shí)現(xiàn)了不需特殊修飾保護(hù)，即具有在活細(xì)胞中的穩(wěn)定性和一定的通透性。其次，我們利用亞克隆技術(shù)在體外將一種酵母菌中亞銅離子結(jié)合蛋白Ace1的關(guān)鍵區(qū)域插入到黃色熒光蛋白（YFP）構(gòu)建4種不同的模型中，有效提高了其熒光強(qiáng)度；我們將此傳感器成功應(yīng)用于檢測細(xì)胞體內(nèi)亞銅離子濃度。通過體外和體內(nèi)的驗(yàn)證，我們研發(fā)的亞銅離子熒光傳感器可以應(yīng)用于研究生物體系內(nèi)銅離子的代謝研究。最后，對(duì)擬南芥根表皮細(xì)胞質(zhì)膜銨轉(zhuǎn)運(yùn)蛋白（AMT1；3）進(jìn)行了活體動(dòng)態(tài)分析。研究發(fā)現(xiàn)：AMT1；3-EGFP在正常供銨狀態(tài)下，在質(zhì)膜上的運(yùn)動(dòng)狀態(tài)主要有兩種：一種是出現(xiàn)后立即消失；另外一種是在膜上停留一段時(shí)間后消失；并且發(fā)現(xiàn)AMT1；3-EGFP在不同銨濃度下，這兩種運(yùn)動(dòng)狀態(tài)的熒光點(diǎn)所占的比例不同，表明銨濃度決定AMT1；3-EGFP的膜表面停留時(shí)間。更重要的是，高銨脅迫下會(huì)引起AMT1；3-EGFP聚合并內(nèi)吞，而這種內(nèi)吞對(duì)于調(diào)控AMT1；3的轉(zhuǎn)運(yùn)活性發(fā)揮著重要的作用；并且高銨所引起的快速內(nèi)吞作用是由銨根離子特異引起的。

關(guān)鍵詞：探針 亞銅離子 銨轉(zhuǎn)運(yùn)蛋白 可變角度的全內(nèi)反射熒光顯微鏡 內(nèi)吞

Abstract： We have developed a novel affinity labeling method based on DNA-templated photocrosslinking chemistry. DPAL uses a modular system to dissect binding from other functions that are usually combined in one probe， therefore simplifying probe design and preparation. Structure-activity relationship information on acceptable modification site on the SM is still required as any affinity probes. Then， we report a genetically encoded copper（I） probe capable of monitoring copper fluctuations inside living cells. We insert the copper regulatory protein Ace1 into a yellow fluorescent protein， which selectively binds copper（I） and generates improved copper（I） probes. To study how AMTs are regulated in the presence of ammonium， we used variable-angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy for single-particle fluorescence imaging of EGFP-tagged AMT1；3 on the plasma membrane of Arabidopsis root cells at various ammonium levels. We demonstrated that AMT1；3-EGFP dynamically appeared and disappeared on the plasma membrane as moving fluorescent spots in low oligomeric states under N-deprived and N-sufficient conditions. Under external high-ammonium stress， however， AMT1；3-EGFPs were found to amass into clusters， which were then internalized into the cytoplasm. A similar phenomenon also occurred in the glutamine synthetase mutant gln1；2 background. Single-particle analysis of AMT1；3-EGFPs in the clathrin heavy chain 2 mutant （chc2 mutant） and Flotllin1 artificial microRNA （Flot1 amiRNA） backgrounds， together with chemical inhibitor treatments， demonstrated that the endocytosis of AMT1；3 clusters induced by high-ammonium stress could occur mainly through clathrin-mediated endocytic pathways， but the contribution of microdomain-associated endocytic pathway cannot be excluded in the internalization. Our results revealed that the clustering and endocytosis of AMT1；3 provides an effective mechanism by which plant cells can avoid accumulation of toxic levels of ammonium by eliminating active AMT1；3 from the plasma membrane.

Key Words： Probe； Copper（I）； AMTs； VA-TIRFM； Endocytosis

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