钙和ROS通路关键信号蛋白的可视化分析研究报告

林金星 李瑞丽

摘 要：我们完成了对多种生物活性分子探针的化学修饰，为下一阶段的应用研究奠定了探针基础。首先，在细胞膜通透性方面，发现我们的核酸分子探针，由于其高度的化学修饰和较短的链长，已经实现了不需特殊修饰保护，即具有在活细胞中的稳定性和一定的通透性。其次，我们利用亚克隆技术在体外将一种酵母菌中亚铜离子结合蛋白Ace1的关键区域插入到黄色荧光蛋白（YFP）构建4种不同的模型中，有效提高了其荧光强度；我们将此传感器成功应用于检测细胞体内亚铜离子浓度。通过体外和体内的验证，我们研发的亚铜离子荧光传感器可以应用于研究生物体系内铜离子的代谢研究。最后，对拟南芥根表皮细胞质膜铵转运蛋白（AMT1；3）进行了活体动态分析。研究发现：AMT1；3-EGFP在正常供铵状态下，在质膜上的运动状态主要有两种：一种是出现后立即消失；另外一种是在膜上停留一段时间后消失；并且发现AMT1；3-EGFP在不同铵浓度下，这两种运动状态的荧光点所占的比例不同，表明铵浓度决定AMT1；3-EGFP的膜表面停留时间。更重要的是，高铵胁迫下会引起AMT1；3-EGFP聚合并内吞，而这种内吞对于调控AMT1；3的转运活性发挥着重要的作用；并且高铵所引起的快速内吞作用是由铵根离子特异引起的。

关键词：探针 亚铜离子 铵转运蛋白 可变角度的全内反射荧光显微镜 内吞

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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