活细胞内以光漂白荧光损失(FLIP)技术分析HuR蛋白的应激动力学行为  

The Analysis on the Dynamic Properties of HuR Granules in Living Cells via FLIP Technology

作　　者：高星杰[1] 张毅[2] 付雪[3] 苏超[1] 张春燕[3] 张桂敏[3] 尹洁[1] 王鑫廷[3] 姚智[3] 杨洁[1,3] 

机构地区：[1]天津医科大学基础医学研究中心,天津300070 [2]天津医科大学药学院,天津300070 [3]天津医科大学基础医学院,天津300070

出　　处：《中国细胞生物学学报》2014年第9期1235-1240,共6页Chinese Journal of Cell Biology

基　　金：国家杰出青年科学基金(批准号:31125012);国家自然科学基金(批准号:21305103;31100967;31170830;31370749);教育部"创新团队发展计划"(批准号:IRT13085);中国博士后科学基金(批准号:2013T60258)资助的课题~~

摘　　要：人类抗原R(human antigen R,HuR)是一种多功能RNA结合蛋白,参与细胞应激颗粒(stress granules,SGs)的构成。SGs是细胞在受到外界环境刺激时在胞浆中形成的颗粒状结构。该研究是利用光漂白荧光损失(fl uorescence loss in photobleaching,FLIP)技术对活细胞内的HuR蛋白颗粒进行应激动力学分析。首先,利用脂质体将RFP-HuR重组质粒瞬时转染入HeLa细胞,以Western blot和细胞免疫荧光实验确定是否实现对于HuR蛋白的红色荧光蛋白(red fl uorecent protein,RFP)标记;然后以405 nm激光束脉冲式重复光漂白HuR应激颗粒,分别监测同一漂白细胞内的其他HuR颗粒以及核内荧光信号,并以邻近的未漂白细胞作为对照组。实验结果表明,转染重组质粒后可有效表达RFP-HuR融合蛋白,且与SGs标记蛋白G3BP存在共定位关系。在第一个光漂白循环,漂白区荧光密度便从2 500 a.u.降低至0 a.u.;而经过约12个漂白循环(240 s)后,邻近HuR颗粒的荧光密度从漂白前的1 800 a.u.左右降低并维持在200 a.u.左右,表明活细胞内的HuR颗粒呈现高度的动态性;而胞核区荧光密度亦从4 400 a.u.降低至2 000 a.u.左右,表明HuR蛋白是一种核浆穿梭蛋白,在SGs、胞浆及胞核之间存在一定的动态平衡。利用FLIP技术可以分析并比较SGs不同成分的应激动力学属性,有助于进行SGs相关临床疾病的分子机制探讨。HuR（human antigen R）, a kind of multifunctional RNA-binding protein, is involved in the SGs（stress granules） assembly. SGs are a type of cytoplasmic RNA foci that accumulate in response to environmental stress stimuli. Here, the fluorescence loss in photobleaching（FLIP） technology was used to study the dynamic nature of HuR granules in living cells. Firstly, the plasmid encoding RFP-HuR fusion protein was transfected into HeLa cells via the liposome reagent. Western blot and immunofluorescence assays were then performed to verify the expression of RFP-HuR protein. For FLIP assay, one HuR granule region ofinterest（ROI） was repeatedly photo-bleached using 405 nm laser beam, other granule or nuclear region were monitored, and the adjacent nonbleached cell was used as a control. The results indicated that RFP-HuR fusion protein was expressed efficiently and co-localized with the G3BP（Ras-GAP SH3 domain-binding protein）（one SGs marker protein） in HeLa cells. In FLIP assay, fluorescence density in the cytosolic pulse bleach region was reduced from 2 500 a.u. to 0 a.u. after the first bleaching. The fluorescence density of the adjacent HuR granule region reduced from 1 800 a.u. and maintained at about 200 a.u. after 12 bleaching cycles（240 s）, suggesting that HuR-containing SGs structure is highly dynamic. The nuclear fluorescence density also reduced from 4 400 a.u. to 2 000 a.u., suggesting that HuR is a nucleocytoplasmic shuttling protein. The nuclear pool of HuR protein is likely to be in equilibrium with cytosolic SGs-routed pool. The FLIP technology can be used to analyze the dynamic properties of different stress associated proteins, helping to investigate the molecular mechanisms underlying the SGs-related diseases.

关 键 词：HuR蛋白 光漂白荧光损失 应激颗粒 活细胞 荧光标记 

分 类 号：R392[医药卫生—免疫学]