Irons Sarah L, Evans David E, Brandizzi Federica
Research School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy Lane, Headington, Oxford OX3 0BP, UK.
J Exp Bot. 2003 Mar;54(384):943-50. doi: 10.1093/jxb/erg102.
In plants, the nuclear envelope (NE) is one of the least characterized cellular structures. In particular, little is known about its dynamics during the cell cycle. This is due to the absence of specific markers for in vivo studies. To generate such an in vivo marker, the suitability of the human lamin B receptor (LBR) was tested. When the first 238 amino acids of the LBR, fused to the green fluorescent protein (GFP), were expressed in tobacco plants, fluorescence accumulated only at the NE of leaf epidermal cells. This was confirmed by electron microscopy. The protein was shown to be membrane-integral by phase separation. Distribution of fluorescence was compared with two ER markers, GFP-calnexin and GFP-HDEL. While co-localization of all three markers was noted at the NE, only LBR-GFP was specific to the NE, while the other two also showed fluorescence of the cortical ER. These results suggest that common targeting mechanisms to those in animals and fungi exist in plants to direct and locate proteins to the NE. This chimaeric construct is the first available fluorescent integral membrane protein marker to be targeted exclusively to the plant NE and it provides a novel opportunity to investigate the dynamics of this membrane system in vivo. With it, the cell cycle was followed in tobacco BY-2 cells stably expressing the fusion protein. The interphase labelling of the NE altered in metaphase into an ER-like meshwork, suggesting the dispersal of the NE to ER as in animal cells. Finally, the meshwork of fluorescent membranes was lost and new fluorescent NE formed around the daughter nuclei.
在植物中，核膜（NE）是细胞结构中了解最少的结构之一。特别是，关于其在细胞周期中的动态变化知之甚少。这是由于缺乏用于体内研究的特异性标记物。为了生成这样一种体内标记物，对人核纤层蛋白B受体（LBR）的适用性进行了测试。当与绿色荧光蛋白（GFP）融合的LBR的前238个氨基酸在烟草植物中表达时，荧光仅在叶表皮细胞的核膜处积累。这通过电子显微镜得到了证实。通过相分离表明该蛋白是膜整合蛋白。将荧光分布与两种内质网标记物GFP-钙连蛋白和GFP-HDEL进行了比较。虽然在核膜处注意到所有三种标记物的共定位，但只有LBR-GFP对核膜具有特异性，而其他两种标记物也显示出皮质内质网的荧光。这些结果表明，植物中存在与动物和真菌中相同的靶向机制，可将蛋白质定向并定位到核膜。这种嵌合构建体是第一个专门靶向植物核膜的可用荧光整合膜蛋白标记物，它为体内研究该膜系统的动态变化提供了新机会。利用它，对稳定表达融合蛋白的烟草BY-2细胞的细胞周期进行了跟踪。核膜的间期标记在中期转变为内质网样网络，表明核膜如在动物细胞中一样分散到内质网。最后，荧光膜网络消失，子细胞核周围形成新的荧光核膜。
