Double labelling of subcellular structures with organelle-targeted GFP mutants in vivo
Double labelling of subcellular structures with organelle-targeted GFP mutants in vivo
复制标题
DOI：
10.1016/s0960-9822(02)00451-7
复制
发表时间：
1996-02-01
期刊：
影响因子：
9.2
通讯作者：
Pozzan, T
中科院分区：
文献类型：
--
作者：
Rizzuto, R;Brini, M;Pozzan, T
背景资料：维多利亚水母的绿色荧光蛋白（GFP）是一种独特的工具，用于监测复杂的现象，如基因表达和细胞器的结构和动态活细胞。最近描述的GFP突变体与修改光谱特性开辟了许多新的应用在细胞生物学。然而，这些GFP突变体在活的真核细胞中的表达和特性尚未得到验证you.Results：在这里，我们证明了有用的GFP突变体的细胞生物学研究在体内，通过使用野生型GFP，一个“明亮”的GFP突变体（S65 T）和突变体与蓝移的激发和发射光谱（Y 66 H/Y145 F）。我们已经构建了两个针对线粒体的GFP嵌合体，mtGFP（S65 T）和mtGFP（Y 66 H/Y145 F），与以前用于mtGFP的相同策略。此外，通过将野生型GFP或S65 T突变体与大鼠糖皮质激素受体融合，构建了两种靶向细胞核的GFP嵌合体nuGFP和nUGFP（S65 T）。通过共转染mtGFP（Y 66 HN/Y145 F）和nuGFP，在活细胞中同时观察到细胞核和线粒体。类似地，mtGFP和mtGFP（Y 66 HN/145 F）转染到不同的细胞群体中，并直接监测细胞融合和线粒体混合和/或融合的事件。细胞器靶向GPP突变体在活的真核生物中的成功表达扩展了这种荧光蛋白在细胞生物学中的用途，允许直接接触关键的生物学问题，例如研究体内不同细胞器的相互作用。这些结果也为其他令人兴奋的应用开辟了道路，例如直接研究活细胞中的蛋白质再分布和蛋白质-蛋白质相互作用。
Background: The green fluorescent protein (GFP) of Aequorea victoria is emerging as a unique tool for monitoring complex phenomena such as gene expression and organelle structure and dynamics in living cells. The recent description of GFP mutants with modified spectral properties opens numerous new applications in cell biology. However, the expression and the characteristics of these GFP mutants in living eukaryotic cells have not been verified yet.Results: Here, we demonstrate the usefulness of the GFP mutants for cell biology studies in vivo, by the use of wild-type GFP, a'bright' GFP mutant (S65T) and a mutant with blue-shifted excitation and emission spectra (Y66H/Y145F). We have constructed two GFP chimeras targeted to mitochondria, mtGFP(S65T) and mtGFP(Y66H/Y145F), with the same strategy used previously for mtGFP. In addition, two GFP chimeras targeted to the nucleus, nuGFP and nUGFP(S65T), were constructed by fusing the wild-type GFP or the S65T mutant to the rat glucocorticoid receptor. By co-transfecting mtGFP(Y66HN/Y145F) and nuGFP, the nucleus and the mitochondria were visualized simultaneously in living cells. Similarly, mtGFP and mtGFP(Y66HN/145F) were transfected into different populations of cells, and the events of cellular fusion, and mitochondrial intermixing and/or fusion, were directly monitored.Conclusions: The successful expression of organelle-targeted GPP mutants in live eukaryotes expands the uses of this fluorescent protein in cell biology, allowing direct access to key biological issues, such as the study of the interactions of different organelles in vivo. These results also open the way to other exciting applications, such as the direct study of protein redistribution and protein-protein interactions in living cells.