Rizzuto R, Brini M, De Giorgi F, Rossi R, Heim R, Tsien R Y, Pozzan T
Department of Biomedical Sciences, University of Padova, Italy.
Curr Biol. 1996 Feb 1;6(2):183-8. doi: 10.1016/s0960-9822(02)00451-7.
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.
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(Y66H/Y145F) and nuGFP, the nucleus and the mitochondria were visualized simultaneously in living cells. Similarly, mtGFP and mtGFP(Y66H/Y145F) were transfected into different populations of cells, and the events of cellular fusion, and mitochondrial intermixing and/or fusion, were directly monitored.
The successful expression of organelle-targeted GFP 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.
维多利亚多管水母的绿色荧光蛋白（GFP）正成为一种独特的工具，用于监测诸如活细胞中的基因表达、细胞器结构及动态等复杂现象。近期对具有改变光谱特性的GFP突变体的描述为细胞生物学开启了众多新应用。然而，这些GFP突变体在活的真核细胞中的表达及特性尚未得到验证。
在此，我们通过使用野生型GFP、一种“明亮的”GFP突变体（S65T）以及一种激发和发射光谱发生蓝移的突变体（Y66H/Y145F），证明了GFP突变体在体内细胞生物学研究中的实用性。我们采用先前用于构建线粒体靶向性GFP（mtGFP）的相同策略，构建了两种靶向线粒体的GFP嵌合体，即mtGFP（S65T）和mtGFP（Y66H/Y145F）。此外，通过将野生型GFP或（S65T）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，即nuGFP和nuGFP（S65T）。通过共转染mtGFP（Y66H/Y145F）和nuGFP，可在活细胞中同时观察到细胞核和线粒体。同样，将mtGFP和mtGFP（Y66H/Y145F）转染到不同细胞群体中，可直接监测细胞融合以及线粒体混合和/或融合事件。
细胞器靶向性GFP突变体在活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应用，使得能够直接研究关键生物学问题，如体内不同细胞器之间的相互作用。这些结果还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重新分布和蛋白质 - 蛋白质相互作用。
