Rizzuto R, Brini M, Pizzo P, Murgia M, Pozzan T
Department of Biomedical Sciences, University of Padova, Italy.
Curr Biol. 1995 Jun 1;5(6):635-42. doi: 10.1016/s0960-9822(95)00128-x.
It has recently been demonstrated that the green fluorescent protein (GFP) of the jellyfish Aequorea victoria retains its fluorescent properties when recombinantly expressed in both prokaryotic (Escherichia coli) and eukaryotic (Caenorhabditis elegans and Drosophila melanogaster) living cells; it can therefore be used as a powerful marker of gene expression in vivo. The specific targeting of recombinant GFP within cells would allow it to be used for even more applications, but no information is yet available on the possibility of targeting GFP to intracellular organelles.
In this study, we show that the GFP cDNA can be expressed at high levels in cultured mammalian cells; the recombinant polypeptide is highly fluorescent and is exclusively localized in the cytosol. Furthermore, we have modified the GFP cDNA to include a mitochondrial targeting sequence (and a strong immunological epitope at the amino terminus of the encoded polypeptide). When transiently transfected into mammalian cells, this construct drives the expression of a strongly fluorescent GFP chimera which selectively localizes to the mitochondria. We also describe two of the many possible applications of this recombinant GFP in physiological studies. The targeted chimera allows the visualization of mitochondrial movement in living cells. Also, unlike dyes such as rhodamine, it reveals morphological changes induced in mitochondria by drugs that collapse the organelle membrane potential. Moreover, when GFP is cotransfected with a membrane receptor, such as the alpha 1-adrenergic receptor, the fluorescence of the GFP in intact cells can be used in recognizing the transfected cells. Thus, specific changes in intracellular Ca2+ concentration that occur in cells expressing the recombinant receptor can be identified using a classical fluorescent Ca2+ indicator.
GFP is an invaluable new tool for studies of molecular biology and cell physiology. As a marker of transfection in vivo, it provides a simple means of identifying genetically modified cells to be used in physiological studies. More importantly, chimeric GFP, which in principle can be targeted to any subcellular location, can be used to monitor complex phenomena in intact living cells, such as changes in shape and distribution of organelles, and it has the potential to be used as a probe of physiological parameters.
最近有研究表明，维多利亚多管水母的绿色荧光蛋白（GFP）在原核生物（大肠杆菌）和真核生物（秀丽隐杆线虫和黑腹果蝇）活细胞中重组表达时仍保留其荧光特性；因此，它可作为体内基因表达的有力标记。重组GFP在细胞内的特异性靶向将使其可用于更多应用，但目前尚无关于将GFP靶向细胞内细胞器可能性的信息。
在本研究中，我们表明GFP cDNA可在培养的哺乳动物细胞中高水平表达；重组多肽具有高度荧光性，且仅定位于细胞质溶胶中。此外，我们对GFP cDNA进行了修饰，使其包含线粒体靶向序列（以及编码多肽氨基末端的强免疫表位）。当瞬时转染到哺乳动物细胞中时，该构建体驱动一种强荧光GFP嵌合体的表达，该嵌合体选择性地定位于线粒体。我们还描述了这种重组GFP在生理学研究中的许多可能应用中的两个。靶向嵌合体可使活细胞中线粒体的运动可视化。此外，与罗丹明等染料不同，它能揭示因药物使细胞器膜电位崩溃而在线粒体中诱导的形态变化。而且，当GFP与膜受体（如α1 - 肾上腺素能受体）共转染时，完整细胞中GFP的荧光可用于识别转染细胞。因此，使用经典的荧光钙指示剂可鉴定表达重组受体的细胞中细胞内Ca2+浓度的特定变化。
GFP是分子生物学和细胞生理学研究中一种非常有价值的新工具。作为体内转染的标记，它提供了一种简单的方法来识别用于生理学研究的基因修饰细胞。更重要的是，原则上可靶向任何亚细胞位置的嵌合GFP可用于监测完整活细胞中的复杂现象，如细胞器形状和分布的变化，并且有潜力用作生理参数的探针。
