Yokoe H, Meyer T
Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.
Nat Biotechnol. 1996 Oct;14(10):1252-6. doi: 10.1038/nbt1096-1252.
We describe a method of monitoring the spatial dynamics of proteins in intact cells by locally enhancing the blue excited fluorescence of green fluorescent protein (GFP) using a spatially focused ultraviolet-laser pulse. GFP fusion proteins were efficiently expressed by micro-electroporation of in vitro synthesized mRNA into adherent mammalian cells. We found that the diffusion coefficient of cycle 3 mutant GFP was 43 microns2/sec, compared to 4 microns2/sec for wild-type GFP, suggesting that cycle 3 GFP diffuses freely in mammalian cells and is ideally suited as a fusion tag. The local fluorescence enhancement method was used to study the membrane dissociation rate of GFP-tagged K-ras, a small GTP binding protein that localizes to plasma membranes by a farnesyl lipid group and a polybasic region. Our data suggest that K-ras exists in a dynamic equilibrium and rapidly switches between a plasma membrane bound form and a cytosolic form with a plasma membrane dissociation time constant of 1.5 sec.
我们描述了一种通过使用空间聚焦的紫外激光脉冲局部增强绿色荧光蛋白（GFP）的蓝色激发荧光来监测完整细胞中蛋白质空间动力学的方法。通过将体外合成的mRNA微电穿孔导入贴壁哺乳动物细胞中，高效表达了GFP融合蛋白。我们发现，第3代突变型GFP的扩散系数为43平方微米/秒，而野生型GFP为4平方微米/秒，这表明第3代GFP在哺乳动物细胞中自由扩散，非常适合作为融合标签。局部荧光增强方法被用于研究GFP标记的K-ras的膜解离速率，K-ras是一种小GTP结合蛋白，通过法尼基脂质基团和多碱性区域定位于质膜。我们的数据表明，K-ras处于动态平衡中，并以1.5秒的质膜解离时间常数在质膜结合形式和胞质形式之间快速转换。
