Nagai T, Sawano A, Park E S, Miyawaki A
Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
Proc Natl Acad Sci U S A. 2001 Mar 13;98(6):3197-202. doi: 10.1073/pnas.051636098. Epub 2001 Mar 6.
To visualize Ca(2+)-dependent protein-protein interactions in living cells by fluorescence readouts, we used a circularly permuted green fluorescent protein (cpGFP), in which the amino and carboxyl portions had been interchanged and reconnected by a short spacer between the original termini. The cpGFP was fused to calmodulin and its target peptide, M13. The chimeric protein, which we have named "pericam," was fluorescent and its spectral properties changed reversibly with the amount of Ca(2+), probably because of the interaction between calmodulin and M13 leading to an alteration of the environment surrounding the chromophore. Three types of pericam were obtained by mutating several amino acids adjacent to the chromophore. Of these, "flash-pericam" became brighter with Ca(2+), whereas "inverse-pericam" dimmed. On the other hand, "ratiometric-pericam" had an excitation wavelength changing in a Ca(2+)-dependent manner. All of the pericams expressed in HeLa cells were able to monitor free Ca(2+) dynamics, such as Ca(2+) oscillations in the cytosol and the nucleus. Ca(2+) imaging using high-speed confocal line-scanning microscopy and a flash-pericam allowed to detect the free propagation of Ca(2+) ions across the nuclear envelope. Then, free Ca(2+) concentrations in the nucleus and mitochondria were simultaneously measured by using ratiometric-pericams having appropriate localization signals, revealing that extra-mitochondrial Ca(2+) transients caused rapid changes in the concentration of mitochondrial Ca(2+). Finally, a "split-pericam" was made by deleting the linker in the flash-pericam. The Ca(2+)-dependent interaction between calmodulin and M13 in HeLa cells was monitored by the association of the two halves of GFP, neither of which was fluorescent by itself.
为了通过荧光读数观察活细胞中依赖钙离子的蛋白质-蛋白质相互作用，我们使用了一种环状排列的绿色荧光蛋白（cpGFP），其氨基和羧基部分已互换，并通过原始末端之间的短间隔重新连接。cpGFP与钙调蛋白及其靶肽M13融合。我们将这种嵌合蛋白命名为“钙成像蛋白”，它具有荧光，其光谱特性随钙离子量的变化而可逆地改变，这可能是由于钙调蛋白与M13之间的相互作用导致发色团周围环境发生改变。通过对发色团附近的几个氨基酸进行突变，获得了三种类型的钙成像蛋白。其中，“闪光钙成像蛋白”在钙离子存在时会变亮，而“反向钙成像蛋白”会变暗。另一方面，“比率型钙成像蛋白”的激发波长以依赖钙离子的方式变化。在HeLa细胞中表达的所有钙成像蛋白都能够监测游离钙离子的动态变化，如细胞质和细胞核中的钙离子振荡。使用高速共聚焦线扫描显微镜和闪光钙成像蛋白进行钙离子成像，可以检测钙离子在核膜上的自由传播。然后，通过使用具有适当定位信号的比率型钙成像蛋白，同时测量细胞核和线粒体中的游离钙离子浓度，结果表明线粒体外的钙离子瞬变会导致线粒体钙离子浓度的快速变化。最后，通过删除闪光钙成像蛋白中的连接子制备了一种“分裂钙成像蛋白”。通过监测GFP两半部分的结合来检测HeLa细胞中钙调蛋白与M13之间依赖钙离子的相互作用，GFP的两半部分本身都不具有荧光。
