Imaging of the intracellular topography of copper with a fluorescent sensor and by synchrotron x-ray fluorescence microscopy
Imaging of the intracellular topography of copper with a fluorescent sensor and by synchrotron x-ray fluorescence microscopy
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DOI：
10.1073/pnas.0406547102
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发表时间：
2005-08-09
影响因子：
11.1
通讯作者：
Fahrni, CJ
中科院分区：
文献类型：
--
作者：
Yang, LC;McRae, R;Fahrni, CJ
铜是一种基本的微量营养素，在广泛的生物过程中发挥着核心作用。尽管有令人信服的证据表明细胞内环境不包含任何游离铜离子，但铜的快速吸收和释放动力学表明细胞内存在不稳定的铜库。为了阐明这个池的亚细胞定位，我们合成并表征了一种膜透过性的铜选择性荧光传感器(CTAP-1)。加入Cu(I)后，该传感器的发射增强了4.6倍，量子产额达到14%。该传感器对铜(I)具有良好的选择性，其发射响应不受毫摩尔浓度的钙(II)或镁(II)离子的影响。变温动态核磁共振研究表明，在室温下，铜(I)存在快速的自交换平衡，其活化势垒与10(5)S(-1)相近，活化势垒为ΔG=44kJ(-1)。与传感器孵育的小鼠成纤维细胞(3T3)产生了铜依赖的核周染色图案，该图案与线粒体和高尔基体的亚细胞位置共存。为了评价和确认该传感器对铜的选择性，我们用同步加速器X射线荧光显微镜测量了铜的亚细胞形貌。此外，不同亚细胞位置的微探针X射线吸收测量显示了一种近边缘特征，这是低配位单价铜的特征，但与已公布的金属硫蛋白或谷胱甘肽的光谱不同。所提供的数据为动力学不稳定的铜池提供了强有力的证据，该铜池主要位于线粒体和高尔基体中。
Copper is an essential micronutrient that plays a central role for a broad range of biological processes. Although there is compelling evidence that the intracellular milieu does not contain any free copper ions, the rapid kinetics of copper uptake and release suggests the presence of a labile intracellular copper pool. To elucidate the subcellular localization of this pool, we have synthesized and characterized a membrane-permeable, copper-selective fluorescent sensor (CTAP-1). Upon addition of Cu(I), the sensor exhibits a 4.6-fold emission enhancement and reaches a quantum yield of 14%. The sensor exhibits excellent selectivity toward Cu(I), and its emission response is not compromised by the presence of millimolar concentrations of Ca(II) or Mg(II) ions. Variable temperature dynamic NMR studies revealed a rapid Cu(I) self-exchange equilibrium with a low activation barrier of Delta G(double dagger) = 44 kJ(.)mol(-1) and k(obs) similar to 10(5) s(-1) at room temperature. Mouse fibroblast cells (3T3) incubated with the sensor produced a copper-dependent perinuclear staining pattern, which colocalizes with the subcellular locations of mitochondria and the Golgi apparatus. To evaluate and confirm the sensor's copper-selectivity, we determined the subcellular topography of copper by synchrotron-based x-ray fluorescence microscopy. Furthermore, microprobe x-ray absorption measurements at various subcellular locations showed a near-edge feature that is characteristic for low-coordinate monovalent copper but does not resemble the published spectra for metallothionein or glutathione. The presented data provide a coherent picture with strong evidence for a kinetically labile copper pool, which is predominantly localized in the mitochondria and the Golgi apparatus.