Neuroscience Institute, National Research Council (CNR), 35131 Padua, Italy.
Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy.
Int J Mol Sci. 2021 Sep 14;22(18):9945. doi: 10.3390/ijms22189945.
Calcium (Ca) exerts a pivotal role in controlling both physiological and detrimental cellular processes. This versatility is due to the existence of a cell-specific molecular Ca toolkit and its fine subcellular compartmentalization. Study of the role of Ca in cellular physiopathology greatly benefits from tools capable of quantitatively measuring its dynamic concentration ([Ca]) simultaneously within organelles and in the cytosol to correlate localized and global [Ca] changes. To this aim, as nucleoplasm Ca changes mirror those of the cytosol, we generated a novel nuclear-targeted version of a Föster resonance energy transfer (FRET)-based Ca probe. In particular, we modified the previously described nuclear Ca sensor, H2BD3cpv, by substituting the donor ECFP with mCerulean3, a brighter and more photostable fluorescent protein. The thorough characterization of this sensor in HeLa cells demonstrated that it significantly improved the brightness and photostability compared to the original probe, thus obtaining a probe suitable for more accurate quantitative Ca measurements. The affinity for Ca was determined in situ. Finally, we successfully applied the new probe to confirm that cytoplasmic and nucleoplasmic Ca levels were similar in both resting conditions and upon cell stimulation. Examples of simultaneous monitoring of Ca signal dynamics in different subcellular compartments in the very same cells are also presented.
钙（Ca）在控制生理和有害的细胞过程中起着关键作用。这种多功能性是由于存在细胞特异性分子 Ca 工具包及其精细的亚细胞区室化。研究 Ca 在细胞生理病理学中的作用极大地受益于能够定量测量其动态浓度([Ca])的工具，同时在细胞器和细胞质内测量，以关联局部和全局[Ca]变化。为此，由于核质 Ca 的变化反映了细胞质的变化，我们生成了一种新型的基于荧光共振能量转移（FRET）的 Ca 探针的核靶向版本。具体来说，我们通过用 mCerulean3 取代供体 ECFP，对先前描述的核 Ca 传感器 H2BD3cpv 进行了修饰，mCerulean3 是一种更亮、更稳定的荧光蛋白。该传感器在 HeLa 细胞中的全面表征表明，与原始探针相比，它显著提高了亮度和光稳定性，从而获得了一种更适合更准确定量 Ca 测量的探针。Ca 的亲和力是在原位确定的。最后，我们成功地应用了新探针来证实，在静息状态和细胞刺激时，细胞质和核质中的 Ca 水平相似。还呈现了在同一细胞中同时监测不同亚细胞区室中 Ca 信号动力学的示例。
