Generation and Characterization of a New FRET-Based Ca²⁺ Sensor Targeted to the Nucleus

Calcium (Ca²⁺) exerts a pivotal role in controlling both physiological and detrimental cellular processes. This versatility is due to the existence of a cell-specific molecular Ca²⁺ toolkit and its fine subcellular compartmentalization. Study of the role of Ca²⁺ in cellular physiopathology greatly benefits from tools capable of quantitatively measuring its dynamic concentration ([Ca²⁺]) simultaneously within organelles and in the cytosol to correlate localized and global [Ca²⁺] changes. To this aim, as nucleoplasm Ca²⁺ changes mirror those of the cytosol, we generated a novel nuclear-targeted version of a Föster resonance energy transfer (FRET)-based Ca²⁺ probe. In particular, we modified the previously described nuclear Ca²⁺ 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 Ca²⁺ measurements. The affinity for Ca²⁺ was determined in situ. Finally, we successfully applied the new probe to confirm that cytoplasmic and nucleoplasmic Ca²⁺ levels were similar in both resting conditions and upon cell stimulation. Examples of simultaneous monitoring of Ca²⁺ signal dynamics in different subcellular compartments in the very same cells are also presented.