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

Abstract

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.

Common methods in mitochondrial research (Review)

“…The closer the two proteins are, the more fluorescence emitted by cFP is received by YFP and the less fluorescence is received by the detector. CFP and YFP are fused to calmodulin and calmodulin-binding peptide, respectively and expressed in the same cell (170)(171)(172)(173)(174)(175). When the intracellular ca 2+ concentration is high, the combination of calmodulin and the calmodulin-binding peptide can induce FRET and the receptor protein YFP emits yellow fluorescence, so the cells appear yellow.…”

“…When the intracellular ca 2+ concentration is high, the combination of calmodulin and the calmodulin-binding peptide can induce FRET and the receptor protein YFP emits yellow fluorescence, so the cells appear yellow. When the intracellular ca 2+ concentration is low, FRET hardly occurs, so cFP is excited and emits green fluorescence during detection and the cells appear green (170,171,175). FRET can detect intracellular ca 2+ , but cannot specifically detect mitochondrial Ca 2+ .…”