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Article

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

by 1,2,†, 1,2,†, 1,2, 1,2, 1,2,3 and 1,2,*
1
Neuroscience Institute, National Research Council (CNR), 35131 Padua, Italy
2
Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy
3
Venetian Institute of Molecular Medicine (VIMM), 35131 Padua, Italy
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Academic Editor: Kiryl D. Piatkevich
Int. J. Mol. Sci. 2021, 22(18), 9945; https://doi.org/10.3390/ijms22189945
Received: 18 August 2021 / Revised: 9 September 2021 / Accepted: 12 September 2021 / Published: 14 September 2021
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. View Full-Text
Keywords: calcium; nucleus; nuclear; FRET-based probe; endoplasmic reticulum; Cameleon; IP3 receptor; SOCE calcium; nucleus; nuclear; FRET-based probe; endoplasmic reticulum; Cameleon; IP3 receptor; SOCE
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MDPI and ACS Style

Galla, L.; Vajente, N.; Pendin, D.; Pizzo, P.; Pozzan, T.; Greotti, E. Generation and Characterization of a New FRET-Based Ca2+ Sensor Targeted to the Nucleus. Int. J. Mol. Sci. 2021, 22, 9945. https://doi.org/10.3390/ijms22189945

AMA Style

Galla L, Vajente N, Pendin D, Pizzo P, Pozzan T, Greotti E. Generation and Characterization of a New FRET-Based Ca2+ Sensor Targeted to the Nucleus. International Journal of Molecular Sciences. 2021; 22(18):9945. https://doi.org/10.3390/ijms22189945

Chicago/Turabian Style

Galla, Luisa, Nicola Vajente, Diana Pendin, Paola Pizzo, Tullio Pozzan, and Elisa Greotti. 2021. "Generation and Characterization of a New FRET-Based Ca2+ Sensor Targeted to the Nucleus" International Journal of Molecular Sciences 22, no. 18: 9945. https://doi.org/10.3390/ijms22189945

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