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.
Highlights
The pleiotropic effects of Ca2+ changes on cell functions depend on their amplitude, duration, and subcellular localization as well as on their origin, i.e., whether they are caused by the influx of Ca2+ across the plasma membrane (PM) or its release from intracellular stores [1]
Starting from H2BD3cpv, the donor was substituted and the linker between the two Ca2+-responsive elements, Calmodulin and M13, was elongated with 16 glycine residues to generate H2BD3mCerulean3+16. This approach has been successfully employed in the recovery of the dynamic range reduction induced by the donor substitution of the mitochondrial Cameleon 4mtD3mCerulean3+16 that we previously generated [16]
In Föster resonance energy transfer (FRET)-based genetically encoded Ca2+ indicators (GECIs), the dynamic range is calculated as Rmax/Rmin, where Rmax is the R obtained under Ca2+ saturating conditions, while Rmin is the R obtained in the absence of Ca2+ and in the presence of ethylene glycol tetra acetic acid (EGTA)
Summary
The pleiotropic effects of Ca2+ changes on cell functions depend on their amplitude, duration, and subcellular localization as well as on their origin, i.e., whether they are caused by the influx of Ca2+ across the plasma membrane (PM) or its release from intracellular stores [1]. Chemical dyes can be in part trapped in the ER lumen and within the NE, and the high [Ca2+] in this compartment can be misinterpreted as a localized high nucleoplasmic [Ca2+] [6] For these reasons, nucleus-targeted genetically encoded Ca2+ indicators (GECIs) appear the best choice to investigate nuclear Ca2+ homeostasis as they ensure a very specific localization. To maximize the probe performance, we corrected some drawbacks of H2BD3cpv, i.e., the low fluorescence of donor ECFP and its photobleaching, by substituting the ECFP with mCerulean and by modifying the D3 domain [16] We thoroughly characterized this new nuclear-targeted Cameleon in situ in HeLa cells, demonstrating that the probe based on mCerluean significantly improved the brightness and photostability. The new probe, together with other sensors targeted to mitochondria and ER, was successfully used to simultaneously monitor nuclear and organelle Ca2+ signaling in response to various cell stimuli
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
