|
ИСТИНА |
Войти в систему Регистрация |
Интеллектуальная Система Тематического Исследования НАукометрических данных |
||
FRCaMP, A Red Fluorescent Genetically Encoded Calcium Indicator Based on Calmodulin from Schizosaccharomyces Pombe Fungusстатья
Статья опубликована в высокорейтинговом журнале
Статья опубликована в журнале из списка Web of Science и/или Scopus- Авторы: Subach 1 Oksana M., Barykina Natalia V., Chefanova Elizaveta S., Vlaskina Anna V., Sotskov Vladimir P., Ivashkina Olga I., Anokhin Konstantin V., Subach Fedor V.
- Журнал: International Journal of Molecular Sciences
- Том: 22
- Номер: 1
- Год издания: 2020
- Издательство: MDPI
- Местоположение издательства: Basel, Switzerland
- DOI: 10.3390/ijms22010111
- Аннотация: Red fluorescent genetically encoded calcium indicators (GECIs) have expanded the available pallet of colors used for the visualization of neuronal calcium activity in vivo. However, their calcium-binding domain is restricted by calmodulin from metazoans. In this study, we developed red GECI, called FRCaMP, using calmodulin (CaM) from Schizosaccharomyces pombe fungus as a calcium binding domain. Compared to the R-GECO1 indicator in vitro, the purified protein FRCaMP had similar spectral characteristics, brightness, and pH stability but a 1.3-fold lower ΔF/F calcium response and 2.6-fold tighter calcium affinity with Kd of 441 nM and 2.4–6.6-fold lower photostability. In the cytosol of cultured HeLa cells, FRCaMP visualized calcium transients with a ΔF/F dynamic range of 5.6, which was similar to that of R-GECO1. FRCaMP robustly visualized the spontaneous activity of neuronal cultures and had a similar ΔF/F dynamic range of 1.7 but 2.1-fold faster decay kinetics vs. NCaMP7. On electrically stimulated cultured neurons, FRCaMP demonstrated 1.8-fold faster decay kinetics and 1.7-fold lower ΔF/F values per one action potential of 0.23 compared to the NCaMP7 indicator. The fungus-originating CaM of the FRCaMP indicator version with a deleted M13-like peptide did not interact with the cytosolic environment of the HeLa cells in contrast to the metazoa-originating CaM of the similarly truncated version of the GCaMP6s indicator with a deleted M13-like peptide. Finally, we generated a split version of the FRCaMP indicator, which allowed the simultaneous detection of calcium transients and the heterodimerization of bJun/bFos interacting proteins in the nuclei of HeLa cells with a ΔF/F dynamic range of 9.4 and a contrast of 2.3–3.5, respectively.Keywords: genetically encoded calcium indicator; protein engineering; calcium imaging; Schizosaccharomyces pombe; FRCaMP; GECI; red fluorescent; fluorescent protein; split; bJun/bFos
- Добавил в систему: Анохин Константин Владимирович