TY - JOUR
T1 - An in-cell NMR study of monitoring stress-induced increase of cytosolic Ca2+ concentration in HeLa cells
AU - Hembram, Dambarudhar Shiba Sankar
AU - Haremaki, Takahiro
AU - Hamatsu, Jumpei
AU - Inoue, Jin
AU - Kamoshida, Hajime
AU - Ikeya, Teppei
AU - Mishima, Masaki
AU - Mikawa, Tsutomu
AU - Hayashi, Nobuhiro
AU - Shirakawa, Masahiro
AU - Ito, Yutaka
N1 - Funding Information:
The authors thank Drs Markus Wälchli and Jonathan Heddle for the critical reading of the manuscript. This work was supported in part by the Funding Program for Next Generation World-Leading Researchers (NEXT Program) from Japan Society for the Promotion of Science (JSPS) and by the Asian Human Resources Fund of Tokyo Metropolitan Government (under Project Asian Net-work for Major Cities 21). DSSH thanks the scholarship from the Asian Human Resources Fund. JH thanks the Research Fellowship for Young Scientists (DC1) from JSPS.
PY - 2013/9/6
Y1 - 2013/9/6
N2 - Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. The lifetime of in-cell NMR samples is however much shorter than that in culture media, presumably because of various stresses as well as the nutrient depletion in the anaerobic environment within the NMR tube. It is well known that Ca2+-bursts occur in HeLa cells under various stresses, hence the cytosolic Ca2+ concentration can be regarded as a good indicator of the healthiness of cells in NMR tubes. In this study, aiming at monitoring the states of proteins resulting from the change of cytosolic Ca2+ concentration during experiments, human calbindin D9k (P47M+C80) was used as the model protein and cultured HeLa cells as host cells.Time-resolved measurements of 2D 1H-15N SOFAST-HMQC experiments of calbindin D9k (P47M+C80) in HeLa cells showed time-dependent changes in the cross-peak patterns in the spectra. Comparison with in vitro assignments revealed that calbindin D9k (P47M+C80) is initially in the Mg2+-bound state, and then gradually converted to the Ca2+-bound state. This conversion process initiates after NMR sample preparation. These results showed, for the first time, that cells inside the NMR tube were stressed, presumably because of cell precipitation, the lack of oxygen and nutrients, etc., thereby releasing Ca2+ into cytosol during the measurements. The results demonstrated that in-cell NMR can monitor the state transitions of stimulated cells through the observation of proteins involved in the intracellular signalling systems. Our method provides a very useful tool for in situ monitoring of the "healthiness" of the cells in various in-cell NMR studies.
AB - Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. The lifetime of in-cell NMR samples is however much shorter than that in culture media, presumably because of various stresses as well as the nutrient depletion in the anaerobic environment within the NMR tube. It is well known that Ca2+-bursts occur in HeLa cells under various stresses, hence the cytosolic Ca2+ concentration can be regarded as a good indicator of the healthiness of cells in NMR tubes. In this study, aiming at monitoring the states of proteins resulting from the change of cytosolic Ca2+ concentration during experiments, human calbindin D9k (P47M+C80) was used as the model protein and cultured HeLa cells as host cells.Time-resolved measurements of 2D 1H-15N SOFAST-HMQC experiments of calbindin D9k (P47M+C80) in HeLa cells showed time-dependent changes in the cross-peak patterns in the spectra. Comparison with in vitro assignments revealed that calbindin D9k (P47M+C80) is initially in the Mg2+-bound state, and then gradually converted to the Ca2+-bound state. This conversion process initiates after NMR sample preparation. These results showed, for the first time, that cells inside the NMR tube were stressed, presumably because of cell precipitation, the lack of oxygen and nutrients, etc., thereby releasing Ca2+ into cytosol during the measurements. The results demonstrated that in-cell NMR can monitor the state transitions of stimulated cells through the observation of proteins involved in the intracellular signalling systems. Our method provides a very useful tool for in situ monitoring of the "healthiness" of the cells in various in-cell NMR studies.
KW - Calbindin D
KW - Cytosolic Ca concentration
KW - HeLa cells
KW - In-cell NMR
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U2 - 10.1016/j.bbrc.2013.07.127
DO - 10.1016/j.bbrc.2013.07.127
M3 - Article
C2 - 23933251
AN - SCOPUS:84883307703
VL - 438
SP - 653
EP - 659
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 4
ER -