An in-cell NMR study of monitoring stress-induced increase of cytosolic Ca2+ concentration in HeLa cells

Dambarudhar Shiba Sankar Hembram, Takahiro Haremaki, Jumpei Hamatsu, Jin Inoue, Hajime Kamoshida, Teppei Ikeya, Masaki Mishima, Tsutomu Mikawa, Nobuhiro Hayashi, Masahiro Shirakawa, Yutaka Ito

Research output: Contribution to journalArticlepeer-review

20 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)653-659
Number of pages7
JournalBiochemical and biophysical research communications
Volume438
Issue number4
DOIs
Publication statusPublished - 2013 Sep 6
Externally publishedYes

Keywords

  • Calbindin D
  • Cytosolic Ca concentration
  • HeLa cells
  • In-cell NMR

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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