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Title: An in-cell NMR study of monitoring stress-induced increase of cytosolic Ca{sup 2+} concentration in HeLa cells

Highlights: •We performed time-resolved NMR observations of calbindin D{sub 9k} in HeLa cells. •Stress-induced increase of cytosolic Ca{sup 2+} concentration was observed by in-cell NMR. •Calbindin D{sub 9k} showed the state-transition from Mg{sup 2+}- to Ca{sup 2+}-bound state in cells. •We provide a useful tool for in situ monitoring of the healthiness of the cells. -- 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 Ca{sup 2+}-bursts occur in HeLa cells under various stresses, hence the cytosolic Ca{sup 2+} 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 Ca{sup 2+} concentration during experiments, human calbindin D{sub 9k} (P47M + C80) was used as the model protein and cultured HeLa cells as host cells. Time-resolved measurements of 2D {sup 1}H–{sup 15}N SOFAST–HMQC experiments of calbindin D{sub 9k} (P47M +more » C80) in HeLa cells showed time-dependent changes in the cross-peak patterns in the spectra. Comparison with in vitro assignments revealed that calbindin D{sub 9k} (P47M + C80) is initially in the Mg{sup 2+}-bound state, and then gradually converted to the Ca{sup 2+}-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 Ca{sup 2+} 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.« less
Authors:
; ; ; ; ; ;  [1] ;  [2] ;  [3] ;  [4] ;  [1]
  1. Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji-shi, Tokyo 192-0373 (Japan)
  2. Cellular and Molecular Biology Unit, RIKEN Advanced Science Institute, Wako-shi, Saitama 351-0198 (Japan)
  3. Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 B-1, Nagatsuda-chou, Midori-ku, Yokohama, Kanagawa 226-8501 (Japan)
  4. Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8510 (Japan)
Publication Date:
OSTI Identifier:
22242077
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 438; Journal Issue: 4; Other Information: Copyright (c) 2013 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; BOUND STATE; CALCIUM IONS; CONCENTRATION RATIO; CULTURE MEDIA; ECOLOGICAL CONCENTRATION; HELA CELLS; IN VITRO; NUCLEAR MAGNETIC RESONANCE; NUTRIENTS; PRECIPITATION; PROTEINS; SAMPLE PREPARATION; TIME DEPENDENCE; TIME RESOLUTION