Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1993 Dec;12(12):4813–4819. doi: 10.1002/j.1460-2075.1993.tb06170.x

Nuclear Ca2+ concentration measured with specifically targeted recombinant aequorin.

M Brini 1, M Murgia 1, L Pasti 1, D Picard 1, T Pozzan 1, R Rizzuto 1
PMCID: PMC413931  PMID: 8223490

Abstract

Activation of nuclear transcription factors, breakdown of nuclear envelope and apoptosis represent a group of nuclear events thought to be modulated by changes in nucleoplasmic Ca2+ concentration, [Ca2+]n. Direct evidence for, or against, this possibility has been, however, difficult to obtain because measurements of [Ca2+]n are hampered by major technical problems. Here we describe a new approach for selectively monitoring Ca2+ concentrations inside the nucleus of living cells, which is based on the construction of a chimeric cDNA encoding a fusion protein composed of the photoprotein aequorin and a nuclear translocation signal derived from the rat glucocorticoid receptor. This modified aequorin (nuAEQ), stably expressed in HeLa cells, was largely confined to the nucleoplasm and thus utilized for monitoring [Ca2+]n in intact cells. No significant differences were observed between [Ca2+]n and cytosolic Ca2+ concentration ([Ca2+]i) under resting conditions. Upon stimulation of surface receptors linked to inositol-1,4,5-trisphosphate (InsP3) generation, and thus to intracellular Ca2+ signalling, the kinetics of [Ca2+]i and [Ca2+]n increases were indistinguishable. However, for the same rise in [Ca2+]i, the amplitude of [Ca2+]n increase was larger when evoked by Ca2+ mobilization from internal stores than when induced by Ca2+ influx across the plasma membrane. The functional significance of these transient nucleus-cytosol Ca2+ gradients is discussed.

Full text

PDF
4813

Images in this article

4814Image
on p.4814
4815Image
on p.4815

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arslan P., Di Virgilio F., Beltrame M., Tsien R. Y., Pozzan T. Cytosolic Ca2+ homeostasis in Ehrlich and Yoshida carcinomas. A new, membrane-permeant chelator of heavy metals reveals that these ascites tumor cell lines have normal cytosolic free Ca2+. J Biol Chem. 1985 Mar 10;260(5):2719–2727. [PubMed] [Google Scholar]
  2. Bading H., Ginty D. D., Greenberg M. E. Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways. Science. 1993 Apr 9;260(5105):181–186. doi: 10.1126/science.8097060. [DOI] [PubMed] [Google Scholar]
  3. Berridge M. J. Inositol trisphosphate and calcium signalling. Nature. 1993 Jan 28;361(6410):315–325. doi: 10.1038/361315a0. [DOI] [PubMed] [Google Scholar]
  4. Berridge M. J., Irvine R. F. Inositol phosphates and cell signalling. Nature. 1989 Sep 21;341(6239):197–205. doi: 10.1038/341197a0. [DOI] [PubMed] [Google Scholar]
  5. Birch B. D., Eng D. L., Kocsis J. D. Intranuclear Ca2+ transients during neurite regeneration of an adult mammalian neuron. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):7978–7982. doi: 10.1073/pnas.89.17.7978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cobbold P. H., Rink T. J. Fluorescence and bioluminescence measurement of cytoplasmic free calcium. Biochem J. 1987 Dec 1;248(2):313–328. doi: 10.1042/bj2480313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Collart M. A., Tourkine N., Belin D., Vassalli P., Jeanteur P., Blanchard J. M. c-fos gene transcription in murine macrophages is modulated by a calcium-dependent block to elongation in intron 1. Mol Cell Biol. 1991 May;11(5):2826–2831. doi: 10.1128/mcb.11.5.2826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Connor J. A. Intracellular calcium mobilization by inositol 1,4,5-trisphosphate: intracellular movements and compartmentalization. Cell Calcium. 1993 Mar;14(3):185–200. doi: 10.1016/0143-4160(93)90066-f. [DOI] [PubMed] [Google Scholar]
  9. Davis R. L., Weintraub H., Lassar A. B. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell. 1987 Dec 24;51(6):987–1000. doi: 10.1016/0092-8674(87)90585-x. [DOI] [PubMed] [Google Scholar]
  10. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  11. Gaido M. L., Cidlowski J. A. Identification, purification, and characterization of a calcium-dependent endonuclease (NUC18) from apoptotic rat thymocytes. NUC18 is not histone H2B. J Biol Chem. 1991 Oct 5;266(28):18580–18585. [PubMed] [Google Scholar]
  12. Garcia-Bustos J., Heitman J., Hall M. N. Nuclear protein localization. Biochim Biophys Acta. 1991 Mar 7;1071(1):83–101. doi: 10.1016/0304-4157(91)90013-m. [DOI] [PubMed] [Google Scholar]
  13. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  14. Himpens B., De Smedt H., Droogmans G., Casteels R. Differences in regulation between nuclear and cytoplasmic Ca2+ in cultured smooth muscle cells. Am J Physiol. 1992 Jul;263(1 Pt 1):C95–105. doi: 10.1152/ajpcell.1992.263.1.C95. [DOI] [PubMed] [Google Scholar]
  15. Inouye S., Noguchi M., Sakaki Y., Takagi Y., Miyata T., Iwanaga S., Miyata T., Tsuji F. I. Cloning and sequence analysis of cDNA for the luminescent protein aequorin. Proc Natl Acad Sci U S A. 1985 May;82(10):3154–3158. doi: 10.1073/pnas.82.10.3154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Klehr D., Schlake T., Maass K., Bode J. Scaffold-attached regions (SAR elements) mediate transcriptional effects due to butyrate. Biochemistry. 1992 Mar 31;31(12):3222–3229. doi: 10.1021/bi00127a025. [DOI] [PubMed] [Google Scholar]
  17. Lang I., Scholz M., Peters R. Molecular mobility and nucleocytoplasmic flux in hepatoma cells. J Cell Biol. 1986 Apr;102(4):1183–1190. doi: 10.1083/jcb.102.4.1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lehrach H., Diamond D., Wozney J. M., Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry. 1977 Oct 18;16(21):4743–4751. doi: 10.1021/bi00640a033. [DOI] [PubMed] [Google Scholar]
  19. Lerea L. S., McNamara J. O. Ionotropic glutamate receptor subtypes activate c-fos transcription by distinct calcium-requiring intracellular signaling pathways. Neuron. 1993 Jan;10(1):31–41. doi: 10.1016/0896-6273(93)90239-n. [DOI] [PubMed] [Google Scholar]
  20. Malgaroli A., Milani D., Meldolesi J., Pozzan T. Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells. J Cell Biol. 1987 Nov;105(5):2145–2155. doi: 10.1083/jcb.105.5.2145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mazzanti M., DeFelice L. J., Cohn J., Malter H. Ion channels in the nuclear envelope. Nature. 1990 Feb 22;343(6260):764–767. doi: 10.1038/343764a0. [DOI] [PubMed] [Google Scholar]
  22. Meldolesi J., Madeddu L., Pozzan T. Intracellular Ca2+ storage organelles in non-muscle cells: heterogeneity and functional assignment. Biochim Biophys Acta. 1990 Nov 12;1055(2):130–140. doi: 10.1016/0167-4889(90)90113-r. [DOI] [PubMed] [Google Scholar]
  23. Nicotera P., McConkey D. J., Jones D. P., Orrenius S. ATP stimulates Ca2+ uptake and increases the free Ca2+ concentration in isolated rat liver nuclei. Proc Natl Acad Sci U S A. 1989 Jan;86(2):453–457. doi: 10.1073/pnas.86.2.453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nomura M., Inouye S., Ohmiya Y., Tsuji F. I. A C-terminal proline is required for bioluminescence of the Ca(2+)-binding photoprotein, aequorin. FEBS Lett. 1991 Dec 16;295(1-3):63–66. doi: 10.1016/0014-5793(91)81385-l. [DOI] [PubMed] [Google Scholar]
  25. Picard D., Yamamoto K. R. Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBO J. 1987 Nov;6(11):3333–3340. doi: 10.1002/j.1460-2075.1987.tb02654.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rink T. J., Tsien R. Y., Pozzan T. Cytoplasmic pH and free Mg2+ in lymphocytes. J Cell Biol. 1982 Oct;95(1):189–196. doi: 10.1083/jcb.95.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rizzuto R., Simpson A. W., Brini M., Pozzan T. Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin. Nature. 1992 Jul 23;358(6384):325–327. doi: 10.1038/358325a0. [DOI] [PubMed] [Google Scholar]
  28. Ross C. A., Meldolesi J., Milner T. A., Satoh T., Supattapone S., Snyder S. H. Inositol 1,4,5-trisphosphate receptor localized to endoplasmic reticulum in cerebellar Purkinje neurons. Nature. 1989 Jun 8;339(6224):468–470. doi: 10.1038/339468a0. [DOI] [PubMed] [Google Scholar]
  29. Satoh T., Ross C. A., Villa A., Supattapone S., Pozzan T., Snyder S. H., Meldolesi J. The inositol 1,4,5,-trisphosphate receptor in cerebellar Purkinje cells: quantitative immunogold labeling reveals concentration in an ER subcompartment. J Cell Biol. 1990 Aug;111(2):615–624. doi: 10.1083/jcb.111.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  31. Stochaj U., Silver P. A. A conserved phosphoprotein that specifically binds nuclear localization sequences is involved in nuclear import. J Cell Biol. 1992 May;117(3):473–482. doi: 10.1083/jcb.117.3.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stricker S. A., Centonze V. E., Paddock S. W., Schatten G. Confocal microscopy of fertilization-induced calcium dynamics in sea urchin eggs. Dev Biol. 1992 Feb;149(2):370–380. doi: 10.1016/0012-1606(92)90292-o. [DOI] [PubMed] [Google Scholar]
  33. Tombes R. M., Simerly C., Borisy G. G., Schatten G. Meiosis, egg activation, and nuclear envelope breakdown are differentially reliant on Ca2+, whereas germinal vesicle breakdown is Ca2+ independent in the mouse oocyte. J Cell Biol. 1992 May;117(4):799–811. doi: 10.1083/jcb.117.4.799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tsien R. Y. Fluorescent probes of cell signaling. Annu Rev Neurosci. 1989;12:227–253. doi: 10.1146/annurev.ne.12.030189.001303. [DOI] [PubMed] [Google Scholar]
  35. Tsien R. Y., Pozzan T., Rink T. J. Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J Cell Biol. 1982 Aug;94(2):325–334. doi: 10.1083/jcb.94.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Waybill M. M., Yelamarty R. V., Zhang Y. L., Scaduto R. C., Jr, LaNoue K. F., Hsu C. J., Smith B. C., Tillotson D. L., Yu F. T., Cheung J. Y. Nuclear calcium gradients in cultured rat hepatocytes. Am J Physiol. 1991 Jul;261(1 Pt 1):E49–E57. doi: 10.1152/ajpendo.1991.261.1.E49. [DOI] [PubMed] [Google Scholar]
  37. Williams D. A., Fogarty K. E., Tsien R. Y., Fay F. S. Calcium gradients in single smooth muscle cells revealed by the digital imaging microscope using Fura-2. Nature. 1985 Dec 12;318(6046):558–561. doi: 10.1038/318558a0. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES