Nuclear Ca2+ concentration measured with specifically targeted recombinant aequorin.
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.
Brini
M
M
Department of Biomedical Sciences, University of Padova, Italy.
Murgia
M
M
Pasti
L
L
Picard
D
D
Pozzan
T
T
Rizzuto
R
R
D000331:Aequorin / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D003001:Cloning, Molecular; D006367:HeLa Cells; D006632:Histamine / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D000331:Aequorin; D006632:Histamine; D002118:Calcium