The proposed studies focus on nuclear calcium and calmodulin - recently identified mediators for gene transcription, DNA replication and repair. Nuclear Ca2+ concentration is independently regulated as suggested by the finding that extracellular stimuli induce nuclear Ca2+ transients that can be much higher or lower than cytosolic ones. Nuclear CaM concentration also appears to be independently controlled as indicated by our recent discovery that the concentration of CaM can be highly enriched in the nucleus. Therefore, our first aim is to investigate the mechanisms that generates nuclear Ca2+ signals in rat basophilic leukemia cells (RBL-cells), a model cell line that triggers Ca2+ transients in response to antigenic stimuli. we have already synthesized an essential tool: a dextran based Ca2+ indicator that is targeted to the nucleus by a viral nuclear localization peptide. The probe is transported into the nucleus within a few minutes after microinjection. We have also synthesized an inhibitor of the IP3 receptor that is excluded from the nucleus. We will use these reagents in conjunction with UV-laser triggered uncaging of IP3 and Ca2+ to determine the rate of Ca2+ diffusion through nuclear pores and to identify the origin of nuclear Ca2+ signals - either from the inner nuclear membrane or form the perinuclear region. Differences in nuclear and cytosolic Ca2+ signals could also result from differential regulation of Ca2+ channels in the nuclear and cytosolic regions. This will be investigated by comparing the regulation of Ca2+ channels in isolated nuclei with those in microsomal fractions using a computer controlled 45Ca2+-flux assay.
Our second aim i s to investigate why CaM is concentrated in the nucleus of RBL-cells. This is relevant, because the strength of nuclear Ca2+/CaM signals can be controlled by regulating nuclear CaM concentration. We will measure the rate of diffusion or transport of CaM across the nuclear membrane using local UV-laser triggered uncaging caged FITC-CaM to the nucleus could also result from higher nuclear concentrations of CaM binding fluorescence anisotropy.
Our third aim i s to use the methods developed in RBL-cells to study nuclear Ca2+ and CaM in hippocampal neurons. Electrical signaling pathways in neuronal networks may be controlled by expression of new genes induced by nuclear Ca2+/CaM signals. We will identify the origin of nuclear Ca2+ signals and find out to what extent ryanodine receptors and IP3-receptors contribute to nuclear Ca2+ signaling. We will examine nuclear distribution and translocation of CaM and will determine whether increased concentrations of CaM and Ca2+ inside the nucleus lead to increased autophosphorylation of nuclear versus cytosolic Ca2+CaM-dependent protein kinase II.
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