Figure 4.

Nuclear Ca²⁺ Oscillations Are Generated by Nuclear InsP₃ Receptors and Are Effective in Maintaining NFAT4 Activity

(A) Immunostaining reveals the presence of type I InsP₃ receptors in the cytoplasm and nucleus. No staining is seen when secondary antibody (Sec. AB) alone is used. Secondary antibody was conjugated with Alexa 488 for visualization.

(B) Western blots of cytoplasmic and nuclear extract show the presence of type I InsP₃ receptors in both compartments. Similar results were obtained in two independent experiments.

(C) Immunogold labeling reveals the presence of type I InsP₃ receptors in the inner nuclear membrane. Left-hand panel denotes a control micrograph and the right-hand one after immunogold labeling of type I InsP₃ receptors. Red arrows denote InsP₃R on the inner nuclear membrane, black arrow shows receptor on the outer nuclear membrane, and gray ones denote receptor in the endoplasmic reticulum.

(D) Comparison of nuclear Ca²⁺ signals to LTC₄ in a control cell and in one in which the cytoplasm and nucleoplasm had been loaded with EGTA via the form EGTA-AM.

(E) Loading the cell with EGTA reduces NFAT4-cherry nuclear migration in response to LTC₄. Migration can be rescued by stimulating cells with ionomycin (2 μM; 20 min).

(F) Aggregate data from several experiments as in (E) are summarized. Open bar above EGTA-AM denotes the extent of rescue by ionomycin. Data are represented as mean ± SEM.

(G) Graph plots NFAT4-cherry nuclear accumulation against the integrated nuclear Ca²⁺ signal in control cells and cells loaded with EGTA. Each point depicts NFAT4 movement and integrated nuclear Ca²⁺ from the same cell. The y axis represents peak NFAT nuclear/cytoplasmic ratio after stimulation with LTC₄ minus the NFAT nuclear/cytoplasmic ratio before stimulation.

(H) Nuclear Ca²⁺ oscillations evoked by LTC₄ run down quickly when Ca²⁺ entry through CRAC channels is blocked with 10 μM BTP2.

(I) Estimated nuclear Ca²⁺ concentration for an oscillatory response in a control cell is compared with a plateau-type response in an EGTA-loaded cell, following stimulation with 160 nM LTC₄. To calibrate the nuclear Ca²⁺ signal in intact cells, following expression of NGCaMP3, Rmin and Rmax were obtained by stimulating cells with 5 μM ionomycin either in Ca²⁺-free external solution (with fluorescence measured ∼15 min later) or 10 mM Ca²⁺ external solution, respectively. The K_D for nuclear GCaMP3 was taken as 660 nM from the literature. The estimated Ca²⁺ concentrations are, at best, only an approximation (see text). Arrow denotes LTC₄ application.

(J) The graph plots the predicted build-up of nuclear Ca²⁺-calmodulin-calcineurin following stimulation with LTC₄ in a control cell (red trace) and in one loaded with EGTA (blue trace).

(K) Contour plot depicts active nuclear calcineurin as a function of nuclear Ca²⁺ concentration and total nuclear calcineurin available.

See also Figure S4, Table S1, and description of the model in Supplemental information.