Fig. 4

Nrz Binds to IP3R1 via Its BH4 Domain and Controls Intracellular Ca²⁺ Levels in Zebrafish Embryos

(A–D) Fluorescence microscopy images of control embryos (A), nrz morphants (B), WT embryos treated with thapsigargin (C), or nrz morphants treated with or 2-APB (D); embryos were stained with Oregon Green BAPTA-1 AM (false colors, oblong-sphere stage). Increased signal in thapsigargin-treated embryos and nrz morphants reveals the release of free Ca²⁺ in the region of the margin from which originates the YSL. In (A′–D′), bright-field images of the same embryos are shown. Scale bar: 100 μm.

(E) Quantification of Oregon Green BAPTA-1 AM staining of control embryos and of nrz morphants injected or not with nrzcytb5 or nrzmaob mRNAs or treated with 2-APB (50 μM); embryos treated with thapsigargin alone (20 μM) were used as positive controls (mean ± SD; three independent experiments; ^*p < 0.01 versus control embryos; #p > 0.9 versus control embryos).

(F) Effect of IP3R inhibition with 2-APB on the mortality of zebrafish embryos. Control embryos (WT) and nrz morphants (MO) were incubated or not with 2-APB (50 μM) from 512 cells to oblong-sphere stage. Mortality was evaluated at indicated times after the beginning of the incubation (mean ± SD; three independent experiments).

(G and H) Immunoprecipitation experiments with protein extracts from transfected HeLa cells: Nrz and NrzCytB5 coimmunoprecipitated with the IP3R1 channel (G); Nrz but not ΔBH4Nrz was able to interact with IP3R1 (H); the Flag-tagged BH4 peptide domain inhibited Nrz/IP3R1 interaction. Bottom: densitometry quantification, Nrz IP/input ratios obtained by calculating the ratio of IP band intensity (lanes “IB:Nrz”) to the input band intensity (lanes “Input Nrz”).

(I) Time course analysis of nrz morphants injected or not with in vitro synthesized ΔBH4NrzCytB5 mRNA. Ectopic expression of ΔBH4NrzCytB5 is unable to prevent nrzMO lethal phenotype (mean ± SD; three independent experiments). See also Figure S3.