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The Bcl-2 Homolog Nrz Inhibits Binding of IP3 to Its Receptor to Control Calcium Signaling During Zebrafish Epiboly

Phosphorylation of a Bcl-2–like protein allows calcium signals needed for the early stages of development. Nrz-IP3 Tug-of-War During early development of zebrafish, cells migrate over the yolk in a process known as epiboly. Epiboly requires Ca2+-dependent myosin contraction along actin filaments at the leading edge of the migratory epithelium. Growth factors stimulate production of inositol trisphosphate (IP3), which binds to and activates the IP3 receptor, a Ca2+ channel on the endoplasmic reticulum (ER). Bonneau et al. found that the Bcl-2–like protein Nrz was phosphorylated in zebrafish during early epiboly and that replacing endogenous Nrz with a mutant that could not be phosphorylated disrupted normal Ca2+ oscillations, actin assembly at the leading edge, and epiboly progression. In human cultured cells, wild-type Nrz, but not Nrz with phosphomimetic mutations, bound to the IP3 receptor, prevented its interaction with IP3, and blocked Ca2+ release from the ER. Thus, these data suggest that phosphorylation of Nrz during early epiboly enables IP3 to bind to its receptor and promote Ca2+ waves that are important for assembly of the actin-myosin ring required for cell migration. Members of the Bcl-2 protein family regulate mitochondrial membrane permeability and also localize to the endoplasmic reticulum where they control Ca2+ homeostasis by interacting with inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs). In zebrafish, Bcl-2–like 10 (Nrz) is required for Ca2+ signaling during epiboly and gastrulation. We characterized the mechanism by which Nrz controls IP3-mediated Ca2+ release during this process. We showed that Nrz was phosphorylated during early epiboly, and that in embryos in which Nrz was knocked down, reconstitution with Nrz bearing mutations designed to prevent its phosphorylation disrupted cyclic Ca2+ transients and the assembly of the actin-myosin ring and led to epiboly arrest. In cultured cells, wild-type Nrz, but not Nrz with phosphomimetic mutations, interacted with the IP3 binding domain of IP3R1, inhibited binding of IP3 to IP3R1, and prevented histamine-induced increases in cytosolic Ca2+. Collectively, these data suggest that Nrz phosphorylation is necessary for the generation of IP3-mediated Ca2+ transients and the formation of circumferential actin-myosin cables required for epiboly. Thus, in addition to their role in apoptosis, by tightly regulating Ca2+ signaling, Bcl-2 family members participate in the cellular events associated with early vertebrate development, including cytoskeletal dynamics and cell movement.