IP₃-dependent Ca²⁺ release dynamics during maturation. A, kinase cascades driving Xenopus oocyte maturation. B, oocytes were injected with caged-IP₃ and Oregan Green 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis 1 before imaging. Maturation was induced with progesterone, and cells were collected at different time points as indicated. Cells were imaged in line scan mode on a Zeiss LSM510 with the near UV 450 nm laser continuously on, at low intensity to produce a slow gradual IP₃ rise. After imaging each cell was lysed and analyzed individually for the activation state of MAPK and MPF. MPF was assayed using an anti-phospho-Tyr-15-cdk1 antibody (arrow). Dephosphorylation is indicative of MPF activation. MAPK activation was detected using a phospho-specific MAPK antibody (arrowhead). Tubulin was the loading control (dash). C, percent of cells at each time point that either exhibit no release for the duration of the line scan (No Rel., black), puffs only (puffs, green), puffs followed by a wave (Puff-Wave, blue), or only a Ca²⁺ wave (Wave, red). For each time point n = 11–23 cells. D, amplitude of the first peak during the line scan as compared with the maximal Ca²⁺ signal. Mean ± S.E. (n = 9–18). E, latency until the first Ca²⁺ signal (Time to first peak) as compared with the time required to reach maximal signal (Time to Max). Mean ± S.E. (n = 9–18). For C–E: oocytes (Ooc); cells treated with progesterone that have not undergone GVBD at 2 or more hours after progesterone (p > 2); cells at GVBD and up to 0.5 h after GVBD (GVBD 0–0.5); cells from 0.5 to 2.5 h after GVBD (GVBD 0.5–2.5); fully mature eggs at 3 or more hours after GVBD (>3 egg).

Potentiation of IP₃-dependent Ca²⁺ release during oocyte maturation. A, cells were collected and imaged before maturation (Ooc); 3 h after progesterone addition with no GVBD (P3hr); at GVBD (GVBD); and >3 h after GVBD (Egg). IP₃ was uncaged using flash photolysis on a Fluoview confocal as previously described (10). A sub-threshold uncaging pulse was empirically determined for each batch of cells in oocytes leading to little or no release and was applied to the other stages of maturation. This uncaging pulse readily induces Ca²⁺ mobilization in eggs and to a lesser extent in cells at GVBD. The inset shows uncaging levels of caged-fluorescein in oocytes and eggs. B, statistics of maximal Ca²⁺ release from 19–24 cells in each group (three donor females). The asterisk indicates significantly different data sets (p ≤ 0.0024). The “Low P-MAPK” group refers to cells treated with progesterone before GVBD where low levels of MAPK phosphorylation were detected as shown in the P3–15h panel in Fig. 3C. C, imaged cells were lysed and analyzed individually to test the activation state of MAPK (arrowhead) and MPF (arrow) in the different groups. Tubulin (Tub, dash) was the loading control. Examples of three individual cells are shown for each time point. For the P3–15h group cells with low MAPK activity (1–2 h before GVBD) are shown, although many cells in that group exhibited no MAPK activation.

Phosphorylation of the IP₃R during oocyte maturation. A, cells were metabolically labeled with [³²P]orthophosphate and left untreated (oocytes, Ooc) or matured with progesterone (Egg). After overnight progesterone incubation cells that did not undergo GVBD were removed, and the remaining cells were incubated for 3 additional hours to ensure full maturation in the entire population (Egg). Lysates were separated by SDS-PAGE and analyzed by autoradiography. The blot was then probed with anti-IP₃R antibody to confirm IP₃R immunoprecipitation. B, the IP₃R was immunoprecipitated from oocyte using either immune or pre-immune antibodies and the IP analyzed by Western using an anti-IP₃R antibody (WB). Immunoprecipitates were incubated with MPF and [γ-³²P]ATP, and the reaction was analyzed by autoradiography. C, map of the peptides detected following mass spectroscopy analysis of the IP₃R. The immunoprecipitation was performed on at least 2000 cells, which typically produced a prominent band after staining with Simply Blue SafeStain. IP₃R bands were excised from the gel, reduced, and alkylated with iodoacetamide and digested with trypsin or chymotrypsin. The peptide pool was analyzed by nano-liquid chromatography/MS/MS, and phosphopeptides were detected as discussed under “Experimental Procedures.” The map shown is for the egg IP with oocyte data yielding practically identical coverage. The red residues indicate peptides that were detected following the MS analysis, and the highlighted peptides indicate phosphorylated peptides. The shaded areas represent IP₃R trans-membrane or lumenal domains. The domain structure of the IP₃R is indicated on the right. D, details of the location and presence in oocytes or eggs of the phosphorylated peptides. The phosphorylated residue is indicated in red. E, sequence alignment of the regions in the type 1 IP₃R surrounding the phosphorylated residues form different vertebrates.

In vivo analysis of IP₃R phosphorylation during meiotic maturation. A, phosphorylation profile of immunoprecipitated endogenous IP₃R from oocytes (O) and eggs (E) using phospho-specific antibodies that recognize the phosphorylated consensus for the indicated kinases. The first panel shows a Western blot using an anti-IP₃R antibody (WB). B, oocytes were injected with mRNA encoding wild type or mutant FLAG-tagged coupling-domain region (565–1895) of the Xenopus IP₃R. In the double mutant (Double) both Thr-931 and Thr-1136 were mutated to Ala. Cells were incubated for 3 days to allow expression of the constructs and lysates collected from oocytes or from cells 2 h after GVBD. Top panel, cell lysates were immunoprecipitated using an anti-FLAG antibody and probed with phospho-Thr MAPK/CDK substrate antibody; middle, the same blot was stripped and blotted with the anti-FLAG antibody; bottom, cell lysates were probed with phospho-MAPK and phospho-cdk1 antibodies as in Fig. 2C. C, modulation of the kinase cascade. Cells were untreated (Ooc) or matured with progesterone (Egg). Mos, cells were injected with MosRNA to activate the maturation kinase cascade. WM: cells were preinjected with Wee1 RNA to inhibit MPF activation before Mos injection. Cy: cells were injected with Cylin B RNA to directly activate MPF. UC: cells were pretreated with U0126 to inhibit the MAPK cascade before Cylcin B injection. Top panel, endogenous IP₃R immunoprecipitated (IP) and probed with phospho-Thr MAPK/CDK substrate antibody; middle, the same blot was stripped and probed with anti-IP₃R antibody; bottom, same as B. D, cell lysates from oocytes (Ooc) and eggs (Egg) were probed with phospho-specific antibodies that specifically recognize phosphorylated Thr-931 or Thr-1136. E, time course of IP₃R phosphorylation. Cells were collected at different time points. P1–5 refers to 1–5 h after progesterone addition. Lysates were probed with the anti-phospho-Thr-931 and Thr-1136 antibodies (top two panels). Both Thr-931 and Thr-1136 are phosphorylated at GVBD. The same blot was stripped and probed with anti-IP₃R antibody (IP₃R WB). The activation state of MAPK and MPF was detected as in Fig. 2C. F, experimental setup is identical to C except that in this case the immunoprecipitates were probed with the anti-phospho-Thr-931 and Thr-1136 antibodies.

Sensitization of IP₃-dependent Ca²⁺ release following modulation of the maturation kinase cascade. Imaging conditions were as in Fig. 2A, and the modulation of the kinase cascade was as in Fig. 4, C and F. A and B, average traces after IP₃ uncaging as indicated by the arrow. The error bars were omitted for clarity. Right panel, mean ± S.E. of the maximal Ca²⁺ signal (right panel). Data are from 11–30 cells per treatment, and the asterisk indicates significantly different datasets (p < 0.003 for A, and p < 0.028 for B). Oocytes (Ooc), eggs (Egg), cells injected with cyclin B RNA (Cy), cells pre-treated with U0126 before cyclin RNA injection (UC), cells injected with MosRNA (Mos), and cells injected with Wee1 RNA followed by MosRNA injection (WM). C, activation state of MAPK and MPF in the different groups as in Fig. 2C. Representative data from three individual cells are shown.

Rate of Ca²⁺ signal decay as a measure of IP₃R sensitization following modulation of the maturation kinase cascade. Imaging conditions were as in supplemental Fig. S1, and the modulation of the kinase cascade was as in Fig. 4 (C and F). A, average traces after gradual IP₃ uncaging using the 405 nm laser as indicated by the gray bar. Error bars were omitted for clarity. B and C, maximal Ca²⁺ release levels immediately after the uncaging duration (B) and the rate of Ca²⁺ signal decay measured as the time required for half-maximal decay (T½) (C) in the different treatment groups. Mean ± S.E. data from 12–28 cells per treatment. Oocytes (Ooc), eggs (Egg), cells injected with cyclin B RNA (Cy), cells pre-treated with U0126 before cyclin RNA injection (U-C), cells injected with MosRNA (Mos), cells injected with Wee1 RNA followed by MosRNA injection (W-M).