Suzuki T , Suzuki E , Yoshida N , Kubo A , Li H , Okuda E , Amanai M , Perry AC .

Wellcome Trust

	Xenopus xEmi2 as meiotic regulatory hub. Diagram showing interactions between principal components of Xenopus meiotic homeostasis and xEmi2. APC, anaphase-promoting complex; xCaMKII, calmodulin kinase II; xCaN, calcineurin; D-box, destruction box; xEmi2, endogenous meiotic inhibitor 2; xMAPK, mitogen-activated protein (xMAP) kinase (xErk); xMAPKK, xMAP kinase kinase (xMek); xMos, Moloney sarcoma oncogene; Plx1, polo-like kinase 1; xRsk, ribosomal s6 kinase 1; ZBR, zinc-binding region.
	Mos establishes Emi2- and MAPK-dependent metaphase arrest but its presence is not required to sustain mII. (A) Histograms for GV oocyte injection with cRNA encoding mCherry (mCh) or mCherry fused to Mos or to the Mos dominant-negative double mutant S3A/S105A (Mosdn). Injections included the Mek inhibitor U0126 or si5′Emi2#1 (Mos+siE), as indicated. Numbers above columns show oocyte numbers. (B) Anti-MAPK and -phospho-MAPK (pMAPK) immunoblotting of non-injected control IVM16, mII and GV oocytes or GV oocytes injected with cRNA as in A and held in IBMX (IB24) (see Fig. S1A in the supplementary material). Upper and lower panels show the same blot. (C) Percentages of oocytes arrested at mI in the IVM16 or si16 systems (see Fig. S1B,C in the supplementary material) following injection with cRNA encoding the Emi2T327A (T327A). Lo, the default [cRNA] throughout (0.5-1.0 mg/ml); Hi, [RNA]=2.0-2.5 mg/ml. (D) Densitometric quantification of anti-Mos immunoblots of oocytes at the times shown post-hCG. See also Fig. S2C in the supplementary material. (E) Paired Hofmann images of mature oocytes at the ages indicated post-hCG (in hours), and (underneath) the same or equivalent oocytes 6 hours after exposure to 10 mM SrCl2. Black arrowheads, Pb; red arrowheads, pronuclei; white arrowheads, protuberances caused by mII spindles/plates. Error bars indicate s.e.m. Scale bar: 100 μm.
	Features of Emi2 species orthologues. (A) Optimized alignment of regions of Emi2 from different species showing positional correspondence to functional domains and residues (red) in Xenopus laevis xEmi2. (p)Deg, (phospho)degron. (B) Fluorescence images after GV oocyte injection with cRNA encoding Emi2-mCherry in the IVM16 system (see Fig. S1B in the supplementary material), showing Emi2 spindle localization. Tubg, γ-Tubulin. DNA is stained with DAPI. (C) GV oocytes injected with cRNA encoding full-length mouse or Xenopus Emi2 and held for 5 hours in IBMX prior to visualization. Insets show Emi2 nuclear localization in mouse, but not in Xenopus. (D) Percentages of mI arrest following GV oocyte injection with cRNA encoding mCherry (mCh), Emi2 or xEmi2 in the si16 system (see Fig. S1C in the supplementary material). Scale bars: 50 μm.
	Mouse Emi2 comprises separable functional domains. (A) Percentages of oocytes arrested at mI in the IVM16 or si16 systems (see Fig. S1B,C in the supplementary material) following injection with cRNAs encoding different Emi2 constructs. Values below the x-axis correspond to Emi2 residue numbers. (B) Immunoblotting of lysates from NIH 3T3 cells transfected (tf) as indicated, in the presence or absence of nocodazole, and subjected to anti-FLAG immunoprecipitation (IP) with beads to capture complexes containing FLAG (uppermost), or endogenous Cdc20 (centre) or Cdc27. (C) Optimal species alignment with mouse Emi2 residues 80-115. Residues that are identical in more than 50% of the sequences are shown in red. (D) Intact mII oocytes were injected with cRNA encoding mCherry (mCh), Emi2-mCherry or Emi2C573A (C573A), exposed to TPEN (10 μM) after 4 hours then scored for Pb2 extrusion 1.5 hours later. (E) Alignment of parts of mouse Emi1 and Emi2 (with Emi2 residue numbering) ZBRs. Varying [cRNA] encoding Emi2 mutants were injected in the si16 system, and fluorescence quantified (x-axis) and plotted against the proportion emitting a Pb (average 14.8 oocytes/point). The wild-type Emi2 profile is shown on each plot for comparison (grey). (F) Western blotting of lysates from NIH 3T3 cells transfected with mCherry or Emi2-mCherry (Emi2) (leftmost) and following IP to capture endogenous Cdc27. Red arrowheads mark complexes containing Cdc27 (left) and Emi2-mCherry. Cells were incubated in 100 μM TPEN prior to collection as indicated. (G) cRNAs encoding Emi2491-Cter (491-Cter), Emi2551-Cter (551-Cter) or Emi2δD (D-box double mutant, R511A, L514A) injected in the IVM16 or si16 systems, showing the percentage arrested at mI. Low (Lo) and high (Hi) RNA concentration are, respectively, 0.5-1.0 and 2.0-2.5 mg/ml; the Lo value corresponds to the default used in other experiments. (H) Mature mII oocytes injected with cRNA encoding mCherry (mCh), Emi2491-Cter (491-Cter), or Emi2551-Cter (551-Cter) or Emi2551-Cter containing the mutations indicated were scored for mII exit as indicated by Pb2 emission 2 hours post-injection. Values above columns show oocyte numbers. Emi2 constructs were all expressed as mCherry fusions.
	Molecular regulation of mII exit by Emi2. (A) Agarose gel electrophoresis of RT-PCR (top) detects transcripts for CaM and the CaMKII γ isoform (g), but not α, β or δ (a, b and g) in sperm (sp) or oocytes (GV and mII). Immunoblotting (centre) with anti-CaMKIIγ reveals two isoforms, γ3 and γJ, in mII oocytes, the variant sequences of which are shown below. IVTT, coupled in vitro transcription/translation. (B) SDS-PAGE of in vitro assays to measure phosphorylation of mouse Emi2 (m) and xEmi2 (X) produced in vitro, by CaMKIIγ mII oocyte isoforms (γ3 and γJ) produced in vitro (A) or a mixture of all CaMKII isoforms from bovine brain (brain). AC, positive control autocamtide peptide. Red arrowheads indicate autophosphorylated CaMKII monomers. (C) Data summary of ectopic Emi2 degradation following oocyte activation. mII oocytes injected with cRNA encoding mCherry fused to Emi2, or the Emi2 mutations indicated were exposed to 10 mM SrCl2 (see Fig. S1D in the supplementary material) and fluorescence levels recorded 1 hour after SrCl2 withdrawal. Complete data sets are presented in Fig. S10A,B in the supplementary material. (D) Fluorescence images (left) and histograms for mII oocytes injected with cRNA encoding xEmi2-mCherry (xEmi2), without (top) or with exposure to SrCl2 as in C. Recordings were made at the times indicated after oocytes had been exposed to SrCl2 for 1.5 hours. Scale bar: 50 μm. Values above columns show oocyte numbers. (E) Fluorescence levels in mII oocytes injected with cRNA encoding Emi2-mCherry and 4 hours later treated with 10 mM SrCl2 (+Sr2+, broken lines) or not treated (−Sr2+, solid lines). Oocytes were from the hybrid B6D2F1 (black plots, wt) or homozygous null Trcpb1−/− mutants (red).
	Mouse Emi2 as meiotic regulatory hub. Diagram showing interactions between principal components of mouse meiotic homeostasis and Emi2. APC, anaphase-promoting complex; CaMKIIg, calmodulin kinase II γ; D-box, destruction box; Emi2, endogenous meiotic inhibitor 2; MAPK, mitogen-activated protein (MAP) kinase; MAPKK, MAP kinase kinase; Mos, Moloney sarcoma oncogene; Plk1, polo-like kinase 1; ZBR, zinc-binding region. A broken line shows a presumptive, possibly indirect, stabilization role for Emi2 residues 80-115. There is no evidence for the indispensable involvement of CaN.

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