Journal: Developmental biology

Article Title: Calcineurin and its regulation by Sra/RCAN is required for completion of meiosis in Drosophila.

doi: 10.1016/j.ydbio.2010.06.011

Figure Lengend Snippet: Fig. 2. Endogenous sra positively regulates calcineurin signaling in the female germline. (A–E) CnADN suppresses the wing phenotypes induced by hyperactivation of calcineurin signaling. Adult wings are shown for en-GAL4/+ (A), en-GAL4/UAS-CnAact (B), en-GAL4/UAS-CnAact; UAS-CnADN/+ (C), en-GAL4/+; UAS-CnADN/+ (D), en-GAL4/UAS-CnAact; UAS-GFP/+ (E). All images were taken with same magnification. Scale bar; 500 μm. (F) Genetic interaction between CnADN and sra in germline cells. Females that express CnAWT, CnADN, or sra by nos-GAL4 and that are wildtype (solid bars) or heterozygous (striped bars) for sraKO were tested. Hatchability of eggs from the mothers of each genotype was scored. UASp-CnADN[6] and UASp-CnADN[5] are independent transgenic lines in which the transgene is located on the 2nd and 3rd chromosomes, respectively. Co-overexpression of Sra was tested only for CnADN[5] and shown on the far right. Error bars indicate 95% confidence intervals. (G–H) Meiotic nuclei of eggs taken from nosNCnADN[5]; sraKO/+ mothers. Chromosomes and spindles are visualized with DAPI (G–H) and anti-αTubulin antibody (G′–H′), respectively, and the merged images are shown in G″–H″. Scale bars; 5 μm.

Article Snippet: More recently, the Ca2 +/CaM-dependent phosphatase calcineurin was demonstrated in Xenopus oocytes to be activated transiently by the Ca2+ increase and to play important roles in meiotic release and probably other events required to begin embryonic development (Mochida and Hunt, 2007; Nishiyama et al., 2007).

Techniques: Transgenic Assay, Over Expression

Journal: Developmental biology

Article Title: Calcineurin and its regulation by Sra/RCAN is required for completion of meiosis in Drosophila.

doi: 10.1016/j.ydbio.2010.06.011

Figure Lengend Snippet: Fig. 3. The SP motif of in Sra is specifically required for positive regulation of calcineurin. (A) The SP motif in Sra and Sra mutant forms. Ser215 and Ser219 which are consensus phosphorylation sites for GSK-3β and its priming kinase MAPK, respectively, were mutated to alanine (SraS215A and SraS219A). SraΔ212–221 lacks 10 amino acids within the SP motif. (B) SraS215A interferes with endogenous Sra. Egg hatchability of females that express SraWT or mutant forms of Sra and that are wildtype (solid bars) or heterozygous (striped bars) for sraKO were tested. Error bars indicate 95% confidence intervals. (C–F) Misexpression of SraS215A causes meiotic defects. Chromosomes (C and blue in D–F) and spindles (C′, and green in D–F) in eggs from nosNsraS215A[10] mothers were visualized. The panel C″ is merged images of C and C′. 62% of the eggs showed a meiotic arrested anaphase I chromosome configuration (C), and 19% meiotic defects at meiosis I or II (D–F). (G) Interaction between Sra variants and CnA in ovaries. Ovary proteins were immunoprecipitated with mouse anti-Myc antibody, and analyzed by Western blot with anti-Sra and rabbit anti-Myc antibodies. An asterisk indicates non-specific signals. Ovary samples were from animals carrying nos-GAL4 and UASp-CnAWT-Myc transgenes, and also sraKO/+ (lane 1), sraKO/sraKO (lane 2), UASp-sraWT/+; sraKO/sraKO (lane 3), UASp-sraS215A/+; sraKO/sraKO (lane 4), UASp- sraS219A/+; sraKO/sraKO (lane 5), and UASp-sraΔ212–221/+; sraKO/sraKO (lane 6). For the negative control of the immunoprecipitation with anti-Myc antibody, ovary extracts from females carrying nos-GAL4 and UASp-sraWT/+; sraKO/sraKO without a UASp-CnAWT-Myc transgene were used (lane 7).

Article Snippet: More recently, the Ca2 +/CaM-dependent phosphatase calcineurin was demonstrated in Xenopus oocytes to be activated transiently by the Ca2+ increase and to play important roles in meiotic release and probably other events required to begin embryonic development (Mochida and Hunt, 2007; Nishiyama et al., 2007).

Techniques: Mutagenesis, Phospho-proteomics, Immunoprecipitation, Western Blot, Negative Control

Journal: Developmental biology

Article Title: Calcineurin and its regulation by Sra/RCAN is required for completion of meiosis in Drosophila.

doi: 10.1016/j.ydbio.2010.06.011

Figure Lengend Snippet: Fig. 4. A model for calcineurin regulation by Sra/RCAN. (A) Sra activates calcineurin during female meiosis. Calcineuerin activity is kept low due to the inhibitory binding of Sra to CnA until egg activation. Sra is sequentially phosphorylated at Ser219 and Ser215 by MAPK and GSK-3β signaling pathways, respectively, during oogenesis, which not only releases inhibitory function of Sra by dissociating from calcineurin complexes or reducing the affinity for CnA, but also modifies calcineurin as its regulator. Upon egg activation, calcineurin is activated by Ca2+/CaM and dephosphorylates as-yet-unidentified substrate(s) to complete meiosis. (B) Overexpression of Sra inhibits active calcineurin. This inhibitory effect is independent of Sra phosphorylation and probably due to competitive blocking of the docking sites for calcineurin substrates.

Article Snippet: More recently, the Ca2 +/CaM-dependent phosphatase calcineurin was demonstrated in Xenopus oocytes to be activated transiently by the Ca2+ increase and to play important roles in meiotic release and probably other events required to begin embryonic development (Mochida and Hunt, 2007; Nishiyama et al., 2007).

Techniques: Activity Assay, Binding Assay, Activation Assay, Protein-Protein interactions, Over Expression, Phospho-proteomics, Blocking Assay