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Activation of ADF/cofilin by phosphorylation-regulated Slingshot phosphatase is required for the meiotic spindle assembly in Xenopus laevis oocytes.

ABSTRACT: We identify Xenopus ADF/cofilin (XAC) and its activator, Slingshot phosphatase (XSSH), as key regulators of actin dynamics essential for spindle microtubule assembly during Xenopus oocyte maturation. Phosphorylation of XSSH at multiple sites within the tail domain occurs just after germinal vesicle breakdown (GVBD) and is accompanied by dephosphorylation of XAC, which was mostly phosphorylated in immature oocytes. This XAC dephosphorylation after GVBD is completely suppressed by latrunculin B, an actin monomer-sequestering drug. On the other hand, jasplakinolide, an F-actin-stabilizing drug, induces dephosphorylation of XAC. Effects of latrunculin B and jasplakinolide are reconstituted in cytostatic factor-arrested extracts (CSF extracts), and XAC dephosphorylation is abolished by depletion of XSSH from CSF extracts, suggesting that XSSH functions as an actin filament sensor to facilitate actin filament dynamics via XAC activation. Injection of anti-XSSH antibody, which blocks full phosphorylation of XSSH after GVBD, inhibits both meiotic spindle formation and XAC dephosphorylation. Coinjection of constitutively active XAC with the antibody suppresses this phenotype. Treatment of oocytes with jasplakinolide also impairs spindle formation. These results strongly suggest that elevation of actin dynamics by XAC activation through XSSH phosphorylation is required for meiotic spindle assembly in Xenopus laevis.

SUBMITTER: Iwase S

PROVIDER: S-EPMC3681698 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Activation of ADF/cofilin by phosphorylation-regulated Slingshot phosphatase is required for the meiotic spindle assembly in Xenopus laevis oocytes.

Iwase Shohei S Sato Ryuhei R De Bock Pieter-Jan PJ Gevaert Kris K Fujiki Saburo S Tawada Toshinobu T Kuchitsu Miyako M Yamagishi Yuka Y Ono Shoichiro S Abe Hiroshi H

Molecular biology of the cell 20130424 12

We identify Xenopus ADF/cofilin (XAC) and its activator, Slingshot phosphatase (XSSH), as key regulators of actin dynamics essential for spindle microtubule assembly during Xenopus oocyte maturation. Phosphorylation of XSSH at multiple sites within the tail domain occurs just after germinal vesicle breakdown (GVBD) and is accompanied by dephosphorylation of XAC, which was mostly phosphorylated in immature oocytes. This XAC dephosphorylation after GVBD is completely suppressed by latrunculin B, a ...[more]

PMID: 23615437

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Project description:Two cDNAs, isolated from a Xenopus laevis embryonic library, encode proteins of 168 amino acids, both of which are 77% identical to chick cofilin and 66% identical to chick actin-depolymerizing factor (ADF), two structurally and functionally related proteins. These Xenopus ADF/cofilins (XADs) differ from each other in 12 residues spread throughout the sequence but do not differ in charge. Purified GST-fusion proteins have pH-dependent actin-depolymerizing and F-actin-binding activities similar to chick ADF and cofilin. Similarities in the developmental and tissue specific expression, embryonic localization, and in the cDNA sequence of the noncoding regions, suggest that the two XACs arise from allelic variants of the pseudotetraploid X. laevis. Immunofluorescence localization of XAC in oocyte sections with an XAC-specific monoclonal antibody shows it to be diffuse in the cortical cytoplasm. After fertilization, increased immunostaining is observed in two regions: along the membrane, particularly that of the vegetal hemisphere, and at the interface between the cortical and animal hemisphere cytoplasm. The cleavage furrow and the mid-body structure are stained at the end of first cleavage. Neuroectoderm derived tissues, notochord, somites, and epidermis stain heavily either continuously or transiently from stages 18-34. A phosphorylated form of XAC (pXAC) was identified by 2D Western blotting, and it is the only species found in oocytes. Dephosphorylation of >60% of the pXAC occurs within 30 min after fertilization. Injection of one blastomere at the 2 cell stage, either with constitutively active XAC or with an XAC inhibitory antibody, blocked cleavage of only the injected blastomere in a concentration-dependent manner without inhibiting nuclear division. The cleavage furrow of eggs injected with constitutively active XAC completely regressed. Blastomeres injected with neutralized antibody developed normally. These results suggest that XAC is necessary for cytokinesis and that its activity must be properly regulated for cleavage to occur.

Dataset Information

Activation of ADF/cofilin by phosphorylation-regulated Slingshot phosphatase is required for the meiotic spindle assembly in Xenopus laevis oocytes.

Publications

Activation of ADF/cofilin by phosphorylation-regulated Slingshot phosphatase is required for the meiotic spindle assembly in Xenopus laevis oocytes.

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