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A CALCIUM-DEPENDENT PROTEIN KINASE FUNCTIONS IN WOUND HEALING IN VENTRICARIA VENTRICOSA (CHLOROPHYTA)

Authors

  • Koh-ichi Sugiyama,

    1. Department of Biology, Tokyo Gakugei University, Nukuikita-machi, Koganei-shi, Tokyo 184-0015, Japan
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    • 2

      Present address: Jumonji High School, Kita-Ohtsuka 1-10-33, Toshima-ku, Tokyo 170-0004, Japan.

  • Izumi C. Mori,

    1. Nagoya University Bioscience Center and Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku,
      Nagoya 464-8601, Japan
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  • Koji Takahashi,

    1. Nagoya University Bioscience Center and Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku,
      Nagoya 464-8601, Japan
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    • 3

      Present address: Unit of Biosystems, School of Informatics and Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan.

  • Shoshi Muto,

    Corresponding author
    1. Nagoya University Bioscience Center and Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku,
      Nagoya 464-8601, Japan
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  • Ikuko Shihira-Ishikawa

    1. Department of Biology, Tokyo Gakugei University, Nukuikita-machi, Koganei-shi, Tokyo 184-0015, Japan
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    • 5

      Present address: 3-60-2-606 Kamiishihara, Chofu-shi, Tokyo 182-0035, Japan.


  Author for correspondence: e-mail h44787a@nucc.cc.nagoya-u.ac.jp.

Abstract

The cytoplasm around a wound made in the multinucleate unicellular green alga Ventricaria ventricosa (  J. Agardh) Olsen et West formed an aggregation-ring surrounding the wound immediately after injury. A contraction of the ring then brought about wound healing in culture medium containing Ca2+. Involvement of a calcium-dependent protein kinase (CDPK) as a regulator of wound healing was examined using an anti-Dunaliella tertiolecta CDPK antibody. A 52-kDa protein cross-reacting with the antibody was detected by Western blotting. Protein kinases of 60 kDa and 52 kDa, which were markedly activated by Ca2+, and a 40-kDa Ca2+-independent protein kinase were detected by an in-gel protein kinase assay using myelin basic protein as the substrate. A 52-kDa band with Ca2+-dependent protein kinase activity was immunoprecipitated from the cytoplasmic extract, indicating that these 52-kDa proteins are identical and possess CDPK activity. Microscopic observation showed that the contraction of the aggregation ring was suppressed by application of the anti-CDPK to the culture medium. A protein kinase inhibitor, K-252a, and the calmodulin inhibitors, calmidazolium and compound 48   /   80, which inhibit CDPK activity, also suppressed the contraction of the aggregation-ring. Immunofluorescence microscopy showed a similar distribution of 52-kDa CDPK to the distribution of f-actin, which was randomly distributed in an intact cell and formed a bundle during wound healing. Further, f-actin was not recruited after injury in the presence of the antibody to CDPK. These results suggest that the 52-kDa CDPK functions as a Ca2+ receptor in wound healing and simultaneously participates in the organization and contraction of f-actin to heal the wound.

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