A calcium-dependent protein kinase functions in wound healing in Ventricaria ventricosa (Chlorophyta)

The cytoplasm around a wound made in the multinucleate unicellular green al ga Ventricaria ventricosa (J. Agardh) Olsen ct West formed an aggregation-r ing surrounding the wound immediately after injury. A contraction of the ri ng then brought about wound healing in culture medium containing Ca2+. Invo lvement of a calcium-dependent protein kinase (CDPK) as a regulator of woun d healing was examined using an anti-Dunaliella tertiolecta CDPK antibody. A 52-kDa protein cross-reacting with the antibody was detected by Western b lotting. Protein kinases of 60 kDa and 52 kDa, which were markedly activate d 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 immunoprecipit ated from the cytoplasmic extract, indicating that these 52-kDa proteins ar e identical and possess CDPK activity. Microscopic observation showed that the contraction of the aggregation ring was suppressed by application of th e 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, Imm unofluorescence microscopy showed a similar distribution of 52-kDa CDPK to the distribution of f-actin, which was randomly distributed in an intact ce ll and formed a bundle during wound healing. Further, f-actin was not recru ited after injury in the presence of the antibody to CDPK. These results su ggest that the 52-kDa CDPK functions as a Ca2+ receptor in wound healing an d simultaneously participates in the organization and contraction of f-acti n to heal the wound.