| Co-Investigator(Kenkyū-buntansha) |
WILLIAMSON R オーストラリア国立大学, 生物教室, シニアフェロー
PALEVITZ A.B ジョージア大学, 生物学教室, 教授
YOKOTA Etsuo Faculty of Science, Himeji Institute of Technology, 理学部, 助手 (80212299)
MIMURA Tetsuro Faculty of Science, Himeji Institute of Technology, 理学部, 助教授 (20174120)
WILLIAMSON Richard E. Research School of Biological Science, Australian National University
BARRY Palevitz A. Department of Botany, The University Georgia
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| Budget Amount *help |
¥5,500,000 (Direct Cost: ¥5,500,000)
Fiscal Year 1993: ¥5,500,000 (Direct Cost: ¥5,500,000)
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| Research Abstract |
The present project aimed to elucidate the mechanism of cytoplasmic streaming and its regulation. In addition, we succeeded in find important facts concerned with reverse fountain streaming in root hair cells of Hydrocharis and the effect of okadaic acid on cytoplasmic streaming in pollen tubes.
1.Mechanism of Ca^<2+> regulation of cytoplasmic streaming in pollen tubes.
Before starting this project, we have succeeded in purifying myosin containing a heavy chain of 170 kD from pollen tubes of lily. We analyzed Calcium sensitivity of the myosin.
In our previous study, we succeeded in inducing sliding movement of pollen tube organelles along characean actin cables. Based on the fact, we concluded that myosin is associated with the organelles and concerned with generation of motive force of cytoplasm streaming in pollen tubes. In addition, we found that the reconstituted, movement was inhibited by calcium, and speculated that pollen tube myosin has inhibitory calcium regulation mechanism. In
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the present study, we found that motility monitored with in vitro motility assay is inhibited by calcium of muM levels. In addition, we found that actin-activated ATPase of the myosin is also inhibited by calcium of similar concentrations. This is the first success in purifying myosin equipped with calcium sensitivity from plant cells. Since the calcium sensitivity of the myosin is lost by partial proteolysis, this system is a suitable system in elucidating the molecular mechanism of calcium regulation of plant myosin.
2.Mechanism of reverse fountain streaming
In root hair cells, typical reverse fountain streaming is observed. We studied the effect of cytochalasin B and micaloride B on the velocity of cytoplasmic streaming and the transvacuolar strand. Cytochalasin inhibited the cytoplasmic streaming in dose-response manner. At 10 muM, cytoplasmic streaming was stopped completely. In parallel with inhibition of cytoplasmic streaming, the transvacuolar strands disappeared. When chtochalasin was removed, both the cytoplasmic streaming and the transvacuolar strand were recovered. Thus, effect of cytochalasin is reversible. Micaloride B also inhibited the cytoplasmic streaming and disrupted the transvacuolar strand. It was discussed that use of cytochalasin and micaloride in combination is a powerful tool in studying structure and function of cytoskeleton in plant cells.
3.Effect of okadaic acid on cytoplasmic streaming and actin filament
Effect of specific inhibitor of protein phosphatase was studied with special reference to cytoplasmic streaming and actin filaments in lily pollen tube. It was found that okadaic acid inhibited without disrupting actin filaments, supporting that okadaic acid inhibit the activity of myosin via inhibition of dephosphorylation. Less
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