| Project/Area Number |
16570033
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
植物生理・分子
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| Research Institution | Osaka University |
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Principal Investigator |
TAKAGI Shingo Osaka University, Graduate School of Science, Associate Professor, 大学院理学研究科, 助教授 (10192626)
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| Project Period (FY) |
2004 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2006: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2005: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2004: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | Actin cytoskeleton / Calcium ion / Chloroplast / Plasma membrane / Plasma membrane ghost / Reconstitution system / Spinach (mesophyll cell) / Villin / ビリン / 細胞質基質 / 細胞内運動 / タバコ培養細胞(BY-2) |
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| Research Abstract |
In plant cells, actin cytoskeleton has been presumed to play pivotal roles in relocation and intracellular positioning of chloroplasts. However, the precise modes of and regulatory mechanisms for such roles of actin cytoskeleton remain to be elucidated.
We prepared plasma membrane (PM) ghosts from enzymatically-produced mesophyll protoplasts of spinach. Even after washing of the cytoplasmic face of the PM with a buffer solution, chloroplasts remained to attach to the PM ghosts. Such chloroplasts were surrounded by actin filaments in an honeycomb array on the PM ghosts. The attachment of chloroplasts to the PM ghosts was sensitive not only to the depolymerizing reagents of actin filaments but also to Ca^<2+> at concentrations higher than 1 μM. Both treatments markedly disrupted the organization of actin filaments on the PM ghosts.
We examined a possible involvement of a Ca^<2+>-sensitive actin depolymerizing protein villin. Using the antibodies raised against plant villins identified in lily pollen tube, we detected plausible candidate polypeptides in spinach leaves by immunofluorescence microscopy and DNase I affinity chromatography.
On the other hand, isolated intact chloroplasts from the spinach leaves could organize actin filaments when mixed with exogenously added skeletal muscle G-actin. This activity was sensitive to treatment of the isolated chloroplasts with high concentrations of salts. The salt-extracted soluble fraction, in turn, retained the organizing activity of actin filaments in vitro.
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