Structure and function of cytoskeleton in plant cells

植物细胞细胞骨架的结构和功能

• 批准号: 05044139
• 负责人: SHIMMEN Teruo
• 金额: $ 3.52万
• 依托单位: Himeji Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05044139/
• 关键词: cytoskeleton; cytoplasmic streaming; myosin; actin; calcium; pollen tube Characeae; tissue cultured cell; 培養細胞

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 … More 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 streamingIn 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 filamentEffect 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

本项目旨在阐明细胞质流的机制及其调控。此外，我们还成功地发现了与水鳅根毛细胞反向喷泉流和冈田酸对花粉管细胞质流的影响有关的重要事实。1.Ca^2+调节花粉管细胞质流的机制。在开始本项目之前，我们已经成功地从水鳖花粉管中纯化出含有170 kD重链的肌球蛋白。 莉莉。我们分析了肌球蛋白的钙敏感性。在我们之前的研究中，我们成功地诱导花粉管细胞器沿着轮藻肌动蛋白电缆滑动。基于此，我们得出结论：肌球蛋白与细胞器相关，与花粉管中细胞质流动动力的产生有关。另外，我们发现重构后的运动受到钙的抑制，推测花粉管肌球蛋白具有抑制性钙调节机制。在本研究中，我们发现通过体外运动测定监测的运动受到μM水平的钙的抑制。此外，我们发现类似浓度的钙也会抑制肌球蛋白的肌动蛋白激活的 ATP 酶。这是首次从植物细胞中成功纯化具有钙敏感性的肌球蛋白。由于肌球蛋白的钙敏感性因部分蛋白水解而丧失，该系统是阐明植物肌球蛋白钙调节分子机制的合适系统。2.反向喷泉流机制在根毛细胞中，观察到典型的反向喷泉流。我们研究了细胞松弛素 B 和米卡洛利 B 对细胞质流和跨液泡链速度的影响。细胞松弛素以剂量反应方式抑制细胞质流动。在10μM时，细胞质流完全停止。在抑制细胞质流动的同时，跨液泡链消失。当去除松弛素时，细胞质流和跨液泡链均恢复。因此，细胞松弛素的作用是可逆的。 Micaloride B 还抑制细胞质流动并破坏跨液泡链。认为细胞松弛素与米卡洛德联用是研究植物细胞骨架结构和功能的有力工具。3.大田酸对细胞质流和肌动蛋白丝的影响研究了蛋白磷酸酶特异性抑制剂的作用，特别针对百合花粉管细胞质流和肌动蛋白丝的影响。研究发现，大田酸在不破坏肌动蛋白丝的情况下进行抑制，这支持大田酸通过抑制去磷酸化来抑制肌球蛋白的活性。较少的

项目成果

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Yokota, E., Mabuchi, I.：“含有来自海胆精鞭毛轴丝的 C 和 A 重链的新型动力蛋白的分离和表征。”J. Cell Sci。

Yokota,E. and Mabuchi,I.: "Isolation and characterization of a novel dynein that contains C and A heavy chains from sea urchin sperm flagellar axonemes." J. Cell Science. (in press).

横田，E.

Shimmen,T.: Iwanami Shoten (in Japanese). Biology of the Cell, 1-180 (1993)

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Beilby.M.J.,Mimura.T.,Shimmen,T.: "The proton pump, high pH channels, and excitation: voltage clamp studies of intact and perfused cells of Nitellopsis obtusa" Protoplasma. 175. 144-152 (1993)

Beilby.M.J.、Mimura.T.、Shimmen,T.：“质子泵、高 pH 通道和激发：完整和灌注 Nitellopsis obtusa 细胞的电压钳研究”原生质体。