Structure and function of cytoskeleton in plant cells

本课题旨在阐明细胞质流动的机制及其调控。此外，我们还成功地发现了与水仙属植物根毛细胞的反向喷泉流动以及冈田酸对花粉管细胞质流动的影响有关的重要事实。1.Ca~(2+)和Gt~(2+)对花粉管细胞质流动的调节机制。我们分析了肌球蛋白的钙敏感性。在我们之前的研究中，我们成功地诱导了花粉管细胞器沿着特征肌动蛋白缆线滑动。根据这一事实，我们认为肌球蛋白与细胞器有关，并与细胞质在花粉管中流动的动力的产生有关。此外，我们还发现重组运动受到钙的抑制，推测花粉管肌球蛋白具有抑制钙调节的机制。在…中在本研究中，我们发现用体外运动性测定监测的运动性受到钙水平的抑制。此外，我们还发现肌球蛋白的肌动蛋白激活的ATPase也被相似浓度的钙抑制。这是首次成功地从植物细胞中提纯具有钙敏感性的肌球蛋白。由于肌球蛋白的钙敏感性被部分蛋白分解而丧失，因此该体系是阐明植物肌球蛋白钙调节的分子机制的合适系统。2.反向喷泉流动的机理在根毛细胞中，观察到典型的反向喷泉流动。我们研究了细胞松弛素B和米卡洛利B对细胞质流速度和跨空泡链的影响。细胞松弛素以剂量-反应方式抑制胞质流动。在10微米时，细胞质流动完全停止。在胞质流动受到抑制的同时，跨空泡链消失。去除Chtochalasin后，细胞质流动和跨空泡链均得到恢复。因此，细胞松弛素的作用是可逆的。米卡洛利B也抑制细胞质流动，并破坏跨空泡链。3.冈田酸对细胞质流和肌动蛋白细丝的影响研究了蛋白磷酸酶特异性抑制剂对百合花粉管中细胞质流和肌动蛋白细丝的影响。研究发现冈田酸在不破坏肌动蛋白细丝的情况下抑制肌球蛋白的活性，支持冈田酸通过抑制肌球蛋白的去磷酸化来抑制肌球蛋白的活性。较少

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