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鳖属根毛细胞逆向喷泉流有关的重要事实，以及冈田酸对花粉管胞质流的影响。1. Ca^<2+>调节花粉管胞质流的机制在本项目开始之前，我们已成功地从百合花粉管中纯化了含有170 kD重链的肌球蛋白。在前期的研究中，我们成功地诱导了花粉管细胞器沿着字符肌动蛋白索滑动。据此，我们认为，肌球蛋白与细胞器有关，并与花粉管中细胞质流动动力的产生有关。此外，我们发现重组运动受到钙离子的抑制，推测花粉管肌球蛋白具有抑制性钙调节机制。在 ...更多信息 在本研究中，我们发现用体外运动分析监测的运动被μ M水平的钙抑制。此外，我们发现肌动蛋白激活的肌球蛋白ATP酶也被类似浓度的钙抑制。这是从植物细胞中纯化具有钙敏感性的肌球蛋白的首次成功。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的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对细胞质流动速度和跨液泡链的影响。细胞松弛素以剂量-反应方式抑制细胞质流动。在10 μ M时，细胞质流动完全停止。与细胞质流动的抑制平行，跨液泡链消失。当chtochalasin被删除，细胞质流和transvacuolar链被恢复。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，破坏跨液泡链。3.冈田酸对百合花粉管细胞质流动和肌动蛋白微丝的影响研究了蛋白磷酸酶特异性抑制剂对百合花粉管细胞质流动和肌动蛋白微丝的影响。研究发现，冈田酸具有抑制作用，但不破坏肌动蛋白丝，这支持冈田酸通过抑制去磷酸化来抑制肌球蛋白的活性。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