Regulation of cytoplasmic streaming by calcium in plant cells

百合花粉管中肌球蛋白的纯化我们成功地从百合花粉管中分离出肌球蛋白。将花粉管在低离子强度溶液中匀浆，离心制备粗提物。用粗提取物处理盖玻片表面。当用荧光染料标记的肌动蛋白丝与Mg·ATP一起应用时，肌动蛋白丝在玻璃表面显示滑动运动（体外运动测定），表明肌球蛋白以活性形式被提取。采用与鸡胸肌肌动蛋白丝共沉淀、羟基磷灰石柱层析、凝胶过滤等方法对肌球蛋白进行纯化。当用SDS-PAGE分析最终样品时，发现肌球蛋白由170 kD的重链组成。我们成功地产生了抗重链的抗体。用该抗体对花粉管粗提液进行免疫印迹，仅170 kD组分与抗体发生反应，加入100 μ mol/L的抗-N-乙酰氨基酚，可使肌球蛋白样品的ATP酶活性提高60倍。 关于我们 从鸡胸肌中纯化肌纤维素。用纯化的肌球蛋白进行的离体运动试验表明，肌动蛋白丝的运动速度为7.7 μ m/sec，这与花粉管中细胞质流动的速度几乎相同。这一事实表明，肌球蛋白参与了细胞质流动动力的产生。我们正在计划一个实验来检查肌球蛋白的钙敏感性。Characean细胞胞质流动的钙调节机制在Characean细胞中，胞质流动在动作电位产生时短暂停止。细胞质内游离钙增加，使细胞质流动停止。据推测，肌球蛋白的磷酸化参与细胞质流动的停止。本研究分析了增加腋生丽藻（Nitellaaxilliformis）胞外钾浓度引起细胞膜去极化时细胞质流动停止的过程，当胞外钾浓度从0.1mM增加到50 mM时，细胞膜去极化并保持在去极化状态。通过降低钾离子浓度，膜复极化。在响应膜的去极化，细胞质流迅速停止，速度停留在一个非常低的水平。在复极的膜通过降低钾浓度，细胞质流的速度在几分钟内恢复。当将5 mM的钙或镁加入到钾培养基中时，细胞质流动停止，如在不存在这些二价阳离子的情况下。然而，在这种情况下，速度逐渐恢复，没有复极化的膜。这表明，这些二价阳离子结合到质膜上，抑制了从膜去极化到胞质流停止的信号处理。利用低温显微镜对钝拟丽藻（Nitellopsis obtusa）节间细胞胞质流的温度依赖性进行了分析。在完整细胞中，在测试的细胞中观察到两种类型的反应。在一组中，速度在25-5 ℃的温度范围内线性下降。在另一组中，速度在25-15 ℃范围内呈线性下降，但在低于10 ℃时受到强烈抑制。同样的实验也在无液泡膜的细胞中进行，其中引入EGTA（钙螯合剂）和PIPES（pH缓冲剂）。在所有测试的无液泡膜细胞中，在25-5 ℃的温度范围内，速度线性下降。在5 ℃时，无液泡膜的细胞中的速度比完整细胞中的速度高3倍。本研究表明，细胞质流动的温度依赖性不仅反映了参与动力产生的肌动球蛋白的温度特性，而且还反映了这些离子在低温下对细胞内稳态的干扰。少

Purification of myosin from lily pollen tube We succeeded in isolating myosin from lily pollen tube. Pollen tube was homogenized in a solution of low ion ic strength and the crude extract was prepared by centrifugation. The surface of cover glass was treated with the crude extract. When actin filament labeled with fluorescent dye was applied together with Mg.ATP, actin filament showed sliding movement at the glass surface(in vitro motility assay), indicating that myosin was extracted in an active from. Myosin was successively purified by co-precipitation with actin filament of chiken breast muscle, hydroxylapatite column and gel filtration. When the final sample was analyzed with SDS-PAGE, myosin was found to be composed of heavy chain of 170 kD. We succeeded in rising antibody against the heavy chain. In immunoblotting of the crude extract of pollen tube using the antibody, only 170 kD component reacted with the antibody.ATPase of the myosin sample was activated to 60 fold by adding a … More ctin filament purified from chiken breast muscle. In in vitro motility assay using purified myosin, actin filament moved at the speed of 7.7 mum/sec, which is almost the same as the speed of cytoplasmic streaming in pollen tube. This fact indicates that the myosin is involved in generation of the motive force of cytoplasmic streaming. We are now planning a experiment to examine the calcium sensitivity of myosin.Mechanism of calcium regulation of cytoplasmic streaming in characean cells In Characean cells, cytoplasmic streamingtransiently stops upon generation of an action potential. Cytoplasm ic streaming stops due to increase of free calcium in the cytoplasm. It has been supposed that phosphorylation of myosin is involved in stopping of cytoplasmic streaming. In the present study, we analyzed the process of stoppage of cytoplasmic streaming upon depolarization of the membrane by increasing potassium concentration of the external medium in Nitella axilliformis.When the extracellular concentration of potassium was increased from 0.1 mM to 50 mM, membrane depolarized and stayed at the depolarized state. By decreasing the potassium concentration, membrane repolarized. In response to depolarization of the membrane, cytoplasmic streaming quickly stopped and the velocity stayed at a very low level. On repolarization of the membrane by decreasing potassium concentration, velocity of cytoplasmic streaming recovered within a few minutes. When calcium or magnesium at 5 mM was added to the potassium medium, cytoplasmic streaming stopped as in the absence of these divalent cation. In this case, however, velocity gradually recovered without repolarization of the membrane. It is suggested that these divalent cations bind to the plasma membrane and inhibit the signal processing from membrane depolarization to stoppage of cytoplasimic streaming.Analysis of temperature dependency of cytoplasmic streaming in Characeae Using cryomicroscope, dependency of velocity of cytoplsmic streaming was analyzed in internodal cells of Nitellopsis obtusa. In intact cells, two types of response was observed among cells tested. In one group, velocity linearly decreased in the temperature range of 25-5ﾟC. In another group, velocity linearly decreased in a range of 25-15ﾟC, but strongly inhibited at the temperature lower than 10ﾟC. Same experiment was also carried out in tonoplast-free cells, in which EGTA, a calcium chelator, and PIPES, a pH buffer, was introduced. In all tonoplast-free cells tested, velocity linearly decreased in temperature range of 25-5ﾟC. At 5ﾟC, velocity in tonoplast-free cells was three times higher than that in intact cells. The present study suggested that temperature dependency of cytoplasmic streaming reflects not only temperature characteristics of actomyosin involved in generation of the motive force but also disturbance of intracellular homeostasis of these ions at low tempeture. Less