Regulation of cytoplasmic streaming by calcium in plant cells

从百合花粉管中分离肌凝蛋白。花粉管在低离子强度溶液中均质，离心制备粗提物。用粗提物对盖板玻璃表面进行处理。用荧光染料标记的肌动蛋白丝与Mg一起应用。ATP、肌动蛋白丝在玻璃表面呈现滑动运动（体外运动测定），表明肌球蛋白以活性形式被提取。分别用鸡胸肌肌动蛋白丝、羟基磷灰石柱、凝胶过滤共沉淀法纯化肌球蛋白。用SDS-PAGE分析最终样品时，发现肌球蛋白是由170 kD的重链组成。我们成功地产生了针对重链的抗体。用该抗体对花粉管粗提物进行免疫印迹时，只有170 kD的组分与该抗体发生反应。加入从鸡胸肌中纯化的肌动蛋白丝，可使肌球蛋白样品的atp酶活性提高60倍。用纯化的肌球蛋白进行体外运动测定，肌动蛋白丝的运动速度为7.7 μ m/s，与花粉管内细胞质流动速度基本一致。这一事实表明肌凝蛋白参与了细胞质流动动力的产生。我们现在正计划做一个实验来检验肌凝蛋白对钙的敏感性。特征细胞中钙对细胞质流动的调节机制特征细胞中，细胞质流动在动作电位产生时短暂停止。由于细胞质中游离钙的增加，细胞质流动停止。据推测，肌凝蛋白的磷酸化参与了细胞质流动的停止。在本研究中，我们分析了通过增加外部介质钾浓度来阻止腋下Nitella axilliformis细胞膜去极化时细胞质流动的过程。当胞外钾浓度从0.1 mM增加到50 mM时，膜去极化并保持去极化状态。通过降低钾离子浓度，膜重极化。由于膜的去极化，细胞质流动迅速停止，速度保持在很低的水平。通过降低钾离子浓度使细胞膜复极化，细胞质流动速度在几分钟内恢复。当在钾培养基中加入5 mM的钙或镁时，细胞质流动停止，因为没有这些二价阳离子。然而，在这种情况下，速度逐渐恢复，膜没有复极化。这些二价阳离子与质膜结合，抑制了从膜去极化到细胞质流动停止的信号处理。利用低温显微镜，研究了黑绒藜节间细胞细胞质流动速度对温度的依赖性。在完整的细胞中，在测试的细胞中观察到两种类型的反应。其中一组在25-5个温度范围内，速度呈线性下降。在另一组中，在25 ~ 15ºC范围内，速度呈线性下降，但在低于10ºC的温度下，速度受到强烈抑制。同样的实验也在无张力细胞中进行，其中引入了EGTA（钙螯合剂）和PIPES （pH缓冲液）。在所有测试的无张力细胞中，在25-5个温度范围内，速度线性下降。在5 C时，无张力质体细胞的速度是完整细胞的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，还反映了这些离子在低温下对胞内稳态的干扰。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