| Research Abstract |
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
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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
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