[Involvement of phosphatidylinositol-4,5-bisphosphate binding proteins in the generation of contractile oscillations in the Physarum polycephalum plasmodium].

Using the Physarum polycephalum, plasmodium, a giant amoeboid cell with the strongly pronounced auto-oscillatory mode of motility, which exhibits regularities of motile behavior common with those of tissue cells and has the same signal systems, the possibility of the participation of phosphatidylinositol-4,5-bisphosphate in the regulation of the contractile activity has been studied. The effect of neomycin as a substrate inhibitor of phospholipase C, which binds with high affinity to phosphatidylinositol-4,5-bisphosphate in the membrane, on force oscillations generated by plasmodial strands under isometric conditions and after the addition of the protein kinase C inhibitors staurosporine, UCN-01, and Ro-318220, separatelyand in combination with the calmodulin inhibitor calmidazolium has been examined. It has been shown that neomycin at pH 7.0 and concentrations of 0.1-5.0 mM stops contractile oscillations for 10-30 min but then they begin to gradually restore; the oscillation period at the initial stage of the restoration is.shorter than it was earlier and then increases due to the elongation of the contraction phase. Analysis of data obtained is in favor of the assumption that the plasmodial membrane contains MARCKS-like proteins and protein kinase C-controlled pools of phosphatidylinositol-4,5-bisphosphate, which can participate in the generation of auto-oscillations observed in the plasmodium.

[Involvement of cyclic adenosine monophosphate in the control of motile behavior of Physarum polycephalum plasmodium].

Possible involvement of autocrine factors into the control of motile behavior via a receptor-mediated mechanism was investigated in Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the auto-oscillatory mode of motility. Cyclic adenosine monophosphate (cAMP) and extracellular cAMP-specific phosphodiesterase, its involvement into the control of plasmodium motile behavior was proved by action of its strong inhibitor, were regarded as putative autocrine factors. It was shown that the plasmodium secreted cAMP. When it was introduced into agar support, 0,1-1 mM cAMP induced a delay of the plasmodium spreading and its transition to migration. When locally applied, cAMP at the same concentrations induced typical for attractant action the increase in oscillation frequency and the decrease of ectoplasm elasticity. The ability to exhibit positive chemotaxis in cAMP gradient and the dependence of its realization were shown to depend on the plasmodium state. Chemotaxis test specimens obtained from the migrating plasmodium, unlike those obtained from growing culture, generate alternative fronts which compete effectively with fronts oriented towards the attractant increment. The results obtained support our supposition stated earlier that advance of the Physarum polycephalum plasmodium leading edge is determined by local extracellular cAMP gradients arising from a time delay between secretion and hydrolysis of the nucleotide.

[Involvement of extracellular cAMP-specific phosphodiesterase in control of motile activity of Physarum polycephalum plasmodium].

Possible involvement of extracellular cAMP-specific phosphodiesterase in the control of cell motile behavior has been investigated in Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the autooscillatory mode of motility. It was found that the rate of the hydrolysis of 10 mM cAMP by a partially purified preparation of cAMP-specific phosphodiesterase secreted by the plasmodium in the course of migration decreases 20-30 times under the action of 1 mM dithiothreitol. In the presence of 1-5 mM of this strong reducing agent, the onset of the plasmodium spreading and the transition to the stage of migration were delayed in a concentration-dependent manner. In accordance with the morphological pattern of motile behavior, the duration of the maintenance of high frequency autooscillations, which normally precede the increase in the rate of the spreading and appear also in response to the application of attractants at spatially uniform concentrations, strongly increased by the action of dithiothreitol. The results obtained suggest that the autocrine production of cAMP and extracellular cAMP-specific phosphodiesterase is an important constituent of the mechanism controlling the motile behavior of the Physarum polycephalum plasmodium.

[The role of phosphoinositide-3-kinase in controlling the shape and directional movement in Physarum polycephalum plasmodium].

The influence of wortmannin and LY294002, specific inhibitors of phosphoinosite-3-kinase, on the shape, motile behavior, and chemotaxis toward glucose has been investigated in Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the autooscillatory mode of motion. Both inhibitors were shown to cause a reduction of the plasmodium frontal edge and a decrease in the efficiency of mass transfer during migration. They also suppress chemotaxis toward glucose and eliminate characteristic changes in autooscillatory behavior normally observed in response to the treatment of the whole plasmodium with glucose. The manifestation of these effects depends on the inhibitor concentration, the duration of treatment, and the size of plasmodium. The involvement of phosphoinosite-3-kinase in creating the frontal edge and in controlling the chemotaxis of Physarum plasmodium suggests that the interrelation of polar shape and directional movement of amoeboid cells with the distribution of phosphoinositides in the plasma membrane has the universal nature.

[Chemotaxis as a method for testing of the biological effects of silver nanoparticles].

A method for assessing the abiotic efficiency of water-dispersed nano-sized silver particles is suggested. Nanoparticles were obtained by the method of biochemical synthesis in reversed micelles made from anionic surfactant bis-(2-ethylhexyl) sodium sulfosuccinate (AOT). A water dispersion of nanoparticles was prepared from the micellar solution in organic solvent by means of a special procedure. The abiotic efficiency assay is based on the capacity of cells to respond by the negative chemotaxis to chemical agents with harmful metabolic action. In plasmodium of myxomicete Physarum polycephalum, the biocide and repellent effects of silver nanoparticles, Ag+ ions, and AOT were tested in order to compare the abiotic efficiency of these substances in water solution and after introduction into agar substrate. The increase in the oscillation period, the reduction in the area of spreading, and the avoidance reaction in the spatial test, taken together, revealed a much higher repellent efficiency of silver nanoparticles as compared with that of Ag+ ions and AOT. The NSP concentrations lethal for Physarum were similar to those found earlier for bacteria and viruses. The chemotaxis-based tests applied in this study allow one to quantitatively assess cell reactions and monitor their time course. Besides, they have a much higher resolving capacity than the tests based on lethal effects of abiotic substances.

Ionomycin and 2,5'-di(tertbutyl)-1,4,-benzohydroquinone elicit Ca2+-induced Ca2+ release from intracellular pools in Physarum polycephalum.

Calcium level in organelles of the slime mold Physarum polycephalum was monitored by chlortetracycline, a low-affinity calcium indicator. It was found that 2,5'-di(tertbutyl)-1,4,-benzohydroquinone (BHQ) at a concentration of 100 microM, but not the highly specific inhibitor of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), thapsigargin (1-10 microM), elicited calcium release from the CTC-stained intracellular calcium pool. Ionomycin also caused a calcium release (23.7+/-5.1%), which was less than that induced by BHQ (30.1+/-6.0%). Procaine (10 mM), a blocker of ryanodine receptor, completely abolished the responses to BHQ and ionomycin. Another blocker, ryanodine (100 microM), only slightly diminished the responses to ionomycin and BHQ. Apparently, BHQ and ionomycin acting as a Ca2+-ATPase inhibitor and an ionophore, respectively, elicit an increase in [Ca2+]i, which in turn triggers a calcium-induced calcium release (CICR) via the ryanodine receptor. Caffeine, an activator of ryanodine receptor, at a concentration of 25-50 mM produced a Ca2+-release (5.6-16.0%), which was not similar in magnitude to CICR. The response to 25 mM caffeine was only moderately inhibited by 25 mM procaine, and almost completely abolished by 50 mM procaine and 100 microM ryanodine.

Effects of Ca2+-ATPase inhibitors, ionomycin, and pharmacological modulators of ryanodine receptor on calcium release from intracellular pools and on oscillatory contractile behavior in Physarum polycephalum.

Changes in calcium levels in organelles of the plasmodium of the myxomycete Physarum polycephalum were analyzed using the fluorescent calcium indicator chlortetracycline (CTC). Both the Ca2+-ATPase inhibitor 2,5;-di(tert-butyl)-1,4-benzohydroquinone (BHQ) (100 microM) and the calcium ionophore ionomycin (1 microM) induce a significant decrease in fluorescence level (by 30%) in CTC-stained microplasmodia; this is caused by release of calcium from intracellular storage compartments. An activator of ryanodine receptors, caffeine (10-50 mM), is less effective on Ca2+ release than BHQ or ionomycin, and their inhibitor, ryanodine (100 microM), almost completely blocks the response to caffeine, but only slightly decreases the effects of BHQ or ionomycin. Procaine, another inhibitor of ryanodine receptors, at 10 mM concentration completely abolishes both the BHQ and the ionomycin responses, but 50 mM is necessary to block the effect of 25 mM caffeine. These results suggest that both the BHQ- and the ionomycin-dependent Ca2+ releases occur through the ryanodine receptor and are to be considered as calcium-induced Ca2+ release (CICR). Both the ionomycin and the BHQ responses persist in the presence of Cd2+, which blocks Ca2+ channels of the plasmalemma. In most cases, Cd2+ itself induces release of Ca2+ from the CTC-stained calcium pool; the more effective Cd2+ is, the less the following ionomycin or BHQ responses occur. This indicates that Ca2+ entry through plasmalemma plays no significant role in the ionomycin- or BHQ-evoked initiation of CICR, and that the Cd2+- and BHQ/ionomycin-depleted Ca2+ stores overlap.