Synergistic effect of heat-shock and UV-irradiation on mitosis and protein synthesis in G2-phase plasmodia of Physarum polycephalum Schw--reduction in UV-induced mitotic delay in a preheat-shocked system.

The synergistic effect of UV irradiation and heat-shock during the last 3 hr of G2 phase of the cell cycle in the plasmodia of P. polycephalum, in terms of mitotic delay and inhibition of protein synthesis, has been evaluated. The mitotic delay due to both perturbers coordinately increased closer to mitosis. Maximum mitotic delay was obtained in plasmodia heat-shocked after UV irradiation, indicating the presence in this system of either a heat-labile mitogenic substance which is comparatively less susceptible to UV or a substance which is made more susceptible to hyperthermia by UV. A protective role for heat-shock applied before irradiation has been observed in that, radiation-induced mitotic delay is significantly reduced in this combination. There was severe inhibition of translation in all the perturbed classes. Organelle level effects which are independent of major protein synthetic activities or different levels of heat-shock protein production could be the reason for the lack of correlation between percentage inhibition of general protein synthesis and the extent of mitotic delay with respect to the two double-perturbed systems.

Photomorphogenesis in Physarum polycephalum. Temporal expression pattern of actin, alpha- and beta-tubulin.

Photo-induced fruiting-body formation of the slime mold Physarum polycephalum can be divided into two stages. The first stage (I) starts with the beginning of illumination (0 h) and ends with the formation of nodules (12 h). The second stage (II) is characterized by culmination, sporangiophore formation and melanization of sporangiophore heads (13-17 h). We investigated the expression of actin, alpha- and beta-tubulin during this differentiation process using Northern blot analysis and run-off transcription in isolated nuclei. Whereas actin mRNA is irreversibly lost during stage I, we observed two peaks in mRNA concentration for alpha-tubulin and one peak for that of beta-tubulin during stage I and a coordinated alpha/beta-tubulin mRNA induction during stage II. Stage II induction appears to be related to a presporangial mitosis. Transcriptional activity of the three genes studied shows two maxima, namely one in the middle of stage I and the other at the end of stage II. Our data suggest that the expression of the three cytoskeletal proteins investigated follows a distinct temporal pattern comprising changes in both mRNA synthesis and decay. We also propose a novel function of tubulin in Physarum which is not related to mitosis.

Mitotic delays and macromolecular synthesis in G2 phase-irradiated plasmodia of Physarum polycephalum.

Mitotically synchronous surface plasmodia of Physarum polycephalum were irradiated at different times of G2 phase with doses of UV ranging from 350 to 2800 Jm-2. The UV sensitivity, measured in terms of delay in progression towards mitosis, gradually declined through G2 phase to almost zero in early prophase, when irradiated with 350 Jm-2 of UV. However, with higher doses, 700 and 1400 Jm-2, late G2 was found to be even more susceptible than early G2. The delay in these cases rises for the last 0.1 part of the cycle up to a UV-transition point in early prophase. Thus, processes which were not sensitive to a lower dose of UV are affected by higher doses. Above 700 Jm-2, delay was not directly proportional to dose. With doses above 1400 Jm-2, the delays obtained were more variable, although some tendency for an increased delay is observed in plasmodia irradiated in late G2 when compared with those irradiated in middle G2. The least UV-sensitive phase is between -0.2 and -0.4 part of the cycle in relation to mitosis (0 point). Both RNA synthesis and protein synthesis are inhibited in plasmodia irradiated at any time during G2 phase with 1400 Jm-2 of UV. However, on an average, irradiation in late G2 caused the most inhibitions.

Cyclic nucleotide responses and radiation-induced mitotic delay in Physarum polycephalum.

The response of the plasmodial levels of cyclic AMP and cyclic GMP in Physarum polycephalum to several putative phosphodiesterase inhibitors and to ionizing radiation has been measured. Isobutylmethylxanthine (2 mM) induces a rapid transient threefold elevation of cyclic AMP alone, with maximum response in about 10 min and return to the base line in about 30 min. Theophylline (2 mM) induces a rapid, sustained twofold elevation of cyclic GMP only. Caffeine (2 mM) and Ro-20-1724 (18 microM) both elicit a rapid transient rise in cyclic AMP, resembling the isobutylmethylxanthine response, and a slow transient elevation of the cyclic GMP level. Of particular interest is the rapid threefold transient elevation of the cyclic AMP, but not of the cyclic GMP, level by gamma radiation. These results (1) emphasize the organism and agent specificity of cyclic nucleotide responses, (2) indicate that the kinetics and apparent refractory response are compatible with the function of previously identified membrane receptor systems, i.e., a blue light receptor and a choleragen receptor mediating cyclic AMP responses, and (3) suggest a relationship between the agent-specific cyclic nucleotide responses and the modification of gamma-radiation-induced delay of mitosis by the same agents, i.e., a decrease in the delay by those which elevate cyclic GMP levels and an increase in the delay by those which elevate only cyclic AMP levels.

The participation of elevated levels of cyclic GMP in the recovery from radiation-induced mitotic delay.

The levels of cyclic AMP and cyclic GMP have been measured in Physarum plasmodia before and after treatment with gamma-radiation, 2 mM caffeine, or combinations of the two agents and compared to the length of the radiation-induced mitotic delay. Caffeine alone produces a rapid transient elevation of cyclic AMP and a slower delayed elevation of cyclic GMP. Irradiation elicits an immediate transient increase in cyclic AMP and a later cyclic GMP increase which accompanies or precedes the delayed mitosis. A composite pattern is produced by combinations of radiation and caffeine, a distinctive feature of which is an elevated level of cyclic GMP near the time of the radiation-delayed and caffeine-promoted mitosis. With pretreatment by caffeine, the least radiation-induced mitotic delay occurs when plasmodia are irradiated during the caffeine-elicited increase in cyclic GMP. The plasmodium becomes refractory to the reduction of mitotic delay by caffeine at approximately the time it becomes refractory to the further elevation of cyclic GMP by caffeine. The data support a role for cyclic AMP in the onset of and for cyclic GMP in the recovery from mitotic delay induced by ionizing radiation.

Blue light influences gene expression and motility in starving microplasmodia of Physarum polycephalum.

The differentiation of starving Physarum polycephalum microplasmodia into resting structures (spherules) was studied. Early events in this differentiation pathway include decreases in both plasmodial motility and protein synthesis. The starving plasmodia show a blue light avoidance response. Blue light (lambda max 450 nm, irradiance 16 W/m2) acts antagonistically to the starvation stimulus so that spherule formation is inhibited [16]. Light affects each of the above mentioned events of the differentiation pathway: the migration rate of illuminated plasmodia is stimulated, the light avoidance response is irreversibly lost. The rate of incorporation of radioactive leucine into plasmodial protein remains at a higher level in illuminated plasmodia as compared to the decreasing rate during spherule formation in the dark. Protein degradation, uptake of external leucine, and the size of the internal leucine pool are not affected by light. Analysis by SDS-polyacrylamide gel electrophoresis of pulse-labelled plasmodial proteins reveals that blue light inhibits the synthesis of distinct starvation-induced proteins and allows continued synthesis of all major plasmodial proteins. Some of the blue light responses described are mimicked by alpha-amanitin suggesting that light might influence gene expression at the level of transcription.

[UV-light-tested protein--DNA interactions in the nuclei during the cell cycle in Physarum polycephalum]. / Testiruemye uf-svetom belok--DNK-kontakty v iadrakh na raznykh stadiiakh kletochnogo tsikla Physarum polysephalum.

The release of DNAs from nuclear preparations isolated at different steps of the cell cycle of the synchronous culture of Physarum polycephalum and irradiated with UV-light for 5 min (lambda 253.7 A, i = 1.8 . 10(2) j . m-2 . min-1) into 1% Na-DS - 1.5 M NaCl solution was studied. The value obtained correlated well with the degree of binding of interacting proteins with DNA in chromatin and was identical for all the preparations tested with the only exception of the nuclei isolated at the end of the S-phase, when a slight but significant (15 +/- 10%) increase in DNA release was observed. The results obtained are discussed in terms of structural transitions and function of chromatin during the cell cycle of the myxomycete Physarum polycephalum.

The pathway of photosensory transduction in Physarum polycephalum.

Irradiation of the plasmodia of Physarum with blue and white light results in a transient change of theie oscillatory contraction frequency. This reaction to light decreases with increasing distance from the illuminated area (Block and Wohlfarth-Bottermann, 1981). The first local appearance of light response in non-illuminated parts of the plasmodia was used to analyse the sensory pathway of the light stimulus modulating the contractile apparatus. Different experimental assays revealed that the direction of photosensory transduction is determined by the momentary direction of protoplasmic shuttle streaming. The endoplasmic flow carriers the signal responsible for photosensory transduction and light reaction to the force generating ectoplasmic tube.

Blue light as a medium to influence oscillatory contraction frequency in Physarum.

Blue light (496 nm; threshold intensity approximately 1500 Lux) induces a transient frequency decrease of oscillatory contraction automaticity in Physarum. Many, but not all specimens react by an increase in the force amplitude of longitudinal contraction. The spectral region of 496 nm provokes a photophobic response of the plasmodia. The blue light reaction of radial and longitudinal contraction activities decreases with increasing distance from the irradiated area. The light-induced decrease in frequency can be used for experimental phase shifting, e.g., when studying the nature and the pathway of signal transmission for the spatial phase synchronization of contractile activities.

Response of Aspergillus nidulans and Physarum polycephalum to microwave irradiation.

The influence of microwaves on genetic processes in Aspergillus nidulans and Physarum polycephalum was investigated. Suspensions of organisms were exposed in the far zone to 2450-MHz waves at 10 mW/cm2 for one hour in both CW and pulsed (1 microsecond, 600 pps) fields. Spores of A. nidulans were irradiated before and during germination. No changes in survival rate or in frequency of morphological mutation were found. Polycephalum under the influence of CW microwaves incorporated 3H-Thymine into DNA at a rate five times that of controls and twice that of thermal controls. The accelerated synthesis may reflect more efficient volume heating by microwaves, or in the presence of microthermal gradients in suspensions, or field-specific influences in concern with focal or volume heating.