Regulation of mitosis onset and thymidine kinase synthesis during the cell cycle of Physarum polycephalum: action of aphidicolin

In Physarum polycephalum (Myxomycetes) aphidicolin has been found to delay metaphase onset when applied to synchronous plasmodia 3 h before control metaphase. In contrast to the action of temperature shifts, aphidicolin treatment did not delay the initiation of the increase of thymidine kinase synthesis (EC 2.7.1.21, ATP-thymidine 5' phosphotransferase) and the decrease of the synthesis of thymidine kinase occurred normally after completion of mitosis in presence of aphidicolin. The amount of thymidine kinase synthesized was larger for aphidicolin treated plasmodia than in the control due to both a longer period of increased synthesis and a higher maximum rate of synthesis. These results were interpreted by postulating the presence of two regulatory pathways. The first one acting on the increase of the synthesis of thymidine kinase and on mitosis onset was sensitive to temperature shifts from 22 to 32 degrees C. The second one acting on mitosis onset only was sensitive to aphidicolin.     

A PSTAIRE CDK-like protein localizes in nuclei and cytoplasm of Physarum polycephalum and functions in the mitosis

CDKs play key roles in controlling cell cycle progression in all eukaryotes. In plants, multiple CDKs are present,among which the best characterized CDKs are PSTAIRE CDKs. In this study, we carried out Western blot,immunoelectron microscopy and antibody treatment with an anti-PSTAIRE monoclonal antibody to explore the subcellular localization and functions of PSTAIRE CDKs in Physarum polycephalum. The results of Western blot and immunoelectron microscopy showed that in P. polycephalum, a PSTAIRE CDK-like protein was 34 kD in molecular weight and located in both nuclei and cytoplasm. In nuclei, the protein was mainly associated with chromosomes and nucleoli. The expression of the PSTAIRE CDK-like protein in both the plasmodia and nuclei showed little fluctuation through the whole cell cycle. When treated with an anti-PSTAIRE monoclonal antibody at early S phase, the cells were arrested in S phase, and the mitotic onset of P. polycephalum was blocked for about 1 h when treated at early G2 phase.Our data indicated that the PSTAIRE CDK-like protein has a direct bearing on the mitosis.     

Adenosine Triphosphate and Synchronous Mitosis in Physarum polycephalum

Synchronous mitoses occur in Physarum polycephalum in the absence of cell division. Nucleoside and nucleotide profiles were prepared from synchronously growing P. polycephalum at intervals throughout the growth cycle. Comparison of these profiles demonstrates that the pool of adenosine triphosphate decreases from a high level at prophase to a minimum through mitosis and increases again in the postmitotic period. These events appear to coincide with changes in the pools of adenosine diphosphate and adenosine but not with that of adenosine monophosphate. This observed decrease in the pool of adenosine triphosphate during mitosis was confirmed by direct enzymatic assay. These results presumably reflect the energy demands of the cell during mitosis.     

Physarum thymidine kinase. A step or a peak enzyme depending upon temperature of growth.

The variations of thymidine kinase or ATP:thymidine 5'-phosphotransferase (EC 2.7.1.21) during the cell cycle of Physarum polycephalum plasmodia have been studied at two extreme physiological temperatures: 22 degrees C and 32 degrees C. At 22 degrees C the enzyme activity increases near mitosis and stays constant during late S and G2 phases, exhibiting the typical pattern of a 'step enzyme'. But at 32 degrees C thymidine kinase activity goes through a maximum 1 h 30 min after mitosis and decreases during the subsequent phases as expected for a 'peak enzyme'. The rate of enzyme degradation and/or inactivation, measured in the presence of metabolic poisons (cycloheximide or dinitrophenol), appears to follow a simple exponential function with a half-life of approximately 3 h and 1 h at 22 degrees C and 32 degrees C respectively. The effect of growth temperature on the decrease of thymidine kinase activity can account entirely for the differences in the pattern of enzyme activity at the two extreme temperatures. Tentative calculations indicate that the rate of enzyme synthesis is nearly constant during the cell cycle except near mitosis, where it is temporarily increased. The results suggest the existence of a regulatory mechanism able to modulate the rate of synthesis of thymidine kinase during the cell cycle.     

Evidence for a homolog of the yeast cell cycle regulatory gene product of cdc2+ in Physarum polycephalum

Evidence for the presence of a Cdc2-like protein in Physarum polycephalum has been obtained using a peptide antibody directed against a highly conserved amino acid sequence near the N-terminal end of Cdc2, Cdc28 and Cdc2HS. The antibody detected a 34 kDa cytoplasmic protein, similar in apparent size to Cdc2 in yeast and Cdc2Hs in HeLa cells. A 60 kDa nuclear band was also detected in Physarum but not in yeast or HeLa. Evidence is presented that this is not related to the 34 kDa protein nor is it found in HeLa nuclei or yeast cells. The Cdc2-like protein level did not fluctuate over more than 10 h of the naturally synchronous cell cycle of Physarum. Several heat-shock experiments using regimens that either: delayed mitosis and S-phase; prevented mitosis or uncoupled S-phase from mitosis were performed. None had any effect on the level of the Cdc2-like protein. The induction of spherulation by starvation was shown to have no effect on the levels of the 34 kDa Cdc2 analog. The invariant level of the 34 kDa protein during the cell cycle and starvation is consistent with previous results obtained with yeast. Three heat-shock regimens which either delay mitosis, eliminate S-phase or uncouple mitosis from S-phase in Physarum also had no effect on the level of the 34 kDa protein. This result emphasizes the stable nature of this protein.     

Evidence For the Presence of A Cdc2-Like Protein Kinase In the Dinoflagellate Crypthecodinium Cohnii

ABSTRACT. The unusual nature of mitosis and ancestral organization of the dinoflagellate nucleus prompted the question of whether the cdc 2-like histone H1 kinase, a presumed ubiquitous cell cycle regulator in eukaryotes, is present in these primitive organisms. Western blotting of Crypthecodinium cohnii protein extracts using antibody against the Pro-Ser-Thr-Ala-Ile-Arg-Glu (=PSTAIRE) amino acid sequence motif, conserved in all cdc 2 homologues known, revealed one prominent band corresponding to a protein with an apparent relative molecular weight ≈ 34,000, identical in mobility to that from HeLa cells and Physarum polycephalum , higher and lower eukaryotic controls, respectively. Incubation of C. cohnii cell lysates with p13 ^{suc 1}-sepharose beads, which preferentially, though not exclusively, bind p34 ^{cdc 2}, resulted in precipitation of a 34-kDa protein which was reactive with anti-PSTAIRE antibody, selectively competed for by the PSTAIRE peptide and able to phosphorylate histone H1 in vitro. We conclude that the dinoflagellate C. cohnii contains a protein very similar to the cdc 2 gene product from fission yeast and its homologues in all eukaryotes studied thus far.     

Ca2+-binding modulator protein in protozoa and myxomycete.

Ca2+-binding protein with the properties of brain modulator protein of 3,5-cyclic nucleotide phosphodiesterase was identified in Physarum polycephalum plasmodia and in Euglena gracilis and Amoeba proteus cells by urea polyacrylamide gel electrophoresis and activation of cyclic nucleotide phosphodiesterase and of myosin light chain kinase.     

An immunological approach to the isolation of factors with mitotic activity from the plasmodial stage of the myxomycete Physarum polycephalum

Nuclear divisions in plasmodia of Physarum polycephalum were advanced by applying immunologically purified plasmodial extracts of late G2 phase on the surface of plasmodia which were 1.5 h before the third mitosis. The purification of G2 extracts was achieved by interaction of antibodies prepared against the antigens of early S phase plasmodia with the antigens of late G2 plasmodia. There was no advancement of mitosis by extracts prepared from early S phase plasmodia. Untreated G2 extracts did not accelerate mitosis with the same effectiveness as did antibody purified G2 extracts.     

Molecular identification of PpHDAC1, the first histone deacetylase fron the slime mold Physarum polycephalum

The dynamic state of post-translational acetylation of eukaryotic histones is maintained by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs and HDACs have been shown to be components of various regulatory protein complexes in the cell. Their enzymatic activities, intracellular localization and substrate specificities are regulated in a complex, cell cycle related manner. In the myxomycete Physarum polycephalum multiple HATs and HDACs can be distinguished in biochemical terms and they exhibit dynamic activity patterns depending on the cell cycle stage. Here we report on the cloning of the first P. polycephalum HDAC (PpHDAC1) related to the S. cerevisiae Rpd3 protein. The expression pattern of PpHDAC1 mRNA was analysed at different time points of the cell cycle and found to be largely constant. Treatment of macroplasmodia with the HDAC inhibitor trichostatin A at several cell cycle stages resulted in a significant delay in entry into mitosis of treated versus untreated plasmodia. No effect of TSA treatment could be observed on PpHDAC1 expression itself.     

Studies of histidine phosphorylation by a nuclear protein histidine kinase show that histidine-75 in histone H4 is masked in nucleosome core particles and in chromatin

Histone H4 is a good substrate in vitro for the protein histidine kinase activity found both in Physarum polycephalum nuclear extracts and in Saccharomyces cerevisiae cell extracts. However, histone H4 in nucleosome core particles is not a substrate for these kinases. Isolated chromatin was also not a substrate for the protein histidine kinase. The results significantly limit possible interpretations of histidine phosphorylation on histone H4 in vivo and provide a new, sharper focus for future work. In addition, a polynucleotide kinase activity was identified in the Physarum extracts.     

Regulation of p34cdc2 protein kinase during mitosis

The cell-cycle timing of mitosis in fission yeast is determined by the cdc25+ gene product activating the p34cdc2 protein kinase leading to mitotic initiation. Protein kinase activity remains high in metaphase and then declines during anaphase. Activation of the protein kinase also requires the cyclin homolog p56cdc13, which also functions post activation at a later stage of mitosis. The continuing function of p56cdc13 during mitosis is consistent with its high level until the metaphase/anaphase transition. At anaphase the p56cdc13 level falls dramatically just before the decline in p34cdc2 protein kinase activity. The behavior of p56cdc13 is similar to that observed for cyclins in oocytes. p13suc1 interacts closely with p34cdc2; it is required during the process of mitosis and may play a role in the inactivation of the p34cdc2 protein kinase. Therefore, the cdc25+, cdc13+, and suc1+ gene products are important for regulating p34cdc2 protein kinase activity during entry into, progress through, and exit from mitosis.     

Fluctuations in S-adenosyl-L-methionine (AdoMet) during mitotic cycle of the myxomycete Physarum polycephalum

A sensitive radioisotope dilution method was used to measure the S-adenosyl-L-methionine (AdoMet) content in macroplasmodia of the slime mold Physarum polycephalum during the mitotic cycle. The AdoMet pool had two maxima, one during mitosis, the other in the middle of G2 phase.     

Thymidine kinase enzyme variants in Physarum polycephalum. In vitro interconversion of the enzyme variants.

Isoelectric focusing of plasmodial extracts of Physarum polycephalum demonstrated the presence of several multiple enzyme variants of thymidine kinase, which appear sequentially during the nuclear division cycle. Variants (A) + (A1) are the only enzyme variants found in the late G2-phase, whereas the variants (C) + (C1) are only present at the time of mitosis and S-phase (1, 2). Evidence is presented that multiple forms of thymidine kinase (A) + (A1) with high pI arise by dephosphorylation of a primary translation product with low pI (C and/or C1). The thymidine kinase fractions (A) + (A1) and (C) + (C1) + (c1) were separated and partially purified by DEAE-cellulose chromatography. The enzyme variants (C) + (C1) are converted in vitro by an endogenous enzymatic factor as well as by bacterial alkaline phosphatase into the variants (A) + (A1).     

Effect of inhibition of the catalytic activity of cyclic AMP-dependent protein kinase on mitosis in PtK1 cells

Evidence has suggested that cyclic AMP, acting through activation of the type II cyclic AMP-dependent protein kinase, may play a role in the regulation of interphase and mitotic microtubules. In order to examine the potential role of the type II cAMP-dependent kinase during mitosis, dividing PtK1 cells were microinjected with two specific inhibitors of the catalytic activity of the type II kinase. These inhibitors were a specific protein inhibitor of cAMP-dependent protein kinase (PKI) and an affinity-purified polyclonal antiserum (anti-C) directed against the catalytic subunit of the kinase. Both have been shown previously to inhibit kinase activity in vitro. Microinjection of PKI during early- to mid-prophase significantly delayed the progression of the cells through mitosis, with the greatest delay occurring in metaphase. PKI injected during prometaphase also delayed progression through mitosis but to a lesser extent. Microinjection of anti-C during early- to mid-prophase also caused a significant delay in the completion of mitosis, with many cells becoming "hung up" in prometaphase. Anti-C injected during prometaphase had little effect on subsequent progression through mitosis. Microinjection of either anti-C or PKI during metaphase had no discernible effect. No effect on anaphase movement of chromosomes was observed with any treatment. These results provide further evidence that cAMP-dependent phosphorylation may be involved in the regulation of mitosis, although whether it acts directly through regulation of mitotic spindle microtubules is unclear.     

Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells

In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.     

Study of pp60v-src protein kinase activity in synchronized chicken embryo fibroblasts infected with Rous sarcoma virus

Chicken embryo fibroblast (CEF) cultures, synchronized by the addition of serum to stationary cells, were exposed to Schmidt-Ruppin strain of Rous Sarcoma Virus (SR-RSV) and the appearance of pp60v-src protein kinase activity was examined through the cell cycle. In cells infected either at the beginning or at the end of G1, the onset of pp60v-src protein kinase activity was coincidental, closely following mitosis, with a delay between the infection of cells with SR-RSV and the appearance of protein kinase activity of about 20 and 16 h, respectively. In cells infected during the S phase this delay was 16 h, as observed for late G1 cells. These experiments show that the activity of pp60v-src protein kinase, which cannot be detected before the first mitosis following infection does not depend on G1. The aphidicolin prevented protein kinase activity if added before or at the beginning of S phase, but not if added later, which is presumably related to the inhibition of S phase, required for provirus integration. The use of colcemid, which suppresses cell division, did not inhibit but delayed the appearance of protein kinase activity. These results show that the synthesis of an active oncogene product, such as pp60v-src protein kinase, depends on both S phase and mitosis.     

Cyclic fluctuations of the mitotic Ca2+-ATPase during the cell cycle of the myxomycete Physarum polycephalum

The occurrence of the mitotic Ca2+-ATPase, resembling the enzyme described for higher organisms, is demonstrated in multinuclear plasmodia of the myxomycete Physarum polycephalum. The activity of this enzyme undergoes cyclic fluctuations during the synchronous nuclear cycle with a minimum in early G2-phase and a maximum around the time of mitosis.     

Staurosporine overrides checkpoints for mitotic onset in BHK cells.

Under normal conditions, mammalian cells will not initiate mitosis in the presence of either unreplicated or damaged DNA. We report here that staurosporine, a tumor promoter and potent protein kinase inhibitor, can uncouple mitosis from the completion of DNA replication and override DNA damage-induced G2 delay. Syrian hamster (BHK) fibroblasts that were arrested in S phase underwent premature mitosis at concentrations as low as 1 ng/ml, with maximum activity seen at 50 ng/ml. Histone H1 kinase activity was increased to approximately one-half the level found in normal mitotic cells. Inhibition of protein synthesis during staurosporine treatment blocked premature mitosis and suppressed the increase in histone H1 kinase activity. In asynchronously growing cells, staurosporine transiently increased the mitotic index and histone H1 kinase activity but did not induce S phase cells to undergo premature mitosis, indicating a requirement for S phase arrest. Staurosporine also bypassed the cell cycle checkpoint that prevents the onset of mitosis in the presence of damaged DNA. The delay in mitotic onset resulting from gamma radiation was reduced when irradiation was followed immediately by exposure to 50 ng/ml of staurosporine. These findings indicate that inhibition of protein phosphorylation by staurosporine can override two important checkpoints for the initiation of mitosis in BHK cells.     

Cycloheximide-induced mitotic delay in Physarum polycephalum

Cycloheximide pulses applied to Physarum polycephalum surface plasmodia delay mitosis. Pulses applied in G2 cause a delay of mitosis which is linearly dependent on the phase in the cell cycle at which the pulse is applied. A 30 min pulse of 10 micrograms/ml cycloheximide starting in G2 at time t after mitosis induces an excess delay (delay in excess of pulse duration) of the next mitosis of (0.55) t-1.3 h. The excess delays induced by 7 h pulses during G2 are at most 1 h larger. Pulses applied less than 30 min before mitosis induce only small delays.