Amphibian oocyte maturation induced by extracts of Physarum polycephalum in mitosis

The orderly progression of eukaryotic cells from interphase to mitosis requires the close coordination of various nuclear and cytoplasmic events. Studies from our laboratory and others on animal cells indicate that two activities, one present mainly in mitotic cells and the other exclusively in G1-phase cells, play a pivotal role in the regulation of initiation and completion of mitosis, respectively. The purpose of this study was to investigate whether these activities are expressed in the slime mold Physarum polycephalum in which all the nuclei traverse the cell cycle in natural synchrony. Extracts were prepared from plasmodia in various phases of the cell cycle and tested for their ability to induce germinal vesicle breakdown and chromosome condensation after microinjection into Xenopus laevis oocytes. We found that extract of cells at 10-20 min before metaphase consistently induced germinal vesicle breakdown in oocytes. Preliminary characterization, including purification on a DNA-cellulose affinity column, indicated that the mitotic factors from Physarum were functionally very similar to HeLa mitotic factors. We also identified a number of mitosis-specific antigens in extracts from Physarum plasmodia, similar to those of HeLa cells, using the mitosis-specific monoclonal antibodies MPM-2 and MPM-7. Interestingly, we also observed an activity in Physarum at 45 min after metaphase (i.e., in early S phase since it has no G1) that is usually present in HeLa cells only during the G1 phase of the cell cycle. These are the first studies to show that maturation-promoting factor activity is present in Physarum during mitosis and is replaced by the G1 factor (or anti-maturation-promoting factor) activity in a postmitotic stage. A comparative study of these factors in this slime mold and in mammalian cells would be extremely valuable in further understanding their function in the regulation of eukaryotic cell cycle and their evolutionary relationship to one another.     

Acceleration of mitosis induced by mitotic stimulators of Physarum polycephalum

Extracts of the myxomycete Physarum polycephalum exhibit an accelerating effect on nuclear division which fluctuates during the synchronous nuclear division cycle. Extracts from late G2 phase plasmodia can advance mitosis in recipient test plasmodia by up to 30% of the length of the control cycle. The advancing capacity of extracts is heat- and ammonium sulphate-precipitable, non-dialysable and destroyed by pronase, suggesting that the active substance is a protein. The advance of mitosis is in strong correlation with the applied dose of stimulatory material.     

Growth of Large Plasmodia of the Myxomycete Physarum polycephalum

A method has been developed for growing Physarum polycephalum plasmodia that are 8 to 10 times larger than those obtained in the petri dish cultures used by Nygaard, Guttes, and Rusch. In the large-scale procedure, plasmodia were grown in metal trays on a membrane supported by filter paper on stainless-steel screen. Plasmodia were started from a ring of inoculum to allow inward and outward migration and were incubated on a rocker so that nutrient medium would flow back and forth, wetting the undersurface of the plasmodium. Rocker and petri dish cultures had similar growth characteristics: (i) the interphase time between mitoses I and II and between II and III was about 8 hr; (ii) ribonucleic acid and protein increased essentially logarithmically throughout the cell cycle; and (iii) deoxyribonucleic acid increased only during early interphase and it doubled in approximately 3 hr after each mitosis. Rocker cultures were not as nearly synchronous as petri dish cultures and had a range in metaphase time (at mitosis III) within individual plasmodia of 15 to 45 min, as compared with 5 to 10 min in petri dish cultures.     

Advancing the onset of mitosis by cell free preparations of Physarum polycephalum

Mitotic divisions in the plasmodia of Physarum polycephalum were advanced by about 1 h by applying to the plasmodial surface extracts of other plasmodia. Advancement of mitosis was greatest when the extracts were prepared from plasmodia harvested at late G2. The activity in the extracts responsible for the advancement of mitosis was found to be heat labile and non-dialysable. It is suggested that this activity belongs to proteins responsible for the regulation of mitosis.     

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.     

Modulation of functional and optimal (Na+-K+)ATPase activity during the cell cycle of neuroblastoma cells

Functional and optimal activities of the (Na+-K+)ATPase, as determined by ouabain-sensitive K+ influx in intact cells and ATP hydrolysis in cell homogenates respectively, have been measured during the cell cycle of neuroblastoma (clone Neuro-2A) cells. The cells were synchronized by selective detachment of mitotic cells. The ouabain-sensitive K+ influx decreased more than fourfold from 1.62 +/- 0.11 nmoles/min/10(6) cells to 0.36 +/- 0.25 nmoles/min/10(6) cells on passing from mitosis to early G1 phase. On entry into S phase a transient sixfold increase to 2.07 +/- 0.30 nmoles/min/10(6) cells was observed, followed by a rapid decline, after which the active K+ influx rose again steadily from 1.03 +/- 0.25 nmoles/min/10(6) cells in early S phase to 2.10 +/- 0.92 nmoles/min/10(6) cells just prior to the next mitosis. The ouabain-insensitive component rose linearly through the cycle in the same manner as the protein content/cell. Combining total K+ influx values with efflux data obtained previously showed that net loss of K+ occurred with transition from mitosis to G1 phase while net accumulation occurred with entry into S. Throughout mid-S phase net K+ flux was virtually zero, but a large net influx occurred again just before the next mitosis. The (Na+-K+)ATPase activity measured in cell homogenates decreased rapidly from mitosis to G1 phase and increased steadily throughout S phase, but the transient activation on entry into S phase was not observed. Complete inhibition of the (Na+-K+)ATPase mediated K+ influx by ouabain (5 mM) prevents the cells from entering S phase, while partial inhibition by lower concentrations of ouabain (0.2 and 0.5 mM; km = 0.17 mM) causes partial blockage in G1 and, to a lesser extent, a reduced rate of progression through the rest of the cell cycle. We conclude that the transient increase in (Na+-K+)ATPase mediated K+ influx at the G1/S transition is a prerequisite for entry into S phase, while maintenance of adequate levels of K+ influx is necessary for normal rate of progression through the rest of the cell cycle.     

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.     

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.     

The effect of heat shock on the cell cycle regulation of tubulin expression in Physarum polycephalum

In the myxomycete Physarum polycephalum, tubulin synthesis is subject to mitotic cycle control. Virtually all tubulin synthesis is limited to a 2-h period immediately preceding mitosis, and the peak of tubulin protein synthesis is accompanied by a parallel increase in the level of tubulin mRNA. The mechanism by which the accumulation of tubulin mRNA is turned on and off is not clear. To probe the relationship between tubulin regulation and cell cycle controls, we have used heat shocks to delay mitosis and have followed the pattern of tubulin synthesis during these delays. Two peaks of tubulin synthesis are observed after a heat shock. One occurs at a time when synthesis would have occurred without a heat shock, and a second peak immediately precedes the eventual delayed mitosis. These results are clearly due to altered cell cycle regulation. No mitotic activity is detected in delayed plasmodia at the time of the control mitosis, and tubulin behavior is shown to be clearly distinct from that of heat shock proteins. We believe that the tubulin family of proteins is subject to regulation by a thermolabile mitotic control mechanism but that once the cell has been committed to a round of tubulin synthesis the "tubulin clock" runs independently of the heat sensitive system. In delayed plasmodia, the second peak of synthesis may be turned on by a repeat of the commitment event.     

Patterns of oscillation during mitosis in plasmodia ofPhysarum polycephalum

Summary The rhythmic contraction pattern in plasmodia ofPhysarum polycephalum was studied to determine whether characteristic changes occur during the synchronized nuclear division. An electrical method that measures the contraction rhythm in situ during several cell cycles was used. Biopsies of the plasmodia were taken at 17 min intervals for precise determination of the cell cycle stages and were correlated with the simultaneously measured contraction rhythm. All measurements were performed in a temperature controlled environment (27 °C) at 100% relative humidity with the plasmodia (less than 24 h old) growing on a semi-defined agar medium. A total of 14 different plasmodia have been examined, and on one occasion the plasmodium was followed through 3 subsequent mitoses. The mitotic stages were identified with aceto-orcein coloring techniques and by fluorescence methods. Except for a few cases where a mitotic asynchrony of 2–3 min was observed, the mitotic events occurred simultaneously in the nuclei within a single plasmodium. Both the occurrence of the first mitosis after inoculation and the intermitotic times were highly variable. Our study indicates that the contraction rhythm in plasmodia ofPhysarum is unperturbed during the synchronized nuclear division. However, in 5 of the 17 examined mitoses an amplitude decay was observed. We discuss possible explanations for the obtained results with emphasis on the applied techniques, interpretation of the oscillation patterns, and possible restrictions in the cell itself.     

Nuclear envelope breakdown and mitosis in sand dollar embryos is inhibited by microinjection of calcium buffers in a calcium-reversible fashion, and by antagonists of intracellular Ca2+ channels

Transient elevations in intracellular free Ca2+ are believed to signal the initiation of mitosis. This model predicts that mitosis might be arrested prior to nuclear envelope breakdown (NEB) or anaphase onset if intracellular Ca2+ concentration is buffered or dampened. Microinjection of a discrete dose of Ca2+ into the cell might then release the cell to resume mitotic cycling. Experimentally, one blastomere of two cell sand dollar (Echinaracnius parma) embryos was microinjected with Ca2+ buffers, Ca2+ solutions, or Ca2+ channel antagonists; the uninjected blastomere was the control. Cells were loaded with 10 pl doses of the Ca2+ buffer antipyrylazo III (ApIII) at specific times in the cell cycle to attempt a competitive inhibition of Ca2+-dependent steps in NEB and initiation of mitosis. Injection of 50 microM ApIII 6 min prior to NEB blocked NEB and further cell cycling. Injections of solutions between 0 and 30 microM ApIII were without observable effect. Control injections had no observable effect on the injected cell. Cells injected with 50 microM ApIII 2 min prior to the onset of anaphase in control cells were blocked in metaphase. Cells were sensitive to Ca2+ buffer injections 6 min prior to NEB (with a 40- to 45-sec duration), and 2 min prior to anaphase onset (with a 10- to 20-sec duration). Vital staining of these cells with H33342 demonstrated that they contained only one nucleus that had the same fluorescence intensity as seen prior to microinjection, and thus did not undergo DNA synthesis following the imposition of the Ca2+ buffer block to mitosis. Cells arrested in this fashion did not spontaneously resume mitotic cycling. This Ca2+ buffer-induced mitotic arrest was, however, experimentally reversible. Cells arrested with 50 microM ApIII 6 min prior to NEB could be returned to mitotic activity by injecting 300 microM CaCl2 5 min after the ApIII injection. The double injected cells resumed cycling, NEB, and mitosis after a delay of one cell cycle period, and remained one cell cycle out of phase with the sister (control) cell. Microinjection of antagonists of endomembrane Ca2+ channels inhibited NEB and anaphase onset in a concentration- and time-dependent fashion. The effective doses of compounds tested were 7 micrograms/ml ryanodine and 500 micrograms/ml TMB-8. These results indicate that a transient elevation of intracellular Ca2+ from endomembrane stores is required to initiate mitotic events, namely NEB and anaphase onset.(ABSTRACT TRUNCATED AT 400 WORDS)     

Condensation-decondensation of the gamma-tubulin containing material in the absence of a structurally visible organelle during the cell cycle of Physarum plasmodia.

Genetic evidence has shown the presence of a common spindle pole organiser in Physarum amoebae and plasmodia. But the typical centrosome and mitosis observed in amoebae are replaced in plasmodia by an intranuclear mitosis devoid of any structurally defined organelle. The fate of gamma-tubulin and of another component (TPH17) of the centrosome of Physarum amoebae was investigated in the nuclei of synchronous plasmodia. These two amoebal centrosomal elements were present in the nuclear compartment during the entire cell cycle and exhibited similar relocalisation from metaphase to telophase. Three preparation methods showed that gamma-tubulin containing material was dispersed in the nucleoplasm during interphase. It constituted an intranuclear thread-like structure. In contrast, the TPH17 epitope exhibited a localisation close to the nucleolus. In late G2-phase, the gamma-tubulin containing elements condensed in a single organelle which further divided. Intranuclear microtubules appeared before the condensation of the gamma-tubulin material and treatment with microtubule poisons suggested that microtubules were required in this process. The TPH17 epitope relocalised in the intranuclear spindle later than the gamma-tubulin containing material suggesting a maturation process of the mitotic poles. The decondensation of the gamma-tubulin material and of the material containing the TPH17 epitope occurred immediately after telophase. Hence in the absence of a structurally defined centrosome homologue, the microtubule nucleating material undergoes a cycle of condensation and decondensation during the cell cycle.     

Resolution of Kinase Activities during the HeLa Cell Cycle: Identification of Kinases with Cyclic Activities

Anin situgel kinase assay was used to resolve kinase activities during the HeLa cell cycle. Three kinase activities changed dramatically during the cell cycle, whereas a number of others remained relatively constant. The former included histone kinases with estimated molecular masses of 46, 87, and 39 kDa. The 46-kDa kinase activity was preferentially expressed at least 10-fold greater in mitotic cells. In contrast, the 87- and 39-kDa kinases were most active early in G1 but their activity declined progressively as cells approached mitosis. The 87- but not the 46- or 39-kDa kinases had autophosphorylation activity. The identities of the kinases are unknown but they may play important roles in regulation of the cell cycle.     

Regulated activity of PP2A–B55δ is crucial for controlling entry into and exit from mitosis in Xenopus egg extracts

Entry into mitosis depends on the activity of cyclin‐dependent kinases (CDKs). Conversely, exit from mitosis occurs when mitotic cyclins are degraded, thereby extinguishing CDK activity. Exit from mitosis must also require mitotic phosphoproteins to revert to their interphase hypophosphorylated forms, but there is a controversy about which phosphatase(s) is/are responsible for dephosphorylating the CDK substrates. We find that PP2A associated with a B55δ subunit is relatively specific for a model mitotic CDK substrate in Xenopus egg extracts. The phosphatase activity measured by this substrate is regulated during the cell cycle—high in interphase and suppressed during mitosis. Depletion of PP2A–B55δ (in interphase) from ‘cycling’ frog egg extracts accelerated their entry into mitosis and kept them indefinitely in mitosis. When PP2A–B55δ was depleted from mitotic extracts, however, exit from mitosis was hardly delayed, showing that other phosphatase(s) are also required for mitotic exit. Increasing the concentration of PP2A–B55δ in extracts by adding recombinant enzyme inhibited the entry into mitosis. This form of PP2A seems to be a key regulator of entry into and exit from mitosis.     

Demonstration of different patterns of microtubule organization in Physarum polycephalum myxamoebae and plasmodia using immunofluorescence microscopy

We have used anti-tubulin antibodies and immunofluorescence microscopy to determine the overall distribution of microtubules during interphase and mitosis in both the myxamoebae and plasmodia of the slime mold Physarum polycephalum. We have paralleled these observations with electron microscopy of the same stages. The myxamoebae possess a network of cytoplasmic microtubules whilst the coenocytic plasmodium does not possess any cytoplasmic microtubules--at either interphase or mitosis. In plasmodia microtubules are, however, elaborated by an intranuclear microtubule organizing centre (MTOC) during prophase of mitosis and these microtubules proceed to form part of the mitotic spindle. There is little difference in the overall distribution and arrangement of microtubules during division of either the myxamoebal or plasmodial nuclei. These findings are discussed in relation to the synthesis of tubulin during the plasmodial cell cycle and the rearrangements of the nuclear envelope during mitosis.     

Cyclic regulation of cytokinesis in amphibian eggs

The effects of amphibian egg cytoplasm extracted at different times after activation and during the first four cleavages on cytokinesis were examined. Extracts of artificially activated or fertilized Xenopus or Pleurodeles eggs taken at the time of activation (T = 0) provoked precocious cleavage furrows in Pleurodeles eggs. Between T = 0.25 and T = 0.75 of the first cell cycle, the period corresponding to interphase, an inhibitory effect was found, and the division of injected eggs was delayed up to 30%. After T = 0.75, that is during mitosis, the cleavage induction effect was observed again. These enhancing and inhibitory effects were also found in the two fractions obtained following gel filtration of the cytoplasmic extracts. These experiments support the hypothesis that two antagonistic factors control cytokinesis. The inhibitory factor is active only during interphase, while the positive factor is present during mitosis and appears to regulate cytokinesis.     

Periodic fluctuations of nuclear high mobility group like proteins during the cell cycle of Physarum polycephalum

High mobility group like (HMG-like) nuclear proteins were isolated from plasmodia of the lower eucaryote Physarum polycephalum and characterized by different types of polyacrylamide gel electrophoresis. The synthesis of these proteins was measured during the naturally synchronous cell cycle of Physarum. The four HMG-like proteins (AS1-4) exhibit a pronounced cell cycle dependent pattern of synthesis: AS1 and AS4 have a clear maximum of synthesis in mid S phase with a basal synthesis during the entire G2 period. In contrast, AS2 and AS3 have little synthesis in S phase but a broad maximum in mid G2 period. The four HMG-like proteins have a very low synthesis in early S phase and late G2 period. In addition, other non-histone proteins, which are coextracted with the HMG proteins, exhibit distinct periodic synthesis patterns. A novel non-histone protein, which is the most abundant protein species in 0.35 M NaCl extracts, was detected. It exhibits a high rate of synthesis around the time of mitosis. In general, the results indicate that, in contrast to the main cytoplasmic proteins, most nuclear proteins are phase-specific with respect to their synthesis in the cell cycle.     

Cell cycle dynamics of an M-phase-specific cytoplasmic factor in Xenopus laevis oocytes and eggs

We have examined the regulation of maturation-promoting factor (MPF) activity in the mitotic and meiotic cell cycles of Xenopus laevis eggs and oocytes. To this end, we developed a method for the small scale extraction of eggs and oocytes and measured MPF activity in extracts by a dilution end point assay. We find that in oocytes, MPF activity appears before germinal vesicle breakdown and then disappears rapidly at the end of the first meiotic cycle. In the second meiotic cycle, MPF reappears before second metaphase, when maturation arrests. Thus, MPF cycling coincides with the abbreviated cycles of meiosis. When oocytes are induced to mature by low levels of injected MPF, cycloheximide does not prevent the appearance of MPF at high levels in the first cycle. This amplification indicates that an MPF precursor is present in the oocyte and activated by posttranslational means, triggered by the low level of injected MPF. Furthermore, MPF disappears approximately on time in such oocytes, indicating that the agent for MPF inactivation is also activated by posttranslational means. However, in the absence of protein synthesis, MPF never reappears in the second meiotic cycle. Upon fertilization or artificial activation of normal eggs, MPF disappears from the cytoplasm within 8 min. For a period thereafter, the inactivating agent remains able to destroy large amounts of MPF injected into the egg. It loses activity just as endogenous MPF appears at prophase of the first mitotic cycle. The repeated reciprocal cycling of MPF and the inactivating agent during cleavage stages is unaffected by colchicine and nocodazole and therefore does not require the effective completion of spindle formation, mitosis, or cytokinesis. However, MPF appearance is blocked by cycloheximide applied before mitosis; and MPF disappearance is blocked by cytostatic factor. In all these respects, MPF and the inactivating agent seem to be tightly linked to, and perhaps participate in, the cell cycle oscillator previously described for cleaving eggs of Xenopus laevis (Hara, K., P. Tydeman, and M. Kirschner, 1980, Proc. Natl. Acad. Sci. USA, 77:462- 466).     

Growth and development in relation to the cell cycle inPhysarum polycephalum

Summary In strain CL ofPhysarum polycephalum, multinucleate, haploid plasmodia form within clones of uninucleate, haploid amoebae. Analysis of plasmodium development, using time-lapse cinematography, shows that binucleate cells arise from uninucleate cells, by mitosis without cytokinesis. Either one or both daughter cells, from an apparently normal amoebal division, can enter an extended cell cycle (28.7 hours compared to the 11.8 hours for vegetative amoebae) that ends in the formation of a binucleate cell. This long cycle is accompanied by extra growth; cells that become binucleate are twice as big as amoebae at the time of mitosis. Nuclear size also increases during the extended cell cycle: flow cytometric analysis indicates that this is not associated with an increase over the haploid DNA content. During the extended cell cycle uninucleate cells lose the ability to transform into flagellated cells and also become irreversibly committed to plasmodium development. It is shown that commitment occurs a maximum of 13.5 hours before binucleate cell formation and that loss of ability to flagellate precedes commitment by 3–5 hours. Plasmodia develop from binucleate cells by cell fusions and synchronous mitoses without cytokinesis.Abbreviations CL Colonia Leicester - DSDM Dilute semi-defined medium - FKB Formalin killed bacterial suspension - IMT Intermitotic time - LIA Liver infusion agar - SBS Standard bacterial suspension - SDM Semi-defined medium