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.     

The intranuclear microtubule organizing centre in the plasmodium of the myxomycete Physarum polycephalum

Physarum possesses two different microtubule cytoskeletons. In amoebae, cytoplasmic and mitotic microtubules are nucleated by a typical centrosome. In contrast, it has been reported that plasmodia have an intranuclear spindle organizing centre (SPOC) devoid of centrioles. We present genetic evidence suggesting that the SPOC located in the centrosome is very similar to the intranuclear plasmodial SPOC. The immunostaining properties of a new monoclonal antibody against Physarum centrosome has been used to compare these different MTOCs. Moreover, a dense plasmodial microtubule network was present in interphase plasmodia and absent in plasmodia undergoing mitosis. MTOCs responsible for the nucleation of the cytoplasmic microtubule network and intranuclear SPOCs were located in two different compartments of the plasmodium.     

A purified cellular extract accelerates the cell cycle in Physarum polycephalum

Plasmodia of the myxomycete Physarum polycephalum (strain Cl) were collected at different times during the cell cycle and extracts were prepared from homogenates using a buffer optimized for microinjection into plasmodial veins. These extracts were injected into plasmodia during the first 3 h of the cell cycle. The time of the following mitosis was monitored and compared with that of the buffer-injected controls. Extracts of plasmodia homogenized 45 min before late telophase accelerated the onset of mitosis in the injected plasmodium up to 70 min, i.e., an advance of 10-14% compared to the 8- to 10-h cell cycle duration of the controls. The accelerating activity vanished completely after heating, freezing, or protease digestion, thus indicating the peptide nature of the active agent. Purification of the active compound by means of gel filtration revealed a molecular mass of about 2500 Da. The active portion of the extract was further fractionated by HPLC and the activity determined in a single peak.     

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.     

Change of contractile behavior in plasmodia ofPhysarum polycephalum during mitosis

Summary Oscillations of ectoplasmic contraction in plasmodia of the myxomycetePhysarum polycephalum growing on agar containing semidefined medium were studied to determine if the contractile force is altered during the synchronous mitosis. In interphase the regular oscillations of contraction in the plasmodial sheet had an average period of 0.93 minutes in plasmodia growing at 24 °C. During mitosis the amplitude of these oscillations gradually decreased, ceasing for an average time of 2.7 minutes in 74% of the 23 plasmodia studied. Cessation of oscillating contractions in mitosis was accompanied by a decrease in the width of the channels embedded in the plasmodial sheet, and a decrease in the velocity of endoplasmic shuttle streaming usually to a complete standstill. Of 13 plasmodia in which the mitotic stage was very accurately determined, the stop in oscillating contractions occurred during metaphase in 10 plasmodia, and in prometaphase, anaphase, telophase in the 3 others. The cessation of contractile oscillations or of streaming did not occur absolutely simultaneously during mitosis in widely separated locations within one plasmodium, indicating mitotic asynchrony over a period of a few minutes within each plasmodium. We suggest that the halt of plasmodial migration during mitosis reported by others is caused by a decrease or cessation at slightly different times in the amplitude of ectoplasmic contractile oscillations in different areas of a plasmodium in mitosis resulting in an overall lack of coordination of endoplasmic flow throughout the plasmodium, thus temporarily halting migration. Possible physiological mechanisms linking a decrease in actomyosin contraction with the metaphase stage of mitosis are discussed.     

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.     

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.     

Katanin Is Responsible for the M-Phase Microtubule-severing Activity in Xenopus Eggs

Microtubules are dynamic structures whose proper rearrangement during the cell cycle is essential for the positioning of membranes during interphase and for chromosome segregation during mitosis. The previous discovery of a cyclin B/cdc2-activated microtubule-severing activity in M-phase Xenopus egg extracts suggested that a microtubule-severing protein might play an important role in cell cycle-dependent changes in microtubule dynamics and organization. However, the isolation of three different microtubule-severing proteins, p56, EF1α, and katanin, has only confused the issue because none of these proteins is directly activated by cyclin B/cdc2. Here we use immunodepletion with antibodies specific for a vertebrate katanin homologue to demonstrate that katanin is responsible for the majority of M-phase severing activity in Xenopus eggs. This result suggests that katanin is responsible for changes in microtubules occurring at mitosis. Immunofluorescence analysis demonstrated that katanin is concentrated at a microtubule-dependent structure at mitotic spindle poles in Xenopus A6 cells and in human fibroblasts, suggesting a specific role in microtubule disassembly at spindle poles. Surprisingly, katanin was also found in adult mouse brain, indicating that katanin may have other functions distinct from its mitotic role.     

A Comparative Study of Mitosis in Amoebae and Plasmodia of the True Slime Mold Didymium nigripes

SYNOPSIS. Studies comparing mitosis in amoebae and plasmodia of the true slime mold Didymium nigripes reveal that at the time of differentiation pronounced changes occur in the mitotic process. Not only does the amount of time required for division of the 2 stages differ, but plasmodial mitosis is characterized by persistence of the nuclear membrane and the apparent lack of centrioles. The origin of multinucleate plasmodia from uninucleate cells which have already undergone cytoplasmic differentiation is described. Division time in a population of amoebae becomes more uniform after those cells which are destined to form plasmodia have differentiated.
The observations and data presented indicated that differences in mitotic behavior also occur between amoebae of 3 stocks with differences in plasmodial structure and behavior. Comparison of mitosis in the plasmodia of these 3 stocks revealed no significant differences.     

Aminopeptidases of Physarum polycephalum. Activity, isoenzyme pattern, and synthesis during differentiation.

In extracts from both growing and differentiating (spherulating) plasmodia of the true slime mold Physarum polycephalum, high aminopeptidase activities were found. The specificity of the aminopeptidases changed during differentiation with a higher relative activity towards hydrophobic NH2-terminal amino acids. This change in specificity was found to be the result of a shift in the isoenzyme spectrum during differentiation as was tested by isoelectric focusing in sucrose gradients. Three different classes of isoenzymes were found: one band which was present in both growing and differentiating cultures; two bands which were found only in growing cultures; and four bands which were detectable only in differentiating plasmodia. If cycloheximide was applied during the induction of differentiation, only one band, the one present in both types of plasmodia, was found in the isoelectric focusing. Density labeling experiments using deuterated amino acids revealed that the bands which are present in differentiated plasmodia only are synthesized de novo during this differentiation.     

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.     

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.     

Regulation of thymidine kinase synthesis during the cell cycle of Physarum polycephalum by the heat-sensitive system which triggers mitosis and S phase

Temperature shifts from 22 to 32 °C perturb one of the systems responsible for mitosis triggering in the plasmodia of Physarum (Myxomycetes). In order to determine if the same regulatory mechanism could also be involved in some other cell cycle events, the effects of temperature shifts on the peak of thymidine kinase (EC 2.7.1.21, ATP : thymidine 5′-phosphotransferase) synthesis have been studied. At 22 °C, the increase in thymidine kinase (tdk) activity begins shortly before mitosis and is thus always associated with the end of the G2 phase, the mitosis and the beginning of the S phase. The consequences of temperature shifts depend upon their position in the cell cycle. In all cases, a peak of tdk occurs concomitantly with the 32 °C mitosis. But, when the temperature shift is applied 90-15 min before the control metaphase at 22 °C, another peak of tdk is observed at 32 °C in absence of mitosis, but at the same time as the control mitosis at 22 °C. These results indicate that the increase in the synthesis of tdk is controlled by the heat-sensitive regulatory system which plays a role in the onset of mitosis and S phase. We further suggest that the increase in the synthesis of tdk and the triggering of mitosis are both controlled by the amount of a heat-sensitive effector. But the former takes place when the amount of the effector reaches a critical value lower than the value necessary to trigger mitosis.     

The effects of anaerobiosis and uncouplers of oxidative phosphorylation on mitotic timing and the induction of intranuclear macrotubules in the slime mold,Physarum polycephalum

Summary Mitotically synchronous plasmodia of the slime moldPhysarum polycephalum were subjected to brief exposures of either pure atmospheres of carbon dioxide or nitrogen gases or to pulsetreatments with respiratory poisons (sodium azide, sodium arsenate, or 2,4-dinitrophenol, DNP) at many different phases of the mitotic cycle to assess their effects on the mechanism(s) controlling the timing of mitosis. Plasmodia were fully viable after a pulse of CO₂ lasting up to 90 minutes or after a N₂-pulse of 30 minutes in duration. Upon return to normal aeration, all treated plasmodia entered a fully synchronous mitosis with a variable excess mitotic delay, which was dependent on the duration of the pulse and time of application in the mitotic cycle. Likewise, plasmodia exposed to 15-minute-pulses of a sublethal dose of sodium arsenate (0.1 mM), sodium azide 0.05 mM) and 2,4-DNP (0.2 mM) yield characteristic patterns of excess mitotic delay upon returnal to normal culture conditions. Two different types of phase response curves (PRC) were generated by these treatments. This suggests that at least two distinct respiratory-linked physiological mechanisms are involved in control of mitosis onset and regulation of mitotic timing inPhysarum.Electron microscope observations of CO₂-treated plasmodia reveal the induction of intranuclear 40–60 nm diameter macrotubules at all stages of the G₂ phase up to and including prometaphase. Both anoxia and sodium azide treatments are effective in macrotubule induction, and both reversibly disrupt the normal tubular cristae organization of mitochondria. In early G₂, macrotubules polymerize in association with both the inner membrane of the nuclear envelope and the nucleolus, while the tubule-organizer region, TOR, serves as the only nucleating site for macrotubules in late G₂ nuclei, coincident with the onset of mitosis and TOR formation.     

Cytoplasmic control of mitosis in true slime mold, Physarum polycephalum

To resolve the problem of whether mitosis is controlled by anuclear or cytoplasmic stimulus, plasmodia from various periodsof the mitotic cycle were fused with one another. Mitosis inthe fused plasmodiura occurred about midway between mitosesof the donors. Treatment with cyclohcximide during the G₂-period.delayed the next mitosis for a period equal to the time of treatment. (Received March 2, 1971; )     

Observations on an ATP-sensitive protein system from the plasmodia of a myxomycete

1. Extracts of the plasmodia of the myxomycete, Physarum polycephalum, exhibit reversible decreases in viscosity in response to the addition of ATP under appropriate conditions. The protoplasm material prepared by extraction with KCl solution can apparently exist in either a high or a low viscosity state. As prepared, it is in the low viscosity condition. Rapid and extensive increases in viscosity of the extract are brought about by addition of AMP, inorganic phosphate, or, under certain conditions, of ATP. Only after the high viscosity state has been attained does addition of appropriate quantities of ATP cause a reversible decrease in viscosity. 2. The active principle of crude plasmodial extracts may be concentrated by fractional precipitation with ammonium sulfate and is found in the fraction precipitated between 30 and 40 per cent saturation. This material possesses a higher viscosity than does the original crude extract and is apparently in the high viscosity state since the addition of ATP causes an immediate reversible decrease in viscosity. 3. The ATP-sensitive fraction of myxomycete plasmodia possesses a viscosity which is dependent upon its previous thermal treatment. Extracts incubated at 0° for a period of a few hours increase greatly in viscosity when they are returned to 24.5°. This increased viscosity is structural in nature, is destroyed by mechanical agitation of the solution, and may be reversibly destroyed by addition of ATP. 4. It is suggested that the ATP-responsive protein of myxomycete plasmodia may be related to sol-gel transformations which have been observed in intact plasmodia and may participate in the protoplasmic streaming of the intact organism. This suggestion is based upon the following facts: (a) the protoplasmic streaming of myxomycete plasmodia is increased by microinjection of ATP; (b) the gel portion of the cytoplasm at the site of the microinjection of ATP is extensively converted to the sol state. The changes in structure of the intact cytoplasm are thus similar in nature to the changes exhibited in response to ATP by the purified ATP-sensitive protein. 5. The ATP-sensitive protein of myxomycete plasmodia appears to undergo reversible aggregation to form a high viscosity state. The function of ATP is to break down the aggregates thus formed. Since a specific ATPase activity is associated with the purified material, added ATP is gradually destroyed and recovery of viscosity attends the spontaneous reconstitution of aggregates.     

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.     

In vitro cell cycle arrest induced by using artificial DNA templates.

In cell extracts of Xenopus eggs which oscillate between S and M phases of the cell cycle, the onset of mitosis is blocked by the presence of incompletely replicated DNA. In this report, we show that several artificial DNA templates (M13 single-stranded DNA and double-stranded plasmid DNA) can trigger this feedback pathway, which inhibits mitosis. Single-stranded M13 DNA is much more effective than double-stranded plasmid DNA at inhibiting the onset of mitosis. Furthermore, we have shown that low levels of M13 single-stranded DNA and high levels of double-stranded plasmid DNA can elevate the tyrosine kinase activity responsible for phosphorylating p34cdc2, thereby inactivating maturation-promoting factor and inhibiting entry into mitosis. This constitutes a simplified system with which to study the signal transduction pathway from the DNA template to the tyrosine kinase responsible for inhibiting p34cdc2 activity.