Phosphorylation of ribosomal proteins during the cell cycle of Physarum polycephalum

Physarum polycephalum has been used as a model system to study the phosphorylation of ribosomal proteins during the cell cycle. The results showed that the phosphate content of S3, the major ribosomal phosphoprotein in this organism, was constant during all phases of the cell cycle. No additional ribosomal phosphoproteins were observed. These results differ significantly from those reported earlier by Rupp, R.G., Humphrey, R.M. and Shaeffer, J.R. (Biochim. Biophys. Acta (1976) 418, 81-92) and suggest that the use of thymidine or hydroxyurea to synchronize cell population may affect the phosphorylation of ribosomal proteins. The results are discussed in relation to protein synthesis and cAMP level during the cell cycle.     

ADP-ribosyltransferase in isolated nuclei during the cell cycle of Physarum polycephalum.

ADP-ribosyltransferase was measured in isolated nuclei of Physarum polycephalum. Activity was determined with and without exogenous DNA and histones. During the synchronous cell cycle the activity measured with exogenous substrates exhibited a typical peak enzyme pattern with a maximum of activity in S-phase, whereas activity measured without exogenous substrates displayed a step enzyme pattern. Both activities doubled in each cell cycle.     

Nuclear pores during the cell cycle in a slime mold,Physarum polycephalum

Freeze-fracture and thin sectioning techniques were used to follow in large synchronous plasmodia of Physarum polycephalum the changes in number and distribution of nuclear pores during the cell cycle. Using freeze-fracture, we determined that average pore frequency rises gradually from 14/μm² of nuclear envelope surface at early S to a value of about 22 just before prophase. Nuclear diameter averaged 3.3 γm at early S and increased to 4.3 μm at late G2. Calculating nuclear volume and average chromatin volume per nucleus with respect to time in the cell cycle leads to the conclusion that number of nuclear pores appears to be most directly related to amount of chromatin present per nucleus and to be independent of nuclear surface area.     

Regulation of tubulin synthesis during the cell cycle in the synchronous plasmodia of Physarum polycephalum

Regulation of alpha- and beta-tubulin isotype synthesis during the cell cycle has been studied in the myxomycete Physarum polycephalum, by subjecting synchronous plasmodia to temperature shifts and pharmacological perturbations. Temperature shifts interfered with the regulation of tubulin synthesis. Inhibition of DNA synthesis prevents tubulin degradation after completion of the cell cycle (Ducommun and Wright, Eur. J. Cell Biol., 50:48-55, 1989) but did not perturb the initiation of tubulin synthesis. The constant increase of tubulin synthesis in the presence of tubulin-sequestering drugs and the decrease of tubulin synthesis during a treatment with aphidicolin in late G2 phase suggest the existence of an autoregulatory mechanism of tubulin synthesis. Moreover, the microtubule poison methyl benzimidazole carbamate dissociated synthesis of the alpha 1-tubulin isotype from the generally strictly coordinated synthesis of all tubulin isotypes during the transient interruption of mitosis. These observations show that a microtubular poison can perturb regulation of the synthesis of specific isotubulins.     

Synthesis and characterization of nuclear matrix proteins during the cell cycle of Physarum polycephalum

Pulse-labelling with [³⁵S]-methionine/cysteine of macroplasmodia of the myxomycete Physarum polycephalum at different time points of the cell cycle reveals that the majority of nuclear matrix proteins is synthesized and assembled into nuclear structures without a pronounced cell cycle periodicity. Bulk nuclear histones on one hand and nuclear matrix associated histones on the other hand assemble with a different cell cycle periodicity suggesting specific functions of nuclear matrix bound chromatin. Characterization of the nuclear matrix by immunoblotting and immunofluorescence techniques with several antisera against vertebrate lamins shows the existence of lamin-homologous proteins in Physarum.     

Changes in phosphorylation of an F-actin capping protein of Physarum polycephalum during the cell cycle

The Cap 42(b), a Ca2+-dependent F-actin capping phosphoprotein of 42,000 daltons, was shown to be localized in the cytosol of Physarum polycephalum by measurements of phosphorylatability in the absence of Ca2+. The phosphorylation of Cap 42(b) in the cytosol changed during the cell cycle: it was high in the S and G2 phase, and low in the M phase and boundary phase between S and G2 phase. When the isolated Cap 42(b) was added to M phase cytosol, the phosphorylation of Cap 42(b) was significantly increased by at least 6-fold. Compared with this result, about 2-fold increase in the phosphorylation of Cap 42(b) was observed when the Cap 42(b) kinase was added to M phase cytosol. Therefore, it is likely that the low level of Cap 42(b) phosphorylation in M phase cytosol is mostly due to the decreased amount of phosphorylatable Cap 42(b) and to a lesser extent due to a low level of the Cap 42(b) kinase activity.     

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.     

Centriole size modifications during the cell cycle of the amoebae of the mxyomycete Physarum polycephalum

Anterior and posterior centrioles of Physarum amoebae are indistinguishable by their size during interphase but there is a correlation between the size of the two centrioles in the same amoeba. The interphase length of centrioles in diploid amoebae possessing only one pair of centrioles was 11% longer than in the case of the haploid strain. Treatment with taxol led to a 23 and 32% increase of the mean length in interphase and blocked mitosis, respectively. Conversely, during control mitosis the parental centrioles showed a 12% decrease of their mean length while the size of the daughter centrioles increased progressively. Neither nocodazole nor cold treatment induce a decrease of centriole length. The mean length of the cartwheel structure (internal proximal part) although constant during mitosis could be increased 24% in the presence of taxol. Similarly there was a correlation between the number of anterior satellites and the centriole length.     

Chromatin remodeling by cell cycle stage-specific extracts from Physarum polycephalum

Remodeling of chromatin is an essential process allowing the establishment of specific genetic programs. The slime mold Physarum polycephalum presents the attractive advantage of natural synchrony of the cell cycle in several million nuclei. Whole-cell extracts prepared at precise stages during the cell cycle were tested for the ability to induce remodeling in erythrocyte nuclei as monitored by microscopy, protamine competition assays, micrococcal nuclease digestions, and release of histone H5. Extracts derived from two specific cell cycle stages caused opposite types of changes in erythrocyte nuclei. An increase in chromatin compaction was imparted by extracts prepared during S-phase while extracts harvested at the end of G2-phase caused increases in nuclear volume, DNA accessibility, and release of linker histone. We also found that late G2 extracts had the ability to alter the DNase I digestion profile of mononucleosomes reconstituted in vitro in a classical nucleosomes remodeling assay. The relevance of these finding to the Physarum cell cycle is discussed.     

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.     

Identification and changes in activity of five thymidine kinase forms during the cell cycle of Physarum polycephalum

Five forms of thymidine kinase have been identified on isoelectric focusing gels of Physarum polycephalum supernatants. Their isoelectric points are 5.9, 6.4, 6.7, 6.9 and 7.1. All are inhibited by deoxythymidine 5′-triphosphate (dTTP). The activity of the pI 7.1 form does not change significantly during the cell cycle. The other four forms change in activity. About 1 h before metaphase the activity of the four more acidic forms is first detected. Their activity peaks during telophase, and by 1 h after metaphase there is a 50% decrease in activity of the 5.9 form. By 3 h after metaphase the activity of the 6.4 form has dropped more sharply than the activity of the 6.7 form. By 6 h after metaphase only the activity of the 6.9 form is present in significant amounts in addition to the 7.1 form. The activity of these new acidic forms probably accounts for the reported increase in total thymidine kinase activity during mitosis and early S phase.     

Change in phosphorylation of nucleolar proteins of Physarum polycephalum during the cell cycle in vivo and in vitro

We compared the phosphorylation of nucleolar proteins during the cell cycle of Physarum polycephalum labeled by pulse and continuous labeling methods in vivo with that obtained by in vitro labeling of isolated nucleoli. Both the phosphorylating activity of nucleoli and total incorporation of radioactive phosphate into nucleolar proteins increased and reached a maximum about 1.5-2.0 h before mitosis, confirming our previous observation. Analyses of labeled nucleolar proteins by SDS-polyacrylamide gel electrophoresis and by autoradiography indicated that most of the phosphoproteins labeled by in vitro labeling were labeled by in vivo pulse labeling. At least 10 nucleolar proteins underwent phosphorylation, which closely followed the cell cycle-dependent changes of the total phosphate incorporation into the nucleolar proteins. When mitosis was delayed by UV-irradiation, the maximal incorporation of radioactive phosphate into nucleolar proteins in vivo was not observed at the usual time, it shifted to about 2 h before the delayed mitosis, and the same set of nucleolar proteins that were phosphorylated without UV-irradiation were most heavily phosphorylated at this time. These results suggest the possibility that the increased phosphorylation of nucleolar proteins of Physarum just before mitosis is related to the onset of subsequent mitosis.     

Expression of ras-family genes in the cell cycle and during differentiation of the lower eukaryote Physarum polycephalum.

Messenger RNA levels of three ras-family genes (Ppras1, Ppras2, and Pprap1) were measured in different life forms and throughout the cell cycle of the slime mold Physarum polycephalum. All three genes are expressed at constant rates in the uninucleate amoebae and flagellates, regardless of the culture conditions (solid or liquid medium, particulate or dissolved nutrients). In the multinucleate stages (micro- and macroplasmodia) Ppras1 and Pprap1 mRNAs are somewhat less abundant, while Ppras2 is not expressed at all. The early stages of the amoeba-plasmodium transition proceed without any drop in Ppras2 expression. During the synchronous cell cycle in macroplasmodia Ppras1 and Pprap1 are expressed at a constant level.