类Cyclin A蛋白在多头绒泡菌细胞周期中的定位研究

采用免疫电镜技术对多头绒泡菌(Physarum polycephalum)是否含有类CyclinA蛋白以及该蛋白在有丝分裂周期各时相的定位进行了研究;并以抗CyclinA抗体封闭细胞内源类CyclinA蛋白的方法,探讨类CyclinA蛋白在多头绒泡菌细胞周期中的作用。免疫电镜结果表明,经抗CyclinA抗体标记的实验组细胞中的金颗粒密度明显高于对照组,说明多头绒泡菌细胞中含有类CyclinA蛋白。实验组样品中,细胞核的金颗粒密度很高,而细胞质的金颗粒密度与对照组的相仿,说明多头绒泡菌细胞中的类CyclinA蛋白是核蛋白。细胞核的金颗粒密度在S期最高,G2期的次之,早中期时明显降低,中期和中期以后与对照组的相近。这种金颗粒密度的变化反映了类CyclinA蛋白在细胞周期中的含量变化。以抗CyclinA抗体分别处理S期和G2期的多头绒泡菌细胞,处理后的细胞分别停滞在原来的时相,细胞核形态变得不规则,核内有空洞现象。处于有丝分裂前期的多头绒泡菌细胞经抗CyclinA抗体处理后,细胞核出现畸变。抗体处理结果说明类CyclinA蛋白是参与多头绒泡菌细胞周期多个转换过程调控的种重要蛋白,主要在S期／G2期和G2期／M期的转换以及走出有丝分裂期的进程中发挥作用。     

多头绒泡菌类cyclin B1蛋白在细胞周期中的变化

以自然同步化的多头绒泡菌(Physarum polycephalum L.)为材料,经抗cyclin B1抗体的免疫印迹和免疫电镜实验观察结果表明,多头绒泡菌中含有类cyclin B1蛋白,该蛋白的含量和细胞内位置在细胞周期进程中存在着动态变化:类cyclin B1蛋白在S期开始合成并在细胞质中积累,G2晚期开始进入细胞核,该蛋白在细胞质和细胞核中含量逐渐增加,有丝分裂中期时达最大值,后末期时骤然消失.在G2晚期到有丝分裂中期期间,类cyclin B1蛋白既是细胞核蛋白又是细胞质蛋白,细胞质是类cyclin B1蛋白的主要存在区域,细胞核中的类cyclin B1蛋白主要结合于染色体和核仁区域.     

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.     

Specific nuclear elimination in polyploid plasmodia of the slime mold Physarum polycephalum

In growing plasmodia of the myxomycete Physarum polycephalum (G2-phase), three distinct classes of nuclei with a relative DNA content of 1x, 2x, and 4x are observed in the presumed haploid strain CL. The 2x and 4x species comprise up to 35% and 5% of the nuclei. Quantitative cytofluorometric studies of nuclei isolated in either G2- or S-phase or after FUDR treatment (G1 arrest) show that the three nuclear populations undergo a synchronous mitotic cycle and that the relative DNA content of the nuclear fractions in G-2 phase reflects the 2c, 4c, and 8c state. The heterogeneity of the nuclear population does, however, seem to be restricted to the growth phase. During a starvation period of 4 days that always preceeds sporulation (and also meiosis), the 4c nuclear population is reduced to 7%, 8c nuclei are no longer detected. These results suggest that a mechanism exists in Physarum for the selective detection and elimination of polyploid nuclei.     

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.     

多头绒泡菌类cyclinB1蛋白在细胞周期中的变化

以自然同步化的多头绒泡菌（Physarum polycephalumL.)为材料,经抗cyclinB1抗体的免疫印迹和免疫电镜实验观察结果表明,多头绒泡菌中含有类cyclinB1蛋白,该蛋白的含量和细胞内位置在细胞周期进程中存在着动态变化。类cyclinB1蛋白在S期开始合成并在细胞质中积累,G2晚期开始进入细胞核,该蛋白在细胞质和细胞核中含量逐渐增加。有丝分裂中期时达最大值。后末期时骤然消失,在G2晚期到有丝分裂中期期间,类cyclinB1蛋白既是细胞核蛋白又是细胞质蛋白,细胞质是类cyclinB1蛋白的主要存在区域,细胞核中的类cyclinB1蛋白主要结合于染色体和核仁区域。     

Kinetics of nuclease digestion of Physarum polycephalum nuclei at different stages of the cell cycle

The kinetics of nuclease digestion of Physarum polycephalum nuclei by staphylococcal nuclease and DNase I has been studied at different stages of the cell cycle. Significant differences in the digestion behaviour of nuclei from metaphase and interphase have been detected with DNase I but not with staphylococcal nuclease. Furthermore the structure of newly replicated DNA in S phase differs from the bulk in that it is more easily degraded to acid-soluble products by either staphylococcal nuclease or by DNAase I. At least four types of chromatin structure can be distinguished by our digestion kinetics experiments.     

The DNA-polymerase inhibiting activity of poly(beta-l-malic acid) in nuclear extract during the cell cycle of Physarum polycephalum.

The naturally synchronous plasmodia of myxomycetes synthesize poly(beta-l-malic acid), which carries out cell-specific functions. In Physarum polycephalum, poly(beta-l-malate) [the salt form of poly(beta-l-malic acid)] is highly concentrated in the nuclei, repressing DNA synthetic activity of DNA polymerases by the formation of reversible complexes. To test whether this inhibitory activity is cell-cycle-dependent, purified DNA polymerase alpha of P. polycephalum was added to the nuclear extract and the activity was measured by the incorporation of [3H]thymidine 5'-monophosphate into acid precipitable nick-activated salmon testis DNA. Maximum DNA synthesis by the reporter was measured in S-phase, equivalent to a minimum of inhibitory activity. To test for the activity of endogenous DNA polymerases, DNA synthesis was followed by the highly sensitive photoaffinity labeling technique. Labeling was observed in S-phase in agreement with the minimum of the inhibitory activity. The activity was constant throughout the cell cycle when the inhibition was neutralized by the addition of spermidine hydrochloride. Also, the concentration of poly(beta-l-malate) did not vary with the phase of the cell cycle [Schmidt, A., Windisch, C. & Holler, E. (1996) Nuclear accumulation and homeostasis of the unusual polymer poly(beta-l-malate) in plasmodia of Physarum polycephalum. Eur. J. Cell Biol. 70, 373-380]. To explain the variation in the cell cycle, a periodic competition for poly(beta-l-malate) between DNA polymerases and most likely certain histones was assumed. These effectors are synthesized in S-phase. By competition they displace DNA polymerase from the complex of poly(beta-l-malate). The free polymerases, which are no longer inhibited, engage in DNA synthesis. It is speculated that poly(beta-l-malate) is active in maintaining mitotic synchrony of plasmodia by playing the mediator between the periodic synthesis of certain proteins and the catalytic competence of DNA polymerases.     

Intrinsic fluctuations of phosphoinositol levels are involved in the progression of the naturally synchronous cell cycle in Physarum polycephalum.

Using [3H]myo-inositol incorporation, changes in phosphoinositol (PI) metabolism at different cell cycle stages in the myxomycete Physarum polycephalum were examined by column chromatography. Two base levels for the inositol trisphosphate (IP3) fraction were determined: a low one for S-phase and a higher one for G2 phase. Two transient increases of IP3 were also observed, one in S-phase, 70 min after mitosis (no G1 phase in the Physarum cell cycle) and another in G2 phase, 90 min before mitosis. It is concluded that the fluctuations in IP3 levels reflect endogenous events in the cell cycle of Physarum, because they occurred in the absence of any exogenous signals. Pulse treatment with Li+ (10 mM) at the points of the cell cycle, characterized by the IP3 transients, had opposite effects: in early S-phase it caused an acceleration while in late G2 phase it caused a prolongation of the cell cycle duration. The pattern of Li(+)-induced changes in PI turnover is also antagonistic: in most cases the IP3 level would decrease, however, Li+ prevents the cell cycle-dependent reduction in IP3 concentration when applied at early S-phase. The possible implications of the autogenous fluctuations in the IP3 fraction on the progression of the cell cycle through several distinct checkpoints are discussed.     

ADP-ribosylation of the nuclear matrix in Physarum polycephalum

Less than 10% of the total ADP-ribosylation in isolated nuclei of Physarum polycephalum are bound to the nuclear matrix. In S-phase the matrix-associated ADP-ribosylation is almost twice as high as compared with the G2-period of the cell cycle. Inhibitors of DNA- and RNA-synthesis and the mutagen N-methyl-N′-nitro-N-nitrosoguanidine increase the percentage of matrix-associated ADP-ribosylation.     

Cell cycle S phase markers are expressed in cerebral neuron nuclei of cats infected by the Feline Panleukopenia Virus

The cell cycle-associated neuronal death hypothesis, which has been proposed as a common mechanism for most neurodegenerative diseases, is notably supported by evidencing cell cycle effectors in neurons. However, in naturally occurring nervous system diseases, these markers are not expressed in neuron nuclei but in cytoplasmic compartments. In other respects, the Feline Panleukopenia Virus (FPV) is able to complete its cycle in mature brain neurons in the feline species. As a parvovirus, the FPV is strictly dependent on its host cell reaching the cell cycle S phase to start its multiplication. In this retrospective study on the whole brain of 12 cats with naturally-occurring, FPV-associated cerebellar atrophy, VP2 capsid protein expression was detected by immunostaining not only in some brain neuronal nuclei but also in neuronal cytoplasm in 2 cats, suggesting that viral mRNA translation was still occurring. In these cats, double immunostainings demonstrated the expression of cell cycle S phase markers cyclin A, cdk2 and PCNA in neuronal nuclei. Parvoviruses are able to maintain their host cells in S phase by triggering the DNA damage response. S139 phospho H2A1, a key player in the cell cycle arrest, was detected in some neuronal nuclei, supporting that infected neurons were also blocked into the S phase. PCR studies did not support a co-infection with an adeno or herpes virus. ERK1/2 nuclear accumulation was observed in some neurons suggesting that the ERK signaling pathway might be involved as a mechanism driving these neurons far into the cell cycle.     

Cell-cycle dependence of two nuclear histone kinase enzyme activities.

Growth-associated histone kinases have been extracted from Physarum polycephalum nuclei and resolved into two components by ion-exchange chromatography. The two component activities have different substrate specificities and different times of appearance in the cell cycle. It is proposed that the enzyme(s) phosphorylate H1 histone in vivo in G2 phase, possibly sequentially in time at different sites in the H1 amino acid sequence.     

The human origin recognition complex protein 1 dissociates from chromatin during S phase in HeLa cells

We investigated the association of human origin recognition complex (ORC) proteins hOrc1p and hOrc2p with chromatin in HeLa cells. Independent procedures including limited nuclease digestion and differential salt extraction of isolated nuclei showed that a complex containing hOrc1p and hOrc2p occurs in a nuclease-resistant compartment of chromatin and can be eluted with moderate high salt concentrations. A second fraction of hOrc2p that dissociates in vitro at low salt conditions was found to occur in a chromatin compartment characterized by its high accessibility to micrococcal nuclease. Functional differences between these two sites become apparent in HeLa cells that synchronously enter the S phase after a release from a double-thymidine block. The hOrc1p/hOrc2p-containing complexes dissociate from their chromatin sites during S phase and reassociate at the end of mitosis. In contrast, the fraction of hOrc2p in nuclease-accessible, more open chromatin remains bound during all phases of the cell cycle. We propose that the hOrc1p/hOrc2p-containing complexes are components of the human origin recognition complex. Thus, the observed cell cycle-dependent release of the hOrc1p/hOrc2p-containing complexes is in line with previous studies with Xenopus and Drosophila systems, which indicated that a change in ORC stability occurs after prereplication complex formation. This could be a powerful mechanism that prevents the rereplication of already replicated chromatin in the metazoan cell cycle.     

Induction of polyploid nuclei in Physarum polycephalum by cycloheximide treatment in prophase

Macroplasmodia of the acellular slime mold Physarum polycephalum were treated with pulses of cycloheximide (10 micrograms/ml medium, for 3 h), initiated 10-20 min before metaphase in the synchronous nuclear division cycle. This treatment interfered with normal division of the nuclei, but permitted DNA synthesis in the next S phase. This interpretation is supported by measurements of the DNA content per nucleus in cycloheximide-treated cultures as compared to control cultures, which show that some nuclei after cycloheximide treatment are polyploid. By this method we can produce polyploid strains of Physarum, but the elevated nuclear DNA content is not stable, and after several months the strains have reverted to the normal diploid DNA content.     

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.     

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.