PLANT MITOSIS PROMOTING FACTOR DISASSEMBLES THE MICROTUBULE PREPROPHASE BAND AND ACCELERATES PROPHASE PROGRESSION IN TRADESCANTIA

The regulation of mitosis in higher plant cells has been investigated by microinjecting protein kinase from the metaphase-arresting (met1) mutant ofChlamydomonas. Biochemical characterization of this enzyme complex confirms the presence of a p34^cdc2/cyclin B-like kinase. The enzyme was injected into living stamen hair cells ofTradescantia virginianain which microtubules (MTs) were visualized using fluorescent analogue cytochemistry and confocal laser scanning microscopy. Microinjection of this p34^cdc2/cyclin B-like kinase caused rapid disassembly of the preprophase band of MTs but not of interphase-cortical, spindle or phragmoplast MTs. Effects of the enzyme on the cytomorphology of live prophase cells were also monitored using video microscopy. We found that injection of this enzyme accelerated chromatin condensation and nuclear envelope breakdown. This indicates the presence and function in plants of an enzyme that can initiate nuclear division similar to the maturation or mitosis promoting factor (MPF) of animal cells. These studies provide the first direct evidence that the mitotically-active form of plant MPF can drive disassembly of preprophase band MTs, chromosome condensation and initiation of mitosis in plant cells.     

Novel alleles ofcdc13 andcdc2 isolated as suppressors of mitotic catastrophe inSchizosaccharomyces pombe

Cell cycle control in the fission yeastSchizosaccharomyces pombe involves interplay amongst a number of regulatory molecules, including thecdc2, cdc13, cdc25, weel, andmik1 gene products. Cdc2, Cdc13, and Cdc25 act as positive regulators of cell cycle progression at the G2/M boundary, while Wee1 and Mik1 play a negative regulatory role. Here, we have screened for suppressors of the lethal premature entry into mitosis, termed mitotic catastrophe, which results from simultaneous loss of function of both Wee1 and Mik1. Through such a screen, we hoped to identify additional components of the cell cycle regulatory network, and/or G2/M-specific substrates of Cdc2. Although we did not identify such molecules, we isolated a number of alleles of bothcdc2 andcdc13, including a novel wee allele ofcdc2, cdc2-5w. Here, we characterizecdc2-5w and two alleles ofcdc13, which have implications for the understanding of details of the interactions amongst Cdc2, Cdc13, and Wee1.     

Phorbol Ester TPA Rapidly Prevents Activation of p34 Histone H1 Kinase and Concomitantly the Transition from G2 Phase to Mitosis in Synchronized HeLa Cells

HeLa cells in G2 phase are temporarily inhibited and prevented from entering mitosis by treatment with the phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate), whereas cells in mitosis are refractory to TPA and divide. In this study the possibility was tested that TPA may interfere with the regulatory cycle of MPF (mitosis promoting factor), the rate-limiting protein kinase for cell division. MPF, consisting of the catalytic subunit p34^cdc2 and the regulatory subunit Cyclin B, is known to be activated at the transition from G2 phase to mitosis through dephosphorylation at Tyr¹⁵ and to become inactivated after metaphase by proteolysis. Treatment of HeLa cells (synchronized around the G2-M transition) with TPA (10^-7M) has now been shown to induce an overall decrease of the histone H1 kinase activity associated with anti-p34^cdc2 immunoprecipitates after about 20 to 30 min. In metaphase cells, the histone H1 kinase activity of p34^cdc2 was shown to remain unaffected by TPA treatment. In cultures enriched in G2 cells neither the amount of p34^cdc2 protein nor that of Cyclin B was influenced by TPA. Moreover, the p34^cdc2/Cyclin B complex formation was also unaffected. However, p34^cdc2 from cultures treated with TPA was more intensely stained by anti-phosphotyrosine antibodies than that of control cells, indicating that TPA treatment probably prevented the tyrosine dephosphorylation required for expression of the histone H1 kinase activity of the complex. The results indicate that TPA treatment of HeLa cultures rapidly stops the G2-M transition because it very rapidly prevents the p34^cdc2/Cyclin B complex in G2 cells from developing histone H1 kinase activity.     

The <Emphasis Type="Italic">alp1-1315</Emphasis> mutation of the tubulin-folding cofactor D gene delays the mitosis initiation in <Emphasis Type="Italic">cdc25-22</Emphasis> mutant cells of <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Tubulin-folding cofactor D is necessary for the assembly of tubulin heterodimers and, possibly, plays additional roles in the cell. The effects of cofactor D, microtubules, and/or tubulin dimers on the mitosis initiation were studied in Schizosaccharomyces pombe. It was found for the first time that S. pombe cells with the alp1-1315 and cdc25-22 mutations remained highly viable at 36°C for 8 h, in contrast to cells with the alp1-1315 mutation alone. The progression of cdc25-22 alp1-1315 cells through mitosis after a cell division arrest at 36°C was described. When transferred to 25°C, cdc25-22 alp1-1315 cells displayed a lag of approximately 30 min in Plo1-GFP appearance in the spindle pole body (SPB), 1 h in chromosome condensation, and 75 min in spindle formation. Thus, the initiation of mitosis in cdc25-22 alp1-1315 cells was delayed as compared with cdc25-22 cells. Since treatment of cdc25-22 cells with a microtubule-destabilizing drug during an arrest is known to cause a premitotic arrest with low activity of the mitosis-promoting factor (MPF), it was assumed that an impaired integrity of microtubules and/or lack of tubulin dimers in the nucleus were responsible for the delayed mitosis initiation in cdc25-22 alp1-1315 cells and in cdc25-22 cells treated with a microtubule-destabilizing drug. The progression through mitosis after a cdc25-22 arrest was extremely slow in cdc25-22 alp1-1315 cells, which was attributed to the de novo formation of tubulin dimers.     

Biochemical Differences between Staurosporine-Induced Apoptosis and Premature Mitosis

Apoptosis is morphologically related to premature mitosis, an aberrant form of mitosis. Staurosporine, a potent protein kinase inhibitor, induces not only apoptotic cell death in a wide variety of mammalian cells but also premature initiation of mitosis in hamster cells that are arrested in S phase by DNA synthesis inhibitors. Here we report on the biochemical differences between the two phenomena commonly caused by staurosporine. Rat 3Y1 fibroblasts that had been arrested in S phase with hydroxyurea underwent apoptosis by treatment with staurosporine, whereas S-phase-arrested CHO cells initiated mitosis prematurely when similarly treated with a low concentration of staurosporine. Chromosome condensation occurred in both apoptosis (3Y1) and premature mitosis (CHO). However, neither formation of mitotic spindles nor mitosis-specific phosphorylation of MPM-2 antigens was observed in apoptosis of 3Y1 cells, unlike premature mitosis of CHO cells. The p34^cdc2kinase activated in normal and prematurely mitotic cells remained inactive in the apoptotic cells, probably because the active cyclin B/p34^cdc2complex was almost absent in the S-phase-arrested 3Y1 cells. The absence of intracellular activation of p34^cdc2in apoptosis was confirmed by immunohistochemical analyses using a specific antibody raised against Ser⁵⁵-phosphorylated vimentin which is specifically phosphorylated by p34^cdc2during M phase. Furthermore, phosphorylation of histones H1 and H3, which is associated with mitotic chromosome condensation, did not occur in the apoptotic cells. These results indicate that the two phenomena, staurosporine-induced apoptosis and premature mitosis, are different in their requirement for p34^cdc2kinase activation and histone phosphorylation.     

Dynamic changes in CCAN organization through CENP-C during cell-cycle progression

The kinetochore is a crucial structure for faithful chromosome segregation during mitosis and is formed in the centromeric region of each chromosome. The 16-subunit protein complex known as the constitutive centromere-associated network (CCAN) forms the foundation for kinetochore assembly on the centromeric chromatin. Although the CCAN can be divided into several subcomplexes, it remains unclear how CCAN proteins are organized to form the functional kinetochore. In particular, this organization may vary as the cell cycle progresses. To address this, we analyzed the relationship of centromeric protein (CENP)-C with the CENP-H complex during progression of the cell cycle. We find that the middle portion of chicken CENP-C (CENP-C^166–324) is sufficient for centromere localization during interphase, potentially through association with the CENP-L-N complex. The C-terminus of CENP-C (CENP-C^601–864) is essential for centromere localization during mitosis, through binding to CENP-A nucleosomes, independent of the CENP-H complex. On the basis of these results, we propose that CCAN organization changes dynamically during progression of the cell cycle.     

Changes in the phosphorylation of non-histone chromatin proteins during the cell cycle of HeLa S 3 cells

The phosphorylation of non-histone chromatin proteins in synchronized HeLa S₃ cells was studied in 5 phases of the cell cycle: mitosis, G₁, early and late S, and G₂. The rate of non-histone chromatin protein phosphorylation was found to be maximal during G₁ and G₂, somewhat decreased during S phase, and almost 90% depressed during mitosis. Analysis of the phosphorylated non-histone chromatin proteins by SDS-acrylamide gel electrophoresis showed a heterogeneous pattern of phosphorylation as measured by labeling with ³²P. Significant variations in the labeling pattern were seen during different stages of the cell cycle, and particular unique species appeared to be phosphorylated selectively during certain stages of the cycle.     

Cdc2-kinases,cyclins, and the switch from proliferation to polyploidization

Summary Almost all organisms, from protists to humans, and from algae to orchids, display somatic polyploidy, including polyteny. In insects and higher plants, nearly all normal, differentiated cells are polyploid, corresponding to the majority of living matter. So far, no universal mechanism controlling the switch from proliferation to polyploidization has been proposed. However, recent progress in understanding regulation of the mitotic cell cycle by protein kinases and cyclins allows some unifying ideas which can be experimentally tested to be put forward. The key events are the abolishment of the dependence of DNA replication on mitosis, and changes in the expression and activity of the complexes formed by cyclin-dependent kinases and cyclins. In addition, repression of further cell cycle control genes may allow underreplication of DNA, characteristic of endo-cycles in many insects and angiosperms. Change to a different checkpoint may be responsible for gene amplification. The switch in cell cycle control is developmentally regulated by signal transduction cascades, which are briefly discussed. Polyploidy is also known from many cancers, where genetic and metabolic disturbances lead to a similar switch to that in normal cells. The related literature is reviewed and some possible lines of future research are suggested.Abbreviations CAK p34^cdc2-activating kinase - cdc2 cell division cycle gene inSchizosaccharomyces pombe (fission yeast), named cdk1 in mammals - CDKs cyclin-dependent kinases - cdk2 S-phase specific CDK gene in higher organisms - MAP kinase mitogen-activated protein kinase - MAPs microtubule-associated proteins - MPF maturation (or mitosis) promoting factor - p34^cdc2 mitosis specific protein kinase     

Novel alleles ofcdc13 andcdc2 isolated as suppressors of mitotic catastrophe inSchizosaccharomyces pombe

Cell cycle control in the fission yeastSchizosaccharomyces pombe involves interplay amongst a number of regulatory molecules, including thecdc2, cdc13, cdc25, weel, andmik1 gene products. Cdc2, Cdc13, and Cdc25 act as positive regulators of cell cycle progression at the G2/M boundary, while Wee1 and Mik1 play a negative regulatory role. Here, we have screened for suppressors of the lethal premature entry into mitosis, termed mitotic catastrophe, which results from simultaneous loss of function of both Wee1 and Mik1. Through such a screen, we hoped to identify additional components of the cell cycle regulatory network, and/or G2/M-specific substrates of Cdc2. Although we did not identify such molecules, we isolated a number of alleles of bothcdc2 andcdc13, including a novel wee allele ofcdc2, cdc2-5w. Here, we characterizecdc2-5w and two alleles ofcdc13, which have implications for the understanding of details of the interactions amongst Cdc2, Cdc13, and Wee1.     

The substrates of the cdc2 kinase.

The eukaryotic cell cycle is characterized by two major events, DNA replication (S phase) and mitosis (M phase). According to the current paradigm of the cell cycle as a cdc2 cycle, both of these events are driven by serine-threonine specific protein kinases encoded by functional homologs of the fission yeast cdc2 gene. To understand how cdc2 kinases function, it is necessary to identify their physiological substrates and to determine how phosphorylation of these substrates promotes cell cycle progression. Definitive information about substrates relevant to early stages of the cell cycle (G1 and S phases) remains scarce, but several likely physiological targets of the mitotic cdc2 kinase have recently been identified. Current evidence indicates that cdc2 kinase may trigger entry of cells into mitosis not only by initiating important regulatory pathways but also by direct phosphorylation of abundant structural proteins.     

Optical tomography analysis of <Emphasis Type="Italic">Amoeba proteus</Emphasis> chromatin organization at various cell cycle stages

An optical tomography investigation of the nuclear cycle in large freshwater amoebae Amoeba proteus has been performed for the first time. Nuclei of cells from a synchronized culture were stained with DAPI and examined using a confocal laser scanning microscope. Detailed analysis of three-dimensional images of the intranuclear chromatin at different stages of the nuclear cycle has been performed. The materials obtained, in combination with the published data, allow for a completely new representation of the dynamics of the structural organization of the A. proteus nucleus during the cell cycle. Two-stage interphase and mitosis of a special type not matching any of the known types in the existing systems of classification of mitosis were found to occur in amoebae. Amplification of chromosomes and/or fragments thereof supposedly occurs during the cell cycle, which is consistent with the available data on nuclear DNA hyperreplication during the cell cycle of A. proteus. The number of chromosomes can vary at different stages of the cycle because of amplification, this being a putative reason for the discordant reports on the number of chromosomes in this species. The elimination of “excess” DNA mainly occurs during the transition from prophase to prometaphase. Finally, specific features of chromosome behavior during mitosis allow conclusion to be drawn that many, if not all, chromosomes are of a holocentric type.     

DNA replication is completed in Saccharomyces cerevisiae cells that lack functional Cdc14, a dual-specificity protein phosphatase

The Cdc14 protein encodes a dual-specificity protein phosphatase which functions in late mitosis, and considerable genetic evidence suggests a role in DNA replication. We find that cdc14 mutants arrested in late mitosis maintain persistent levels of mitotic kinase activity, suggesting that Cdc14 controls inactivation of this kinase. Overexpression of Sic1, a cyclin-dependent protein kinase inhibitor, is able to suppress telophase mutants such as dbf2, cdc5 and cdc15, but not cdc14. It does, however, force cdc14-arrested cells into the next cell cycle, in which an apparently normal S phase occurs as judged by FACS and pulsed-field gel electrophoretic analysis. Furthermore, in a promoter shut-off experiment, cells lacking Cdc14 appear to carry out a normal S phase. Thus Cdc14 functions mainly in late mitosis and it has no essential role in S phase. Received: 9 January 1998 / Accepted: 22 January 1998     

Isolation and characterization of cDNA clones encoding cdc2 homologues from Oryza sativa: a functional homologue and cognate variants.

Summary Using probes obtained by PCR amplification, we have isolated two cognate rice cDNAs (cdc2Os-1 andcdc2Os-2) encoding structural homologues of thecdc2 ⁺/CDC28(cdc2) protein kinase from a cDNA library prepared from cultured rice cells. Comparison of the deduced amino acid sequences of cdc2Os-1 and cdc2Os-2 showed that they are 83 % identical. They are 62 % identical toCDC28 ofSaccharomyces cerevisiae and much more similar to the yeast and mammalian p34^cdc2 kinases than to riceR2, acdc2-related kinase isolated previously by screening the same rice cDNA library with a different oligonucleotide probe. Southern blot analysis indicated that the three rice clones (cdc2Os-1,cdc2Os-2 andR2) are derived from distinct genes and are each found in a single copy per rice haploid genome. RNA blot analysis revealed that these genes are expressed in proliferating rice cells and in young rice seedlings.cdc2Os-1 could complement a temperature-sensitive yeast mutant ofcdc28. However, despite the similarity in structure, bothcdc2Os-2 andR2 were unable to complement the same mutant. Thus, the present results demonstrate the presence of structurally related, but functionally distinct cognates of thecdc2 cell cycle kinase in rice.The nucleotide sequence data in this paper have been deposited in the EMBL database under accession number X60374 (cdc2Os-1) and X60375 (cdc2Os-2)     

14-3-3 family members act coordinately to regulate mitotic progression

The mitosis promoting phosphatase, cdc25C, is a target of both the DNA replication and DNA damage checkpoint pathways. These pathways regulate cdc25C function, in part, by promoting the association of cdc25C with 14-3-3 proteins, which results in the retention of cdc25C in the cytoplasm. To determine which 14-3-3 proteins were required to regulate cdc25C function, we tested the ability of various 14-3-3 family members to form a complex with and negatively regulate cdc25C in human cells. Two 14-3-3 family members, 14-3-3epsilon and 14-3-3gamma specifically formed a complex with cdc25C but not with the 14-3-3 binding defective cdc25C mutant, S216A. In addition, 14-3-3epsilon and 14-3-3gamma inhibited the ability of cdc25C, but not the S216A mutant, to induce premature chromatin condensation (PCC) in U-2OS cells. These results suggested that the reduction in PCC by 14-3-3epsilon and 14-3-3gamma was due to inhibition of cdc25C function. In contrast, 14-3-3sigma was unable to form a complex with cdc25C, but was able to inhibit the ability of both wild type cdc25C and S216A to induce PCC. This suggests that 14-3-3sigma regulates entry into mitosis independently of cdc25C and 14-3-3epsilon and 14-3-3gamma. Thus, specific members of the 14-3-3 family of proteins may act coordinately to maintain the DNA replication checkpoint by regulating the activity of different cell cycle proteins.