Towards understanding the control of the division cycle in animal cells

1992 ◽  
Vol 70 (10-11) ◽  
pp. 920-945 ◽  
Author(s):  
Yoshio Masui
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Mammalian Cells ◽  
Microtubule Assembly ◽  
Nuclear Transplantation ◽  
Animal Cells ◽  
Cytoplasmic Factor ◽  
Molecular Control ◽  
Cytostatic Factor ◽  
Maturation Promoting Factor

The author reviewed the historical process by which classical knowledge of cell division accumulated, to give rise to the molecular biology of the cell cycle, and discussed the perspective of this field of research. The study of the control of cell division began at the turn of the century. It was hypothesized that cell division was a physiological regulation necessary for growing cells to maintain a proper nucleocytoplasmic ratio to survive, which was later substantiated by the finding that amoeba cells could be prevented from dividing by repeated excision of the cytoplasm. However, the observation in Tetrahymena that heat-shocked cells grow exceedingly, but fail to divide, suggested that the cell required the accumulation of a labile "division protein" to initiate division. Mechanisms that control the cell cycle were studied in oocytes by nuclear transplantation and cytoplasmic transfer, and in cultured mammalian cells, protozoa, and Physarum Plasmodia by cell fusion. These experiments demonstrated the existence of cytoplasmic factors that control the cell cycle. Maturation promoting factor (MPF) thus discovered in frog oocytes became known to be an ubiquitous cytoplasmic factor that causes the transition from interphase to metaphase in all organisms. The insight into the molecular control of cell growth and division was gained from yeast cell genetics. For biochemical analysis of the cell cycle control, the method to observe the cell cycle in vitro was developed using frog egg extracts. Thus, MPF was identified as a cdc2 – cyclin protein complex. Its activity was found to depend on synthesis and phosphorylation of these proteins. However, recently it was found that there were cell cycle phenomena that were difficult to explain in these terms. Various other cellular factors, including nucleocytoplasmic ratio and microtubule assembly, were also found to control MPF, as well as the cell cycle. It remained open to future investigation how these factors control MPF to alter the pattern of the cell cycle.Key words: cell cycle, cytostatic factor, maturation promoting factor, nucleocytoplasmic relation.

Overexpression Phenotypes of Plk1 and Ndrg2 in Schizosaccharomyces Pombe: Fission Yeast System for Mammalian Gene Study

2005 ◽  
Vol 277-279 ◽  
pp. 1-6 ◽  
Author(s):  
Young Joo Jang ◽  
Young Sook Kil ◽  
Jee Hee Ahn ◽  
Jae Hoon Ji ◽  
Jong Seok Lim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Rna Splicing ◽  
Mammalian Cells ◽  
Protein Modification ◽  
Genetic System ◽  
Functional Study ◽  
Mammalian Genes

The fission yeast, Schizosaccharomyces pombe is a single-celled free-living fungus that shares many features with cells of more complicated eukaryotes. Many of the genes required for the cell-cycle control, proteolysis, protein modification, and RNA splicing are highly conserved with those of higher eukaryotes. Moreover, fission yeast has the merit of genetics and its genetic system is already well characterized. As such, the current study evaluated the use of a fission yeast system as a tool for the functional study of mammalian genes and attempted to set up an assay system for novel genes. Since the phenotypes of a deletion mutant and the overexpression of a gene are generally analyzed for a functional study of specific genes in yeast, the present study used overexpression phenotypes to study the functions of mammalian genes. Therefore, based on using a thiamine-repressive promoter, two mammalian genes were expressed in fission yeast, and their overexpressed phenotypes compared with those in mammalian cells. The phenotypes resulting from overexpression were analyzed using a FACS, which analyzes the DNA contents, and a microscope. One of the selected genes was the mammalian Polo-like kinase 1 (Plk1), which is activated and plays a role in the mitotic phase of the cell division cycle. The overexpression of various constructs of Plk1 in the HeLa cells caused cell cycle defects, suggesting that the ectopic Plk1s blocked the endogenous Plk1 in the cells. As expected, when the constructs were overexpressed in the fission yeast system, the cells were arrested in mitosis and defected at the end of mitosis. As such, this data suggests that the Plk1-overexpressed phenotypes were similar in the mammalian cells and the fission yeast, thereby enabling the mammalian Plk1 functions to be approximated in the fission yeast. The other selected gene was the N-Myc downstream-regulated gene 2 (ndrg2), which is upregulated during cell differentiation, yet still not well characterized. When the ndrg2 gene was overexpressed in the fission yeast, the cells contained multi-septa. The septa were positioned well, yet their number increased per cell. Therefore, this gene was speculated to block cell division in the last stage of the cell cycle, making the phenotype potentially useful for explaining cell growth and differentiation in mammalian cells. Accordingly, fission yeast is demonstrated to be an appropriate species for the functional study of mammalian genes.

Nocodazole and cytochalasin D induce tetraploidy in mammalian cells

1984 ◽  
Vol 246 (1) ◽  
pp. C154-C156 ◽  
Author(s):  
G. W. Zieve
Keyword(s):  
Cell Division ◽  
Mammalian Cells ◽  
Cytochalasin D ◽  
Microtubule Assembly ◽  
Reversible Inhibitor ◽  
Cleavage Furrow ◽  
Synchronized Cells ◽  
Cells In Culture ◽  
Wide Range

Nocodazole, a rapidly reversible inhibitor of microtubule assembly is useful for preparing mammalian cells synchronized at all stages of mitosis. When synchronized cells are allowed to progress through mitosis in the presence of cytochalasin D, the cleavage furrow is inhibited and dikaryon cells are formed. These cells become homogeneous populations of stable mononuclear tetraploid cells after the following cell division. This procedure is applicable to a wide range of mammalian cells in culture.

scholarly journals Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division

2018 ◽  
Author(s):  
Evgeny Zatulovskiy ◽  
Daniel F. Berenson ◽  
Benjamin R. Topacio ◽  
Jan M. Skotheim
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Cell Growth ◽  
Cell Size ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Inverse Correlation ◽  
Cell Types ◽  
Similar Amount ◽  
Cell Cycle Inhibitor

Cell size is fundamental to function in different cell types across the human body because it sets the scale of organelle structures, biosynthesis, and surface transport1,2. Tiny erythrocytes squeeze through capillaries to transport oxygen, while the million-fold larger oocyte divides without growth to form the ~100 cell pre-implantation embryo. Despite the vast size range across cell types, cells of a given type are typically uniform in size likely because cells are able to accurately couple cell growth to division3–6. While some genes whose disruption in mammalian cells affects cell size have been identified, the molecular mechanisms through which cell growth drives cell division have remained elusive7–12. Here, we show that cell growth acts to dilute the cell cycle inhibitor Rb to drive cell cycle progression from G1 to S phase in human cells. In contrast, other G1/S regulators remained at nearly constant concentration. Rb is a stable protein that is synthesized during S and G2 phases in an amount that is independent of cell size. Equal partitioning to daughter cells of chromatin bound Rb then ensures that all cells at birth inherit a similar amount of Rb protein. RB overexpression increased cell size in tissue culture and a mouse cancer model, while RB deletion decreased cell size and removed the inverse correlation between cell size at birth and the duration of G1 phase. Thus, Rb-dilution by cell growth in G1 provides a long-sought cell autonomous molecular mechanism for cell size homeostasis.

Subcellular localisation of human wee1 kinase is regulated during the cell cycle

1995 ◽  
Vol 108 (6) ◽  
pp. 2425-2432
Author(s):  
V. Baldin ◽  
B. Ducommun
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Tyrosine Phosphatase ◽  
Cyclin B ◽  
Microtubule Assembly ◽  
Subcellular Localisation ◽  
Temporal Regulation ◽  
Wee1 Kinase ◽  
Cdc2 Activity ◽  
Intracellular Localisation

Wee1 kinase-dependent phosphorylation of cdc2 maintains the cdc2/cyclin B complex in an inert form until it is activated by the cdc25 tyrosine phosphatase at the end of G2. As described for cdc25, cell cycle-linked changes in the intracellular localisation of wee1 may constitute an important aspect of the temporal regulation of cdc2 activity. Here we report that the subcellular distribution of human wee1 changes during the cell cycle in HeLa and IMR90 cells. During interphase, wee1 is found almost exclusively in the nucleus. When the cell enters mitosis, wee1 is relocalised into the cytoplasm. During cell division, wee1 becomes restricted to the mitotic equator and by the end of mitosis it is found exclusively in association with midbody bridges, a phenomenon that is dependent on microtubule assembly. The relocalisations of wee1 and its association with subcellular structures may play key regulatory roles at different stages of the cell cycle and during mitosis.

scholarly journals Nonperiodic Activity of the Human Anaphase-Promoting Complex–Cdh1 Ubiquitin Ligase Results in Continuous DNA Synthesis Uncoupled from Mitosis

2000 ◽  
Vol 20 (20) ◽  
pp. 7613-7623 ◽  
Author(s):  
Claus Storgaard Sørensen ◽  
Claudia Lukas ◽  
Edgar R. Kramer ◽  
Jan-Michael Peters ◽  
Jiri Bartek ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Kinase Inhibitor ◽  
Cyclin B1 ◽  
S Phase ◽  
Anaphase Promoting Complex

ABSTRACT Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccharomyces cerevisiae andDrosophila spp., triggers exit from mitosis and during G1 prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference with the APC-Cdh1 dissociation at the G1/S transition resulted in an inability to accumulate a surprisingly broad range of critical mitotic regulators including cyclin B1, cyclin A, Plk1, Pds1, mitosin (CENP-F), Aim1, and Cdc20. Unexpectedly, although constitutively assembled APC-Cdh1 also delayed G1/S transition and lowered the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcription and a rapid turnover of the p27Kip1 cyclin-dependent kinase inhibitor. Consequently, failure to inactivate APC-Cdh1 beyond the G1/S transition not only inhibited productive cell division but also supported slow but uninterrupted DNA replication, precluding S-phase exit and causing massive overreplication of the genome. Our data suggest that timely oscillation of the APC-Cdh1 ubiquitin ligase activity represents an essential step in coordinating DNA replication with cell division and that failure of mechanisms regulating association of APC with the Cdh1 activating subunit can undermine genomic stability in mammalian cells.

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

1984 ◽  
Vol 98 (4) ◽  
pp. 1247-1255 ◽  
Author(s):  
J Gerhart ◽  
M Wu ◽  
M Kirschner
Keyword(s):  
Cell Cycle ◽  
Xenopus Laevis ◽  
Germinal Vesicle ◽  
Small Scale ◽  
Xenopus Laevis Oocytes ◽  
Germinal Vesicle Breakdown ◽  
Cytoplasmic Factor ◽  
M Phase ◽  
Cytostatic Factor ◽  
Meiotic Cycle

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).

A human nuclear protein with sequence homology to a family of early S phase proteins is required for entry into S phase and for cell division

1994 ◽  
Vol 107 (1) ◽  
pp. 253-265 ◽  
Author(s):  
I.T. Todorov ◽  
R. Pepperkok ◽  
R.N. Philipova ◽  
S.E. Kearsey ◽  
W. Ansorge ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Amino Acid ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Nuclear Protein ◽  
Amino Acid Level ◽  
S Phase ◽  
Significant Similarity

Molecular cloning and characterisation of a human nuclear protein designated BM28 is reported. On the amino acid level this 892 amino acid protein, migrating on SDS-gels as a 125 kDa polypeptide, shares areas of significant similarity with a recently defined family of early S phase proteins. The members of this family, the Saccharomyces cerevisiae Mcm2p, Mcm3p, Cdc46p/Mcm5p, the Schizosaccharomyces pombe Cdc21p and the mouse protein P1 are considered to be involved in the onset of DNA replication. The highest similarity was found with Mcm2p (42% identity over the whole length and higher than 75% over a conservative region of 215 amino acid residues), suggesting that BM28 could represent the human homologue of the S. cerevisiae MCM2. Using antibodies raised against the recombinant BM28 the corresponding antigen was found to be localised in the nuclei of various mammalian cells. Microinjection of anti-BM28 antibody into synchronised mouse NIH3T3 or human HeLa cells presents evidence for the involvement of the protein in cell cycle progression. When injected in G1 phase the anti-BM28 antibody inhibits the onset of subsequent DNA synthesis as tested by the incorporation of bromodeoxyuridine. Microinjection during the S phase had no effect on DNA synthesis, but inhibits cell division. The data suggest that the nuclear protein BM28 is required for two events of the cell cycle, for the onset of DNA replication and for cell division.

scholarly journals A novel mammalian protein, p55CDC, present in dividing cells is associated with protein kinase activity and has homology to the Saccharomyces cerevisiae cell division cycle proteins Cdc20 and Cdc4.

1994 ◽  
Vol 14 (5) ◽  
pp. 3350-3363 ◽  
Author(s):  
J Weinstein ◽  
F W Jacobsen ◽  
J Hsu-Chen ◽  
T Wu ◽  
L G Baum
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Division ◽  
Protein Kinase ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Yeast Cells ◽  
Protein Kinase Activity ◽  
Growing Cell ◽  
Dividing Cells

A novel protein, p55CDC, has been identified in cycling mammalian cells. This transcript is readily detectable in all exponentially growing cell lines but disappears when cells are chemically induced to fall out of the cell cycle and differentiate. The p55CDC protein appears to be essential for cell division, since transfection of antisense p55CDC cDNA into CHO cells resulted in isolation of only those cells which exhibited a compensatory increase in p55CDC transcripts in the sense orientation. Immunoprecipitation of p55CDC yielded protein complexes with kinase activity which fluctuated during the cell cycle. Since p55CDC does not have the conserved protein kinase domains, this activity must be due to one or more of the associated proteins in the immune complex. The highest levels of protein kinase activity were seen with alpha-casein and myelin basic protein as substrates and demonstrated a pattern of activity distinct from that described for the known cyclin-dependent cell division kinases. The p55CDC protein was also phosphorylated in dividing cells. The amino acid sequence of p55CDC contains seven repeats homologous to the beta subunit of G proteins, and the highest degree of homology in these repeats was found with the Saccharomyces cerevisiae Cdc20 and Cdc4 proteins, which have been proposed to be involved in the formation of a functional bipolar mitotic spindle in yeast cells. The G beta repeat has been postulated to mediate protein-protein interactions and, in p55CDC, may modulate its association with a unique cell cycle protein kinase. These findings suggest that p55CDC is a component of the mammalian cell cycle mechanism.

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