Transition of the blastomere cell cycle from cell size-independent to size-dependent control at the midblastula stage in Xenopus laevis

Dissociated animal cap blastomeres of Xenopus laevis blastulae were cultured at a low Ca level (1 microM) from 9th to 18th cell cycle at 22 +/- 1 degrees C and observed by a time-lapse video recorder. Blastomeres cleaved unequally to increase variability in cell size as cell cycles progressed, but synchronously at a constant cell cycle time of about 30 min up to the 12th cleavage in diploid cells, and up to the 13th cleavage in haploid cells, regardless of their cell sizes. Thereafter, blastomeres cleaved asynchronously at varying cell cycle times in proportion to the inverse square of their radii. The transition from the cell size-independent to -dependent cell cycles occurred at the critical cell radius, 37.5 microm for the diploid and 27.9 microm for the haploid. While the protein synthesis inhibitor, cycloheximide (CHX) lengthened cell cycle times two- to six-fold, epidermal growth factor (EGF) had no significant effect on the cell cycle. CHX-treated blastomeres synchronously cleaved at a constant cell cycle time of 60 min up to the 12th cleavage. Thereafter, cell cycle times became variable in proportion to the inverse square of radii in the presence of CHX at 0.10-0.14 microg/ml, but to the inverse cube of radii at 0.18 microg/ml. The critical cell size of CHX-treated blastomeres for the transition from cell size-independent to -dependent cell cycles remained the same as that of untreated blastomeres. Frequency distributions of cell cycle times of synchronous cell cycles were monomodal with the peak at 30 min, except for CHX-treated blastomeres with the peak at 60 min. In contrast, frequency distributions of asynchronous cell cycles were polymodal with peaks at multiples of a unit time of 30-35 min. To explain these results, we propose that blastomere cytoplasm has 30-min cycles that repeatedly produce mitosis promoting factor (MPF) in a quantity proportional to the cell surface area. MPF is neutralized when it titrates a nuclear inhibitor present in a quantity proportional to the genome size, and sequestered in the nucleus. When the total amount of MPF produced exceeds the threshold required to titrate all of the inhibitor, mitosis is initiated.     

Flow cytometric fluorescence pulse width analysis of etoposide-induced nuclear enlargement in HCT116 cells

Fluorescence pulse width can provide size information on the fluorescence-emitting particle, such as the nuclei of propidium iodide-stained cells. To analyze nuclear size in the present study, rather than perform the simple doublet discrimination approach usually employed in flow cytometric DNA content analyses, we assessed the pulse width of the propidium iodide fluorescence signal. The anti-cancer drug etoposide is reportedly cytostatic, can induce a strong G2/M arrest, and results in nuclear enlargement. Based on these characteristics, we used etoposide-treated HCT116 cells as our experimental model system. The fluorescence pulse widths (FL2-W) of etoposide-treated (10 μM, 48 h) cells were distributed at higher positions than those of vehicle control, so the peak FL2-W value of etoposide-treated cells appeared at 400 while those of vehicle control cells appeared at 200 and 270. These results were consistent with our microscopic observations. This etoposide-induced increase in FL2-W was more apparent in G2/M phase than other cell cycle phases, suggesting that etoposide-induced nuclear enlargement preferentially occurred in G2/M phase cells rather than in G0/G1 or S phase cells.     

The durations and compositions of cell cycles in embryos of the leech, Helobdella triserialis

When tritiated thymidine triphosphate ([(3)H]TTP) or its immunohistochemically detectable analogue, bromodeoxyuridine triphosphate (BrdUTP), is injected into blastomeres of leech embryos it passes throughout the entire embryo and is rapidly incorporated (within 2 min after injection) into nuclei of cells synthesizing DNA (S phase). In the same embryos a DNA-specific stain can be used to identify cells in mitosis (M phase) or nonreplicative interphase (G(1) or G(2) phase) on the basis of nuclear or chromosomal morphology. Using this procedure, we have determined the lengths and compositions of the mitotic cell cycles of identifiable cells in early embryos of the leech, Helobdella triserialis, and have analysed how the cell cycles change during the first seven stages of development. The relatively short cell cycles of the early blastomeres comprise not only phases of M and S, but also postreplicative gap (G(2)) phases. The lengthening of the cell cycles that occurs as development progresses is primarily accomplished by an increase in the length of G(2) and secondarily by an increase in the length of S and,in some instances, the addition of a prereplicative gap(G(1)) phase; M phase remains relatively constant. These data suggest that the durations of the cell cycles of embryonic cells are regulated by a variety of mechanisms.     

The difference in murine CDC2 kinase activity between cytoplasmic and nuclear fractions during the cell cycle

The mouse analog of yeast CDC2+ kinase was detected in the cytoplasmic and nuclear fractions of cultured mouse FM3A cells. Its activity in the nuclear fraction increased in the G2/M phase became seven times higher than that in the G1/S phase, while the activity in the cytoplasmic fraction remained was almost constant from the G1/S to G1 phases. The activity in the cytoplasmic fraction was similar to that in the nuclear fraction in the G2/M phase. The amount of the enzyme remained almost constant during the cell cycle in both the nuclear and cytoplasmic fractions. These findings suggest that the cytoplasmic enzyme might play an independent role in the cell cycle.     

Bromodeoxyuridine labeling and flow cytometric identification of replicating Saccharomyces cerevisiae cells: lengths of cell cycle phases and population variability at specific cell cycle positions.

An immunofluorescent staining procedure has been developed to identify, with flow cytometry, replicating cells of Saccharomyces cerevisiae after incorporation of bromodeoxyuridine (BrdUrd) into the DNA. Incorporation of BrdUrd is made possible by using yeast strains with a cloned thymidine kinase gene from the herpes simplex virus. An exposure time of 4 min to BrdUrd results in detectable labeling of the DNA. The BrdUrd/DNA double staining procedure has been optimized and the flow cytometry measurements yield histograms comparable to data typically obtained for mammalian cells. On the basis of the accurate assessment of cell fractions in individual cell cycle phases of the asynchronously growing cell population, the average duration of the cell cycle phases has been evaluated. For a population doubling time of 100 min it was found that cells spend in average 41 min in the replicating phase and 24 min in the G2+M cell cycle period. Assuming that mother cells immediately reenter the S phase after cell division, daughter cells spend 65 min in the G1 cell cycle phase. Together with the single cell fluorescence parameters, the forward-angle light scattering intensity (FALS) has been determined as an indicator of cell size. Comparing different temporal positions within the cell cycle, the determined FALS distributions show the lowest variability at the beginning of the S phase. The developed procedure in combination with multiparameter flow cytometry should be useful for studying the kinetics and regulation of the budding yeast cell cycle.     

Cell cycle transition in early embryonic development of Xenopus laevis

This article reviews cell cycle changes that occur during midblastula transition (MBT) in Xenopus laevis based on research carried out in the authors' laboratory. Blastomeres dissociated from the animal cap of blastulae, as well as those in an intact embryo, divide synchronously with a constant cell cycle duration in vitro, up to the 12th cell cycle regardless of their cell sizes. During this synchronous cleavage, cell sizes of blastomeres become variable because of repeated unequal cleavage. After the 12th cell cycle blastomeres require contact with an appropriate protein substrate to continue cell division. When nucleocytoplasmic (N/C) ratios of blastomeres reach a critical value during the 13th cycle, their cell cycle durations lengthen in proportion to the reciprocal of cell surface areas, and cell divisions become asynchronous due to variations in cell sizes. The same changes occur in haploid blastomeres with a delay of one cell cycle. Thus, post-MBT cell cycle control becomes dependent not only on the N/C relation but also on cell surface activities of blastomeres. Unlike cell cycle durations of pre-MBT blastomeres, which show monomodal frequency distributions with a peak at about 30 min, those of post-MBT blastomeres show polymodal frequency distributions with peaks at multiples of about 30 min, suggesting 'quantisement' of the cell cycle. Thus, we hypothesised that MPF is produced periodically during its unit cycle with 30 min period, but it titrates, and is neutralized by, an inhibitor contained in the nucleus in a quantity proportional to the genome size; however, when all of the inhibitor has been titrated, excess MPF during the last cycle triggers mitosis. At MBT, cell cycle checkpoint mechanisms begin to operate. While the operation of S phase checkpoint to monitor DNA replication is initiated by N/C relation, the initiation of M phase checkpoint operation to monitor chromosome segregation at mitosis is regulated by an age-dependent mechanism.     

Apical migration of nuclei during G2 is a prerequisite for all nuclear motion in zebrafish neuroepithelia

Nuclei in the proliferative pseudostratified epithelia of vastly different organisms exhibit a characteristic dynamics - the so-called interkinetic nuclear migration (IKNM). Although these movements are thought to be intimately tied to the cell cycle, little is known about the relationship between IKNM and distinct phases of the cell cycle and the role that this association plays in ensuring balanced proliferation and subsequent differentiation. Here, we perform a quantitative analysis of modes of nuclear migration during the cell cycle using a marker that enables the first unequivocal differentiation of all four phases in proliferating neuroepithelial cells in vivo. In zebrafish neuroepithelia, nuclei spend the majority of the cell cycle in S phase, less time in G1, with G2 and M being noticeably shorter still in comparison. Correlating cell cycle phases with nuclear movements shows that IKNM comprises rapid apical nuclear migration during G2 phase and stochastic nuclear motion during G1 and S phases. The rapid apical migration coincides with the onset of G2, during which we find basal actomyosin accumulation. Inhibiting the transition from G2 to M phase induces a complete stalling of nuclei, indicating that IKNM and cell cycle continuation cannot be uncoupled and that progression from G2 to M is a prerequisite for rapid apical migration. Taken together, these results suggest that IKNM involves an actomyosin-driven contraction of cytoplasm basal to the nucleus during G2, and that the stochastic nuclear movements observed in other phases arise passively due to apical migration in neighboring cells.     

Analysis of the fifth cell cycle of mouse development

The 5th cell cycle of mouse development was analyzed to determine the lengths of each cell cycle phase. The DNA content of Feulgen-stained blastomere nuclei was measured at various times throughout the cell cycle by microdensitometry. To achieve precise timing of the start of the 5th cell cycle, experiments utilized isolated 16-cell blastomeres and cell pairs obtained by in-vitro division of isolated 8-cell blastomeres. The following estimates were made for a mixed population of polar and apolar 16-cell blastomeres: G1, less than or equal to 2 h; S, 8-9 h; G2 + M, 2 h. No significant difference was found in the timing of DNA synthesis between polar and apolar cells or between cell pairs and whole embryos.     

Analysis of the third and fourth cell cycles of mouse early development

The third (4-cell) and fourth (8-cell) cell cycles of early mouse development have been analysed in populations of blastomeres synchronized to the preceding cleavage division. DNA content was measured microdensitometrically. The entry of blastomeres into these cell cycles showed considerable heterogeneity both within and between individual embryos. This heterogeneity was greater in the fourth than in the third cell cycle. The component phases of the third cell cycle were estimated as G1 = 1 h, S = 7 h, and G2 + M = 2-5 h, and those of the fourth cell cycle as G1 = 2 h, S = 7 h, and G2 + M = 1-3 h.     

Dominant negative E2F inhibits progression of the cell cycle after the midblastula transition in Xenopus

The cleavage cycle, which is initiated by fertilization, consists of only S and M phases, and the gap phases (G1 and G2) appear after the midblastula transition (MBT) in the African clawed frog, Xenopus laevis. During early development in Xenopus, we examined the E2F activity, which controls transition from the G1 to S phase in the somatic cell cycle. Gel retardation and transactivation assays revealed that, although the E2F protein was constantly present throughout early development, the E2F transactivation activity was induced in a stage-specific manner, that is, low before MBT and rapidly increased after MBT. Introduction of the recombinant dominant negative E2F (dnE2F), but not the control, protein into the 2-cell stage embryos specifically suppressed E2F activation after MBT. Cells in dnE2F-injected embryos appeared normal before MBT, but ceased to proliferate and eventually died at the gastrula. These cells contained decreased cdk activity with enhanced inhibitory phosphorylation of Cdc2 at Tyr15. Thus, E2F activity is required for cell cycle progression and cell viability after MBT, but not essential for MBT transition and developmental progression during the cleavage stage.     

Characteristics of the cell cycle of matrix cells in the mouse embryo during histogenesis of telencephalon

The cell cycle of matrix cells in the telencephalon of the mouse embryo at different stages at day 10, 13, and 17 of gestation was investigated by means of ³H-thymidine autoradiography.The cell cycle time of matrix cells in the day 10 group was found to be 7.0 h, and lengthened linearly with embryonic age. The cell cycle times of day 13 and 17 groups were 15.5 and 26.0 h, respectively.The duration of G1 and S phases also lengthened linearly with embryonic age. The durations of G1 phase were 0.1, 6.8, and 13.8 h, for day 10, 13, and 17 groups, respectively, and those of S phase were 5.1, 6.9, and 10.4 h, for day 10, 13, and 17 groups, respectively. On the other hand, the durations of both G2 and M phases remained unchanged and these were 1.0 and 0.8 h, respectively, throughout the embryonic stages.It was a characteristic of the alteration of the cell cycle of the telencephalon during mouse embryonic life that not only G1 but also S phases lengthened linearly with embryonic age and both G2 and M phases remained constant.     

GROWTH CHARACTERISTICS OF MARINE PHYTOPLANKTON DETERMINED BY CELL CYCLE PROTEINS: THE CELL CYCLE OF ETHMODISCUS REX (BACILLARIOPHYCEAE) IN THE SOUTHWESTERN NORTH ATLANTIC OCEAN AND CARIBBEAN SEA1

Ethmodiscus spp. is an important contributor to oceanic tropical-ooze sediments and thus might be an important transport vehicle of carbon from the ocean surface to sediments. The knowledge of its cell cycle and growth rate, which is still lacking, is necessary to evaluate the importance of Ethmodiscus in nutrient cycling and to solve the discrepancy between its high sedimentary abundance and rarity in the plankton. We used immunofluorescence of a cell cycle protein, prolqerating cell nuclear antigen (PCNA), and DNA-specific staining to study the progression of the cell cycle and roughly estimate the growth rate for E. rex (Rattray) Wiseman and Hendey in the southwestern North Atlantic Ocean and Caribbean Sea in June 1994 and January 1995. During the cell division cycle, the chloroplasts appeared to synthesize DNA before the nucleus (S phase). Following the S phase, the nucleus moved from one end of the cell toward the center underneath the midline of the girdle band (G2 phase) where it divided (M phase). During a very brief period, the parent cell split and moved apart from the girdle midline, and two new valves were produced (late M phase). The two daughter nuclei apparently remained attached at the joint of the two newly produced valves, where they appeared to be responsible for coordinating the symmetrical formation of the new valves. The morphologically complete daughter cells remained joined for a short period of time before separating into solitary cells whose nucleus was located at one end of the cell. Derived from the phase fraction curves, the duration of the cell cycle phases decreased in the order from G1, S, G2, to M. A conservative estimate of the growth rate in the study area obtained by using PCNA immunostaining was 0.39–0.46 d^?1 in June and 0.15 d^?1 in January. The validity and implication of the growth rate estimates are discussed.     

Lineage-specific alteration in cell cycle structure in early Tubifex embryos

Embryos of the freshwater oligochaete Tubifex exhibit asynchrony in division timing as early as the second cleavage; this cleavage asynchrony becomes pronounced as development proceeds. The present study was undertaken to elucidate the composition and duration of the cell cycles of early Tubifex embryos, with special reference to their cell lineages. No significant variations in lengths of cleavage cycles were found among early embryos. In all blastomeres up to the eighth cleavage cycle, the M phase was followed directly by a 30 min S phase, which suggested that early embryos lack G1 phase. The durations of the M phase did not change during this period of development, but did differ between cell lines. The M phase in the A and B cell lines lasted for about 130 min, while the M phase in the C and D cell lines lasted for about 95 min. An examination of chromosome cycles showed that this difference in M phase durations resulted from a longer stay by the A/B cell lines in prometaphase. Only G2 phase lengthened during early development. After several rounds of G2 phase extension, three classes of G2 phase duration were established: the most extended G2 phase (∼6 h) in the first quartette of micromeres (cells 1 a–1 d), the shortest G2 phase (∼1.58 h) in teloblasts, and an intermediate G2 phase (∼2.4 h) in the progeny of macromeres (i.e. endodermal cells). Experiments with syncytial blastomeres showed that the timing of entry into the M phase, hence the duration of the G2 phase, was affected by cytoplasmic compositions. The shortest G2 phase correlated closely with the presence of yolk-free cytoplasm called pole plasm.     

Apoptosis in erythroid progenitors deprived of erythropoietin occurs during the G1 and S phases of the cell cycle without growth arrest or stabilization of wild-type p53.

Erythropoietin (Epo) inhibits apoptosis in murine proerythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells). We have shown that the apoptotic process in FVA cell populations deprived of Epo is asynchronous as a result of a heterogeneity in Epo dependence among individual cells. Here we investigated whether apoptosis in FVA cells correlated with cell cycle phase or stabilization of p53 tumor suppressor protein. DNA analysis in nonapoptotic FVA cell subpopulations cultured without Epo demonstrated little change in the percentages of cells in G1,S, and G2/M phases over time. Analysis of the apoptotic subpopulation revealed high percentages of cells in G1 and S, with few cells in G2/M at any time. When cells were sorted from G1 and S phases prior to culture without Epo, apoptotic cells appeared at the same rate in both populations, indicating that no prior commitment step had occurred in either G1 or S phase. Steady-state wild-type p53 protein levels were very low in FVA cells compared with control cell lines and did not accumulate in Epo-deprived cultures; however, p53 protein did accumulate when FVA cells were treated with the DNA-damaging agent actinomycin D. These data indicate that erythroblast apoptosis caused by Epo deprivation (i) occurs throughout G1 and S phases and does not require cell cycle arrest, (ii) does not have a commitment event related to cell cycle phase, and (iii) is not associated with conformational changes or stabilization of wild-type p53 protein.     

Polyplex-mediated gene transfer and cell cycle: effect of carrier on cellular uptake and intracellular kinetics, and significance of glycosaminoglycans

BACKGROUND: Here we report on studies that probe whether the intracellular kinetics of plasmid DNA (pDNA) and cell surface glycosaminoglycans (GAGs) are modified during the cell cycle in a way that can be correlated with changes in gene transfer efficiency with poly(ethyleneimine) (PEI) and poly-L-lysine (PLL) polyplexes. METHODS: Synchronized D407 retinal cells were transfected with PEI and PLL polyplexes using a luciferase reporter. The free and/or loosely complexed nuclear pDNA was determined by real-time PCR, and compared with transgene expression, the rate of pinocytosis by FITC-dextran uptake and the content of cell surface GAGs. RESULTS: The amount of free and/or loosely complexed nuclear pDNA between cell cycle phases varied approximately 4-20 times (G1 < S < G2/M). Both carriers delivered pDNA in a similar way into the nucleus (PLL vs. PEI < or = 3.5-fold), but PEI was approximately 10-100 times more efficient in gene expression than PLL (G1 < G2/M < S). The rate of pinocytosis increased up to 70-fold from G1 to middle S phase. Cell surface heparan and chondroitin sulfate increased 50-80%, and hyaluronan decreased 50% when the cells went from G1 through S to G2/M. CONCLUSIONS: The data obtained indicates that no single parameter (pinocytosis, cell surface GAGs, nuclear uptake) solely accounts for the differential pDNA uptake or expression during cell cycle, and that the main difference in PLL- and PEI-mediated transfections seems to be at the nuclear level.     

Influence of cell cycle stage of the donor nucleus on development of nuclear transplant rabbit embryos.

We evaluated the influence of the stage of the cell cycle of the donor nucleus on development in vitro of nuclear transplant rabbit embryos. The developmental potential of nuclei in early, mid-, and late stages of the cell cycle was determined. Duration of the G1 phase in early embryos was determined, and a procedure for reversibly synchronizing donor embryos in the G1 phase was developed. In addition, the extent of development in vitro of nuclear transplant embryos with donor nuclei synchronized in the G1 phase was evaluated. Development to blastocysts was greatly affected by the stage of the cycle of the donor nucleus. Use of early-stage nuclei led to 59% nuclear transplant blastocysts, whereas 32% and 3% were obtained with mid- and late-stage nuclear donors, respectively (p less than 0.001). The short duration of the G1 phase in 16- and 32-cell-stage embryos (approximately 30 min) necessitated a procedure for synchronizing blastomeres in the G1 phase. This entailed, first, a 10-h incubation in 0.5 micrograms/ml colcemid to arrest embryos in metaphase. After release from colcemid, embryos were allowed to cleave in 0.1 microgram/ml of the DNA synthesis inhibitor, aphidicolin, and remained blocked at the G1/S transition. This treatment was reversible, as assessed by the resumption of DNA synthesis, cleavage rate, and development to blastocysts of treated embryos. The beneficial effect of using early-stage donor blastomeres was confirmed by the enhanced rate of development of manipulated embryos to blastocysts with donor nuclei in the G1 phase (71%), as opposed to the late S phase (15%, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of human Mcm10 is spatially and temporally regulated during the S phase

Mcm10 (Dna43) is an essential protein for the initiation of DNA replication in Saccharomyces cerevisiae. Recently, we identified a human Mcm10 homolog and found that it is regulated by proteolysis and phosphorylation in a cell cycle-dependent manner and that it binds chromatin exclusively during the S phase of the cell cycle. However, the precise roles that Mcm10 plays are still unknown. To study the localization dynamics of human Mcm10, we established HeLa cell lines expressing green fluorescent protein (GFP)-tagged Mcm10. From early to mid-S phase, GFP-Mcm10 appeared in discrete nuclear foci. In early S phase, several hundred foci appeared throughout the nucleus. In mid-S phase, the foci appeared at the nuclear periphery and nucleolar regions. In the late S and G phases, GFP-Mcm10 was localized to nucleoli. Although (2)the distributions of GFP-Mcm10 during the S phase resembled those of replication foci, GFP-Mcm10 foci did not colocalize with sites of DNA synthesis in most cases. Furthermore, the transition of GFP-Mcm10 distribution patterns preceded changes in replication foci patterns or proliferating cell nuclear antigen foci patterns by 30-60 min. These results suggest that human Mcm10 is temporarily recruited to the replication sites 30-60 min before they replicate and that it dissociates from chromatin after the activation of the prereplication complex.     

Naphthol AS-BI (7-bromo-3-hydroxy-2-naphtho-o-anisidine) phosphatase and naphthol AS-BI beta-D-glucuronidase in Chinese hamster ovary cells: biochemical and flow cytometric studies.

Conditions for the biochemical and flow cytometric assay of 7-bromo-3-hydroxy-2-naphtho-o-anisidine phosphatase and beta-D-glucuronidase activities in Chinese hamster ovary cells were studied. In the biochemical assay, the pH optimum for the phosphatase activity was pH 4.6 with a Km of 10(-5) M; the pH optimum for beta-D-glucuronidase activity was pH 5.0 with a Km of 2 x 10(-5) M. For intact cells the derived constants were 3 to 10 times higher. The rate of hydrolysis of both substrates was also examined by flow cytometry. Cellular fluorescence increased linearly for only about 15 min. Diffusion of the fluorescent product probably caused nonlinearity of the fluorescence increase and was demonstrated by mixing cells incubated with substrate with those that had not been incubated. After 15 min, cells that had not been exposed previously to product or substrate contained the fluorescent product. Cells fractionated into size classes by centrifugal elutriation also were analyzed by flow cytometry for beta-D-glucuronidase activity. The activity increased linearly with the increase in cell size corresponding to the progression from G1 through S and into G2-M phases of the cell cycle.     

He—Ne激光照射对成纤维细胞内[Ca^2＋]i浓度影响的流式细胞计测定

目的：Ｈｅ－Ｎｅ激光照射治疗的机理不明,激光照射引起细胞内Ｃａ＾２＋水平变化,为治疗机理提供理论依据。方法：Ｈｅ－Ｎｅ激光照射引起鼠成纤维细胞Ｌ９２９内［Ｃａ＾２＋］ｉ的变化,用ＨＯ３４２对细胞ＤＮＡ活性染色,Ｆｌｕｏ－３ＡＭ对细胞内Ｃａ＾２＋染色,利用ＦＣＭ同时定量分析细胞ＤＮＡ和细胞内Ｃａ＾２＋的变化。结果：激光照射１５ｍｉｎ（光剂量１１．８１Ｊ／ｃｍ＾２后,ＦＣＭ分析可见ＤＮＡ分布直方图右移