Midblastula transition (MBT) of the cell cycles in the yolk and pigment granule-free translucent blastomeres obtained from centrifuged Xenopus embryos

We obtained translucent blastomeres free of yolk and pigment granules from Xenopus embryos which had been centrifuged at the beginning of the 8-cell stage with cellular integrity. They divided synchronously regardless of their cell size until they had decreased to 37.5 microm in radius; those smaller than this critical size, however, divided asynchronously with cell cycle times inversely proportional to the square of the cell radius after midblastula transition (MBT). The length of the S phase was determined as the time during which nuclear DNA fluorescence increased in Hoechst-stained blastomeres. When the cell cycle time exceeded 45 min, S and M phases were lengthened; when the cell cycle times exceeded 70 min, the G2 phase appeared; and after cell cycle times became longer than 150 min, the G1 phase appeared. Lengths of G1, S and M phases increased linearly with increasing cell cycle time. Enhanced green fluorescent protein (EGFP)-tagged proliferating cell nuclear antigen (PCNA) expressed in the blastomeres appeared in the S phase nucleus, but suddenly dispersed into the cytoplasm at the M phase. The system developed in this study is useful for examining the cell cycle behavior of the cell cycle-regulating molecules in living Xenopus blastomeres by fluorescence microscopy in real time.     

The cell cycle in the shoot apex ofSilene during the first day of floral induction

Summary The length of the cell cycle was measured in the shoot apical meristem ofSilene coeli-rosa during the first day of an inductive photoperiod. The length of the cell cycle in the shoot apex of vegetative controls (those in short days) was about 18–20 hours. Exposure of plants to the long day resulted in an immediate shortening of the cell cycle to about 13 hours, roughly two thirds of that in short days. Measurements of the component phases of the cell cycle revealed that the shortened cycle in long days was the result of a decrease in the length of G 1 and perhaps S, whilst G 2 and M remained constant.     

A dual‐color marker system for in vivo visualization of cell cycle progression in Arabidopsis

Visualization of the spatiotemporal pattern of cell division is crucial to understand how multicellular organisms develop and how they modify their growth in response to varying environmental conditions. The mitotic cell cycle consists of four phases: S (DNA replication), M (mitosis and cytokinesis), and the intervening G1 and G2 phases; however, only G2/M‐specific markers are currently available in plants, making it difficult to measure cell cycle duration and to analyze changes in cell cycle progression in living tissues. Here, we developed another cell cycle marker that labels S‐phase cells by manipulating Arabidopsis CDT1a, which functions in DNA replication origin licensing. Truncations of the CDT1a coding sequence revealed that its carboxy‐terminal region is responsible for proteasome‐mediated degradation at late G2 or in early mitosis. We therefore expressed this region as a red fluorescent protein fusion protein under the S‐specific promoter of a histone 3.1‐type gene, HISTONE THREE RELATED2 (HTR2), to generate an S/G2 marker. Combining this marker with the G2/M‐specific CYCB1‐GFP marker enabled us to visualize both S to G2 and G2 to M cell cycle stages, and thus yielded an essential tool for time‐lapse imaging of cell cycle progression. The resultant dual‐color marker system, Cell Cycle Tracking in Plant Cells (Cytrap), also allowed us to identify root cells in the last mitotic cell cycle before they entered the endocycle. Our results demonstrate that Cytrap is a powerful tool for in vivo monitoring of the plant cell cycle, and thus for deepening our understanding of cell cycle regulation in particular cell types during organ development.     

Cell cycle phase expansion in nitrogen-limited cultures of Saccharomyces cerevisiae

The time and coordination of cell cycle events were examined in the budding yeast Saccharomyces cerevisiae. Whole-cell autoradiographic techniques and time-lapse photography were used to measure the duration of the S, G1, and G2 phases, and the cell cycle positions of "start" and bud emergence, in cells whose growth rates were determined by the source of nitrogen. It was observed that the G1, S, and G2 phases underwent a proportional expansion with increasing cell cycle length, with the S phase occupying the middle half of the cell cycle. In each growth condition, start appeared to correspond to the G1 phase/S phase boundary. Bud emergence did not occur until mid S phase. These results show that the rate of transit through all phases of the cell cycle can vary considerably when cell cycle length changes. When cells growing at different rates were arrested in G1, the following synchronous S phase were of the duration expected from the length of S in each asynchronous population. Cells transferred from a poor nitrogen source to a good one after arrest in G1 went through the subsequent S phase at a rate characteristic of the better medium, indicating that cells are not committed in G1 to an S phase of a particular duration.     

Kinetics of the nuclear division cycle of Aspergillus nidulans.

We have analyzed the cell cycle kinetics of Aspergillus nidulans by using the DNA synthesis inhibitor hydroxyurea (HU) and a temperature-sensitive cell cycle mutant nimT that blocks in G2. HU rapidly inhibits DNA synthesis (S), and as a consequence progression beyond S to mitosis (M) is blocked. Upon removal of HU the inhibition is rapidly reversible. Conidia (asexual spores) of nimT were germinated at restrictive temperature to synchronize germlings in G2 and then downshifted to permissive temperature in the presence of HU. This procedure synchronizes the germlings at the beginning of S in the second cell cycle after spore germination. We have measured the total duration of S, G2, and M as the time required for these cells to recover from the HU block and undergo the next nuclear division. The duration of S was defined by the time course of sensitivity to reintroduction of HU during recovery from the initial HU block. The cell cycle time was measured as the nuclear doubling time, and the duration of mitosis was determined from the mitotic index. The duration of G1 was calculated by subtracting the combined durations of S, G2, and M from the nuclear doubling time, and the length of G2 was calculated by subtracting S and M from the aggregate length of S, G2, and M. We have also determined the duration of the phases of the cell cycle during the first cycle after spore germination. In these experiments spores were germinated directly in HU without first being blocked in G2. Because the durations of G1, S, G2, and M for the first cell cycle after spore germination were identical with those previously determined for spores presynchronized at the beginning of S in the second cell cycle, we conclude that dormant conidia of A. nidulans are arrested at, or before, the start of S.     

Kinetic heterogeneity of an experimental tumour revealed by BrdUrd incorporation and mathematical modelling

In the present paper we propose a method of analysis of the cell kinetic characteristics of in vivo experimental tumours, that uses DNA-BrdUrd flow cytometry data at various times after the bromodeoxyuridine (BrdUrd) injection and mathematical modelling. The model of the cell population takes into account the cell-cell heterogeneity of the progression rate across cell cycle phases within the tumour, and assumes a strict correlation between the durations of S and G2M phases. The model also allows for a nonconstant DNA synthesis rate across S phase. In addition, the measurement process is modelled, considering the possibility of nonimpulsive labelling and providing a representation of the time course of the bivariate DNA-BrdUrd fluorescence distribution. Sequential DNA-BrdUrd distributions were obtained in vivo from a human ovarian carcinoma transplanted in mice and, for comparison, in vitro from a cell line of the same origin. From these data, that included the fractional density and the mean BrdUrd-fluorescence of BrdUrd-positive cells as a function of the DNA-fluorescence, kinetic parameters such as the potential doubling time (T _pot) and the mean and variance of the transit times in S and G2M phases, were estimated. This study revealed the presence of a substantial heterogeneity in S and G2M phases within the in vivo cell population and of a lower heterogeneity in the in vitro population. Moreover, our analysis suggests a nonnegligible effect of the BrdUrd pharmacokinetics in the in vivo cell labelling.     

The effect of cell cycle on GFPuv gene expression in the baculovirus expression system.

The effects of cell cycle on recombinant protein production and infection yield in the baculovirus-insect cell expression system (BES) were investigated. When, at any cell cycle phase, the host cell was infected by baculovirus, the cell cycle was finally arrested at the S or G(2)/M phase with 4n DNA. In the case of G(1) or S phase-infection, cell cycle of virus-infected cells began to be arrested at S phase from 8 h post-infection or at G(2)/M phase from 4 h post-infection, respectively; while, in the case of M phase-infection, cell cycle was arrested at S phase after 12 h post-infection. When the host cell was infected at the G(1) phase, average intracellular GFPuv fluorescence intensity was 1.3-fold higher than that at G(2)/M phase at 24 h post-infection. The GFPuv expression corresponded to the profile of the G(1) cell cycle in the BES. Infection yield was measured by detection of intracellular DNA binding protein using immunohistochemical method within 7 h post-infection. The infection yield at G(1) or S phase-infection was 1.5-1.8-fold higher than that at G(2)/M phase-infection.     

Changes in cell-cycle duration and growth fraction in the shoot meristem of Sinapis during floral transition

The cell-cycle duration and the growth fraction were estimated in the shoot meristem of Sinapis alba L. during the transition from the vegetative to the floral condition. Compared with the vegetative meristem, the cell-cycle length was reduced from 86 to 32 h and the growth fraction, i.e. the proportion of rapidly cycling cells, was increased from 30–40% to 50–60%. These changes were detectable as early as 30 h after the start of the single inductive long day. The faster cell cycle in the evoked meristem was achieved by a shortening of the G1 (pre-DNA synthesis), S (DNA synthesis) and G2 (post-DNA synthesis) phases of the cycle. In both vegetative and evoked meristems, both-the central and peripheral zones were mosaics of rapidly cycling and non-cycling cells, but the growth fraction was always higher in the peripheral zone.Abbreviations G1 pre-DNA synthesis phase - G2 post-DNA synthesis phase - GF growth fraction - M mitosis phase - PLM percentage-labelled-mitoses method - S DNA synthesis phase - TdR thymidine     

Cell cycle parameters inAedes albopictus mosquito cells

Summary We have analyzed cell cycle parameters for theAedes albopictus C7-10 mosquito cell line, which has been systematically developed for somatic cell genetics, expression of transfected genes, and synthesis of hormone-inducible proteins. In rapidly cycling cells, we measured a generation time of 10–12 h. The duration of mitosis (M) was ≤1 h, and the DNA synthesis phase (S) required 6 h. UnlikeDrosophila melanogaster Kc cells, in which the G2 gap is substantially longer than G1, in C7-10 cells G1 and G2 each lasted approximately 2h. In these cells, the duration of both S and G2 was independent of the population doubling time, and the increase in population doubling time as cells approached confluency was due to prolongation of G1. When treated with the insect steroid hormone, 20-hydroxyecdysone, C7-10 mosquito cells complete the cycle in progress before undergoing a reversible arrest.     

Insulin delays the progression of Drosophila cells through G2/M by activating the dTOR/dRaptor complex

In Drosophila and mammals, insulin signalling can increase growth, progression through G1/S, cell size and tissue size. Here, we analyse the way insulin affects cell size and cell-cycle progression in two haemocyte-derived Drosophila cell lines. Surprisingly, we find that although insulin increases cell size, it slows the rate at which these cells increase in number. By using BrdU pulse-chase to label S-phase cells and follow their progression through the cell cycle, we show that insulin delays progression through G2/M, thereby slowing cell division. The ability of insulin to slow progression through G2/M is independent of its ability to stimulate progression through G1/S, so is not a consequence of feedback by the cell-cycle machinery to maintain cell-cycle length. Insulin's effects on progression through G2/M are mediated by dTOR/dRaptor signalling. Partially inhibiting dTOR/dRaptor signalling by dsRNAi or mild rapamycin treatment can increase cell number in cultured haemocytes and the Drosophila wing, respectively. Thus, insulin signalling can influence cell number depending on a balance between its ability to accelerate progression through G1/S and delay progression through G2/M.     

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.     

A new method for rapid and sensitive detection of bromodeoxyuridine in DNA-replicating cells

A new flow cytometric technique, involving differential fluorescence analysis of two DNA-binding fluorochromes, was used to quantify cellular incorporation of the base analog, bromodeoxyuridine (BrdU), into DNA over short time periods. During analysis of stained cells, the blue fluorescence signal of Hoechst 33342, which is quenched by BrdU-substituted DNA, was subtracted, on a cell by cell basis, from the green-yellow fluorescence signal of mithramycin, which remained stoichiometric to cellular DNA content. Bivariate contour profiles obtained for CHO cells pulse-labeled for 30 min showed that fluorescence quenching of Hoechst 33342 in BrdU-labeled, S phase cells produced fluorescence difference signals that were significantly greater than the difference signals from G1 and G2 + M phase cells. Analysis of L1210 cells demonstrated that the amount of BrdU detected was proportional to the length of the labeling period. The novel technique is simple, rapid, and mild; it produces minimal cell loss and does not significantly affect cellular moieties such as DNA, chromatin, or RNA.     

Analysis of cytokinetics by means of Feulgen cytofluorometry combine with 3H-thymidine autoradiography

DNA cytofluorometry combined with autoradiography after pulse-labelling with ³H-TdR visualizes movement of the labelled cells along the cell cycle. If the specimen is fixed after an adequate waiting time, this method enables us to measure the absolute length of the S phase in a cell population with a single sampling. The method was applied for Yoshida sarcoma cells proliferating in the rat ascites. Using a single specimen fixed after 4 h waiting time, the shortest, the average, and the longest durations of S phase in the population were estimated at 5.8, 7.5 and 13 h, respectively. Measurement on a flash-labelled specimen gives the relative durations of G 1, S, G 2 and M. It is shown that, using these 2 samples, a complete cell cycle time analysis can be performed.     

A Detailed Study of the Complex Cell Cycle of the Dinoflagellate Crypthecodinium cohnii Biecheler and Evidence for Variation in Histone H1 Kinase Activity

ABSTRACT By adding the protein synthesis inhibitor, emetine (10^-4 M) to a highly synchronized population of Crypthecodinium cohnii Biecheler 1938 at different phases of its cycle, we were able to determine: 1. The existence and the lengthening of the G2-Phase (30 min) in the first cycle (cycle with swimming G1 phase). 2. The time of the second cell cycle phases (cycle in the cyst): G1, 30 min; S, 1.5 h; G2, 2 h and M, 2 h. These results, together with the estimation of the cell volume of the two and four swimming daughter cells emerging from the cysts, allowed us to state the existence of two transition points: G1/S and G2/M, which are necessary for completion of mitosis. We completed this refined approach of the cell cycle in studying the activities of the histone H1 kinase either in dividing or in non-dividing Crypthecodinium cohnii cells with either total soluble proteins or the isolated mitotic kinase complex. The H1 kinase activity of this purified complex is noticeably higher (twice as high) in the dividing cells than in the non-dividing ones. These data are discussed in the light of the basic characteristics of the dinokaryon, and also compared with recent biochemical observations on the same organism and studies on other higher eukaryotic protists and metazoa.     

Mode of estrogen action on cell proliferation in CAMA-1 cells: II. Sensitivity of G1 phase population

The mammary cancer cell line CAMA-1 synchronized at the G1/S boundary by thymidine block or at the G1/M boundary by nocodazole was used to evaluate 1) the sensitivity of a specific cell cycle phase or phases to 17 beta-estradiol (E2), 2) the effect of E2 on cell cycle kinetics, and 3) the resultant E2 effect on cell proliferation. In synchronized G1/S cells, E2-induced 3H-thymidine uptake, which indicated a newly formed S population, was observed only when E2 was added during, but not after, thymidine synchronization. Synchronized G2/M cells, enriched by Percoll gradient centrifugation to approximately 90% mitotic cells, responded to E2 added immediately following selection; the total E2-treated population traversed the cycle faster and reached S phase approximately 4 hr earlier than cells not exposed to E2. When E2 was added during the last hour of synchronization (ie, at late G2 or G2/M), or for 1 hr during mitotic cell enrichment, a mixed response occurred: a small portion had an accelerated G1 exit, while the majority of cells behaved the same as controls not incubated with E2. When E2 addition was delayed until 2 hr, 7 hr, or 12 hr following cell selection, to allow many early G1 phase cells to miss E2 exposure, the response to E2 was again mixed. When E2 was added during the 16 hr of nocodazole synchronization, when cells were largely at S or possibly at early G2, it inhibited entry into S phase. The E2-induced increase or decrease of S phase cells in the nocodazole experiments also showed corresponding changes in mitotic index and cell number. These results showed that the early G1 phase and possibly the G2/M phase are sensitive to E2 stimulation, late G1, G1/S, or G2 are refractory; the E2 stimualtion of cell proliferation is due primarily to an increased proportion of G1 cells that traverse the cell cycle and a shortened G1 period, E2 does not facilitate faster cell division; and estrogen-induced cell proliferation or G1/S transition occurs only when very early G1 phase cells are exposed to estrogen. These results are consistent with the constant transition probability hypothesis, that is, E2 alters the probability of cells entering into DNA synthesis without significantly affecting the duration of other cell cycle phases. Results from this study provide new information for further studies aimed at elucidating E2-modulated G1 events related to tumor growth.     

Cell cycle parameters of Chinese hamster ovary cells during exponential, polyamine-limited growth.

We have previously shown that Chinese hamster ovary cells made polyamine deficient by treatment with alpha-methylornithine, an inhibitor of ornithine decarboxylase, grow exponentially in culture at low densities at one-half the rate observed in untreated (control) cultures. In this study, the cell cycle of polyamine-limited cells was examined by using thymidine autoradiography, mitotic index analysis, and fraction labeled mitoses analysis. We found that the longer doubling time of inhibitor-treated cultures was a consequence of increases in the lengths of the G1 and S phases. The expansion of the S phase was proportional to the increase in doubling time (twofold), whereas the G1 phase was lengthened by slightly more than a factor of 2. The lengths of the G2 and M phases were essentially unchanged. Putrescine stimulated the growth of inhibitor-treated cultures and restored the cell cycle parameters to those of untreated cells.