Synchronization in chains of pacemaker cells by phase resetting action potential effects

Interactions between pacemaker cells in a chain were calculated according to a "phase-reset" model. It is based on effects of action potentials in the cells on the cycle lengths of neighbouring cells. These effects were defined for each cell by a latency-phase curve (LPC), giving the latency time (L) until the onset of the next action potential in that cell, as a function of the phase (phi) at which a neighbour cell fired an action potential. Neighbour cells with simultaneous action potentials did not influence each others cycle length. We investigated how stable synchronization depends on the shape of the LPC's of the pacemaker cells and on chain length. Three types of interactive behaviour were distinguished. First, anti-phase synchrony, in which neighbouring cells fired with large phase differences with respect to the synchronized period Ps. Second, asynchrony, in which the periods of the cells did not become equal and constant. Third, in-phase synchrony, in which the phase differences between the neighbouring cells were zero or much smaller than the synchronized period Ps, depending on the differences between the intrinsic periods. Asynchrony and anti-phase synchrony may be seen as cardiophysiological arrhythmias, while in-phase synchrony represents the physiological type of synchrony in the heart. In-phase synchrony appeared to be strongly favoured by LPC's, which have a no-effect (refractory) part at early phases, a lengthened latency (or phase delay) part at intermediate phases and a shortened latency (or phase advance) part at late phases in the cycle. Such LPC-shapes are commonly found in preparations of cardiac pacemaker cells. When the pacemaker cells were identical, the synchronized period Ps during in-phase synchrony was equal to their intrinsic period P*i. For different intrinsic periods, Ps was equal to the intrinsic period of the fastest cell if the LPC's contained a sufficiently long initial no-effect period at early phases and a shortened latency part at late phases. When, on the other hand, such cell chains had a linear gradient in their intrinsic periods, "action potentials" started from the fast end and traveled along the chain. The propagation of an action potential wave slowed down as it reached the slower cells. When the gradient in the intrinsic periods was too steep, only the intrinsically fast end of the chain developed synchrony.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synchrony induced by double phosphate starvation in a suspension culture of Catharanthus roseus

A synchronous cell division system was established using the double phosphate starvation method, based on the observation that one of the limiting factors in the growth of a suspension culture of Catharanthus roseus (L.) G. Don cells in the medium of Murashige and Skoog was phosphate. In the system, an increase in cell number took place in a short period of only 4 h, while the cell number remained almost constant during other periods of the cell cycle. The synchrony of the culture was confirmed by changes in mitotic index, which increased sharply prior to the increase in cell number. The S phase was determined by measuring incorporation of [³H]-thymidine into the DNA fraction during the cell cycle and synchrony of DNA synthesis was verified likewise. Synchronization by phosphate starvation is discussed in relation to the function of phosphate as a nutrient. The synchronous system thus established will be useful in biochemical studies of the cell cycle in higher plants.     

Timing of swarmer cell cycle morphogenesis and macromolecular synthesis by Hyphomicrobium neptunium in synchronous culture.

The swarmer cycle of Hyphomicrobium neptunium consists of a temporal sequence of discrete developmental events. To time morphogenesis and to investigate modulations in macromolecular synthesis, we attempted methods for synchronous culture. During synchrony, swarmer maturation occurred over 32%, hyphal growth occurred over 36%, and bud maturation occurred over 32% of the time required to complete the swarmer cycle. Daughter cells were released after 265 min. Deoxyribonucleic acid replication was discontinuous, having a G1 period of approximately 180 min. In addition, ribonucleic acid and protein syntheses were depressed during the earlier phases of development.     

Periodic forcing of microbial cultures: a model for induction synchrony

Periodic environmental shifts have been used to induce synchrony in many different microbial populations. In this article, the induction synchrony phenomenon is analyzed using an age distribution model in which the age at which the cells divide is subjected to periodic forcing. It is found that synchrony will occur whenever the period of the forcing lies in the interval between the youngest and the oldest division age that occur in the population during the forcing. The analysis also predicts that under certain conditions it should be possible to obtain a multimodal synchrony in which cells in the population are distributed among a set of discrete, synchronized cell lines. The behavior of the age distribution when the conditions for synchrony are not satisfied is briefly explored. It is found that the age distribution model is able to exhibit a very rich spectrum of possible dynamic behavior. Many of the phenomena observed can be thought of in terms that are familiar from nonlinear analysis, such as stable and unstable limit cycles, period doubling, halving, and chaos. The richness of dynamic behavior opens the possibility that environmental shifts or periodic forcing could be used as a powerful tool in discriminating models of microbial kinetics and cell cycle control.     

A quantitative theory of liver regeneration in the rat. II: matching an improved mitotic inhibitor model to the data

A model of liver regeneration is put forward in which the rate of liver growth is controlled both by a liver-produced mitotic inhibitor and by the availability of parenchymal cells to enter the mitotic cycle. The model can be expressed as a pair of coupled differential equations, the first describing the dependance of inhibitor concentration on liver size and inhibitor decay and the second specifying the dependance of liver growth on inhibitor concentration and entry of cells into the mitotic cycle. The model is tested by comparing its solutions to the published data on mitotic indices following partial hepatectomy. For such a comparison, it is necessary to specify the cell-cycle time and the inhibitor dose-response function and half-life. If a negative exponential dose-response function, an inhibitor half-life of 11·4 h, and a cycle time of 18·25 h are postulated, the solutions match the data of Fabrikant (1968) who found that there were two waves of mitosis with a period of quiescence between them. The data of Grisham (1962), characterized by a single peak of mitosis, is matched by the theory using similar inhibitor properties but a shorter cell-cycle time (13·25 h); this causes the two peaks to overlap. In both cases, a better fit is obtained if the second cell cycle is longer than the first by 2–3 h. This suggests that cells enter a G₀ period after mitosis. A mechanism for littoral cell division, which occurs some 24 h after parenchymal cell division, is put forward in which the former cells depend on the enlargement of the latter for the stimulus to divide.     

Sporulation Synchrony of Saccharomyces cerevisiae Grown in Various Carbon Sources

Sporulation of several strains of Saccharomyces cerevisiae grown in a variety of carbon sources that do not repress the tricarboxylic acid cycle enzymes was more synchronous than the sporulation of cells grown in medium containing dextrose which does repress those enzymes. Dextrose-grown cells showed optimal sporulation synchrony when inoculated into sporulation medium from early stationary phase when the dextrose in the medium is exhausted. Logarithmic-phase cells grown in either non-fermentable carbon sources (acetate and glycerol) or a fermentable carbon source that does not repress tricarboxylic acid cycle enzymes (galactose) sporulated more synchronously than the early stationary-phase dextrose cells. Attempts were made to sporulate cells taken from both complex and semidefined media. The semidefined acetate medium failed to support the growth of a number of strains. However, cells grown in the complex acetate medium, as well as both complex and semidefined glycerol and galactose media, sporulated with better synchrony than did the dextrose-grown cells.     

Wall structure of a bitunicate ascus

Summary Ultraviolet light-induced, bleached Euglena clones exhibit synchronous steps of cell division in response to daily cycles of light and dark. The cyclic division activity, in the bleached cells, will persist in constant lighting conditions with a period, independent of temperature, of about 24 h. This persisting rhythm of cell division supports the hypothesis that this phase of the cell cycle may be coupled to the fluctuations of the endogenous circadian clock of the cell.Newly isolated bleached clones are sensitive to light in their growth rates and metabolic characteristics, showing light induced difference in substrate-stimulated respiration, and production of the polyglucan, paramylon. After repeated subculturing of a bleached clone the photosensitivity of the metabolic characteristics and of the growth rate are diminished along with the ability to photo-entrain division synchrony. Division control and the induction of cell synchrony in this organism apparently involve both the temporal influence of the endogenous cell clock and one or more other photosensitive reactions in the metabolism of the cell.A unique culture mixing technique utilizing the bleached Euglena, failed to support the hypothesis of the involvement of intercellular communication in the maintenance of cell synchrony in constant lighting conditions.     

Cell synchrony techniques. I. A comparison of methods

Selected cell synchrony techniques, as applied to asynchronous populations of Chinese hamster ovary (CHO) cells, have been compared. Aliquots from the same culture of exponentially growing cells were synchronized using mitotic selection, mitotic selection and hydroxyurea block, centrifugal elutriation, or an EPICS V cell sorter. Sorting of cells was achieved after staining cells with Hoechst 33258. After synchronization by the various methods the relative distribution of cells in G1, S, or G2 + M phases of the cell cycle was determined by flow cytometry. Fractions of synchronized cells obtained from each method were replated and allowed to progress through a second cell cycle. Mitotic selection gave rise to relatively pure and unperturbed early G1 phase cells. While cell synchrony rapidly dispersed with time, cells progressed through the cell cycle in 12 hr. Sorting with the EPICS V on the modal G1 peak yielded a relatively pure but heterogeneous G1 population (i.e. early to late G1). Again, synchrony dispersed with time, but cell-cycle progression required 14 hr. With centrifugal elutriation, several different cell populations synchronized throughout the cell cycle could be rapidly obtained with a purity comparable to mitotic selection and cell sorting. It was concluded that, either alone or in combination with blocking agents such as hydroxyurea, elutriation and mitotic selection were both excellent methods for synchronizing CHO cells. Cell sorting exhibited limitations in sample size and time required for synchronizing CHO cells. Its major advantage would be its ability to isolate cell populations unique with respect to selected cellular parameters.     

Phase of the circadian clock is accurately transferred from mother to daughter cells in the dinoflagellate Gonyaulax polyedra.

In the first cycle following transfer from a 12 h light-12 h dark cycle (LD12:12) to constant darkness (DD), the standard deviation in circadian phase among individual clocks in populations of Gonyaulax polyedra is approximately 60 min. When a culture is transferred to constant light conditions (LL) from an LD 12:12 cycle, the standard deviation increases in the first 2-3 d, but then remains unchanged, suggesting a lack of observable desynchronization in LL after the transient period. The synchrony in a cell population is preserved even after several cell divisions. The results indicate that variations in period among cells are small, that the period of an individual clock does not fluctuate randomly from day to day, and that the circadian phase of a mother cell is faithfully passed to the clocks of the daughter cells.     

The basis of synchronization by repetitive dilution of a growing culture

Cultures of Escherichia coli have been synchronized by periodic dilution with fresh growth medium in the laboratory of Francois Kepes. When diluted by a large factor into complete test medium, the treated cultures undergo up to 12 synchronous divisions. This long term synchrony must result from an adjustment process during the periodic dilution procedure so that all cells have nearly identical biochemical properties. Robert Pritchard (University of Leicester, personal communication) suggested that this phasing would happen if the uptake of a critical nutrient was limited by the surface area of the cell during a portion of the dilution cycle. If his suggestion is valid, a general method for synchronization of almost any organism that grows exponentially and divides by binary fission into equal sized daughters should be achievable. A computer program was devised to simulate the growth of an initially asynchronous culture under periodic dilution with medium containing a single limiting nutrient. Various models of cell shape and growth were tested along with various models for the growth-limiting substrate uptake.     

SYNCHRONIZED GROWTH OF THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE) PROVIDES NOVEL INSIGHTS INTO CELL‐WALL SYNTHESIS PROCESSES IN RELATION TO THE CELL CYCLE1

Cell‐cycle effects in phytoplankton have both general and specific influences over a variety of cellular processes. Understanding these effects requires that the majority of cells in a culture are progressing through the same cell‐cycle stage, which requires synchronous growth. We report the development of a silicon starvation–recovery synchrony for the first diatom with a sequenced genome, Thalassiosira pseudonana Hasle et Heimdale, which provides several novel insights into the process of cell‐wall formation. After 24 h of silicate starvation, flow cytometry measurements indicated that 80% of the cells were arrested in the early G1 phase of the cell cycle and then upon silicate replenishment progressed synchronously through the cycle. An early G1‐arrest point was not previously documented in diatoms. After silicate replenishment, girdle‐band synthesis was confined to a particular period in G1, and cells did not lengthen in accordance with each girdle band added, which has implications related to cell growth and separation processes in diatoms. Measurements of silicic acid uptake, intracellular pools, and silica incorporation into the cell wall, coupled with fluorescence visualization of newly synthesized cell‐wall structures, provide the first direct measurements of silica amounts in individual girdle bands and valves in a diatom. Fluorescence imaging indicated why valves in T. pseudonana do not have to reduce in size with each generation and enabled visualization of intermediates in structure formation. The development of a synchrony procedure for T. pseudonana enables correlation of cellular events with the cell cycle, which should facilitate the use of genomic information.     

Effect of lectins on auxin-induced elongation and wall loosening in oat coleoptile and azuki bean epicotyl segments

A marine unicellular aerobic nitrogen-fixing cyanobacterium Synechococcus sp. strain Miarni BG 043511 was pretreated with different light and dark regimes in order to induce higher growth synchrony. A pretreatment of two dark and light cycles of 16 h each yielded good synchrony for 3 cell division cycles. Longer dark treatments decreased the degree of synchrony and shorter dark treatments caused irregular cell division. Once synchronous culture was established, distinct phases of cellular carbohydrate accumulation and cellular carbohydrate degradation were observed even under continuous illumination. Changes in carbohydrate content were repeated in a cyclic manner with approximately 20 h intervals, the same as the cell division cycle. This change in carbohydrate metabolism provided a good index of growth synchrony under nitrogen-fixing conditions.
Photosynthetic oxygen evolution and nitrogen fixation capabilities and their activities in near, in situ, culture conditions were measured in well synchronized cultures of this strain under continuous illumination. Distinct oscillations of both photosynthetic oxygen evolution and nitrogen fixation capabilities with ca 20-h intervals, similar to the interval of the cell division cycle, were observed for three cycles. However, the activities of photosynthetic oxygen evolution were inversely correlated with those of nitrogen fixation. During the nitrogen fixation period, net oxygen consumption was observed even in the light under conditions approximating in situ culture conditions. The phase of temporal appearance of nitrogenase activity during the cell division cycle coincided with the phase of carbohydrate net degradation. These data indicate that this unicellular cyanobacterium can grow diazotrophically under conditions of continuous illumination by the segregation of photosynthesis and nitrogen fixation within a cell division cycle.     

Cell cycle synchronization of Cupriavidus necator by continuous phasing measured via flow cytometry

The continuous phasing technique was successfully used to obtain a high degree of cell cycle synchrony in cultures of the model organism Ralstonia eutropha JMP 134 (today reclassified into Cupriavidus necator). The responses of the organism were evaluated with flow cytometric determinations of DNA contents and cell size (by fluorescence and forward scatter measurements, respectively, after staining with the DNA-binding dye 4',6-diamidino-2'-phenylindole, DAPI), and cell concentration, after staining with the nucleic acid binding dye LDS-751. The strain was cultivated on a mineral medium with pyruvic acid sodium salt as the limiting carbon and energy source. Famine conditions, and thus cell dormancy, were achieved in every cycle. The best synchronization, according to the determination of DNA contents, was induced with phasing cycle durations of at least 4 h. The method allows the induction of synchrony for an indefinite period if the medium is exchanged rapidly and precisely. The results show that the time required for a complete cell cycle of Cupriavidus necator JMP 134 is independent of the chosen phasing cycle duration, provided that each process cycle lasts at least 3 h which is much longer than the time needed for a single DNA replication cycle. With shorter cycling periods DNA-synthesis is carried out in an uncoupled manner and only weak cell cycle synchrony can be attained. The results also show that DNA-synthesis can only be undertaken by cells when they have exceeded a critical size.     

Estrous synchrony in a group of African elephants (Loxodonta africana) under human care

Synchrony of estrous, and consequently of conception and birth of young, may be of adaptive significance for certain mammals. Among the species in which estrous synchrony has been suspected several times are elephants, but clear evidence is still missing. We determined estrous cycles of African elephants (Loxodonta africana) (n=4) at the Vienna Zoo, Austria, between June 2003 and January 2006 by measuring serum progesterone levels from weekly blood samples. Except for the dominant female when she was intensively lactating, all animals showed clear cycles or progesterone release with a mean period of 105.3+/-15.37 days. For most of the study period, estrous cycles were asynchronous between females. However, after re-occurrence of the progesterone cycle in the dominant female following the first period of lactation, all four females showed high synchrony of progesterone release over the two subsequent cycles. Large changes in individual period lengths indicated that synchronization was due to the adjustment of cycle length in subdominants to that of the dominant female. We used a bootstrap procedure, based on resampling measured times of progesterone peaks, to determine if this apparent synchrony could have been caused by chance alone. This statistical analysis indicated that between-individual variances of the timing of progesterone peaks were much smaller that to be expected by chance (P=0.009). This finding represents the first evidence for estrous synchrony between elephants. We discuss various hypotheses to explain the biological function of cycle synchrony in elephants.     

A model of cell cycle control: effects of thymidine on synchronous cell cultures.

Further evidence is presented in support of a model for growth control in which commitment for cell division is determined by an event in the preceding cell cycle. A study was made of conditions affecting synchronous growth following treatment of murine mastocytoma cells with excess thymidine at different phases of the cell cycle. Cells were synchronized by a physical procedure involving velocity sedimentation in a zonal rotor. Pulse treatment of such cultures with thymidine at times corresponding to the S, G2, and M periods had no effect on further growth. However, addition at G1, although having no immediate effect, arrested cell growth in the next cell cycle. This temporal effect may account for the decay of synchrony observed during double thymidine blockade or thymidine-FUdR blockade. When the time interval between two such blocks was 7 hr or less, P815Y cells were arrested after one synchronous division. At this critical time a majority of cells were at, or near, G1. It is suggested that thymidine exerts a hitherto unrecognized effect at the G1 interval.     

Cell Synchrony Techniques. I. A Comparison of Methods

Abstract Selected cell synchrony techniques, as applied to asynchronous populations of Chinese hamster ovary (CHO) cells, have been compared. Aliquots from the same culture of exponentially growing cells were synchronized using mitotic selection, mitotic selection and hydroxyurea block, centrifugal elutriation, or an EPICS V cell sorter. Sorting of cells was achieved after staining cells with Hoechst 33258. After synchronization by the various methods the relative distribution of cells in G₁ S, or G₂+ M phases of the cell cycle was determined by flow cytometry. Fractions of synchronized cells obtained from each method were replated and allowed to progress through a second cell cycle. Mitotic selection gave rise to relatively pure and unperturbed early G₁ phase cells. While cell synchrony rapidly dispersed with time, cells progressed through the cell cycle in 12 hr. Sorting with the EPICS V on the modal G₁ peak yielded a relatively pure but heterogeneous G₁ population (i.e. early to late G₁). Again, synchrony dispersed with time, but cell-cycle progression required 14 hr. With centrifugal elutriation, several different cell populations synchronized throughout the cell cycle could be rapidly obtained with a purity comparable to mitotic selection and cell sorting. It was concluded that, either alone or in combination with blocking agents such as hydroxyurea, elutriation and mitotic selection were both excellent methods for synchronizing CHO cells. Cell sorting exhibited limitations in sample size and time required for synchronizing CHO cells. Its major advantage would be its ability to isolate cell populations unique with respect to selected cellular parameters.     

SYNCHRONOUS GROWTH OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE): A REVIEW OF OPTIMAL CONDITIONS1

Chlamydomonas reinhardtii Dangeard was synchronized at optimal growth conditions under a 12:4 LD regime at 35 C and 20,000 lx with serial dilution to a standard starting cell density of (1.4 ± 0.2) × 10⁶ cells/ml. Synchronous growth and division were characterized by measuring cell number, cell volume and size distribution, dry weight, protein, carbon, nitrogen, chlorophyll, carotenoids, nucleic acids, nuclear and cytoplasmic division during the vegetative life cycle. The main properties of the present system are: Exponential growth with high productivity, high degrees of synchrony and reproducibility during repeated life cycles. The degree of synchrony of this light-dark synchronization system was evaluated and compared with those described in the literature using probit analysis of the time course of DNA synthesis, nuclear and cytoplasmic division and sporulation (increase in cell number). The results showed that the degree of synchrony is highest for cells grown under optimal conditions.     

Stabilizers and destabilizers controlling cell cycle oscillators

Various destabilizing factors of the ubiquitin system contribute to the synchrony and unidirectionality of the cell cycle clock by finely tuning the activity of various CDKs. The recent findings of hierarchical and connected waves of cyclin stabilizers highlight the complexity of this network.     

Analysis of the schedule of DNA replication in heat-synchronized Tetrahymena

The temporal schedule of DNA replication in heat-synchronized Tetrahymena was studied by autoradiographic and cytofluorometric methods. It was shown that some cells, which were synchronized by selection of individual dividing cells or by temporary thymidine starvation, incorporated [³H]thymidine into macronuclei in a periodic fashion during the heat-shock treatment. It was concluded that supernumerary S periods occurred while cell division was blocked by high temperature. The proportion of cells which initiated supernumerary S periods was found to be dependent on the duration of the heat-shock treatment and on the cell cycle stage when the first heat shock was applied. Cytofluorometric measurements of Feulgen-stained macronuclei during the heat-shock treatment indicated that the DNA complement of these cells was substantially increased and probably duplicated during the course of each S period. Estimates of DNA content also suggested that the rate of DNA synthesis progressively declined during long heat-shock treatments. These results indicate that the mechanism which brings about heat-induced division synchrony is not an interruption of the process of DNA replication. Further experiments were concerned with the regulation of DNA synthesis during the first synchronized division cycle. It was shown that participation in DNA synthesis at this time increased as more cells were able to conclude the terminal S period during the preceding heat-shock treatment. It is suggested that a discrete period of time is necessary after the completion of DNA synthesis before another round of DNA synthesis can be initiated.