Cellular autolytic activity in synchronized populations of Streptococcus faecium.

The autolytic capacity of Streptococcus faecium (S. faecalis ATCC 9790) varied during synchronous cell division. This phenomenon was initially observed in rapidly dividing populations (TD=30 to 33 min) synchronized by a combination of induction and size selection techniques. To minimize the problems inherent in studies of cells containing overlapping chromosome cycles and possible artifacts generated by induction techniques, the autolytic capacities of slowly dividing populations (TD=60 to 110 min) synchronized by selection only were examined. Although the overall level of cellular autolytic capacity was observed to decline with decreasing growth rate, sharp, periodic fluctuations in cellular autolytic capacity were seen during synchronous growth at all growth rates examined. On the basis of similar patterns of cyclic fluctuations in autolytic capacity of cultures synchronized by (i) selection, (ii) amino acid starvation followed by size selection, and (iii) amino acid starvation followed by inhibition of DNA synthesis, a link of such fluctuations with the cell division cycle has been postulated.     

Gene expression heterogeneities in embryonic stem cell populations: origin and function

Stem and progenitor cells are populations of cells that retain the capacity to populate specific lineages and to transit this capacity through cell division. However, attempts to define markers for stem cells have met with limited success. Here we consider whether this limited success reflects an intrinsic requirement for heterogeneity with stem cell populations. We focus on Embryonic Stem (ES) cells, in vitro derived cell lines from the early embryo that are considered both pluripotent (able to generate all the lineages of the future embryo) and indefinitely self renewing. We examine the relevance of recently reported heterogeneities in ES cells and whether these heterogeneities themselves are inherent requirements of functional potency and self renewal.     

Intracytoplasmic membrane synthesis in synchronous cell populations of Rhodopseudomonas sphaeroides. Fate of "old" and "new" membrane.

A nonspecific density labeling technique has been employed to monitor the synthesis of intracytoplasmic membrane in synchronously dividing populations of Rhodopseudomonas sphaeroides. The intracytoplasmic membranes of cells synchronized in D2O-based medium were found to undergo discontinuous decreases in specific density during synchronous cell growth following transfer to H2O-based medium. These abrupt decreases in membrane specific density occurred immediately prior to cell division and were not observed with intracytoplasmic membranes prepared from asynchronously dividing cells (see also Kowakowski, H., and Kaplan, S. (1974) J. Bacteriol. 118, 1144-1157). Discontinuous increases in the net accumulation of cellular phospholipid were also observed during the synchronous growth of R. sphaeroides. This is to be contrasted to the continuous insertion of protein and the photopigment components of the photosynthetic apparatus into the intracytoplasmic membrane during the cell division cycle (Fraley, R.T., Lueking, D.R., and Kaplan, S. (1978) J. Biol. Chem. 253, 458-464; Wraight, C.A., Lueking, D.R., Fraley, R.T., and Kaplan, S. (1978) J. Biol. Chem. 253, 465-471). Further, examination of the protein/phospholipid ratios of purified intracytoplasmic membrane preparations revealed that this ratio undergoes cyclical changes of 35 to 40% during a normal cycle of cell division. In contrast to the results of Ferretti and Gray ((1968) J. Bacteriol, 95, 1400-1406), DNA synthesis was found to occur in a stepwise manner in synchronously dividing cell populations of R. sphaeroides.     

Contributions of Cell Growth and Biochemical Reactions to Nongenetic Variability of Cells

Cell-to-cell variability in the molecular composition of isogenic, steady-state growing cells arises spontaneously from the inherent stochasticity of intracellular biochemical reactions and cell growth. Here, we present a general decomposition of the total variance in the copy number per cell of a particular molecule. It quantifies the individual contributions made by processes associated with cell growth, biochemical reactions, and their control. We decompose the growth contribution further into variance contributions of random partitioning of molecules at cell division, mother-cell heterogeneity, and variation in cell-cycle progression. The contribution made by biochemical reactions is expressed in variance generated by molecule synthesis, degradation, and their regulation. We use this theory to study the influence of different growth and reaction-related processes, such as DNA replication, variable molecule-partitioning probability, and synthesis bursts, on stochastic cell-to-cell variability. Using simulations, we characterize the impact of noise in the generation-time on cell-to-cell variability. This article offers a widely-applicable theory on the influence of biochemical reactions and cellular growth on the phenotypic variability of growing, isogenic cells. The theory aids the design and interpretation of experiments involving single-molecule counting or real-time imaging of fluorescent reporter constructs.     

Stochastic nucleoid segregation dynamics as a source of the phenotypic variability in E. coli

Segregation of the replicating chromosome from a single to two nucleoid bodies is one of the major processes in growing bacterial cells. The segregation dynamics is tuned by intricate interactions with other cellular processes such as growth and division, ensuring flexibility in a changing environment. We hypothesize that the internal stochasticity of the segregation process may be the source of cell-to-cell phenotypic variability, in addition to the well-established gene expression noise and uneven partitioning of low copy number components. We compare dividing cell lineages with filamentous cells, where the lack of the diffusion barriers is expected to reduce the impact of other factors on the variability of nucleoid segregation dynamics. The nucleoid segregation was monitored using time-lapse microscopy in live E. coli cells grown in linear grooves. The main characteristics of the segregation process, namely, the synchrony of partitioning, rates of separation, and final positions, as well as the variability of these characteristics, were determined for dividing and filamentous lineages growing under the same conditions. Indeed, the gene expression noise was considerably homogenized along filaments as determined from the distribution of CFP and YFP stochastically expressed from the chromosome. We find that 1) the synchrony of nucleoid partitioning is progressively decreasing during consecutive cell cycles, but to a significantly lesser degree in filamentous than in dividing cells; 2) the mean partitioning rate of nucleoids is essentially the same in dividing and filamentous cells, displaying a substantial variability in both; and 3) nucleoids segregate to the same distances in dividing and filamentous cells. Variability in distances is increasing during successive cell cycles, but to a much lesser extent in filamentous cells. Our findings indicate that the variability of the chromosome segregation dynamics is reduced upon removal of boundaries between nucleoids, whereas the remaining variability is essentially inherent to the nucleoid itself.     

Contributions of Cell Growth and Biochemical Reactions to Nongenetic Variability of Cells

Cell-to-cell variability in the molecular composition of isogenic, steady-state growing cells arises spontaneously from the inherent stochasticity of intracellular biochemical reactions and cell growth. Here, we present a general decomposition of the total variance in the copy number per cell of a particular molecule. It quantifies the individual contributions made by processes associated with cell growth, biochemical reactions, and their control. We decompose the growth contribution further into variance contributions of random partitioning of molecules at cell division, mother-cell heterogeneity, and variation in cell-cycle progression. The contribution made by biochemical reactions is expressed in variance generated by molecule synthesis, degradation, and their regulation. We use this theory to study the influence of different growth and reaction-related processes, such as DNA replication, variable molecule-partitioning probability, and synthesis bursts, on stochastic cell-to-cell variability. Using simulations, we characterize the impact of noise in the generation-time on cell-to-cell variability. This article offers a widely-applicable theory on the influence of biochemical reactions and cellular growth on the phenotypic variability of growing, isogenic cells. The theory aids the design and interpretation of experiments involving single-molecule counting or real-time imaging of fluorescent reporter constructs.     

SYNCHRONIZATION OF CHLOROPLAST DIVISION IN THE ULTRAMICROALGA CYANIDIOSCHYZON MEROLAE (RHODOPHYTA) BY TREATMENT WITH LIGHT AND APHIDICOLIN

A system of highly synchronized chloroplast divisions was developed in the unicellular red alga Cyanidioschyzon merolae De Luca, Taddei, & Varano. Chloroplast divisions were examined by epifluorescence microscopy following treatments with light and inhibitors. When the cells during stationary phase were transferred into a new medium under a 12:12 h LD cycle, chloroplasts, mitochondria, and cell nuclei divided synchronously in that order soon after the initiation of dark periods. More than 40% of the cells contained dividing chloroplasts. To obtain a system of highly synchronized cell division and chloroplast division, the cells synchronized by a 12:12 h LD cycle were treated with various inhibitors. Nocodazole and propyzamide did not affect cell and organelle divisions, whereas aphidicolin markedly inhibited cell-nuclear divisions and cytokinesis and induced a delay in chloroplast division. More than 80% of the cells contained dividing chloroplasts when cells synchronized by light were treated with aphidicolin for 12 h. This synchronized system will be useful for studies of the molecular and cellular mechanisms of organelle divisions .     

Changes in calcium and magnesium levels during heat-shock synchronized cell division in Tetrahymena

Calcium and magnesium contents were measured in cells of Tetrahymena pyriformis induced to divide synchronously by a multi-heat-shock procedure. During free-running synchronized cell division in complex proteose peptone medium, significant peaks of both calcium and magnesium were observed at points in the cell cycle just prior to division. No such peaks were detected in cells dividing asynchronously in proteose peptone. When synchronized cell division was followed after transfer to an inorganic medium, cell calcium and magnesium levels were observed to decrease in relation to the corresponding cell number increase, indicating that in concentration terms, calcium and magnesium remain fairly constant. This latter result suggests that neither calcium nor magnesium influxes act as triggers for cell division in Tetrahymena and that the fluctuations of these metals seen during the synchronized division cycle in complex medium represent an effect rather than a cause.     

Development and regeneration in the central nervous system

As part of our attempts to understand principles that underly organism development, we have been studying the development of the rat optic nerve. This simple tissue is composed of three glial cell types derived from two distinct cellular lineages. Type-1 astrocytes appear to be derived from a monopotential neuroepithelial precursor, whereas type-2 astrocytes and oligodendrocytes are derived from a common oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell. Type-1 astrocytes modulate division and differentiation of O-2A progenitor cells through secretion of platelet-derived growth factor, and can themselves be stimulated to divide by peptide mitogens and through stimulation of neurotransmitter receptors. In vitro analysis indicates that many dividing O-2A progenitors derived from optic nerves of perinatal rats differentiate symmetrically and clonally to give rise to oligodendrocytes, or can be induced to differentiate into type-2 astrocytes. O-2Aperinatal progenitors can also differentiate to form a further O-2A lineage cell, the O-2Aadult progenitor, which has properties specialized for the physiological requirements of the adult nervous system. In particular, O-2Aadult progenitors have many of the features of stem cells, in that they divide slowly and asymmetrically and appear to have the capacity for extended self-renewal. The apparent derivation of a slowly and asymmetrically dividing cell, with properties appropriate for homeostatic maintenance of existing populations in the mature animal, from a rapidly dividing cell with properties suitable for the rapid population and myelination of central nervous system (CNS) axon tracts during early development, offers novel and unexpected insights into the possible origin of self-renewing stem cells and also into the role that generation of stem cells may play in helping to terminate the explosive growth of embryogenesis. Moreover, the properties of O-2Aadult progenitor cells are consistent with, and may explain, the failure of successful myelin repair in conditions such as multiple sclerosis, and thus seem to provide a cellular biological basis for understanding one of the key features of an important human disease.     

Modeling the impact of single-cell stochasticity and size control on the population growth rate in asymmetrically dividing cells

Microbial populations show striking diversity in cell growth morphology and lifecycle; however, our understanding of how these factors influence the growth rate of cell populations remains limited. We use theory and simulations to predict the impact of asymmetric cell division, cell size regulation and single-cell stochasticity on the population growth rate. Our model predicts that coarse-grained noise in the single-cell growth rate λ decreases the population growth rate, as previously seen for symmetrically dividing cells. However, for a given noise in λ we find that dividing asymmetrically can enhance the population growth rate for cells with strong size control (between a “sizer” and an “adder”). To reconcile this finding with the abundance of symmetrically dividing organisms in nature, we propose that additional constraints on cell growth and division must be present which are not included in our model, and we explore the effects of selected extensions thereof. Further, we find that within our model, epigenetically inherited generation times may arise due to size control in asymmetrically dividing cells, providing a possible explanation for recent experimental observations in budding yeast. Taken together, our findings provide insight into the complex effects generated by non-canonical growth morphologies.     

Preferential expulsion of dividing algal cells as a mechanism for regulating algal-cnidarian symbiosis

A wide range of both intrinsic and environmental factors can influence the population dynamics of algae in symbiosis with marine cnidarians. The present study shows that loss of algae by expulsion from cnidarian hosts is one of the primary regulators of symbiont population density. Because there is a significant linear correlation between the rate of algal expulsion and the rate of algal division, factors that increase division rates (e.g., elevated temperature) also increase expulsion rates. Additionally, 3H-thymidine is taken up to a greater extent by algae destined to be expelled than by algae retained in the host cnidarians. Taken together, data for rates of expulsion, rates of division at different temperatures, and uptake of 3H-thymidine suggest that dividing algal cells are preferentially expelled from their hosts. The preferential expulsion of dividing cells may be a mechanism for regulation of algal population density, where the rate of expulsion of algae may be an inverse function of the ability of host cells to accommodate new algal daughter cells. This kind of regulation is present in some cnidarian species (e.g., Aiptasia pulchella, Pocillopora damicornis), but not in all (e.g., Montipora verrucosa, Porites compressa, and Fungia scutaria).     

A quantitative method for the analysis of mammalian cell proliferation in culture in terms of dividing and non-dividing cells

The application of the exponential growth equation is the standard method employed in the quantitative analyses of mammalian cell proliferation in culture. This method is based on the implicit assumption that, within a cell population under study, all division events give rise to daughter cells that always divide. When a cell population does not adhere to this assumption, use of the exponential growth equation leads to errors in the determination of both population doubling time and cell generation time. We have derived a more general growth equation that defines cell growth in terms of the dividing fraction of daughter cells. This equation can account for population growth kinetics that derive from the generation of both dividing and non-dividing cells. As such, it provides a sensitive method for detecting non-exponential division dynamics. In addition, this equation can be used to determine when it is appropriate to use the standard exponential growth equation for the estimation of doubling time and generation time.     

Flow cytometric studies of the host-regulated cell cycle in algae symbiotic with green paramecium

Summary. Paramecium bursaria (green paramecium) possesses endosymbiotically growing chlorella-like green algae. An aposymbiotic cell line of P. bursaria (MBw-1) was prepared from the green MB-1 strain with the herbicide paraquat. The SA-2 clone of symbiotic algae was employed to reinfect MBw-1 cells and thus a regreened cell line (MBr-1) was obtained. The regreened paramecia were used to study the impact of the hosts growth status on the life cycle of the symbiotic algae. Firstly, the relationship between the timing of algal propagation and the host cell division was investigated by counting the algal cells in single host cells during and after the host cell division and also in the stationary phase. Secondly, the changes in the endogenous chlorophyll level, DNA content, and cell size in the symbiotic algae were monitored by flow cytometry and fluorescence microscopy. The number of algae was shown to be doubled prior to or during the host cell division and the algal population in the two daughter cells is maintained at constant level until the host cell cycle reenters the cytokinesis, suggesting that algal propagation and cell cycle are dependent on the hosts cell cycle. During the hosts stationary growth, unicellular algal vegetatives with low chlorophyll content were dominant. In contrast, complexes of algal cells called sporangia (containing 1–4 autospores) were present in the logarithmically growing hosts, indicating that algal cell division leading to the formation of sporangia with multiple autospores is active in the dividing paramecia.Correspondence and reprints: Graduate School of Environmental Engineering, University of Kitakyushu, 1-1 Hibikino, Wakamatsu-ku, 808-0135 Kitakyushu, Japan.     

Inhibition of cell division in Escherichia coli K-12 by the R-factor R1 and copy mutants of R1.

The effect of the copy number of plasmid R1drd-19 on cell division of Escherichia coli K-12 was studied in populations growing as steady-state cultures at different growth rates, the growth rate being varied by use of different carbon sources. The plasmid copy number was also varied by using copy mutants of the R-factor. The mean cell size was larger in populations carrying an R-factor than in R-factorless populations, an effect that was more pronounced at low growth rates and in populations carrying R-factor copy mutants. The increased cell size was due to formation of elongated cells in a fraction of the population and to an increase in the diameter of all cells. The majority of the cells divided at a normal cell length, but the presence of an R-factor caused some cells to elongate, probably by the uncoupling of chromosome replication and cell division. This can be explained as a competition between the chromosome and plasmid replicons for some replication factor(s), presumably acting on both initiation and elongation of replication. The formation of elongated cells was a reversible process, but occasionally some of the elongated cells reached lengths 20 times that of newborn cells. If cell division did not occur at the normal cell size, the septum was not formed until the cell size was four times that of a newborn cell. When an elongated cell divided, it usually formed a polar septum, thus producing a newborn cell of normal cell length. The ability of plasmid-containing cells to omit one cell division but to retain the capacity of dividing one mass doubling later is compatible with a mechanical model for septum formation and cell division.     

Light-induced division and genomic synchrony in phototrophically growing cultures of Rhodopseudomonas sphaeroides.

An experimental procedure for rapidly obtaining cell populations of phototrophically growing Rhodopseudomonas sphaeroides which display division and genomic synchrony has been developed. The basis of the procedure resides with the normal physiological response displayed by cells of R. sphaeroides that have been subjected to an immediate decrease in incident light intensity. After an abrupt high- to low-light transition of an asynchronously dividing cell population, an immediate cessation of increases in culture turbidity, total cell number, and net accumulations of culture deoxyribonucleic acid and phospholipid occurs. Total cell number remains constant for 2.5 h after the transition to low light, after which time, it undergoes a sharp increase. Reinitiation of high-light conditions of growth 1 h subsequent to this increase in total cell number results in a cell population possessing a high degree of division and genomic synchrony. A characterization of this procedure, together with a demonstration of its utility for studies on intracytoplasmic membrane assembly, is presented.     

A novel assay based on fluorescence microscopy and image processing for determining phenotypic distributions of rod-shaped bacteria

Cell population balance (CPB) models can account for the phenotypic heterogeneity that characterizes isogenic cell populations. To utilize the predictive power of these models, however, we must determine the single-cell reaction and division rates as well as the partition probability density function of the cell population. These functions can be obtained through the Collins-Richmond inverse CPB modeling methodology, if we know the phenotypic distributions of (a) the overall cell population, (b) the dividing cell subpopulation, and (c) the newborn cell subpopulation. This study presents the development of a novel assay that combines fluorescence microscopy and image processing to determine these distributions. The method is generally applicable to rod-shaped cells dividing through the formation of a characteristic constriction. Morphological criteria were developed for the automatic identification of dividing cells and validated through direct comparison with manually obtained measurements. The newborn cell subpopulation was obtained from the corresponding dividing cell subpopulation by collecting information from the two compartments separated by the constriction. The method was applied to E. coli cells carrying the genetic toggle network with a green fluorescent marker. Our measurements for the overall cell population were in excellent agreement with the distributions obtained via flow cytometry. The new assay constitutes a powerful tool that can be used in conjunction with inverse CPB modeling to rigorously quantify single-cell behavior from data collected from highly heterogeneous cell populations.     

温度休克法诱导草履虫的同步分裂

运用细胞周期原理,采用温度休克法,对尾草履虫进行分裂周期同步化的研究,实验中草履虫经过3－5h的处理后,就能观察到大量不同阶段的无性生殖横分裂状态,并获得了大量处于分裂阶段的草履虫。运用这种技术取材容易,获取率稳定,可达61％,可为细胞生理学等领域的研究提供大量的同步分裂个体。     

Control of proliferation in the retina: temporal changes in responsiveness to FGF and TGF alpha.

Proliferation in the rat retina, as in other parts of the nervous system, occurs during a restricted period of development. In addition to regulating cell number, the mechanisms that control proliferation influence the patterning of tissues, and may affect the determination of cell type. To begin to determine how proliferation is controlled, several growth factors found in the retina were tested for effects on progenitor cell division in culture. Proliferation was enhanced by TGF alpha, bFGF and aFGF, and many of the dividing cells later differentiated into cells with the antigenic phenotypes of retinal neurons and glial cells. The mitotic response of retinal cells to these factors changed during development: progenitor cells from younger retinas (embryonic day 15 to 18; E15-E18) were more responsive to FGF's, while progenitor cells from older retinas (greater than E20) were more responsive to TGF alpha. Progenitor cells stopped dividing in vitro, even when treated with excess mitogen. These observations suggest that proliferation in the retina may be stimulated by multiple mitogenic signals provided by TGF alpha, FGF, or related factors, and that proliferation is not controlled by limiting concentrations of mitogen alone. Rather, these data demonstrate that retinal cells change during development in their responsiveness to mitogenic signals. Such changes may contribute to the regulation of proliferation.     

The phased division of the freshwater dinoflagellate Ceratium hirundinella and its use as a method of assessing growth in natural populations

SUMMARY. The nocturnal phasing and partial synchrony of cell division in Ceratium hirundinella was investigated on four occasions for dense planktonic populations in a small productive lake (Esthwaite Water). The population growth rates deduced from the proportion of cells dividing per day are compared with the rates of increase of cell density in the lake. The maximum proportion of Ceratium cells found dividing at any time was 5.8 ± 1.0%, and the time of optimum division was 03.00 hours G.M.T. The daily rate of division during the main phase of population increases was similar to that deduced from the overall population increments at that time, although during the week of collection the increase had apparently ceased temporarily. On the other three occasions, either increase of cell numbers had ceased or the population was declining, but a continued low rate of division ( c . 3% day⁻¹) was found.
The nocturnal division of Ceratium in Esthwaite Water is compared with the division phasing of dinaflagellates described from elsewhere. Some general problems associated with the derivation of estimates of population growth rate from division frequency are also considered.     

Induction and stabilization of synchrony in the cell division of Escherichia coli

Escherichia coli strains B5 and B/r/1 were grown under conditions of periodic glucose starvation in a minimal medium. Such conditions of growth give rise to two synchronous populations that are out of phase regarding their time of division, one dividing shortly after a new supply of fresh medium and the other at a later stage of the feeding cycle. Preferential selection of one of the two populations using heat treatment resulted in a homogeneous synchronized culture that exhibited in a non-limiting medium a high degree of synchrony that was long lasting. Synchrony and its persistence could survive preservation of such a synchronized culture by freeze drying. An explanation of the synchrony persistence was put forward and the practical implications of these findings were discussed.