A Millifluidic Study of Cell-to-Cell Heterogeneity in Growth-Rate and Cell-Division Capability in Populations of Isogenic Cells of Chlamydomonas reinhardtii

To address possible cell-to-cell heterogeneity in growth dynamics of isogenic cell populations of Chlamydomonas reinhardtii, we developed a millifluidic drop-based device that not only allows the analysis of populations grown from single cells over periods of a week, but is also able to sort and collect drops of interest, containing viable and healthy cells, which can be used for further experimentation. In this study, we used isogenic algal cells that were first synchronized in mixotrophic growth conditions. We show that these synchronized cells, when placed in droplets and kept in mixotrophic growth conditions, exhibit mostly homogeneous growth statistics, but with two distinct subpopulations: a major population with a short doubling-time (fast-growers) and a significant subpopulation of slowly dividing cells (slow-growers). These observations suggest that algal cells from an isogenic population may be present in either of two states, a state of restricted division and a state of active division. When isogenic cells were allowed to propagate for about 1000 generations on solid agar plates, they displayed an increased heterogeneity in their growth dynamics. Although we could still identify the original populations of slow- and fast-growers, drops inoculated with a single progenitor cell now displayed a wider diversity of doubling-times. Moreover, populations dividing with the same growth-rate often reached different cell numbers in stationary phase, suggesting that the progenitor cells differed in the number of cell divisions they could undertake. We discuss possible explanations for these cell-to-cell heterogeneities in growth dynamics, such as mutations, differential aging or stochastic variations in metabolites and macromolecules yielding molecular switches, in the light of single-cell heterogeneities that have been reported among isogenic populations of other eu- and prokaryotes.     

Epiplastome variation of the number of chloroplasts in stomata guard cells of sugar beet (Beta vulgaris L.)

In stomata guard cells of sugar beet, variation in the number of chloroplasts was studied in successive generations: (1) hybrid generation; (2) generation yielded by uniparental apozygotic seed reproduction; (3) generation obtained after seed treatment with a colchicine solution; (4) generation obtained after seed treatment with 5-azacytidine. As compared to hybrid generation, uniparental seed reproduction increases the average number of chloroplasts in stomata guard cells (from 13.5 to 15.0) and decreases distribution variance of this trait by a factor of 3 to 4. Colchicine increases both average number of chloroplasts in stomata guard cells (from 13.5 to 18.2) and distribution variance (about twice). 5-Azacytindine reduces the number of chloroplasts in cells (from 15.0 to 12.9) but enhances distribution variance (about 1.5 times). Variation in the number of chromosomes in stomata cells is related to myxoploidy in meristem tissue, on the one hand, and to the rate of cell division, on the other. Uniparental seed reproduction is suggested to enhance the number of organelles per cell due to high myxoploidy in cell populations, which is typical of inbred plants. Colchicine blocks spindle division and sharply increases the level of myxoploidy in cell populations and the number of organelles per cell. 5-Azacytidine hypomethylates chromosome DNA, increases the rate of cell divisions, and reduces the number of organelles per cell. The described changes in the number of chloroplasts are inherited in cell lineage ("cell hereditary memory") and successive sporophyte generations.     

Approximation of the cell cycle in synchronized populations of Streptococcus faecium.

Slowly growing populations (TD = 70 to 80 min) of Streptococcus faecium (S. faecalis ATCC 9790) were synchronized by selection after sucrose gradient fractionation. The cell cycle was approximated by correlating the patterns of DNA accumulation and cell division. More specifically, the beginning of cell cycle was equated with the beginning of a rapid linear increase in DNA accumulation. The DNA content of the culture approximately doubled during the period of accumulation, which lasted about 51 min. The period of rapid DNA accumulation, was followed by a period of reduced accumulation that lasted about 24 min. During synchronized growth, cell numbers increased rapidly in coordination with the period of rapid DNA accumulation and exhibited a plateau during the period of reduced DNA accumulation. In contrast, RNA and protein appeared to accumulate exponentially throughout the cell cycle at the same rate as culture mass.     

Widespread RNA segregation in a spiralian embryo

Asymmetric cell divisions are a crucial mode of cell fate specification in multicellular organisms, but their relative contribution to early embryonic patterning varies among taxa. In the embryo of the mollusc Ilyanassa, most of the early cell divisions are overtly asymmetric. During Ilyanassa early cleavage, mRNAs for several conserved developmental patterning genes localize to interphase centrosomes, and then during division they move to a portion of the cortex that will be inherited by one daughter cell. Here we report an unbiased survey of RNA localization in the Ilyanassa embryo, and examine the overall patterns of centrosomal localization during early development. We find that 3-4% of RNAs are specifically localized to centrosomes during early development, and the remainder are either ubiquitously distributed throughout the cytoplasm or weakly enriched on centrosomes compared with levels in the cytoplasm. We observe centrosomal localization of RNAs in all cells from zygote through the fifth cleavage cycle, and asymmetric RNA segregation in all divisions after the four-cell stage. Remarkably, each specifically localized message is found on centrosomes in a unique subset of cells during early cleavages, and most are found in unique sets of cells at the 24-cell stage. Several specifically localized RNAs are homologous to developmental regulatory proteins in other embryos. These results demonstrate that the mechanisms of localization and segregation are extraordinarily intricate in this system, and suggest that these events are involved in cell fate specification across all lineages in the early Ilyanassa embryo. We propose that greater reliance on segregation of determinants in early cleavage increases constraint on cleavage patterns in molluscs and other spiralian groups.     

Epiplastome Variation of the Number of Chloroplasts in Stomata Guard Cells of Sugar Beet (Beta vulgaris L.)

In stomata guard cells of sugar beet, variation in the number of chloroplasts was studied in successive generations: (1) hybrid generation; (2) generation yielded by uniparental apozygotic seed reproduction; (3) generation obtained after seed treatment with a colchicine solution; (4) generation obtained after seed treatment with 5-azacytidine. As compared to hybrid generation, uniparental seed reproduction increases the average number of chloroplasts in stomata guard cells (from 13.5 to 15.0) and decreases distribution variance of this trait by a factor of 3 to 4. Colchicine increases both average number of chloroplasts in stomata guard cells (from 13.5 to 18.2) and distribution variance (about twice). 5-Azacytindine reduces the number of chloroplasts in cells (from 15.0 to 12.9) but enhances distribution variance (about 1.5 times). Variation in the number of chromosomes in stomata cells is related to myxoploidy in meristem tissue, on the one hand, and to the rate of cell division, on the other. Uniparental seed reproduction is suggested to enhance the number of organelles per cell due to high myxoploidy in cell populations. Colchicine blocks spindle division and sharply increases the level of myxoploidy in cell populations and the number of organelles per cell. 5-Azacytidine hypomethylates chromosome DNA, increases the rate of cell divisions, and reduces the number of organelles per cell. The described changes in the number of chloroplasts are inherited in cell lineage (cell hereditary memory) and successive sporophyte generations.     

Erythropoietic progenitors capable of colony formation in culture: response of normal and genetically anemic W-W-V mice to manipulations of the erythron

The macromolecular reguirements for the initiation and maintenance of macronuclear DNA replication were studied in heat synchronized Tetrahymena pyriformis GL-C. Previous work had established that macronuclear S periods could occur in a consecutive fashion without intervening cell divisions during a multiple heat shock treatment, as well as immediately following the synchronized cell divisions. Cycloheximide treatment prior to or during the S period which follows the first synchronized cell division resulted in abolition of the initiation of DNA synthesis or an almost immediate cessation of DNA synthesis in progress. Temporary inhibition of DNA synthesis occurred when cycloheximide was added late in the S period. Treatment with actinomycin D was found to block the initiation of DNA synthesis but did not appreciably affect the continuation of the S period. It was concluded that RNA synthesis was required for the initiation but not the maintenance of DNA replication, whereas protein synthesis was necessary for both processes. The dependency of the initiation of an S period on prior RNA and protein synthesis was also shown to exist when a second consecutive S period was initiated without a preceding cell division. Treatment with actinomycin or cycloheximide prior to a supernumerary S period during a multiple heat shock treatment completely abolished the initiation of DNA synthesis. In T. pyriformis the synthesis of RNA and protein related to the initiation of the S period is tightly coupled to each cycle of DNA replication.     

Rate of virus-specific RNA synthesis in synchronized chicken embryo fibroblasts infected with avian leukosis virus.

The rate of avian leukosis virus (ALV)-specific RNA synthesis has been examined in bot- uninfected and ALV-infected synchronized chicken embryo fibroblasts. RNA from cells labeled for 2h with [3H]uridine was hybridized with avian myeloblastosis virus poly(dC)-DNA, and the hybridized RNA was analyzed with poly(I)-spephadex chromatography. Approximately 0.5% of the RNA synthesized in ALV-infected cells was detected as virus specific, and no more than a twofold variation in the rate of synthesis was detected at different times in the cell cycle. In synchronized uninfected chicken embryo fibroblasts, approximately 0.03% of the RNA synthesized was detected as virus specific, and no significant variation in the rate of synthesis was observed during the cell cycle. Treatment of ALV-infected chicken embryo fibroblasts with cytosine arabinoside or colchicine was used to block cells at different stages in the cell cycle. The rates of virus-specific RNA synthesis in cells so treated did not differ significantly from the rates in either stationary or unsynchronized virus-infected chicken embryo fibroblasts. These findings support the conclusion that after the initial division of an ALV-infected chicken embryo fibroblast and the initiation of virus RNA synthesis, the rate of virus-specific RNA synthesis is independent of the cell cycle.     

Studies of Messenger RNA during the Cell Cycle in Synchronized Mass Cultures of Tetrahymena pyriformis GL by Hybridization

SYNOPSIS RNA isolated from free ribosomes, from cell structures and from soluble cell phase after indole lysis of synchronized Tetrahymena cells showed different abilities to hybridize with DNA. The supraoptimal temperature (34 C) caused a decrease in the ability to hybridize in all 3 RNA fractions. The same effect was noted at the time of cell division. Synthesized messenger RNA as a proportion of the total quantity of RNA was roughly constant during the whole cell cycle. However, in contrast to synchronized mammalian cells the messenger RNA synthesis did not proceed at a constant rate thruout the cell cycle.     

Age Distribution of Human Diploid Fibroblasts: A Stochastic Model for In Vitro Aging

Variation in the lifespan of mass cultures and clones of human diploid fibroblasts can be explained on the basis of variation in the length of the mitotic cycle. This variation is of biological significance; the intrinsic standard deviation of culture lifespan is equal to about 10% of the mean. We constructed a two-parameter stochastic model based on the following assumptions: the time between successive divisions of a given cell is of random duration; cells divide or lose the ability to divide independently of one another; the probability that a cell can undergo further division is constant up to some maximum number of divisions and zero thereafter. We determined numerically the proportion of nondividing cells and the distribution of cell generations. Samples taken by Monte Carlo means from a hypothetical in vitro population were compared with clonal survival data obtained experimentally. The fit between experimental and theoretical findings was within the range of sampling variation. If we accept our model as being applicable to human diploid cell culture, we can draw the following conclusions: the proportion of dividing cells is an inadequate index of a population's age; even in populations in which almost all cells are still capable of division, a majority of the cells have less than eight generations remaining to them. At each subcultivation the ultimate fate of a culture is determined by the disposition of a relatively small number of “young” cells.     

RNA dependence in the cell cycle of V79 cells

The cell cycle of V79 Chinese hamster lung cells synchronized by hydroxyurea was investigated by flow cytometry. The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA of V79 cells. Green and red fluorescence from individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry. Periodic changes of cellular DNA and RNA contents were observed over nine cell cycles. The duration of G1, S, and G2 + M phases of synchronized V79 cells whose RNA content was close to that of the cells in balanced growth was 3, 4.5, and 1.5 hours, respectively. The duration of G1 and S phases of cells containing RNA above a certain threshold was inversely proportional to the RNA content. The RNA content of cells containing RNA above the normal level regressed to normal after a few generations. Coefficients of variation for RNA content were significantly larger than those for DNA. An explanation for the decay of synchrony in a synchronized cell population is proposed.     

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 .     

From single-cell genetic architecture to cell population dynamics: quantitatively decomposing the effects of different population heterogeneity sources for a genetic network with positive feedback architecture

Phenotypic cell-to-cell variability or cell population heterogeneity originates from two fundamentally different sources: unequal partitioning of cellular material at cell division and stochastic fluctuations associated with intracellular reactions. We developed a mathematical and computational framework that can quantitatively isolate both heterogeneity sources and applied it to a genetic network with positive feedback architecture. The framework consists of three vastly different mathematical formulations: a), a continuum model, which completely neglects population heterogeneity; b), a deterministic cell population balance model, which accounts for population heterogeneity originating only from unequal partitioning at cell division; and c), a fully stochastic model accommodating both sources of population heterogeneity. The framework enables the quantitative decomposition of the effects of the different population heterogeneity sources on system behavior. Our results indicate the importance of cell population heterogeneity in accurately predicting even average population properties. Moreover, we find that unequal partitioning at cell division and sharp division rates shrink the region of the parameter space where the population exhibits bistable behavior, a characteristic feature of networks with positive feedback architecture. In addition, intrinsic noise at the single-cell level due to slow operator fluctuations and small numbers of molecules further contributes toward the shrinkage of the bistability regime at the cell population level. Finally, the effect of intrinsic noise at the cell population level was found to be markedly different than at the single-cell level, emphasizing the importance of simulating entire cell populations and not just individual cells to understand the complex interplay between single-cell genetic architecture and behavior at the cell population level.     

Nicotiana tabacum actin-depolymerizing factor 2 is involved in actin-driven,auxin-dependent patterning

Polar transport of auxin has been identified as a central element of pattern formation. To address the underlying cellular mechanisms, we use the tobacco cell line (Nicotiana tabacum L. cv. Bright Yellow 2; BY-2) as model. We showed previously that cell divisions within a cell file are synchronized by polar auxin flow, linked to the organization of actin filaments (AF) which, in turn, is modified via actin-binding proteins (ABPs). From a preparatory study for disturbed division synchrony in cell lines overexpressing different ABPs, we identified the actin depolymerizing factor 2 (ADF2). A cell line overexpressing GFP-NtADF2 was specifically affected in division synchrony. The cell division pattern could be rescued by addition of Phosphatidylinositol 4,5-bisphosphate (PIP₂) or by phalloidin. These observations allow to draw first conclusions on the pathway linking auxin signalling via actin reorganization to synchronized cell division placing the regulation of cortical actin turnover by ADF2 into the focus.     

Colonic stem cell data are consistent with the immortal model of stem cell division under non-random strand segregation

Objectives:  Colonic stem cells are thought to reside towards the base of crypts of the colon, but their numbers and proliferation mechanisms are not well characterized. A defining property of stem cells is that they are able to divide asymmetrically, but it is not known whether they always divide asymmetrically (immortal model) or whether there are occasional symmetrical divisions (stochastic model). By measuring diversity of methylation patterns in colon crypt samples, a recent study found evidence in favour of the stochastic model, assuming random segregation of stem cell DNA strands during cell division. Here, the effect of preferential segregation of the template strand is considered to be consistent with the 'immortal strand hypothesis', and explore the effect on conclusions of previously published results.
Materials and methods:  For a sample of crypts, it is shown how, under the immortal model, to calculate mean and variance of the number of unique methylation patterns allowing for non-random strand segregation and compare them with those observed.
Results:  The calculated mean and variance are consistent with an immortal model that incorporates non-random strand segregation for a range of stem cell numbers and levels of preferential strand segregation.
Conclusions:  Allowing for preferential strand segregation considerably alters previously published conclusions relating to stem cell numbers and turnover mechanisms. Evidence in favour of the stochastic model may not be as strong as previously thought.     

Heritable stochastic switching revealed by single-cell genealogy

The partitioning and subsequent inheritance of cellular factors like proteins and RNAs is a ubiquitous feature of cell division. However, direct quantitative measures of how such nongenetic inheritance affects subsequent changes in gene expression have been lacking. We tracked families of the yeast Saccharomyces cerevisiae as they switch between two semi-stable epigenetic states. We found that long after two cells have divided, they continued to switch in a synchronized manner, whereas individual cells have exponentially distributed switching times. By comparing these results to a Poisson process, we show that the time evolution of an epigenetic state depends initially on inherited factors, with stochastic processes requiring several generations to decorrelate closely related cells. Finally, a simple stochastic model demonstrates that a single fluctuating regulatory protein that is synthesized in large bursts can explain the bulk of our results.     

Stochastic and deterministic simulations of heterogeneous cell population dynamics

A Monte Carlo algorithm, which can accurately simulate the dynamics of entire heterogeneous cell populations, was developed. The algorithm takes into account the random nature of cell division as well as unequal partitioning of cellular material at cell division. Moreover, it is general in the sense that it can accommodate a variety of single-cell, deterministic reaction kinetics as well as various stochastic division and partitioning mechanisms. The validity of the algorithm was assessed through comparison of its results with those of the corresponding deterministic cell population balance model in cases where stochastic behavior is expected to be quantitatively negligible. Both algorithms were applied to study: (a) linear intracellular kinetics and (b) the expression dynamics of a genetic network with positive feedback architecture, such as the lac operon. The effects of stochastic division as well as those of different division and partitioning mechanisms were assessed in these systems, while the comparison of the stochastic model with a continuum model elucidated the significance of cell population heterogeneity even in cases where only the prediction of average properties is of primary interest.     

Pole age affects cell size and the timing of cell division in Methylobacterium extorquens AM1

A number of recent experiments at the single-cell level have shown that genetically identical bacteria that live in homogeneous environments often show a substantial degree of phenotypic variation between cells. Often, this variation is attributed to stochastic aspects of biology-the fact that many biological processes involve small numbers of molecules and are thus inherently variable. However, not all variation between cells needs to be stochastic in nature; one deterministic process that could be important for cell variability in some bacterial species is the age of the cell poles. Working with the alphaproteobacterium Methylobacterium extorquens, we monitored individuals in clonally growing populations over several divisions and determined the pole age, cell size, and interdivision intervals of individual cells. We observed the high levels of variation in cell size and the timing of cell division that have been reported before. A substantial fraction of this variation could be explained by each cell's pole age and the pole age of its mother: cell size increased with increasing pole age, and the interval between cell divisions decreased. A theoretical model predicted that populations governed by such processes will quickly reach a stable distribution of different age and size classes. These results show that the pole age distribution in bacterial populations can contribute substantially to cellular individuality. In addition, they raise questions about functional differences between cells of different ages and the coupling of cell division to cell size.     

Hypermutation rate normalized by chronological time

It is generally believed that in cells undergoing Ig somatic hypermutation, more cell divisions result in more mutations. This is because DNA synthesis and replication is thought to play roles in the known mechanisms-cytidine deamination and subsequent conversion to thymidine, uracil-DNA glycosylase-mediated repair, mismatch repair, and DNA synthesis by error-prone polymerases. In this study, we manipulated the number of cell generations by varying the rate at which cultures of a mouse cell line were replenished with fresh medium. We found that the frequency of mutants does not necessarily increase with the number of cell generations. On the contrary, a greater number of divisions can lead to a lower frequency of mutants, indicating that cell division is not a rate-limiting step in the hypermutation process. Thus, when comparing mutation rates, we suggest that rates are more appropriately expressed as mutations per day than per cell generation.     

Remote changes in the course of mitosis in synchronized cultures of Chinese hamster cells following inhibition of RNA and protein synthesis

The effect of inhibition of the synthesis of some types of RNA and proteins was determined in the synchronized culture of Chinese hamster cells at various stages of the interphase on the course of remote mitosis. Analysis of MI and some forms of pathological cell division showed that the action of different doses of AMD and pyromycin during the first part of the interphase provoked an identical effect--C-mitosis at the immediate and remote waves of cell division. Suppression of the synthesis of whole cell RNA and proteins during the second half of the interphase was accompanied by a metaphase cell delay with scattering chromosomes, this indicating derangement of the synthesis of the division spindle component. It is suggested that proteins (tubulins) and RNA participate in the organization of the division spindle during remote mitosis as a "reserve pool".     

Epigenetic memory transmission through mitosis and meiosis in plants

Gene activities can be regulated by epigenetic modifications of nucleotides and chromatin that are stably propagated through somatic cell divisions and, in some cases, across generations. The mechanisms that control epigenetic marks have recently been uncovered using model organisms, such as the flowering plant Arabidopsis thaliana. In Arabidopsis, perturbation of epigenetic gene activity often results in heritable developmental phenotypes. Stable, but potentially reversible, changes in epigenetic status can also be sources for phenotypic variations in natural plant populations.