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.     

Stochasticity of Intranuclear Biochemical Reaction Processes Controls the Final Decision of Cell Fate Associated with DNA Damage

A massive integrative mathematical model of DNA double-strand break (DSB) generation, DSB repair system, p53 signaling network, and apoptosis induction pathway was constructed to explore the dominant factors of unknown criteria of cell fate decision. In the proposed model, intranuclear reactions were modeled as stochastic processes and cytoplasmic reactions as deterministic processes, and both reaction sets were simulated simultaneously. The simulated results at the single-cell level showed that the model generated several sustained oscillations (pulses) of p53, Mdm2, ATM, and Wip1, and cell-to-cell variability in the number of p53 pulses depended on IR intensity. In cell populations, the model generated damped p53 oscillations, and IR intensity affected the amplitude of the first p53 oscillation. Cells were then subjected to the same IR dose exhibiting apoptosis induction variability. These simulated results are in quantitative agreement with major biological findings observed in human breast cancer epithelial MCF7, NIH3T3, and fibrosarcoma cells, demonstrating that the proposed model was concededly biologically appropriate. Statistical analysis of the simulated results shows that the generation of multiple p53 pulses is a prerequisite for apoptosis induction. Furthermore, cells exhibited considerable individual variability in p53 dynamics, which correlated with intrinsic apoptosis induction. The simulated results based on the proposed model demonstrated that the stochasticity of intranuclear biochemical reaction processes controls the final decision of cell fate associated with DNA damage. Applying stochastic simulation to an exploration of intranuclear biochemical reaction processes is indispensable in enhancing the understanding of the dynamic characteristics of biological multi-layered systems of higher organisms.     

Cellular heterogeneity: yeast-side story

A major challenge for cells lies in their ability to detect, respond and adapt to changing environments that may threaten their survival. Among the numerous evolutionary strategies, cell-to-cell heterogeneity allows the emergence of different phenotypes within a population. This variability in cellular behaviors can be essential for a small fraction of cells to adapt and survive in various environments. Analyses at the single-cell level have allowed to highlight the great variability that is present between cells within an isogenic population. Numerous molecular mechanisms have been uncovered, allowing to understand the emergence and the role of cellular heterogeneity. These attempts at identifying the source of cellular noise have also provided clues for strategies needed to control heterogeneity. In this review, S. cerevisiae is used as an example to illustrate the different factors leading to cell heterogeneity, ranging from intracellular processes to environmental constraints. In addition, some recent strategies developed to modulate cell-to-cell variability are discussed.     

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.     

Influence of molecular noise on the growth of single cells and bacterial populations

During the last decades experimental studies have revealed that single cells of a growing bacterial population are significantly exposed to molecular noise. Important sources for noise are low levels of metabolites and enzymes that cause significant statistical variations in the outcome of biochemical reactions. In this way molecular noise affects biological processes such as nutrient uptake, chemotactic tumbling behavior, or gene expression of genetically identical cells. These processes give rise to significant cell-to-cell variations of many directly observable quantities such as protein levels, cell sizes or individual doubling times. In this study we theoretically explore if there are evolutionary benefits of noise for a growing population of bacteria. We analyze different situations where noise is either suppressed or where it affects single cell behavior. We consider two specific examples that have been experimentally observed in wild-type Escherichia coli cells: (i) the precision of division site placement (at which molecular noise is highly suppressed) and (ii) the occurrence of noise-induced phenotypic variations in fluctuating environments. Surprisingly, our analysis reveals that in these specific situations both regulatory schemes [i.e. suppression of noise in example (i) and allowance of noise in example (ii)] do not lead to an increased growth rate of the population. Assuming that the observed regulatory schemes are indeed caused by the presence of noise our findings indicate that the evolutionary benefits of noise are more subtle than a simple growth advantage for a bacterial population in nutrient rich conditions.     

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.     

We are all individuals: causes and consequences of non-genetic heterogeneity in mammalian cells

The human body is formed by trillions of individual cells. These cells work together with remarkable precision, first forming an adult organism out of a single fertilized egg, and then keeping the organism alive and functional for decades. To achieve this precision, one would assume that each individual cell reacts in a reliable, reproducible way to a given input, faithfully executing the required task. However, a growing number of studies investigating cellular processes on the level of single cells revealed large heterogeneity even among genetically identical cells of the same cell type. Here we discuss the sources of heterogeneity in mammalian systems; how cells ensure reliable processing of information despite fluctuations in their molecular components; and what could be the benefit of cell-to-cell variability for mammalian cells.     

Origin and evolution of peptide-protein bioregulators

Possible evolutionary pathways of cellular regulatory systems are discussed. Analysis of animal evolution suggests that peptide and protein bioregulators emerged at an early stage during formation of biochemical systems in prokaryotic cells involving protein synthesis on ribosomes, the processes of exo- and endocytosis and limited proteolysis reactions. Primary autocrine bioregulators are compared with growth factors. Models for cellular bioregulation are discussed in which both cell receptors and peptide/protein ligands, primarily immunoglobins, act as prehormones. Their internalization and limited proteolysis can lead to formation of low-molecular peptides (tetines) acting as autocrine or paracrine bioregulators. Basing on the concept of biochemical universality, it is suggested that the effects of many growth factors, hormones, immunoglobulins, mono- and lymphokins are mediated by identical or similar (carrying the same signatures) fragments which are produced in cells due to limited proteolysis reactions and which are directly involved in activation of biochemical systems in these cells.     

Centriole ciliation and cell cycle variability during G1 phase of BALB/c 3T3 cells

Although variability in the duration of the cell cycle is thought to reflect growth-regulatory processes that control cell cycle progression, the precise timing of the variable period within the G1 phase of the cell cycle has not been defined. In particular, the timing of cell cycle variability in relation to the cell's commitment (R point) to the initiation of DNA synthesis remains controversial. In order to investigate cell cycle variability, indirect immunofluorescence was used to measure the formation of the primary cilium as a possible marker of G1 events in both stimulated quiescent and exponentially growing cells. The primary cilium, an internal "9 + 0" nonmotile structure formed by one of the interphase centrioles, was first detected in postmitotic BALB/c 3T3 cells 5 hr before the initiation of DNA synthesis, an interval similar to that for the reassembly of the primary cilium in serum-stimulated quiescent fibroblasts. This similarity in the timing of ciliation suggests that serum-stimulated quiescent cells reenter the cell cycle in early G1 and recapitulate much of G1. Moreover, the rate of cilia formation in both postmitotic and serum-stimulated quiescent cells was identical to the rate of DNA synthesis initiation. Thus, cell cycle variability occurs before ciliation in both stimulated quiescent and exponentially growing cells. Furthermore, since ciliation also precedes the R point, variability in the centriole cycle occurs before the R point and thus may reflect processes controlling the cell's commitment to the initiation of DNA synthesis.     

Composition of cell-polymer cartilage implants

Cartilage implants for potential in vivo use for joint repair or reconstructive surgery can be created in vitro by growing chondrocytes on biodegradable polymer scaffolds. Implants 1 cm in diameter by 0.176 cm thick were made using isolated calf chondrocytes and polyglucolic acid (PGA). By 6 weeks, the total amount of glycosaminoglycan (GAG) and collagen (types I and II) increased to 46% of the implant dry weight; there was a corresponding decrease in the mass of PGA. Implant biochemical and histological compositions depended on initial cell density, scaffold thickness, and the methods of cell seeding and implant culture. Implants seeded at higher initial cell densities reached higher GAG contents (total and per cell), presumably due to cooperative cell-to-cell interactions. Thicker implants had lower GAG and collagen contents due to diffusional limitations.Implants that were seeded and cultured under mixed conditions grew to be thicker and more spatially uniform with respect to the distribution of cells, matrix, and remaining polymer than those seeded and/or cultured statically. Implants from mixed cultures had a 20-40-mum thick superficial zone of flat cells and collagen oriented parallel to the surface and a deep zone with perpendicular columns of cells surrounded by GAG Mixing during cell seeding and culture resulted in a more even cell distribution ad enhanced nutrient diffusion which could be related to a more favorable biomechanical environment for chondrogenesis. Cartilage with appropriate for and function for in vivo implantation ca thus be created by selectively stimulating the growth and differentiated function of chondrocytes (i.e., GAG and collagen synthesis) through optimization of the in vitro culture environment. (c) 1994 John Wiley & Sons, Inc.     

Emergence of bimodal cell population responses from the interplay between analog single-cell signaling and protein expression noise

ABSTRACT: BACKGROUND: Cell-to-cell variability in protein expression can be large, and its propagation through signaling networks affects biological outcomes. Here, we apply deterministic and probabilistic models and biochemical measurements to study how network topologies and cell-to-cell protein abundance variations interact to shape signaling responses. RESULTS: We observe bimodal distributions of extracellular signal-regulated kinase (ERK) responses to epidermal growth factor (EGF) stimulation, which are generally thought to indicate bistable or ultrasensitive signaling behavior in single cells. Surprisingly, we find that a simple MAPK/ERK-cascade model with negative feedback that displays graded, analog ERK responses at a single cell level can explain the experimentally observed bimodality at the cell population level. Model analysis suggests that a conversion of graded input--output responses in single cells to digital responses at the population level is caused by a broad distribution of ERK pathway activation thresholds brought about by cell-to-cell variability in protein expression. CONCLUSIONS: Our results show that bimodal signaling response distributions do not necessarily imply digital (ultrasensitive or bistable) single cell signaling, and the interplay between protein expression noise and network topologies can bring about digital population responses from analog single cell dose responses. Thus, cells can retain the benefits of robustness arising from negative feedback, while simultaneously generating population-level on/off responses that are thought to be critical for regulating cell fate decisions.     

Origins of regulated cell-to-cell variability

Single-cell measurements and lineage-tracing experiments are revealing that phenotypic cell-to-cell variability is often the result of deterministic processes, despite the existence of intrinsic noise in molecular networks. In most cases, this determinism represents largely uncharacterized molecular regulatory mechanisms, which places the study of cell-to-cell variability in the realm of molecular cell biology. Further research in the field will be important to advance quantitative cell biology because it will provide new insights into the mechanisms by which cells coordinate their intracellular activities in the spatiotemporal context of the multicellular environment.     

New motion analysis system for characterization of the chemosensory response kinetics of Rhodobacter sphaeroides under different growth conditions

We developed a new set of software tools that enable the speed and response kinetics of large numbers of tethered bacterial cells to be rapidly measured and analyzed. The software provides precision, accuracy, and a good signal-to-noise ratio combined with ease of data handling and processing. The software was tested on the single-cell chemosensory response kinetics of large numbers of Rhodobacter sphaeroides cells grown under either aerobic or photoheterotrophic conditions and either in chemostats or in batch cultures, allowing the effects of growth conditions on responses to be accurately measured. Aerobically and photoheterotrophically grown R. sphaeroides exhibited significantly different chemosensory response kinetics and cell-to-cell variability in their responses to 100 μM propionate. A greater proportion of the population of aerobically grown cells responded to a 100 μM step decrease in propionate; they adapted faster and showed less cell-to-cell variability than photosynthetic populations. Growth in chemostats did not significantly reduce the measured cell to cell variability but did change the adaptation kinetics for photoheterotrophically grown cells.     

The art of cellular communication: tunneling nanotubes bridge the divide

The ability of cells to receive, process, and respond to information is essential for a variety of biological processes. This is true for the simplest single cell entity as it is for the highly specialized cells of multicellular organisms. In the latter, most cells do not exist as independent units, but are organized into specialized tissues. Within these functional assemblies, cells communicate with each other in different ways to coordinate physiological processes. Recently, a new type of cell-to-cell communication was discovered, based on de novo formation of membranous nanotubes between cells. These F-actin-rich structures, referred to as tunneling nanotubes (TNT), were shown to mediate membrane continuity between connected cells and facilitate the intercellular transport of various cellular components. The subsequent identification of TNT-like structures in numerous cell types revealed some structural diversity. At the same time it emerged that the direct transfer of cargo between cells is a common functional property, suggesting a general role of TNT-like structures in selective, long-range cell-to-cell communication. Due to the growing number of documented thin and long cell protrusions in tissue implicated in cell-to-cell signaling, it is intriguing to speculate that TNT-like structures also exist in vivo and participate in important physiological processes.     

A membrane model of plant cell extension

A theory is presented for the mechanics of plant cell wall extension and is based on the analogy of the cell wall with a membrane structure made of material capable of large non-linear deformations. These wall deformations may be elastic, elastic-plastic or visco-elastic. Mathematical analyses of such membrane structures show that there is, generally, a critical internal pressure at which dimensional instability occurs. This instability is characterized by a sudden drop in internal pressure accompanied by a large increase in the physical proportions of the membrane structure. The theory proposes that cell wall extension occurs when the cell turgor pressure reaches this critical instability value. The cell wall thus stretched is fixed by biochemical synthesis of wall material. Osmotic regulation re-establishes the turgor pressure and the instability cycle repeats itself as long as the critical instability pressure of the cell is below the osmotic pressure of the cell contents. Equalization of these pressures stops cell extension. The rate of cell extension depends on the frequency of the instability cycle and is thus dependent on the various rate processes associated with the instability cycle. The theory appears to be able to explain most of the known facts regarding cell extension such as the influence of temperature and the action of some growth substances.     

Formation of multicellular aggregates under different conditions of microenvironment

Multicellular aggregates (spheroids) represent an intermittent level between monolayer growing cells and tissue culture. Spheroids are rather objective model of the three-dimensional growth and organization, the cell-to-cell interactions and influence of microenvironmental conditions on tumour microaggre-gates. In our work formation and growth of spheroids depends on concentration of CMC and FCS. Conditions of microenvironment influence on intensiveness of proliferation as well as on cells adhesiveness and formation of microaggregates.