Cellular Growth Rate Research Articles

Yeast lifespan variation correlates with cell growth and SIR2 expression.

Isogenic wild type yeast cells raised in controlled environments display a significant range of lifespan variation. Recent microfluidic studies suggest that differential growth or gene expression patterns may explain some of the heterogeneity of aging assays. Herein, we sought to complement this work by similarly examining a large set of replicative lifespan data from traditional plate assays. In so doing, we reproduced the finding that short-lived cells tend to arrest at senescence with a budded morphology. Further, we found that wild type cells born unusually small did not have an extended lifespan. However, large birth size and/or high inter-generational growth rates significantly correlated with a reduced lifespan. Finally, we found that SIR2 expression levels correlated with lifespan and intergenerational growth. SIR2 expression was significantly reduced in large cells and increased in small wild type cells. A moderate increase in SIR2 expression correlated with reduced growth, decreased proliferation and increased lifespan in plate aging assays. We conclude that cellular growth rates and SIR2 expression levels may contribute to lifespan variation in individual cells.

Abstract 2260: Exploring drug response metrics for biomarker discovery using the OncoPanel™ cell-based profiling service

Abstract Using drug response data, generated by the OncoPanelTM cell-based profiling service, we investigated the differences between traditional and normalized growth rate inhibition (GR) metrics in drug sensitivity classification and predictive genomic biomarker discovery. Traditional cellular response parameters can be confounded by numerous growth conditions, especially when the mechanism of action of a drug is coupled to cell cycle processes. Metrics such as IC50 or Emax can be influenced by cellular growth rate, adding bias to the apparent sensitivity to or efficacy of the drug. Use of GR metrics aims to eliminate this bias by normalizing the response data to a per-cell division rate. In this study, we analyzed various parameters for a selection of standard of care compounds when tested against a large panel of cancer cell lines. Compounds were tested against between 213 and 297 different human cancer cell lines. Cells were seeded in 384-well plates and allowed to incubate for 24 hours before acoustic energy-based compound addition was made over 10 half-log concentrations, in triplicate. Initial cell density was captured in a parallel plate to enable cell doubling and end-point GR calculations. After 3 or 10 days of incubation, the cells were fixed, stained with DAPI, and imaged using a high-content imaging platform. Cell proliferation dose response curves were fitted using a 4-parameter log-logistic model within a custom curve-fitting software platform. These same data were then re-analyzed using the GR metrics package developed for the statistical programming language R, to determine GR metrics (GR50 and GRmax). Exploratory analysis was conducted on four parameters: IC50, GR50, GRmax, and number of doublings. IC50 and GR50 were used to classify compounds into drug-resistant or sensitive groups, and GRmax was used to classify each as cytostatic, partially cytostatic, or cytotoxic. Each parameter was used in a predictive genomic biomarker search, using baseline mRNA expression data and mutation data from the COSMIC/Sanger and Broad/CCLE databases. Correlation analysis for doublings and IC50, or GR50, suggests that cell lines with faster doubling rates are associated with drug sensitivity, especially with compounds that target cell cycle processes, such as doxorubicin, carboplatin, cisplatin, and oxaliplatin. Genomic analyses revealed different predictive biomarkers for each choice of parameter. The relationships between significant biomarkers were explored across compounds and cancer cell types. In summary, the methods employed in the OncopanelTM cell-based profiling service enable various dose response parameter estimations, including GR metrics. Through careful use of growth-normalized data, we have expanded our capacity to discover predictive genomic biomarkers that are less influenced by cellular growth conditions. Citation Format: Charles R. Wageman, Jesse J. Parry, Lee R. Cavedine, Alyssa M. Croff, Steven M. Garner, Brogan A. Epkins, Natiya E. Robinson, Kristin C. Dempsey, Vanessa L. Norman, Usha Warrior, Alastair J. King. Exploring drug response metrics for biomarker discovery using the OncoPanel™ cell-based profiling service [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2260.

Abstract 340: CTDP1 regulates FANCI activation and DNA repair

Abstract Objective: The modular BRCA1 C-terminal (BRCT) domain plays a central role in the regulation of DNA damage repair (DDR). RNA Pol II C-terminal domain phosphatase 1 (CTDP1), the only phosphatase encoding a BRCT domain, is one of the few BRCT domain-containing proteins without a formal role ascribed to DDR. Thus, the goal of this study is to characterize the DDR-specific role of CTDP1 through interrogation of the BRCT domain-protein interaction with FANCI and the repair of DNA interstrand cross-links (ICL) through the Fanconi anemia pathway. Methods: Novel CTDP1 BRCT domain-protein interactions were identified with mass spectrometry and analyzed with gene ontology enrichment. Molecular biology experiments were conducted to examine the effects of CTDP1 knockdown or overexpression on cell growth, survival, apoptosis, regulation of FANCI activation and localization, FANCD2 foci formation and ubiquitination, and homologous recombination repair. Mass spectrometry was used to determine both CTDP1-regulated FANCI phosphorylation sites and FANCI protein interactions. Results: The BRCT domain of CTDP1 was found to interact with three Fanconi anemia proteins-FANCA, FANCD2, and FANCI. This interaction remains unchanged upon mitomycin C treatment. Loss of CTDP1 induces a four-fold decrease in homologous recombination repair efficiency. CTDP1 prevents ICL-induced apoptosis. Knockdown of CTDP1 negatively affects plating efficiency and decreases the rate of cellular growth in breast cancer cell lines, but not the untransformed MCF-10A breast cell line. CTDP1 prevents sensitivity to DNA-damaging agents repaired by homologous recombination, but not UV damage or treatment with paclitaxel. CTDP1 knockdown negatively impacts FANCD2 activation measured by DNA damage-induced foci formation and monoubiquitination, which is critical for the onset of ICL repair by the ID2 complex and downstream effector proteins of the Fanconi anemia pathway. CTDP1 regulates 15 phosphorylation sites in FANCI determined by mass spectrometry profiling of cells overexpressing wild-type CTDP1 compared to phosphatase-dead D302K mutant. Paradoxically, CTDP1 phosphatase significantly increases SQ motif phosphorylation of S559 and S556, which is important for dormant origin firing and replication fork restart. This suggests that phosphorylation regulation of FANCI at non-SQ motif residues could regulate FANCI activation potential, resulting in increased phosphorylation at the SQ motif. Significance: This is the first demonstration of a formal role of CTDP1 in DDR. CTDP1 is implicated in the regulation of the specific ICL-repair Fanconi anemia pathway, and regulates breast cancer cell survival in vitro and in vivo. Delineating the mechanism by which CTDP1 regulates FANCI activation can provide the basis for preclinical studies targeting CTDP1 in breast cancer to promote sensitivity to DNA-damaging therapies. Citation Format: Kimiko L. Krieger, Wen-Feng Hu, Dragana Lagundzin, Nicholas T. Woods. CTDP1 regulates FANCI activation and DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 340.

Colony and Single Cell Level Analysis of the Heterogeneous Response of Cryptococcus neoformans to Fluconazole.

Cryptococcus neoformans is a human fungal pathogen that can cause fatal meningitis in immunocompromised individuals. Fluconazole (FLC) is a fungistatic drug administered to treat cryptococcosis. When exposed to the inhibitory concentration of FLC, C. neoformans exhibits heteroresistance where a small subpopulation of cells develops into FLC-resistant colonies. FLC-resistant cells are aneuploids with regard to specific beneficial chromosomal regions. Factors underlying the potential for only certain C. neoformans cells in a genetically isogenic population to become FLC-resistant are unknown. In this study, we systematically examine the heterogeneous response of C. neoformans to FLC at a colony and individual cell level. We find that the heterogeneity in response to FLC is reflected by variable diminishment of the ergosterol at the plasma membrane. A population of C. neoformans spread on a semi-solid medium displays two types of outcomes following FLC exposure. The first outcome is colonies consisting of non-resistant cells (survivors). The size of colonies consisting of survivors ranges from a few cells to visible colonies, which reflects intrinsic phenotypic heterogeneity of the C. neoformans population. The second outcome is FLC-resistant cells forming colonies of sizes significantly larger as compared to colonies made of survivors. We propose a model that describes how a distribution of these types of cellular responses within a population changes depending on FLC concentration and factors that influence the rate of cellular growth including temperature, media type, growth phase, and the age of cells. Our findings highlight a complex nature of the response to a fungistatic drug and provide insights that may help to optimize FLC therapy.

Cell size sensing in animal cells coordinates anabolic growth rates and cell cycle progression to maintain cell size uniformity.

Cell size uniformity in healthy tissues suggests that control mechanisms might coordinate cell growth and division. We derived a method to assay whether cellular growth rates depend on cell size, by monitoring how variance in size changes as cells grow. Our data revealed that, twice during the cell cycle, growth rates are selectively increased in small cells and reduced in large cells, ensuring cell size uniformity. This regulation was also observed directly by monitoring nuclear growth in live cells. We also detected cell-size-dependent adjustments of G1 length, which further reduce variability. Combining our assays with chemical/genetic perturbations confirmed that cells employ two strategies, adjusting both cell cycle length and growth rate, to maintain the appropriate size. Additionally, although Rb signaling is not required for these regulatory behaviors, perturbing Cdk4 activity still influences cell size, suggesting that the Cdk4 pathway may play a role in designating the cell's target size.

Enhanced expression of soluble antibody fragments by low-temperature and overdosing with a nitrogen source

Escherichia coli has been a primary host for the prokaryotic production of antibody fragments (Fabs) and has contributed to several successes in the pharmaceutical industry. Nevertheless, the requirement of disulfide bonds often results in low-yield fermentation and a lack of cost-effectiveness. Despite the improved production of functional Fabs by fermentation below 30 °C, the limited cellular growth needs further work. To address these issues, we investigated the effect of nitrogen supply on the cellular growth and the Fab productivity. We used the anti-human VEGF-A Fab as a model that exhibited poor expression at 37 °C regardless of the amount of nitrogen supplied during fermentation. In stark contrast, the expression yield of soluble Fab with a gross nitrogen supply of 6.91 g/L of broth throughout the fermentation at 25 °C was 332 mg/L. Furthermore, and increased nitrogen supply of 10.9 g/L significantly improved the yield of active form by 59.7% and the cellular growth rate by 39.3%. These results indicate that overdosing of a nitrogen source at low temperature is critical to Fab productivity in E. coli.

Rerouting of Metabolism into Desired Cellular Products by Nutrient Stress: Fluxes Reveal the Selected Pathways in Cyanobacterial Photosynthesis.

Boosting cellular growth rates while redirecting metabolism to make desired products are the preeminent goals of gene engineering of photoautotrophs, yet so far these goals have been hardly achieved owing to lack of understanding of the functional pathways and their choke points. Here we apply a 13C mass isotopic method (INST-MFA) to quantify instantaneous fluxes of metabolites during photoautotrophic growth. INST-MFA determines the globally most accurate set of absolute fluxes for each metabolite from a finite set of measured 13C-isotopomer fluxes by minimizing the sum of squared residuals between experimental and predicted mass isotopomers. We show that the widely observed shift in biomass composition in cyanobacteria, demonstrated here with Synechococcus sp. PCC 7002, favoring glycogen synthesis during nitrogen starvation is caused by (1) increased flux through a bottleneck step in gluconeogenesis (3PG → GAP/DHAP), and (2) flux overflow through a previously unrecognized hybrid gluconeogenesis-pentose phosphate (hGPP) pathway. Our data suggest the slower growth rate and biomass accumulation under N starvation is due to a reduced carbon fixation rate and a reduced flux of carbon into amino acid precursors. Additionally, 13C flux from α-ketoglutarate to succinate is demonstrated to occur via succinic semialdehyde, an alternative to the conventional TCA cycle, in Synechococcus 7002 under photoautotrophic conditions. We found that pyruvate and oxaloacetate are synthesized mainly by malate dehydrogenase with minimal flux into acetyl coenzyme-A via pyruvate dehydrogenase. Nutrient stress induces major shifts in fluxes into new pathways that deviate from historical metabolic pathways derived from model bacteria.

Multiple Transceptors for Macro- and Micro-Nutrients Control Diverse Cellular Properties Through the PKA Pathway in Yeast: A Paradigm for the Rapidly Expanding World of Eukaryotic Nutrient Transceptors Up to Those in Human Cells.

The nutrient composition of the medium has dramatic effects on many cellular properties in the yeast Saccharomyces cerevisiae. In addition to the well-known specific responses to starvation for an essential nutrient, like nitrogen or phosphate, the presence of fermentable sugar or a respirative carbon source leads to predominance of fermentation or respiration, respectively. Fermenting and respiring cells also show strong differences in other properties, like storage carbohydrate levels, general stress tolerance and cellular growth rate. However, the main glucose repression pathway, which controls the switch between respiration and fermentation, is not involved in control of these properties. They are controlled by the protein kinase A (PKA) pathway. Addition of glucose to respiring yeast cells triggers cAMP synthesis, activation of PKA and rapid modification of its targets, like storage carbohydrate levels, general stress tolerance and growth rate. However, starvation of fermenting cells in a glucose medium for any essential macro- or micro-nutrient counteracts this effect, leading to downregulation of PKA and its targets concomitant with growth arrest and entrance into G0. Re-addition of the lacking nutrient triggers rapid activation of the PKA pathway, without involvement of cAMP as second messenger. Investigation of the sensing mechanism has revealed that the specific high-affinity nutrient transporter(s) induced during starvation function as transporter-receptors or transceptors for rapid activation of PKA upon re-addition of the missing substrate. In this way, transceptors have been identified for amino acids, ammonium, phosphate, sulfate, iron, and zinc. We propose a hypothesis for regulation of PKA activity by nutrient transceptors to serve as a conceptual framework for future experimentation. Many properties of transceptors appear to be similar to those of classical receptors and nutrient transceptors may constitute intermediate forms in the development of receptors from nutrient transporters during evolution. The nutrient-sensing transceptor system in yeast for activation of the PKA pathway has served as a paradigm for similar studies on candidate nutrient transceptors in other eukaryotes and we succinctly discuss the many examples of transceptors that have already been documented in other yeast species, filamentous fungi, plants, and animals, including the examples in human cells.

Importin-β Directly Regulates the Motor Activity and Turnover of a Kinesin-4

Spatiotemporal regulation of kinesins is essential formicrotubule-dependent intracellular transport. In plants, cell wall deposition depends on the FRA1 kinesin, whose abundance and motility are tightly controlled to match cellular growth rate. Here, we show that an importin-β, IMB4, regulates FRA1 activity in a developmental manner. IMB4 physically interacts with a PY motif in the FRA1 motor domain and inhibits its motility by preventing microtubule binding, while also protecting FRA1 against proteasome-mediated degradation, thus providing a mechanism to couple the motility and stability of FRA1. This regulatory mechanism is likely to be broadly applicable, based on the conservation of the PY motif in the motor domains of plant and animal kinesins and the direct interaction of multiple plant kinesins with IMB4. Together, our data establish IMB4 as a multi-functional regulator of FRA1 and reveal a mechanism for how plants control the magnitude of cargo transport needed for cell wall assembly.

Effect of Biofield Energy Treated Cell Growth Medium on Human Fibroblast Cell Growth for Assessment of Skin Health Promotion Potential

Cell growth and proliferation plays an important role in wound healing, which is a complex and dynamic process. Wound management and recovery is currently done using various antiseptics and antibiotics in order to prevent or treat infection. The objective of the present study was to assess the effect of Biofield Energy (Consciousness Energy Healing Treatment - The Trivedi Effect®) Treated cell growth medium on the proliferation of human dermal fibroblast cells (HFF-1) for the assessment of skin health promotion potential. An aliquot of HFF-1 cells and DMEM were divided into two parts, one was treated with the Biofield Energy Treatment and denoted as the treated test samples, while other part was coded as the untreated test samples. MTT assay was used to study the cellular proliferation, while cell growth rate was estimated under phase contrast microscope by monitoring the cell growth at 24, 48, and 72 hours interval and photomicrographs were captured. The results of HFF-1 cellular proliferation rate showed a significant (p≤0.001) increased by 80% in the Biofield Energy Treated DMEM, while positive controls, ascorbic acid (10 µM) and FBS (15% fetal bovine serum) showed increased cell proliferation by 21% and 44%, respectively. In addition, cell growth rate was evaluated at 24, 48, and 72 hours and the results were observed with a significant growth in the Biofield Energy Treated DMEM group compared with the Biofield Energy Treated HFF-1 cells. Thus, on the basis of the experimental results, it can be concluded that The Trivedi Effect®-Consciousness Energy Healing Treatment has the significant capacity to improve the cellular proliferation and cell growth rate of human skin cells in the Biofield Energy Treated media (DMEM) for improving the overall skin health. It can be used as a complementary and alternative approach with respect to the various skin-related disorders, anti-aging, wound healing, and many more.

Poly-β-hydroxybutyrate (PHB) production by Synechocystis PCC6803 from CO2: Model development

The biosynthesis of poly-β-hydroxybutyrate (PHB) by bioconversion of CO2 is a sustainable alternative to the non-renewable, petroleum-based polymer production. Indeed, the PHB production by conversion of CO2 contributes to the reduction of the greenhouse-gas concentration in the atmosphere. A kinetic dynamic model of PHB production by autotrophic cultures of Synechocystis PCC6803 was proposed and developed by means of the biochemical networks simulator COPASI.Two classes of cells were assumed to be present in the broth: growing cells, PHB producing cells. The model included the two classes of cells and their nitrogen and phosphate internal quota. The dynamics of the cell growth and PHB production were described taking into account: cellular growth rate; lysis rate; nitrate and phosphate utilization rate; PHB production rate. The assessment of the kinetic parameters and of the yields (model calibration) was carried out by the regression of experimental data. Tests were carried out in photobioreactors under dynamic light system (light/dark cycle) using media characterized by initial nitrate concentration ranging between 0 and 1.5g/L. The developed model was validated with respect to independent experimental set.The proposed model successfully reproduced the experimental data (cell concentration, nitrogen and phosphate concentration and PHB content): the square correlation coefficient of the investigated variable concentrations ranged between 0.81 and 0.99. Parameter sensitivity analysis was carried out to assess the role of the selected parameters on cell growth and PHB accumulation. The dynamics of cellular growth were not significantly affected by a ±20% variation of maximum specific growth rate, of velocity of conversion to PHB producing cells, and of maximum uptake rate of nitrate. The PHB accumulation dynamics were particularly sensitive to the variation of the value of the investigated parameters.The proposed model may support the design and the optimization of a PHB production process by means of autotrophic cultures.

Proteome-wide modulation of degradation dynamics in response to growth arrest

In dividing cells, cytoplasmic dilution is the dominant route of clearance for long-lived proteins whose inherent degradation is slower than the cellular growth rate. Thus, as cells transition from a dividing to a nondividing state, there is a propensity for long-lived proteins to become stabilized relative to short-lived proteins, leading to alterations in the abundance distribution of the proteome. However, it is not known if cells mount a compensatory response to counter this potentially deleterious proteostatic disruption. We used a proteomic approach to demonstrate that fibroblasts selectively increase degradation rates of long-lived proteins as they transition from a proliferating to a quiescent state. The selective degradation of long-lived proteins occurs by the concurrent activation of lysosomal biogenesis and up-regulation of macroautophagy. Through this mechanism, quiescent cells avoid the accumulation of aged long-lived proteins that would otherwise result from the absence of cytoplasmic dilution by cell division.

212 - Thresholds and Limitations of Adaptive Homeostasis in Stressed and Unstressed Cells

In response to stress-related signaling cells transiently and reversibly expand a wide suite of defense mechanisms, as part of the process of adaptive homeostasis. Included in the array of adaptive responses are alterations to existing proteasomes and increased de novo synthesis of 20S proteasomes, immuno-proteasomes, and PA28 (11S) proteasome regulators that enable more efficient and rapid removal of oxidatively damaged proteins from cells. Our laboratory has shown that this proteasomal adaptive response confers greater survival capacity against oxidative stress in cultured mammalian cells, Caenorhabditis elegans nematode worms, and Drosophila melanogaster fruit flies. There are, however, there are limits and thresholds to adaptive homeostasis stress responses. If cells (or organisms) are already experiencing a degree of increased stress, they may have only a limited ability to further adapt to additional stresses. When we started to explore the adaptive homeostasis phenomenon in mammalian cells, for example, we found that cells grown in room air with 20% ambient O2 were much less able to adapt to H2O2 than were cells grown at 5% ambient O2; importantly, however, the cells grown 20% O2 were initially more H2O2 resistant than were the cells grown at 5% O2. In practice, of course, many cell types used in laboratories throughout the world were derived from tissues where physiological normoxia is 3 - 5% O2, yet they are cultured and studied at ambient 20% O2. This study examined the adaptive response to H2O2 and cellular growth rate of Mouse Embryonic Fibroblasts (MEFs), cultured continuously at either 5% O2, 20% O2, or 40% O2. Our goal was to define thresholds and limitations to adaptive homeostasis imposed by pre-existing oxidative stress levels. Our results show that adaptive homeostasis responses in general, and proteasome induction as a specific marker, decrease with increasing O2 culturing concentrations: in other words, with increasing baseline pre-existing oxidative stress.

Influence of imidazolium‐based ionic liquids on steroid biotransformation by Arthrobacter simplex

AbstractBACKGROUNDIonic liquids (ILs) have become increasingly attractive as alternatives for organic solvents. In this study, the influences of ILs on the cellular growth rate, cellular activity, cortisone acetate (CA) conversion, CA solubility, and cell membrane permeability of Arthrobacter simplex CPCC 140451 (ASP) were examined.RESULTSResults showed that ILs with ≥3 carbon atoms in their side chains increased CA conversion, CA solubility, and cell membrane permeability of ASP and universally inhibited the cellular growth rate and activity, with ILs with [PF6]‐anion exhibiting the most severe inhibition activity. Inhibition was also more serious using ILs with longer alkyl side chains and at higher concentrations. CA conversion, CA solubility, and leakage of cell inclusions showed higher increase under ILs with [PF6]‐anion than those of ILs with [BF4]‐anion and lower increase for ILs with longer side chains.CONCLUSIONThis study improved the understanding of the influence and primary selection of imidazolium‐based ILs when applied in steroid biotransformation with Arthrobacter simplex whole cells. The data provided a foundation for the selection of suitable ILs for various applications. © 2017 Society of Chemical Industry

Bisphosphoglycerate mutase controls serine pathway flux via 3-phosphoglycerate

Lower glycolysis involves a series of reversible reactions, which interconvert intermediates that also feed anabolic pathways. 3-phosphoglycerate (3-PG) is an abundant lower glycolytic intermediate that feeds serine biosynthesis via the enzyme phosphoglycerate dehydrogenase, which is genomically amplified in several cancers. Phosphoglycerate mutase (PGAM1) catalyzes the isomerization of 3-PG into the downstream glycolytic intermediate 2-phosphoglycerate (2-PG). Catalytic activity of PGAM1 requires its histidine phosphorylation. We show that the primary PGAM1 histidine phosphate donor is 2,3-bisphosphoglycerate (2,3-BPG), which is made from the glycolytic intermediate 1,3-bisphosphoglycerate (1,3-BPG) by bisphosphoglycerate mutase (BPGM). When BPGM is knocked out, 1,3-BPG can directly phosphorylate PGAM1. In this case, PGAM1 phosphorylation and activity are decreased, but nevertheless sufficient to maintain normal glycolytic flux and cellular growth rate. 3-PG, however, accumulates, leading to increased serine synthesis. Thus, one biological function of BPGM is to control glycolytic intermediate levels and thereby serine biosynthetic flux.

Modeling mechanical interactions in growing populations of rod-shaped bacteria

Advances in synthetic biology allow us to engineer bacterial collectives with pre-specified characteristics. However, the behavior of these collectives is difficult to understand, as cellular growth and division as well as extra-cellular fluid flow lead to complex, changing arrangements of cells within the population. To rationally engineer and control the behavior of cell collectives we need theoretical and computational tools to understand their emergent spatiotemporal dynamics. Here, we present an agent-based model that allows growing cells to detect and respond to mechanical interactions. Crucially, our model couples the dynamics of cell growth to the cell’s environment: Mechanical constraints can affect cellular growth rate and a cell may alter its behavior in response to these constraints. This coupling links the mechanical forces that influence cell growth and emergent behaviors in cell assemblies. We illustrate our approach by showing how mechanical interactions can impact the dynamics of bacterial collectives growing in microfluidic traps.

Effects of growth rate and promoter activity on single-cell protein expression

Protein expression in a single cell depends on its global physiological state. Moreover, genetically-identical cells exhibit variability (noise) in protein expression, arising from the stochastic nature of biochemical processes, cell growth and division. While it is well understood how cellular growth rate influences mean protein expression, little is known about the relationship between growth rate and noise in protein expression. Here we quantify this relationship in Bacillus subtilis by a novel combination of experiments and theory. We measure the effects of promoter activity and growth rate on the expression of a fluorescent protein in single cells. We disentangle the observed protein expression noise into protein-specific and systemic contributions, using theory and variance decomposition. We find that noise in protein expression depends solely on mean expression levels, regardless of whether expression is set by promoter activity or growth rate, and that noise increases linearly with growth rate. Our results can aid studies of (synthetic) gene circuits of single cells and their condition dependence.

Abstract 514: Genetic load in cancer cell populations

Abstract Populations accumulate deleterious mutations that have yet to be removed by natural selection. This mutated portion of the population that does not contribute to its fitness is called the genetic load and is proportional to the deleterious mutation rate. Given the genomic instability of cancer cells, the genetic load could be substantial if the instability indeed leads to loss of cell fitness. We hypothesize that because aneuploidy and copy number variations (CNVs) are the most common forms of genome instability, their fitness consequences may determine the genetic load in cancer cells. To test this hypothesis, we randomly selected single cells from a HeLa cell line and measured the cell growth rate via daily cell counting and an MTT cell proliferation assay. We then measured the genetic changes and resulting growth rate variation. We observed rapid generation of heterogeneity in the growth rate within the population. The growth rate of different progeny cells was stable indicating that it is genetically determined. Next, using whole genome sequencing of recently descended clones of a single HeLa cell, we found that cells with fewer newly acquired CNVs have higher proliferation rates vice versa. This indicated that CNV accumulation decreased cell fitness systematically. We next estimated the rate of CNV mutation by measuring the growth rate of two daughter cells from single proliferation and found that there is approximately 1 deleterious mutation in every 4 cell divisions. We speculated that due to a high, deleterious CNV mutation rate, tumor cells inevitably accumulate deleterious CNVs and a large percentage of tumor cells are genetically defective. Accordingly, we observed that the average growth rate of tumor cell populations decreased in the short term since defective cells accumulated in the population and the variation of cellular growth rates within the population increased. By modeling the process of mutation accumulation and measuring cell growth rate, we estimated that the deleterious mutation rate in HeLa cells is about 0.26-0.31 per cell division, and that HeLa cells reduce roughly 5%-6% of fitness for every cell division. The observations of a high proliferation rate and high genetic load in this representative tumor cell line indicates a “high risk, high reward” evolution strategy for tumor cells and suggests that increasing the level of genomic instability may cause the meltdown of tumor cell population by forcing cells to accelerate the cell cycle. Citation Format: Yuezheng Zhang, Xu Shen, Yawei Li, Tianqi Zhu, Yong Tao, Tao Li, Di Wang, Xueying Li, Qin Ma, Xuemei Lu, Hurng-Yi Wang, Chung-I Wu. Genetic load in cancer cell populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 514. doi:10.1158/1538-7445.AM2017-514

Analysis of Noise Mechanisms in Cell-Size Control

At the single-cell level, noise arises from multiple sources, such as inherent stochasticity of biomolecular processes, random partitioning of resources at division, and fluctuations in cellular growth rates. How these diverse noise mechanisms combine to drive variations in cell size within an isoclonal population is not well understood. Here, we investigate the contributions of different noise sources in well-known paradigms of cell-size control, such as adder (division occurs after adding a fixed size from birth), sizer (division occurs after reaching a size threshold), and timer (division occurs after a fixed time from birth). Analysis reveals that variation in cell size is most sensitive to errors in partitioning of volume among daughter cells, and not surprisingly, this process is well regulated among microbes. Moreover, depending on the dominant noise mechanism, different size-control strategies (or a combination of them) provide efficient buffering of size variations. We further explore mixer models of size control, where a timer phase precedes/follows an adder, as has been proposed in Caulobacter crescentus. Although mixing a timer and an adder can sometimes attenuate size variations, it invariably leads to higher-order moments growing unboundedly over time. This results in a power-law distribution for the cell size, with an exponent that depends inversely on the noise in the timer phase. Consistent with theory, we find evidence of power-law statistics in the tail of C. crescentus cell-size distribution, although there is a discrepancy between the observed power-law exponent and that predicted from the noise parameters. The discrepancy, however, is removed after data reveal that the size added by individual newborns in the adder phase itself exhibits power-law statistics. Taken together, this study provides key insights into the role of noise mechanisms in size homeostasis, and suggests an inextricable link between timer-based models of size control and heavy-tailed cell-size distributions.

Effects of iron limitation on silicon uptake kinetics and elemental stoichiometry in two Southern Ocean diatoms, Eucampia antarctica and Proboscia inermis, and the temperate diatom Thalassiosira pseudonana

AbstractWe investigated the effects of iron (Fe) limitation on the elemental stoichiometry, silicic acid (Si(OH)4) uptake kinetics and cell morphology in two Southern Ocean diatoms Eucampia antarctica and Proboscia inermis and the temperate diatom Thalassiosira pseudonana. An increase in Fe‐stress resulted in reductions in specific growth rate and decreases in cellular nitrogen (N) and carbon (C) content relative to cellular biogenic silica (BSi) in both Southern Ocean diatoms and a reduction in growth rate only for T. pseudonana. Both E. antarctica and P. inermis exhibited an increase in cell volume in response to Fe‐limitation resulting in a decrease in the cell surface to volume ratio, while normalization of BSi content to cell surface area suggests these diatoms do not become more heavily silicified under Fe limitation. Kinetic Si(OH)4 uptake experiments performed on all three diatom species show that Si(OH)4 uptake is reduced under Fe‐limited conditions. For Southern Ocean diatoms, this was manifested through a decrease in the maximum specific uptake rate of Si(OH)4 (VSi‐max), along with a decrease in the half‐saturation constant for Si(OH)4 uptake (KSi) under Fe stress for E. antarctica. Our data also show that when normalized to cell surface area, VSi‐max of the three diatoms species exhibited a linear relationship with cellular growth rate, and was independent of cell morphological variations. Our results suggest that the morphological adaptations of Southern Ocean diatoms in response to Fe‐stress have the potential to affect phytoplankton community dynamics and Si(OH)4 : NO3 uptake and export ratios in Southern Ocean waters.