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The heat shock response (HSR) controls expression of molecular chaperones to maintain protein homeostasis. Previously, we proposed a feedback loop model of the HSR in which heat-denatured proteins sequester the chaperone Hsp70 to activate the HSR, and subsequent induction of Hsp70 deactivates the HSR (Krakowiak et al., 2018; Zheng et al., 2016). However, recent work has implicated newly synthesized proteins (NSPs) - rather than unfolded mature proteins - and the Hsp70 co-chaperone Sis1 in HSR regulation, yet their contributions to HSR dynamics have not been determined. Here, we generate a new mathematical model that incorporates NSPs and Sis1 into the HSR activation mechanism, and we perform genetic decoupling and pulse-labeling experiments to demonstrate that Sis1 induction is dispensable for HSR deactivation. Rather than providing negative feedback to the HSR, transcriptional regulation of Sis1 by Hsf1 promotes fitness by coordinating stress granules and carbon metabolism. These results support an overall model in which NSPs signal the HSR by sequestering Sis1 and Hsp70, while induction of Hsp70 - but not Sis1 - attenuates the response.
Assuntos
Proteínas de Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ligação Proteica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Resposta ao Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Choque Térmico HSP40/metabolismoRESUMO
Conjugative plasmids drive genetic diversity and evolution in microbial populations. Despite their prevalence, plasmids can impose long-term fitness costs on their hosts, altering population structure, growth dynamics, and evolutionary outcomes. In addition to long-term fitness costs, acquiring a new plasmid introduces an immediate, short-term perturbation to the cell. However, due to the transient nature of this plasmid acquisition cost, a quantitative understanding of its physiological manifestations, overall magnitudes, and population-level implications, remains unclear. To address this, here we track growth of single colonies immediately following plasmid acquisition. We find that plasmid acquisition costs are primarily driven by changes in lag time, rather than growth rate, for nearly 60 conditions covering diverse plasmids, selection environments, and clinical strains/species. Surprisingly, for a costly plasmid, clones exhibiting longer lag times also achieve faster recovery growth rates, suggesting an evolutionary tradeoff. Modeling and experiments demonstrate that this tradeoff leads to counterintuitive ecological dynamics, whereby intermediate-cost plasmids outcompete both their low and high-cost counterparts. These results suggest that, unlike fitness costs, plasmid acquisition dynamics are not uniformly driven by minimizing growth disadvantages. Moreover, a lag/growth tradeoff has clear implications in predicting the ecological outcomes and intervention strategies of bacteria undergoing conjugation.
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Bactérias , Transferência Genética Horizontal , Plasmídeos , Bactérias/genéticaRESUMO
Stochastic variation in gene products ("noise") is an inescapable by-product of gene expression. Noise must be minimized to allow for the reliable execution of cellular functions. However, noise cannot be suppressed beyond an intrinsic lower limit. For constitutively expressed genes, this limit is believed to be Poissonian, meaning that the variance in mRNA numbers cannot be lower than their mean. Here, we show that several cell division genes in fission yeast have mRNA variances significantly below this limit, which cannot be explained by the classical gene expression model for low-noise genes. Our analysis reveals that multiple steps in both transcription and mRNA degradation are essential to explain this sub-Poissonian variance. The sub-Poissonian regime differs qualitatively from previously characterized noise regimes, a hallmark being that cytoplasmic noise is reduced when the mRNA export rate increases. Our study re-defines the lower limit of eukaryotic gene expression noise and identifies molecular requirements for ultra-low noise which are expected to support essential cell functions.
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It has become increasingly clear in the last few years that gene expression in eukaryotes is not a linear process from mRNA synthesis in the nucleus to translation and degradation in the cytoplasm, but works as a circular one where the mRNA level is controlled by crosstalk between nuclear transcription and cytoplasmic decay pathways. One of the consequences of this crosstalk is the approximately constant level of mRNA. This is called mRNA buffering and happens when transcription and mRNA degradation act at compensatory rates. However, if transcription and mRNA degradation act additively, enhanced gene expression regulation occurs. In this work, we analyzed new and previously published genomic datasets obtained for several yeast mutants related to either transcription or mRNA decay that are not known to play any role in the other process. We show that some, which were presumed only transcription factors (Sfp1) or only decay factors (Puf3, Upf2/3), may represent examples of RNA-binding proteins (RBPs) that make specific crosstalk to enhance the control of the mRNA levels of their target genes by combining additive effects on transcription and mRNA stability. These results were mathematically modeled to see the effects of RBPs when they have positive or negative effects on mRNA synthesis and decay rates. We found that RBPs can be an efficient way to buffer or enhance gene expression responses depending on their respective effects on transcription and mRNA stability.
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Type I interferon (IFN-I)-mediated antiviral responses are central to host defense against viral infections. Crucial is the tight and well-orchestrated control of cellular decision-making leading to the production of IFN-Is. Innovative single-cell approaches revealed that the initiation of IFN-I production is limited to only fractions of 1-3% of the total population, both found in vitro, in vivo, and across cell types, which were thought to be stochastically regulated. To challenge this dogma, we addressed the influence of various stochastic and deterministic host-intrinsic factors on dictating early IFN-I responses, using a murine fibroblast reporter model. Epigenetic drugs influenced the percentage of responding cells. Next, with the classical Luria-Delbrück fluctuation test, we provided evidence for transient heritability driving responder fates, which was verified with mathematical modeling. Finally, while studying varying cell densities, we substantiated an important role for cell density in dictating responsiveness, similar to the phenomenon of quorum sensing. Together, this systems immunology approach opens up new avenues to progress the fundamental understanding on cellular decision-making during early IFN-I responses, which can be translated to other (immune) signaling systems.
When we start to develop a cold, influenza or another viral infection, some of our cells produce signaling molecules known as type I interferons (or IFN-Is for short). These early IFN-I signals establish defenses against viruses in both infected and as yet uninfected cells. If the cells produce too much IFN-Is, however, it can result in uncontrolled inflammation that may harm the body and cause life threatening illness. Individual cells need to tightly control how much IFN-Is they produce and match this with the course of the viral infection. They also need to assess how much IFN-I their neighbors are producing and adjust their behavior accordingly. Cells have evolved a myriad of mechanisms to ensure the right amounts of IFN-Is are produced in different circumstances. Broadly, these mechanisms can be divided into two categories: stochastic regulation and deterministic regulation. Stochastic regulation occurs when individual cells receive the exact same information, but this leads to different outcomes, such as, different cells producing various quantities of IFN-Is. In contrast, deterministic regulation causes the same outcome in different cells independent on the information they receive. It was thought that stochastic regulation is the main driver of early IFN-1 responses, but recently a handful of studies have reported deterministic regulation being primarily responsible, instead. Here, Van Eyndhoven et al. explored the roles of both types of regulation in the early IFN-I responses of mouse cells. Van Eyndhoven et al. used genetic approaches and mathematical modelling to show that the fraction of cells that initiate early IFN-I responses can be considered deterministic. Moreover, this deterministic feature turned out to be heritable, such that the fate to produce IFN-I gets passed on for several generations of cells. Additionally, the experiments suggest that cell density, that is, how tightly packed together the cells are, plays an important role in controlling how many cells make IFN-I, with a lower cell density resulting in a higher fraction of cells producing IFN-Is. The findings of Van Eyndhoven et al. add to a growing body of evidence reporting heritable states that can guide decision-making in individual cells. Furthermore, it revises our view on how individual immune cells coordinate population-wide responses.
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Interferon Tipo I , Viroses , Camundongos , Animais , Percepção de Quorum/genética , Interferon Tipo I/metabolismo , Antivirais , Transdução de SinaisRESUMO
Bacteriophage burst size is the average number of phage virions released from infected bacterial cells, and its magnitude depends on the duration of an intracellular progeny accumulation phase. Burst size is often measured at the population level, not the single-cell level, and consequently, statistical moments are not commonly available. In this study, we estimated the bacteriophage lambda (λ) single-cell burst size mean and variance following different intracellular accumulation period durations by employing Escherichia coli lysogens bearing lysis-deficient λ prophages. Single lysogens can be isolated and chemically lysed at desired times following prophage induction to quantify progeny intracellular accumulation within individual cells. Our data showed that λ phage burst size initially increased exponentially with increased lysis time (i.e., period between induction and chemical lysis) and then saturated at longer lysis times. We also demonstrated that cell-to-cell variation, or "noise," in lysis timing did not contribute significantly to burst size noise. The burst size noise remained constant with increasing mean burst size. The most likely explanation for the experimentally observed constant burst size noise was that cell-to-cell differences in burst size originated from intercellular heterogeneity in cellular capacities to produce phages. The mean burst size measured at different lysis times was positively correlated to cell volume, which may determine the cellular phage production capacity. However, experiments controlling for cell size indicated that there are other factors in addition to cell size that determine this cellular capacity. IMPORTANCE Phages produce offspring by hijacking a cell's replicative machinery. Previously, it was noted that the variation in the number of phages produced by single infected cells far exceeded cell size variation. It was hypothesized that this variation is a consequence of variation in the timing of host cell lysis. Here, we show that cell-to-cell variation in lysis timing does not significantly contribute to the burst size variation. We suggest that the constant burst size variation across different host lysis times results from cell-to-cell differences in capacity to produce phages. We found that the mean burst size measured at different lysis times was positively correlated to cell volume, which may determine the cellular phage production capacity. However, experiments controlling for cell size indicated that there are other factors in addition to cell size that determine this cellular capacity.
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Bacteriófago lambda , Escherichia coli , Humanos , PrófagosRESUMO
Gene expression heterogeneity underlies cell states and contributes to developmental robustness. While heterogeneity can arise from stochastic transcriptional processes, the extent to which it is regulated is unclear. Here, we characterize the regulatory program underlying heterogeneity in murine embryonic stem cell (mESC) states. We identify differentially active and transcribed enhancers (DATEs) across states. DATEs regulate differentially expressed genes and are distinguished by co-binding of transcription factors Klf4 and Zfp281. In contrast to other factors that interact in a positive feedback network stabilizing mESC cell-type identity, Klf4 and Zfp281 drive opposing transcriptional and chromatin programs. Abrogation of factor binding to DATEs dampens variation in gene expression, and factor loss alters kinetics of switching between states. These results show antagonism between factors at enhancers results in gene expression heterogeneity and formation of cell states, with implications for the generation of diverse cell types during development.
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Células-Tronco Embrionárias , Fatores de Transcrição , Camundongos , Animais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Cromatina/genética , Cromatina/metabolismo , Elementos Facilitadores GenéticosRESUMO
Expression of the transcriptional transactivator protein Tax, encoded on the proviral plus-strand of human T-cell leukaemia virus type 1 (HTLV-1), is crucial for the replication of the virus, but Tax-expressing cells are rarely detected in fresh blood ex vivo. The dynamics and consequences of the proviral plus-strand transcriptional burst remain insufficiently characterised. We combined time-lapse live-cell imaging, single-cell tracking and mathematical modelling to study the dynamics of Tax expression at single-cell resolution in two naturally-infected, non-malignant T-cell clones transduced with a short-lived enhanced green fluorescent protein (d2EGFP) Tax reporter system. Five different patterns of Tax expression were observed during the 30-hour observation period; the distribution of these patterns differed between the two clones. The mean duration of Tax expression in the two clones was 94 and 417 hours respectively, estimated from mathematical modelling of the experimental data. Tax expression was associated with a transient slowing in cell-cycle progression and proliferation, increased apoptosis, and enhanced activation of the DNA damage response pathways. Longer-term follow-up (14 days) revealed an increase in the proportion of proliferating cells and a decrease in the fraction of apoptotic cells as the cells ceased Tax expression, resulting in a greater net expansion of the initially Tax-positive population. Time-lapse live-cell imaging showed enhanced cell-to-cell adhesion among Tax-expressing cells, and decreased cell motility of Tax-expressing cells at the single-cell level. The results demonstrate the within-clone and between-clone heterogeneity in the dynamics and patterns of HTLV-1 plus-strand transcriptional bursts and the balance of positive and negative consequences of the burst for the host cell.
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Intracellular reaction rates depend on concentrations and hence their levels are often regulated. However classical models of stochastic gene expression lack a cell size description and cannot be used to predict noise in concentrations. Here, we construct a model of gene product dynamics that includes a description of cell growth, cell division, size-dependent gene expression, gene dosage compensation, and size control mechanisms that can vary with the cell cycle phase. We obtain expressions for the approximate distributions and power spectra of concentration fluctuations which lead to insight into the emergence of concentration homeostasis. We find that (i) the conditions necessary to suppress cell division-induced concentration oscillations are difficult to achieve; (ii) mRNA concentration and number distributions can have different number of modes; (iii) two-layer size control strategies such as sizer-timer or adder-timer are ideal because they maintain constant mean concentrations whilst minimising concentration noise; (iv) accurate concentration homeostasis requires a fine tuning of dosage compensation, replication timing, and size-dependent gene expression; (v) deviations from perfect concentration homeostasis show up as deviations of the concentration distribution from a gamma distribution. Some of these predictions are confirmed using data for E. coli, fission yeast, and budding yeast.
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Escherichia coli , Schizosaccharomyces , Divisão Celular/genética , Homeostase , Modelos Biológicos , RNA Mensageiro/genética , Schizosaccharomyces/genéticaRESUMO
Timely progression of a genetic program is critical for embryonic development. However, gene expression involves inevitable fluctuations in biochemical reactions leading to substantial cell-to-cell variability (gene expression noise). One of the important questions in developmental biology is how pattern formation is reproducibly executed despite these unavoidable fluctuations in gene expression. Here, we studied the transcriptional variability of two paired zebrafish segmentation clock genes (her1 and her7) in multiple genetic backgrounds. Segmentation clock genes establish an oscillating self-regulatory system, presenting a challenging yet beautiful system in studying control of transcription variability. In this study, we found that a negative feedback loop established by the Her1 and Her7 proteins minimizes uncorrelated variability whereas gene copy number affects variability of both RNAs in a similar manner (correlated variability). We anticipate that these findings will help analyze the precision of other natural clocks and inspire the ideas for engineering precise synthetic clocks in tissue engineering.
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Sound localization involves information analysis in the lateral superior olive (LSO), a conspicuous nucleus in the mammalian auditory brainstem. LSO neurons weigh interaural level differences (ILDs) through precise integration of glutamatergic excitation from the cochlear nucleus (CN) and glycinergic inhibition from the medial nucleus of the trapezoid body (MNTB). Sound sources can be localized even during sustained perception, an accomplishment that requires robust neurotransmission. Virtually nothing is known about the sustained performance and the temporal precision of MNTB-LSO inputs after postnatal day (P)12 (time of hearing onset) and whether acoustic experience guides development. Here we performed whole-cell patch-clamp recordings to investigate neurotransmission of single MNTB-LSO fibres upon sustained electrical stimulation (1-200 Hz/60 s) at P11 and P38 in wild-type (WT) and deaf otoferlin (Otof) knock-out (KO) mice. At P11, WT and KO inputs performed remarkably similarly. In WTs, the performance increased drastically between P11 and P38, e.g. manifested by an 8 to 11-fold higher replenishment rate (RR) of synaptic vesicles and action potential robustness. Together, these changes resulted in reliable and highly precise neurotransmission at frequencies ≤100 Hz. In contrast, KO inputs performed similarly at both ages, implying impaired synaptic maturation. Computational modelling confirmed the empirical observations and established a reduced RR per release site for P38 KOs. In conclusion, acoustic experience appears to contribute massively to the development of reliable neurotransmission, thereby forming the basis for effective ILD detection. Collectively, our results provide novel insights into experience-dependent maturation of inhibitory neurotransmission and auditory circuits at the synaptic level. KEY POINTS: Inhibitory glycinergic inputs from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO) are involved in sound localization. This brainstem circuit performs reliably throughout life. How such reliability develops is unknown. Here we investigated the role of acoustic experience on the functional maturation of MNTB-LSO inputs at juvenile (postnatal day P11) and young adult ages (P38) employing deaf mice lacking otoferlin (KO). We analysed neurotransmission at single MNTB-LSO fibres in acute brainstem slices employing prolonged high-frequency stimulation (1-200 Hz/60 s). At P11, KO inputs still performed normally, as manifested by normal synaptic attenuation, fidelity, replenishment rate, temporal precision and action potential robustness. Between P11 and P38, several synaptic parameters increased substantially in wild-type mice, collectively resulting in high-fidelity and temporally precise neurotransmission. In contrast, maturation of synaptic fidelity was largely absent in KOs after P11. Collectively, reliable neurotransmission at inhibitory MNTB-LSO inputs develops under the guidance of acoustic experience.
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Surdez , Localização de Som , Potenciais de Ação/fisiologia , Animais , Vias Auditivas/fisiologia , Proteínas de Membrana , Camundongos , Núcleo Olivar/fisiologia , Reprodutibilidade dos Testes , Localização de Som/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
There is rich literature on using continuous-time and discrete-time models for studying population dynamics of consumer-resource interactions. A key focus of this contribution is to systematically compare between the two modeling formalisms the stabilizing/destabilizing impacts of diverse ecological processes that result in a density-dependent consumer attack rate. Inspired by the Nicholson-Bailey/Lotka-Volterra models in discrete-time/continuous-time, respectively, we consider host-parasitoid interactions with an arbitrary parasitoid attack rate that is a function of both the host/parasitoid population densities. Our analysis shows that a Type II functional response is stabilizing in both modeling frameworks only when combined with other mechanisms, such as mutual interference between parasitoids. A Type III functional response is by itself stabilizing, but the extent of attack-rate acceleration needed is much higher in the discrete-time framework, and its stability regime expands with increasing host reproduction. Finally, our results show that while mutual parasitoid interference can stabilize population dynamics, cooperation between parasitoids to handle hosts is destabilizing in both frameworks. In summary, our comparative analysis systematically characterizes diverse ecological processes driving stable population dynamics in discrete-time and continuous-time consumer-resource models.
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Modelos Biológicos , Reprodução , Interações Hospedeiro-Parasita , Densidade Demográfica , Dinâmica PopulacionalRESUMO
Single-cells grow by increasing their biomass and size. Here, we report that while mass and size accumulation rates of single Escherichia coli cells are exponential, their density and, thus, the levels of macromolecular crowding fluctuate during growth. As such, the average rates of mass and size accumulation of a single cell are generally not the same, but rather cells differentiate into increasing one rate with respect to the other. This differentiation yields a density homeostasis mechanism that we support mathematically. Further, we observe that density fluctuations can affect the reproduction rates of single cells, suggesting a link between the levels of macromolecular crowding with metabolism and overall population fitness. We detail our experimental approach and the "invisible" microfluidic arrays that enabled increased precision and throughput. Infections and natural communities start from a few cells, thus, emphasizing the significance of density-fluctuations when taking non-genetic variability into consideration.
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Escherichia coli , Reprodução , Escherichia coli/metabolismo , Homeostase , Substâncias Macromoleculares/metabolismoRESUMO
Optimality models have a checkered history in evolutionary biology. While optimality models have been successful in providing valuable insight into the evolution of a wide variety of biological traits, a common objection is that optimality models are overly simplistic and ignore organismal genetics. We revisit evolutionary optimization in the context of a major bacteriophage life history trait, lysis time. Lysis time refers to the period spanning phage infection of a host cell and its lysis, whereupon phage progenies are released. Lysis time, therefore, directly determines phage fecundity assuming progeny assembly does not exhaust host resources prior to lysis. Noting that previous tests of lysis time optimality rely on batch culture, we implemented a quasi-continuous culture system to observe productivity of a panel of isogenic phage λ genotypes differing in lysis time. We report that under our experimental conditions, λ phage productivity is maximized around optimal lysis times ranging from 60 to 100 min, and λ wildtype strain falls within this range. It would appear that natural selection on phage λ lysis time uncovered a set of genetic solutions that optimized progeny production in its ecological milieu relative to alternative genotypes. We discuss this finding in light of recent results that lysis time variation is also minimized in the strains with lysis times closer to the λ wild-type strain. IMPORTANCE Optimality theory presents the idea that natural selection acts on organismal traits to produce genotypes that maximize organismal survival and reproduction. As such, optimality theory is a valuable tool in guiding our understanding of the genetic constraints and tradeoffs organisms experience as their genotypes are selected to produce optimal solutions to biological problems. However, optimality theory is often critiqued as being overly simplistic and ignoring the roles of chance and history in the evolution of organismal traits. We show here that the wild-type genotype of a popular laboratory model organism, the bacteriophage λ, produces a phenotype for a major life history trait, lysis time, that maximizes the reproductive success of bearers of that genotype relative to other possible genotypes. This result demonstrates, as is rarely shown experimentally, that natural selection can achieve optimal solutions to ecological challenges.
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Bacteriófago lambda , Bacteriófago lambda/genética , Morte Celular/genética , Genótipo , Fenótipo , Fatores de TempoRESUMO
Stochastic protein accumulation up to some concentration threshold sets the timing of many cellular physiological processes. Here we obtain the exact distribution of first threshold crossing times of protein concentration, in either Laplace or time domain, and its associated cumulants: mean, variance, and skewness. The distribution is asymmetric, and its skewness nonmonotonically varies with the threshold. We study lysis times of E. coli cells for holin gene mutants of bacteriophage-λ and find a good match with theory. Mutants requiring higher holin thresholds show more skewed lysis time distributions as predicted. The theory also predicts a linear relationship between infection delay time and host doubling time for lytic viruses, that has recently been experimentally observed.
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Escherichia coli , Modelos Biológicos , Proteínas Virais , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virologia , Proteínas Virais/metabolismoRESUMO
Cellular signaling responses show substantial cell-to-cell heterogeneity, which is often ascribed to the inherent randomness of biochemical reactions, termed molecular noise, wherein high noise implies low signaling fidelity. Alternatively, heterogeneity could arise from differences in molecular content between cells, termed molecular phenotypic variability, which does not necessarily imply imprecise signaling. The contribution of these two processes to signaling heterogeneity is unclear. Here, we fused fibroblasts to produce binuclear syncytia to distinguish noise from phenotypic variability in the analysis of cytokine signaling. We reasoned that the responses of the two nuclei within one syncytium could approximate the signaling outcomes of two cells with the same molecular content, thereby disclosing noise contribution, whereas comparison of different syncytia should reveal contribution of phenotypic variability. We found that ~90% of the variance in the primary response (which was the abundance of phosphorylated, nuclear STAT) to stimulation with the cytokines interferon-γ and oncostatin M resulted from differences in the molecular content of individual cells. Thus, our data reveal that cytokine signaling in the system used here operates in a reproducible, high-fidelity manner.
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Interferon gama , Transdução de Sinais , Variação Biológica da População , Oncostatina M/genética , Transdução de Sinais/fisiologiaRESUMO
Unlike many single-celled organisms, the growth of fission yeast cells within a cell cycle is not exponential. It is rather characterized by three distinct phases (elongation, septation, and reshaping), each with a different growth rate. Experiments also showed that the distribution of cell size in a lineage can be bimodal, unlike the unimodal distributions measured for the bacterium Escherichia coli. Here we construct a detailed stochastic model of cell size dynamics in fission yeast. The theory leads to analytic expressions for the cell size and the birth size distributions, and explains the origin of bimodality seen in experiments. In particular, our theory shows that the left peak in the bimodal distribution is associated with cells in the elongation phase, while the right peak is due to cells in the septation and reshaping phases. We show that the size control strategy, the variability in the added size during a cell cycle, and the fraction of time spent in each of the three cell growth phases have a strong bearing on the shape of the cell size distribution. Furthermore, we infer all the parameters of our model by matching the theoretical cell size and birth size distributions to those from experimental single-cell time-course data for seven different growth conditions. Our method provides a much more accurate means of determining the size control strategy (timer, adder or sizer) than the standard method based on the slope of the best linear fit between the birth and division sizes. We also show that the variability in added size and the strength of size control in fission yeast depend weakly on the temperature but strongly on the culture medium. More importantly, we find that stronger size homeostasis and larger added size variability are required for fission yeast to adapt to unfavorable environmental conditions.
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Ciclo Celular/fisiologia , Tamanho Celular , Modelos Biológicos , Schizosaccharomyces/citologia , Schizosaccharomyces/crescimento & desenvolvimento , Biologia ComputacionalRESUMO
Modulatory mechanisms of neurotransmitter release and clearance are highly controlled processes whose finely tuned regulation is critical for functioning of the nervous system. Dysregulation of the monoamine neurotransmitter dopamine can lead to several neuropathies. Synaptic modulation of dopamine is known to involve pre-synaptic D2 auto-receptors and acid sensing ion channels. In addition, the dopamine membrane transporter (DAT), which is responsible for clearance of dopamine from the synaptic cleft, is suspected to play an active role in modulating release of dopamine. Using functional imaging on the Caenorhabditis elegans model system, we show that DAT-1 acts as a negative feedback modulator to neurotransmitter vesicle fusion. Results from our fluorescence recovery after photo-bleaching (FRAP) based experiments were followed up with and reaffirmed using swimming-induced paralysis behavioral assays. Utilizing our numerical FRAP data we have developed a mechanistic model to dissect the dynamics of synaptic vesicle fusion, and compare the feedback effects of DAT-1 with the dopamine auto-receptor. Our experimental results and the mechanistic model are of potential broader significance, as similar dynamics are likely to be used by other synaptic modulators including membrane transporters for other neurotransmitters across species.
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Proteínas de Caenorhabditis elegans , Dopamina , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Homeostase , Neurotransmissores , Receptores DopaminérgicosRESUMO
Resistance to targeted therapies is an important clinical problem in HER2-positive (HER2+) breast cancer. "Drug-tolerant persisters" (DTP), a subpopulation of cancer cells that survive via reversible, nongenetic mechanisms, are implicated in resistance to tyrosine kinase inhibitors (TKI) in other malignancies, but DTPs following HER2 TKI exposure have not been well characterized. We found that HER2 TKIs evoke DTPs with a luminal-like or a mesenchymal-like transcriptome. Lentiviral barcoding/single-cell RNA sequencing reveals that HER2+ breast cancer cells cycle stochastically through a "pre-DTP" state, characterized by a G0-like expression signature and enriched for diapause and/or senescence genes. Trajectory analysis/cell sorting shows that pre-DTPs preferentially yield DTPs upon HER2 TKI exposure. Cells with similar transcriptomes are present in HER2+ breast tumors and are associated with poor TKI response. Finally, biochemical experiments indicate that luminal-like DTPs survive via estrogen receptor-dependent induction of SGK3, leading to rewiring of the PI3K/AKT/mTORC1 pathway to enable AKT-independent mTORC1 activation. SIGNIFICANCE: DTPs are implicated in resistance to anticancer therapies, but their ontogeny and vulnerabilities remain unclear. We find that HER2 TKI-DTPs emerge from stochastically arising primed cells ("pre-DTPs") that engage either of two distinct transcriptional programs upon TKI exposure. Our results provide new insights into DTP ontogeny and potential therapeutic vulnerabilities. This article is highlighted in the In This Issue feature, p. 873.
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Neoplasias da Mama , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Transdução de SinaisRESUMO
Fission yeast cells prevent mitotic entry until a threshold cell surface area is reached. The protein kinase Cdr2 contributes to this size control system by forming multiprotein nodes that inhibit Wee1 at the medial cell cortex. Cdr2 node anchoring at the cell cortex is not fully understood. Through a genomic screen, we identified the conserved GTPase Arf6 as a component of Cdr2 signaling. Cells lacking Arf6 failed to divide at a threshold surface area and instead shifted to volume-based divisions at increased overall size. Arf6 stably localized to Cdr2 nodes in its GTP-bound but not GDP-bound state, and its guanine nucleotide exchange factor (GEF), Syt22, was required for both Arf6 node localization and proper size at division. In arf6Δ mutants, Cdr2 nodes detached from the membrane and exhibited increased dynamics. These defects were enhanced when arf6Δ was combined with other node mutants. Our work identifies a regulated anchor for Cdr2 nodes that is required for cells to sense surface area.
