The modern evolutionary synthesis assumes that mutations occur at random, independently of the environment in which they confer an advantage. However, there are indications that cells facing challenging conditions can adapt rapidly, utilizing processes beyond selection of pre-existing genetic variation. Here, we show that a strong regulatory challenge can induce mutations in many independent yeast cells, in the absence of general mutagenesis. Whole genome sequencing of cell lineages reveals a repertoire of independent mutations within a single lineage that arose only after the cells were exposed to the challenging environment, while other cells in the same lineage adapted without any mutation in their genomes. Thus, our experiments uncovered multiple alternative routes for heritable adaptation that were all induced in the same lineage during a short time period. Our results demonstrate the existence of adaptation mechanisms beyond random mutation, suggesting a tight connection between physiological and genetic processes.
Frequently during evolution, new phenotypes evolved due to novelty in gene regulation, such as that caused by genome rewiring. This has been demonstrated by comparing common regulatory sequences among species and by identifying single regulatory mutations that are associated with new phenotypes. However, while a single mutation changes a single element, gene regulation is accomplished by a regulatory network involving multiple interactive elements. Therefore, to better understand regulatory evolution, we have studied how mutations contributed to the adaptation of cells to a regulatory challenge. We created a synthetic genome rewiring in yeast cells, challenged their gene regulation, and studied their adaptation. HIS3, an essential enzyme for histidine biosynthesis, was placed exclusively under a GAL promoter, which is induced by galactose and strongly repressed in glucose. Such rewired cells were faced with significant regulatory challenges in a repressive glucose medium. We identified several independent mutations in elements of the GAL system associated with the rapid adaptation of cells, such as the repressor GAL80 and the binding sites of the activator GAL4. Consistent with the extraordinarily high rate of cell adaptation, new regulation emerged during adaptation via multiple trajectories, including those involving mutations in elements of the GAL system. The new regulation of HIS3 tuned its expression according to histidine requirements with or without these significant mutations, indicating that additional factors participated in this regulation and that the regulatory network could reorganize in multiple ways to accommodate different mutations. This study, therefore, stresses network plasticity as an important property for regulatory adaptation and evolution.
Neo-Darwinian evolution has presented a paradigm for population dynamics built on random mutations and selection with a clear separation of time-scales between single-cell mutation rates and the rate of reproduction. Laboratory experiments on evolving populations until now have concentrated on the fixation of beneficial mutations. Following the Darwinian paradigm, these experiments probed populations at low temporal resolution dictated by the rate of rare mutations, ignoring the intermediate evolving phenotypes. Selection however, works on phenotypes rather than genotypes. Research in recent years has uncovered the complexity of genotype-to-phenotype transformation and a wealth of intracellular processes including epigenetic inheritance, which operate on a wide range of time-scales. Here, by studying the adaptation dynamics of genetically rewired yeast cells, we show a novel type of population dynamics in which the intracellular processes intervene in shaping the population structure. Under constant environmental conditions, we measure a wide distribution of growth rates that coexist in the population for very long durations (>100 generations). Remarkably, the fastest growing cells do not take over the population on the time-scale dictated by the width of the growth-rate distributions and simple selection. Additionally, we measure significant fluctuations in the population distribution of various phenotypes: the fraction of exponentially-growing cells, the distributions of single-cell growth-rates and protein content. The observed fluctuations relax on time-scales of many generations and thus do not reflect noisy processes. Rather, our data show that the phenotypic state of the cells, including the growth-rate, for large populations in a constant environment is metastable and varies on time-scales that reflect the importance of long-term intracellular processes in shaping the population structure. This lack of time-scale separation between the intracellular and population processes calls for a new framework for population dynamics which is likely to be significant in a wide range of biological contexts, from evolution to cancer.
"Mitotic cell rounding" describes the rounding of mammalian cells before dividing into two daughter cells. This shape change requires coordinated cytoskeletal contraction and changes in osmotic pressure. While considerable research has been devoted to understanding mechanisms underlying cytoskeletal contraction, little is known about how osmotic gradients are involved in cell division. Here we describe cytoplasmic condensation preceding cell division, termed "premitotic condensation" (PMC), which involves cells extruding osmotically active Cl(-) via ClC-3, a voltage-gated channel/transporter. This leads to a decrease in cytoplasmic volume during mitotic cell rounding and cell division. Using a combination of time-lapse microscopy and biophysical measurements, we demonstrate that PMC involves the activation of ClC-3 by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in human glioma cells. Knockdown of endogenous ClC-3 protein expression eliminated CaMKII-dependent Cl(-) currents in dividing cells and impeded PMC. Thus, kinase-dependent changes in Cl(-) conductance contribute to an outward osmotic pressure in dividing cells, which facilitates cytoplasmic condensation preceding cell division.
The responses of five North American frog species that were exposed in an aqueous system to the original formulation of Roundup were compared. Carefully designed and un-confounded laboratory toxicity tests are crucial for accurate assessment of potential risks from the original formulation of Roundup to North American amphibians in aquatic environments. The formulated mixture of this herbicide as well as its components, isopropylamine (IPA) salt of glyphosate and the surfactant MON 0818 (containing polyethoxylated tallowamine (POEA)) were separately tested in 96 h acute toxicity tests with Gosner stage 25 larval anurans. Rana pipiens, R. clamitans, R. catesbeiana, Bufo fowleri, and Hyla chrysoscelis were reared from egg masses and exposed to a series of 11 concentrations of the original formulation of Roundup herbicide, nine concentrations of MON 0818 and three concentrations of IPA salt of glyphosate in static (non-renewal) aqueous laboratory tests. LC50 values are expressed as glyphosate acid equivalents (ae) or as mg/L for MON 0818 concentrations for comparison between the formulation and components. R. pipiens was the most sensitive of five species with 96 h-LC50 values for formulation tests, for the five species, ranging from 1.80 to 4.22 mg ae/L, and MON 0818 exposures with 96 h-LC50 values ranging from 0.68 to 1.32 mg/L. No significant mortality was observed during exposures of 96 h for any of the five species exposed to glyphosate IPA salt at concentrations up to 100 times the predicted environmental concentration (PEC). These results agree with previous studies which have noted that the surfactant MON 0818 containing POEA contributes the majority of the toxicity to the herbicide formulations for fish, aquatic invertebrates, and amphibians. These study results suggest that anurans are among the most sensitive species, and emphasize the importance of testing the herbicide formulation in addition to its separate components to accurately characterize the toxicity and potential risk of the formulation.
The toxicity of two glyphosate formulations (the original formulation of Roundup® and Roundup WeatherMAX®) to six species of North American larval anurans was evaluated by using 96-h static, nonrenewal aqueous exposures. The 96-h median lethal concentration values (LC50) ranged from 1.80 to 4.22 mg acid equivalent (ae)/L and 1.96 to 3.26 mg ae/L for the original formulation of Roundup and Roundup WeatherMAX, respectively. Judged by LC50 values, four species were more sensitive to Roundup WeatherMAX exposures, and two species were more sensitive to the original formulation. Two of six species, Bufo fowleri (p < 0.05, F = 14.89, degrees of freedom [df] = 1) and Rana clamitans (p < 0.05, F = 18.46, df = 1), had significantly different responses to the two formulations tested. Increased sensitivity to Roundup WeatherMAX likely was due to differences in the surfactants or relative amounts of the surfactants in the two formulations. Potency slopes for exposures of the original formulation ranged from 24.3 to 92.5% mortality/mg ae/L. Thresholds ranged from 1.31 to 3.68 mg ae/L, showing an approximately three times difference in the initiation of response among species tested. For exposures of Roundup WeatherMAX, slopes ranged from 49.3 to 84.2% mortality/mg ae/L. Thresholds ranged from 0.83 to 2.68 mg ae/L. Margins of safety derived from a simulated direct overspray were above 1, except for one species in exposures of Roundup WeatherMAX. Laboratory data based on aqueous exposures are conservative because of the lack of environmental ligands; however, these tests provide information regarding the relative toxicity between these two Roundup formulations.
The copy number of any protein fluctuates among cells in a population; characterizing and understanding these fluctuations is a fundamental problem in biophysics. We show here that protein distributions measured under a broad range of biological realizations collapse to a single non-gaussian curve under scaling by the first two moments. Moreover, in all experiments the variance is found to depend quadratically on the mean, showing that a single degree of freedom determines the entire distribution. Our results imply that protein fluctuations do not reflect any specific molecular or cellular mechanism, and suggest that some buffering process masks these details and induces universality.
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