Autorepression-Based Conditional Gene Expression System in Yeast for Variation-Suppressed Control of Protein Dosage.

Conditional control of gene expression allows an experimenter to investigate many aspects of a gene's function. In the model organism Saccharomyces cerevisiae, a number of methods to control gene expression are widely practiced, including induction by metabolites, small molecules, and even light. However, all current methods suffer from at least one of a set of drawbacks, including need for specialized growth conditions, leaky expression, or requirement of specialized equipment. Here we describe protocols using two transformations to construct strains that carry a new controller in which all these drawbacks are overcome. In these strains, the expression of a controlled gene of interest is repressed by the bacterial repressor TetR and induced by anhydrotetracycline. TetR also regulates its own expression, creating an autorepression loop. This autorepression allows tight control of gene expression and protein dosage with low cell-to-cell variation in expression. A second repressor, TetR-Tup1, prevents any leaky expression. We also present a protocol showing a particular workhorse application of such strains to generate synchronized cell populations. We turn off expression of the cell cycle regulator CDC20 completely, arresting the cell population, and then we turn it back on so that the synchronized cells resume cell cycle progression. This control system can be applied to any endogenous or exogenous gene for precise expression. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Generating a parent WTC846 strain Basic Protocol 2: Generating a WTC846 strain with controlled expression of the targeted gene Alternate Protocol: CRISPR-mediated promoter replacement Basic Protocol 3: Cell cycle synchronization/arrest and release using the WTC846- K3 ::CDC20 strain.

A precisely adjustable, variation-suppressed eukaryotic transcriptional controller to enable genetic discovery.

Conditional expression of genes and observation of phenotype remain central to biological discovery. Current methods enable either on/off or imprecisely controlled graded gene expression. We developed a 'well-tempered' controller, WTC846, for precisely adjustable, graded, growth condition independent expression of genes in Saccharomyces cerevisiae. Controlled genes are expressed from a strong semisynthetic promoter repressed by the prokaryotic TetR, which also represses its own synthesis; with basal expression abolished by a second, 'zeroing' repressor. The autorepression loop lowers cell-to-cell variation while enabling precise adjustment of protein expression by a chemical inducer. WTC846 allelic strains in which the controller replaced the native promoters recapitulated known null phenotypes (CDC42, TPI1), exhibited novel overexpression phenotypes (IPL1), showed protein dosage-dependent growth rates and morphological phenotypes (CDC28, TOR2, PMA1 and the hitherto uncharacterized PBR1), and enabled cell cycle synchronization (CDC20). WTC846 defines an 'expression clamp' allowing protein dosage to be adjusted by the experimenter across the range of cellular protein abundances, with limited variation around the setpoint.

Chaperone biomarkers of lifespan and penetrance track the dosages of many other proteins.

Many traits vary among isogenic individuals in homogeneous environments. In microbes, plants and animals, variation in the protein chaperone system affects many such traits. In the animal model C. elegans, the expression level of hsp-16.2 chaperone biomarkers correlates with or predicts the penetrance of mutations and lifespan after heat shock. But the physiological mechanisms causing cells to express different amounts of the biomarker were unknown. Here, we used an in vivo microscopy approach to dissect different contributions to cell-to-cell variation in hsp-16.2 expression in the intestines of young adult animals, which generate the most lifespan predicting signal. While we detected both cell autonomous intrinsic noise and signaling noise, we found both contributions were relatively unimportant. The major contributor to cell-to-cell variation in biomarker expression was general differences in protein dosage. The hsp-16.2 biomarker reveals states of high or low effective dosage for many genes.

In vivo measurements reveal a single 5'-intron is sufficient to increase protein expression level in Caenorhabditis elegans.

Introns can increase gene expression levels using a variety of mechanisms collectively referred to as Intron Mediated Enhancement (IME). IME has been measured in cell culture and plant models by quantifying expression of intronless and intron-bearing reporter genes in vitro. We developed hardware and software to implement microfluidic chip-based gene expression quantification in vivo. We altered position, number and sequence of introns in reporter genes controlled by the hsp-90 promoter. Consistent with plant and mammalian studies, we determined a single, natural or synthetic, 5'-intron is sufficient for the full IME effect conferred by three synthetic introns, while a 3'-intron is not. We found coding sequence can affect IME; the same three synthetic introns that increase mcherry protein concentration by approximately 50%, increase mEGFP by 80%. We determined IME effect size is not greatly affected by the stronger vit-2 promoter. Our microfluidic imaging approach should facilitate screens for factors affecting IME and other intron-dependent processes.

Recipes and Tools for Culture of Escherichia coli.

In this article, we provide information about culture media, including minimal liquid media, rich liquid media, solid media, top agar, and stab agar. We also provide descriptions and useful information about tools used with growth media such as inoculating loops, sterile toothpicks, and spreaders. © 2018 by John Wiley & Sons, Inc.

Growth of E. coli in Liquid Medium.

We describe the procedure for inoculating overnight (starter) cultures of E. coli from a single colony, along with considerations for growing larger cultures. We also include two methods for monitoring the number of cells per unit volume (density) of liquid cultures using a spectrophotometer and a hemacytometer or "count slide." © 2018 by John Wiley & Sons, Inc.

Growth of E. coli on Solid Media.

We provide protocols for titering and isolating bacterial colonies from single cells by serial dilutions, for streaking agar plates, and for spreading suspensions of cells on plates. Support protocols describe replica plating and methods for storing strains as agar stabs and frozen stocks. © 2018 by John Wiley & Sons, Inc.

Transferring information without distortion.

Despite employing diverse molecular mechanisms, many different cell signaling systems avoid losing information by transmitting it in a linear manner.

Single-cell profiling screen identifies microtubule-dependent reduction of variability in signaling.

Populations of isogenic cells often respond coherently to signals, despite differences in protein abundance and cell state. Previously, we uncovered processes in the Saccharomyces cerevisiae pheromone response system (PRS) that reduced cell-to-cell variability in signal strength and cellular response. Here, we screened 1,141 non-essential genes to identify 50 "variability genes". Most had distinct, separable effects on strength and variability of the PRS, defining these quantities as genetically distinct "axes" of system behavior. Three genes affected cytoplasmic microtubule function: BIM1, GIM2, and GIM4 We used genetic and chemical perturbations to show that, without microtubules, PRS output is reduced but variability is unaffected, while, when microtubules are present but their function is perturbed, output is sometimes lowered, but its variability is always high. The increased variability caused by microtubule perturbations required the PRS MAP kinase Fus3 and a process at or upstream of Ste5, the membrane-localized scaffold to which Fus3 must bind to be activated. Visualization of Ste5 localization dynamics demonstrated that perturbing microtubules destabilized Ste5 at the membrane signaling site. The fact that such microtubule perturbations cause aberrant fate and polarity decisions in mammals suggests that microtubule-dependent signal stabilization might also operate throughout metazoans.

Environmental Canalization of Life Span and Gene Expression in Caenorhabditis elegans.

Animals, particularly poikilotherms, exhibit distinct physiologies at different environmental temperatures. Here, we hypothesized that temperature-based differences in physiology could affect the amount of variation in complex quantitative traits. Specifically, we examined, in Caenorhabditis elegans, how different temperatures (15°C, 20°C, and 25°C) affected the amount of interindividual variation in life span and also expression of three reporter genes-transcriptional reporters for vit-2, gpd-2, and hsp-16.2 (a life-span biomarker). We found the expected inverse relationship between temperature and average life span. Surprisingly, we found that at the highest temperature, there were fewer differences between individuals in life span and less interindividual variation in expression of all three reporters. We suggest that growth at 25°C might canalize (reduce interindividual differences in) life span and expression of some genes by eliciting a small constitutive heat shock response. Growth at 25°C requires wild-type hsf-1, which encodes the main heat shock response transcriptional activator. We speculate that increased chaperone activity at 25°C may reduce interindividual variation in gene expression by increasing protein folding efficiency. We hypothesize that reduced variation in gene expression may ultimately cause reduced variation in life span.

Caenorhabditis elegans Genes Affecting Interindividual Variation in Life-span Biomarker Gene Expression.

Genetically identical organisms grown in homogenous environments differ in quantitative phenotypes. Differences in one such trait, expression of a single biomarker gene, can identify isogenic cells or organisms that later manifest different fates. For example, in isogenic populations of young adult Caenorhabditis elegans, differences in Green Fluorescent Protein (GFP) expressed from the hsp-16.2 promoter predict differences in life span. Thus, it is of interest to determine how interindividual differences in biomarker gene expression arise. Prior reports showed that the thermosensory neurons and insulin signaling systems controlled the magnitude of the heat shock response, including absolute expression of hsp-16.2. Here, we tested whether these regulatory signals might also influence variation in hsp-16.2 reporter expression. Genetic experiments showed that the action of AFD thermosensory neurons increases interindividual variation in biomarker expression. Further genetic experimentation showed the insulin signaling system acts to decrease interindividual variation in life-span biomarker expression; in other words, insulin signaling canalizes expression of the hsp-16.2-driven life-span biomarker. Our results show that specific signaling systems regulate not only expression level, but also the amount of interindividual expression variation for a life-span biomarker gene. They raise the possibility that manipulation of these systems might offer means to reduce heterogeneity in the aging process.

Push-Pull and Feedback Mechanisms Can Align Signaling System Outputs with Inputs.

Many cell signaling systems, including the yeast pheromone response system, exhibit "dose-response alignment" (DoRA), in which output of one or more downstream steps closely matches the fraction of occupied receptors. DoRA can improve the fidelity of transmitted dose information. Here, we searched systematically for biochemical network topologies that produced DoRA. Most networks, including many containing feedback and feedforward loops, could not produce DoRA. However, networks including "push-pull" mechanisms, in which the active form of a signaling species stimulates downstream activity and the nominally inactive form reduces downstream activity, enabled perfect DoRA. Networks containing feedbacks enabled DoRA, but only if they also compared feedback to input and adjusted output to match. Our results establish push-pull as a non-feedback mechanism to align output with variable input and maximize information transfer in signaling systems. They also suggest genetic approaches to determine whether particular signaling systems use feedback or push-pull control.

Phasor plotting with frequency-domain flow cytometry.

Interest in time resolved flow cytometry is growing. In this paper, we collect time-resolved flow cytometry data and use it to create polar plots showing distributions that are a function of measured fluorescence decay rates from individual fluorescently-labeled cells and fluorescent microspheres. Phasor, or polar, graphics are commonly used in fluorescence lifetime imaging microscopy (FLIM). In FLIM measurements, the plotted points on a phasor graph represent the phase-shift and demodulation of the frequency-domain fluorescence signal collected by the imaging system for each image pixel. Here, we take a flow cytometry cell counting system, introduce into it frequency-domain optoelectronics, and process the data so that each point on a phasor plot represents the phase shift and demodulation of an individual cell or particle. In order to demonstrate the value of this technique, we show that phasor graphs can be used to discriminate among populations of (i) fluorescent microspheres, which are labeled with one fluorophore type; (ii) Chinese hamster ovary (CHO) cells labeled with one and two different fluorophore types; and (iii) Saccharomyces cerevisiae cells that express combinations of fluorescent proteins with different fluorescence lifetimes. The resulting phasor plots reveal differences in the fluorescence lifetimes within each sample and provide a distribution from which we can infer the number of cells expressing unique single or dual fluorescence lifetimes. These methods should facilitate analysis time resolved flow cytometry data to reveal complex fluorescence decay kinetics.

Overview of Post Cohen-Boyer Methods for Single Segment Cloning and for Multisegment DNA Assembly.

In 1973, Cohen and coworkers published a foundational paper describing the cloning of DNA fragments into plasmid vectors. In it, they used DNA segments made by digestion with restriction enzymes and joined these in vitro with DNA ligase. These methods established working recombinant DNA technology and enabled the immediate start of the biotechnology industry. Since then, "classical" recombinant DNA technology using restriction enzymes and DNA ligase has matured. At the same time, researchers have developed numerous ways to generate large, complex, multisegment DNA constructions that offer advantages over classical techniques. Here, we provide an overview of "post-Cohen-Boyer" techniques used for cloning single segments into vectors (T/A, Topo cloning, Gateway and Recombineering) and for multisegment DNA assembly (BioBricks, Golden Gate, Gibson, yeast homologous recombination in vivo, and ligase cycling reaction). We compare and contrast these methods and also discuss issues that researchers should consider before choosing a particular multisegment DNA assembly method. © 2016 by John Wiley & Sons, Inc.

Using measures of single-cell physiology and physiological state to understand organismic aging.

Genetically identical organisms in homogeneous environments have different lifespans and healthspans. These differences are often attributed to stochastic events, such as mutations and 'epimutations', changes in DNA methylation and chromatin that change gene function and expression. But work in the last 10 years has revealed differences in lifespan- and health-related phenotypes that are not caused by lasting changes in DNA or identified by modifications to DNA or chromatin. This work has demonstrated persistent differences in single-cell and whole-organism physiological states operationally defined by values of reporter gene signals in living cells. While some single-cell states, for example, responses to oxygen deprivation, were defined previously, others, such as a generally heightened ability to make proteins, were, revealed by direct experiment only recently, and are not well understood. Here, we review technical progress that promises to greatly increase the number of these measurable single-cell physiological variables and measureable states. We discuss concepts that facilitate use of single-cell measurements to provide insight into physiological states and state transitions. We assert that researchers will use this information to relate cell level physiological readouts to whole-organism outcomes, to stratify aging populations into groups based on different physiologies, to define biomarkers predictive of outcomes, and to shed light on the molecular processes that bring about different individual physiologies. For these reasons, quantitative study of single-cell physiological variables and state transitions should provide a valuable complement to genetic and molecular explanations of how organisms age.

Single Cell Quantification of Reporter Gene Expression in Live Adult Caenorhabditis elegans Reveals Reproducible Cell-Specific Expression Patterns and Underlying Biological Variation.

In multicellular organisms such as Caenorhabditis elegans, differences in complex phenotypes such as lifespan correlate with the level of expression of particular engineered reporter genes. In single celled organisms, quantitative understanding of responses to extracellular signals and of cell-to-cell variation in responses has depended on precise measurement of reporter gene expression. Here, we developed microscope-based methods to quantify reporter gene expression in cells of Caenorhabditis elegans with low measurement error. We then quantified expression in strains that carried different configurations of Phsp-16.2-fluorescent-protein reporters, in whole animals, and in all 20 cells of the intestine tissue, which is responsible for most of the fluorescent signal. Some animals bore more recently developed single copy Phsp-16.2 reporters integrated at defined chromosomal sites, others, "classical" multicopy reporter gene arrays integrated at random sites. At the level of whole animals, variation in gene expression was similar: strains with single copy reporters showed the same amount of animal-to-animal variation as strains with multicopy reporters. At the level of cells, in animals with single copy reporters, the pattern of expression in cells within the tissue was highly stereotyped. In animals with multicopy reporters, the cell-specific expression pattern was also stereotyped, but distinct, and somewhat more variable. Our methods are rapid and gentle enough to allow quantification of expression in the same cells of an animal at different times during adult life. They should allow investigators to use changes in reporter expression in single cells in tissues as quantitative phenotypes, and link those to molecular differences. Moreover, by diminishing measurement error, they should make possible dissection of the causes of the remaining, real, variation in expression. Understanding such variation should help reveal its contribution to differences in complex phenotypic outcomes in multicellular organisms.

Measuring and sorting cell populations expressing isospectral fluorescent proteins with different fluorescence lifetimes.

Study of signal transduction in live cells benefits from the ability to visualize and quantify light emitted by fluorescent proteins (XFPs) fused to different signaling proteins. However, because cell signaling proteins are often present in small numbers, and because the XFPs themselves are poor fluorophores, the amount of emitted light, and the observable signal in these studies, is often small. An XFP's fluorescence lifetime contains additional information about the immediate environment of the fluorophore that can augment the information from its weak light signal. Here, we constructed and expressed in Saccharomyces cerevisiae variants of Teal Fluorescent Protein (TFP) and Citrine that were isospectral but had shorter fluorescence lifetimes, ∼ 1.5 ns vs ∼ 3 ns. We modified microscopic and flow cytometric instruments to measure fluorescence lifetimes in live cells. We developed digital hardware and a measure of lifetime called a "pseudophasor" that we could compute quickly enough to permit sorting by lifetime in flow. We used these abilities to sort mixtures of cells expressing TFP and the short-lifetime TFP variant into subpopulations that were respectively 97% and 94% pure. This work demonstrates the feasibility of using information about fluorescence lifetime to help quantify cell signaling in living cells at the high throughput provided by flow cytometry. Moreover, it demonstrates the feasibility of isolating and recovering subpopulations of cells with different XFP lifetimes for subsequent experimentation.

Utilization of extracellular information before ligand-receptor binding reaches equilibrium expands and shifts the input dynamic range.

Cell signaling systems sense and respond to ligands that bind cell surface receptors. These systems often respond to changes in the concentration of extracellular ligand more rapidly than the ligand equilibrates with its receptor. We demonstrate, by modeling and experiment, a general "systems level" mechanism cells use to take advantage of the information present in the early signal, before receptor binding reaches a new steady state. This mechanism, pre-equilibrium sensing and signaling (PRESS), operates in signaling systems in which the kinetics of ligand-receptor binding are slower than the downstream signaling steps, and it typically involves transient activation of a downstream step. In the systems where it operates, PRESS expands and shifts the input dynamic range, allowing cells to make different responses to ligand concentrations so high as to be otherwise indistinguishable. Specifically, we show that PRESS applies to the yeast directional polarization in response to pheromone gradients. Consideration of preexisting kinetic data for ligand-receptor interactions suggests that PRESS operates in many cell signaling systems throughout biology. The same mechanism may also operate at other levels in signaling systems in which a slow activation step couples to a faster downstream step.

Assigning quantitative function to post-translational modifications reveals multiple sites of phosphorylation that tune yeast pheromone signaling output.

Cell signaling systems transmit information by post-translationally modifying signaling proteins, often via phosphorylation. While thousands of sites of phosphorylation have been identified in proteomic studies, the vast majority of sites have no known function. Assigning functional roles to the catalog of uncharacterized phosphorylation sites is a key research challenge. Here we present a general approach to address this challenge and apply it to a prototypical signaling pathway, the pheromone response pathway in Saccharomyces cerevisiae. The pheromone pathway includes a mitogen activated protein kinase (MAPK) cascade activated by a G-protein coupled receptor (GPCR). We used published mass spectrometry-based proteomics data to identify putative sites of phosphorylation on pheromone pathway components, and we used evolutionary conservation to assign priority to a list of candidate MAPK regulatory sites. We made targeted alterations in those sites, and measured the effects of the mutations on pheromone pathway output in single cells. Our work identified six new sites that quantitatively tuned system output. We developed simple computational models to find system architectures that recapitulated the quantitative phenotypes of the mutants. Our results identify a number of putative phosphorylation events that contribute to adjust the input-output relationship of this model eukaryotic signaling system. We believe this combined approach constitutes a general means not only to reveal modification sites required to turn a pathway on and off, but also those required for more subtle quantitative effects that tune pathway output. Our results suggest that relatively small quantitative influences from individual phosphorylation events endow signaling systems with plasticity that evolution may exploit to quantitatively tailor signaling outcomes.