Live-cell analysis of IMPDH protein levels during yeast colony growth provides insights into the regulation of GTP synthesis.

The purine nucleotides ATP and GTP are made from the common precursor inosine monophosphate (IMP). Maintaining the correct balance of these nucleotides for optimal cell growth is controlled in part by the enzyme IMP dehydrogenase (IMPDH), which catalyzes the first dedicated step of GTP biosynthesis. The regulation of IMPDH mRNA and protein levels in the yeast S. cerevisiae grown in liquid culture has been studied in some detail, but regulation of IMPDH protein under conditions of cellular crowding on a solid substrate has not been examined. Here, we report real-time, live-cell analysis of the accumulation of the Imd2 isoform of IMPDH in yeast cells forming a monolayer colony in a microfluidic device over a 50-hour time course. We observe two distinct phases of increased Imd2 accumulation: a guanine-insensitive phase early in outgrowth and a guanine-sensitive phase later, when cells become crowded. We show that the IMPDH inhibitor mycophenolic acid enhances both phases of increase. Deletion of a transcription attenuator upstream of the mRNA start site that decreases Imd2 mRNA synthesis in the presence of high GTP increases the baseline level of Imd2 protein 10-fold and abolishes guanine-sensitive but not guanine-insensitive induction. Our results suggest that at least two mechanisms of yeast Imd2 regulation exist, the known GTP-dependent attenuation of RNA polymerase II elongation and a GTP concentration-independent pathway that may be controlled by cell growth state. Live-cell analysis of IMPDH protein levels in a growing yeast colony confirms a known mechanism of regulation and provides evidence for an additional mode of regulation. IMPORTANCE: This study used live-cell microscopy to track changes in the level of a key enzyme in GTP nucleotide biosynthesis, inosine monophosphate dehydrogenase (IMPDH), during growth of a brewers yeast colony over 2 days in a microfluidic device. The results show that feedback regulation via transcription attenuation allows cells to adapt to nutrient limitation in the crowded environs of a yeast colony. They also identify a novel mode of regulation of IMPDH level that is not driven by guanine nucleotide availability.

Tuning interdomain conjugation to enable in situ population modification in yeasts.

The ability to modify and control natural and engineered microbiomes is essential for biotechnology and biomedicine. Fungi are critical members of most microbiomes, yet technology for modifying the fungal members of a microbiome has lagged far behind that for bacteria. Interdomain conjugation (IDC) is a promising approach, as DNA transfer from bacterial cells to yeast enables in situ modification. While such genetic transfers have been known to naturally occur in a wide range of eukaryotes and are thought to contribute to their evolution, IDC has been understudied as a technique to control fungal or fungal-bacterial consortia. One major obstacle to the widespread use of IDC is its limited efficiency. In this work, we manipulated metabolic and physical interactions between genetically tractable Escherichia coli and Saccharomyces cerevisiae to control the incidence of IDC. We test the landscape of population interactions between the bacterial donors and yeast recipients to find that bacterial commensalism leads to maximized IDC, both in culture and in mixed colonies. We demonstrate the capacity of cell-to-cell binding via mannoproteins to assist both IDC incidence and bacterial commensalism in culture and model how these tunable controls can predictably yield a range of IDC outcomes. Furthermore, we demonstrate that these controls can be utilized to irreversibly alter a recipient yeast population, by both "rescuing" a poor-growing recipient population and collapsing a stable population via a novel IDC-mediated CRISPR/Cas9 system.IMPORTANCEFungi are important but often unaddressed members of most natural and synthetic microbial communities. This work highlights opportunities for modifying yeast microbiome populations through bacterial conjugation. While conjugation has been recognized for its capacity to deliver engineerable DNA to a range of cells, its dependence on cell contact has limited its efficiency. Here, we find "knobs" to control DNA transfer, by engineering the metabolic dependence between bacterial donors and yeast recipients and by changing their ability to physically adhere to each other. Importantly, we functionally validate these "knobs" by irreversibly altering yeast populations. We use these controls to "rescue" a failing yeast population, demonstrate the capacity of conjugated CRISPR/Cas9 to depress or collapse populations, and show that conjugation can be easily interrupted by disrupting cell-to-cell binding. These results offer building blocks toward in situ mycobiome editing, with significant implications for clinical treatments of fungal pathogens and other fungal system engineering.

Dynamic Multiplexed Control and Modeling of Optogenetic Systems Using the High-Throughput Optogenetic Platform, Lustro.

The ability to control cellular processes using optogenetics is inducer-limited, with most optogenetic systems responding to blue light. To address this limitation, we leverage an integrated framework combining Lustro, a powerful high-throughput optogenetics platform, and machine learning tools to enable multiplexed control over blue light-sensitive optogenetic systems. Specifically, we identify light induction conditions for sequential activation as well as preferential activation and switching between pairs of light-sensitive split transcription factors in the budding yeast, Saccharomyces cerevisiae. We use the high-throughput data generated from Lustro to build a Bayesian optimization framework that incorporates data-driven learning, uncertainty quantification, and experimental design to enable the prediction of system behavior and the identification of optimal conditions for multiplexed control. This work lays the foundation for designing more advanced synthetic biological circuits incorporating optogenetics, where multiple circuit components can be controlled using designer light induction programs, with broad implications for biotechnology and bioengineering.

Enhancing high-throughput optogenetics: Integration of LITOS with Lustro enables simultaneous light stimulation and shaking.

Optogenetics is a powerful tool that uses light to control cellular behavior. Here we enhance high-throughput characterization of optogenetic experiments through the integration of the LED Illumination Tool for Optogenetic Stimulation (LITOS) with the previously published automated platform Lustro. Lustro enables efficient high-throughput screening and characterization of optogenetic systems. The initial iteration of Lustro used the optoPlate illumination device for light induction, with the robot periodically moving the plate over to a shaking device to resuspend cell cultures. Here, we designed a 3D-printed adaptor, rendering LITOS compatible with the BioShake 3000-T ELM used in Lustro. This novel setup allows for concurrent light stimulation and culture agitation, streamlining experiments. Our study demonstrates comparable growth rates between constant and intermittent shaking of Saccharomyces cerevisiae liquid cultures. While the light intensity of the LITOS is not as bright as the optoPlate used in the previous iteration of Lustro, the constant shaking increased the maturation rate of the mScarlet-I fluorescent reporter used. Only a marginal increase in temperature was observed when using the modified LITOS equipped with the 3D-printed adaptor. Our findings show that the integration of LITOS onto a plate shaker allows for constant culture shaking and illumination compatible with laboratory automation platforms, such as Lustro.

Tuning Interdomain Conjugation Toward in situ Population Modification in Yeast.

The ability to modify and control natural and engineered microbiomes is essential for biotechnology and biomedicine. Fungi are critical members of most microbiomes, yet technology for modifying the fungal members of a microbiome has lagged far behind that for bacteria. Interdomain conjugation (IDC) is a promising approach, as DNA transfer from bacterial cells to yeast enables in situ modification. While such genetic transfers have been known to naturally occur in a wide range of eukaryotes, and are thought to contribute to their evolution, IDC has been understudied as a technique to control fungal or fungal-bacterial consortia. One major obstacle to widespread use of IDC is its limited efficiency. In this work, we utilize interactions between genetically tractable Escherichia coli and Saccharomyces cerevisiae to control the incidence of IDC. We test the landscape of population interactions between the bacterial donors and yeast recipients to find that bacterial commensalism leads to maximized IDC, both in culture and in mixed colonies. We demonstrate the capacity of cell-to-cell binding via mannoproteins to assist both IDC incidence and bacterial commensalism in culture, and model how these tunable controls can predictably yield a range of IDC outcomes. Further, we demonstrate that these lessons can be utilized to lastingly alter a recipient yeast population, by both "rescuing" a poor-growing recipient population and collapsing a stable population via a novel IDC-mediated CRISPR/Cas9 system.

High-Throughput Optogenetics Experiments in Yeast Using the Automated Platform Lustro.

Optogenetics offers precise control over cellular behavior by utilizing genetically encoded light-sensitive proteins. However, optimizing these systems to achieve the desired functionality often requires multiple design-build-test cycles, which can be time-consuming and labor-intensive. To address this challenge, we have developed Lustro, a platform that combines light stimulation with laboratory automation, enabling efficient high-throughput screening and characterization of optogenetic systems. Lustro utilizes an automation workstation equipped with an illumination device, a shaking device, and a plate reader. By employing a robotic arm, Lustro automates the movement of a microwell plate between these devices, allowing for the stimulation of optogenetic strains and the measurement of their response. This protocol provides a step-by-step guide on using Lustro to characterize optogenetic systems for gene expression control in the budding yeast Saccharomyces cerevisiae. The protocol covers the setup of Lustro's components, including the integration of the illumination device with the automation workstation. It also provides detailed instructions for programming the illumination device, plate reader, and robot, ensuring smooth operation and data acquisition throughout the experimental process.

Lustro: High-Throughput Optogenetic Experiments Enabled by Automation and a Yeast Optogenetic Toolkit.

Optogenetic systems use genetically encoded light-sensitive proteins to control cellular processes. This provides the potential to orthogonally control cells with light; however, these systems require many design-build-test cycles to achieve a functional design and multiple illumination variables need to be laboriously tuned for optimal stimulation. We combine laboratory automation and a modular cloning scheme to enable high-throughput construction and characterization of optogenetic split transcription factors in Saccharomyces cerevisiae. We expand the yeast optogenetic toolkit to include variants of the cryptochromes and enhanced Magnets, incorporate these light-sensitive dimerizers into split transcription factors, and automate illumination and measurement of cultures in a 96-well microplate format for high-throughput characterization. We use this approach to rationally design and test an optimized enhanced Magnet transcription factor with improved light-sensitive gene expression. This approach is generalizable to the high-throughput characterization of optogenetic systems across a range of biological systems and applications.

Lustro: High-throughput optogenetic experiments enabled by automation and a yeast optogenetic toolkit.

Optogenetic systems use genetically-encoded light-sensitive proteins to control cellular processes. This provides the potential to orthogonally control cells with light, however these systems require many design-build-test cycles to achieve a functional design and multiple illumination variables need to be laboriously tuned for optimal stimulation. We combine laboratory automation and a modular cloning scheme to enable high-throughput construction and characterization of optogenetic split transcription factors in Saccharomyces cerevisiae . We expand the yeast optogenetic toolkit to include variants of the cryptochromes and Enhanced Magnets, incorporate these light-sensitive dimerizers into split transcription factors, and automate illumination and measurement of cultures in a 96-well microplate format for high-throughput characterization. We use this approach to rationally design and test an optimized Enhanced Magnet transcription factor with improved light-sensitive gene expression. This approach is generalizable to high-throughput characterization of optogenetic systems across a range of biological systems and applications.

Transcription factor localization dynamics and DNA binding drive distinct promoter interpretations.

Environmental information may be encoded in the temporal dynamics of transcription factor (TF) activation and subsequently decoded by gene promoters to enact stimulus-specific gene expression programs. Previous studies of this behavior focused on the encoding and decoding of information in TF nuclear localization dynamics, yet cells control the activity of TFs in myriad ways, including by regulating their ability to bind DNA. Here, we use light-controlled mutants of the yeast TF Msn2 as a model system to investigate how promoter decoding of TF localization dynamics is affected by changes in the ability of the TF to bind DNA. We find that yeast promoters directly decode the light-controlled localization dynamics of Msn2 and that the effects of changing Msn2 affinity on that decoding behavior are highly promoter dependent, illustrating how cells could regulate TF localization dynamics and DNA binding in concert for improved control of gene expression.

Strain-dependent differences in coordination of yeast signalling networks.

The yeast mitogen-activated protein kinase pathways serve as a model system for understanding how network interactions affect the way in which cells coordinate the response to multiple signals. We have quantitatively compared two yeast strain backgrounds YPH499 and ∑1278b (both of which have previously been used to study these pathways) and found several important differences in how they coordinate the interaction between the high osmolarity glycerol (HOG) and mating pathways. In the ∑1278b background, in response to simultaneous stimulus, mating pathway activation is dampened and delayed in a dose-dependent manner. In the YPH499 background, only dampening is dose-dependent. Furthermore, leakage from the HOG pathway into the mating pathway (crosstalk) occurs during osmostress alone in the ∑1278b background only. The mitogen-activated protein kinase Hog1p suppresses crosstalk late in an induction time course in both strains but does not affect the early crosstalk seen in the ∑1278b background. Finally, the kinase Rck2p plays a greater role suppressing late crosstalk in the ∑1278b background than in the YPH499 background. Our results demonstrate that comparisons between laboratory yeast strains provide an important resource for understanding how signalling network interactions are tuned by genetic variation without significant alteration to network structure.

Dynamic Multiplexed Control and Modeling of Optogenetic Systems Using the High-Throughput Optogenetic Platform, Lustro.

The ability to control cellular processes using optogenetics is inducer-limited, with most optogenetic systems responding to blue light. To address this limitation we leverage an integrated framework combining Lustro, a powerful high-throughput optogenetics platform, and machine learning tools to enable multiplexed control over blue light-sensitive optogenetic systems. Specifically, we identify light induction conditions for sequential activation as well as preferential activation and switching between pairs of light-sensitive spit transcription factors in the budding yeast, Saccharomyces cerevisiae . We use the high-throughput data generated from Lustro to build a Bayesian optimization framework that incorporates data-driven learning, uncertainty quantification, and experimental design to enable the prediction of system behavior and the identification of optimal conditions for multiplexed control. This work lays the foundation for designing more advanced synthetic biological circuits incorporating optogenetics, where multiple circuit components can be controlled using designer light induction programs, with broad implications for biotechnology and bioengineering.

Modeling single-cell phenotypes links yeast stress acclimation to transcriptional repression and pre-stress cellular states.

Stress defense and cell growth are inversely related in bulk culture analyses; however, these studies miss substantial cell-to-cell heterogeneity, thus obscuring true phenotypic relationships. Here, we devised a microfluidics system to characterize multiple phenotypes in single yeast cells over time before, during, and after salt stress. The system measured cell and colony size, growth rate, and cell-cycle phase along with nuclear trans-localization of two transcription factors: stress-activated Msn2 that regulates defense genes and Dot6 that represses ribosome biogenesis genes during an active stress response. By tracking cells dynamically, we discovered unexpected discordance between Msn2 and Dot6 behavior that revealed subpopulations of cells with distinct growth properties. Surprisingly, post-stress growth recovery was positively corelated with activation of the Dot6 repressor. In contrast, cells lacking Dot6 displayed slower growth acclimation, even though they grow normally in the absence of stress. We show that wild-type cells with a larger Dot6 response display faster production of Msn2-regulated Ctt1 protein, separable from the contribution of Msn2. These results are consistent with the model that transcriptional repression during acute stress in yeast provides a protective response, likely by redirecting translational capacity to induced transcripts.

Cellular context alters EGF-induced ERK dynamics and reveals potential crosstalk with GDF-15.

Cellular signaling dynamics are sensitive to differences in ligand identity, levels, and temporal patterns. These signaling patterns are also impacted by the larger context that the cell experiences (i.e., stimuli such as media formulation or substrate stiffness that are constant in an experiment exploring a particular variable but may differ between independent experiments which explore that variable) although the reason for different dynamics is not always obvious. Here, we compared extracellular-regulated kinase (ERK) signaling in response to epidermal growth factor treatment of human mammary epithelial cells cultures in either well culture or a microfluidic device. Using a single-cell ERK kinase translocation reporter, we observed extended ERK activation in well culture and only transient activity in microfluidic culture. The activity in microfluidic culture resembled that of the control condition, suggesting that shear stress led to the early activity and a loss of autocrine factors dampened extended signaling. Through experimental analysis we identified growth differentiation factor-15 as a candidate factor that led to extended ERK activation through a protein kinase C-α/ß dependent pathway. Our results demonstrate that context impacts ERK dynamics and that comparison of distinct contexts can be used to elucidate new aspects of the cell signaling network.

Evaluation of Benzinger et al.: Optogenetic circuits for dynamic signal processing.

One snapshot of the peer review process for "Synthetic gene networks recapitulate dynamic signal decoding and differential gene expression" (Benzinger et al., 2022).

Design and implementation of a microfluidic device capable of temporal growth factor delivery reveal filtering capabilities of the EGFR/ERK pathway.

Utilizing microfluidics to mimic the dynamic temporal changes of growth factor and cytokine concentrations in vivo has greatly increased our understanding of how signal transduction pathways are structured to encode extracellular stimuli. To date, these devices have focused on delivering pulses of varying frequency, and there are limited cell culture models for delivering slowly increasing concentrations of stimuli that cells may experience in vivo. To examine this setting, we developed and validated a microfluidic device that can deliver increasing concentrations of growth factor over periods ranging from 6 to 24 h. Using this device and a fluorescent biosensor of extracellular-regulated kinase (ERK) activity, we delivered a slowly increasing concentration of epidermal growth factor (EGF) to human mammary epithelial cells and surprisingly observed minimal ERK activation, even at concentrations that stimulate robust activity in bolus delivery. The cells remained unresponsive to subsequent challenges with EGF, and immunocytochemistry suggested that the loss of an epidermal growth factor receptor was responsible. Cells were then challenged with faster rates of change of EGF, revealing an increased ERK activity as a function of rate of change. Specifically, both the fraction of cells that responded and the length of ERK activation time increased with the rate of change. This microfluidic device fills a gap in the current repertoire of in vitro microfluidic devices and demonstrates that slower, more physiological changes in growth factor presentation can reveal new regulatory mechanisms for how signal transduction pathways encode changes in the extracellular growth factor milieu.

Optogenetic Tools for Control of Public Goods in Saccharomyces cerevisiae.

Microorganisms live in dense and diverse communities, with interactions between cells guiding community development and phenotype. The ability to perturb specific intercellular interactions in space and time provides a powerful route to determining the critical interactions and design rules for microbial communities. Approaches using optogenetic tools to modulate these interactions offer promise, as light can be exquisitely controlled in space and time. We report new plasmids for rapid integration of an optogenetic system into Saccharomyces cerevisiae to engineer light control of expression of a gene of interest. In a proof-of-principle study, we demonstrate the ability to control a model cooperative interaction, namely, the expression of the enzyme invertase (SUC2) which allows S. cerevisiae to hydrolyze sucrose and utilize it as a carbon source. We demonstrate that the strength of this cooperative interaction can be tuned in space and time by modulating light intensity and through spatial control of illumination. Spatial control of light allows cooperators and cheaters to be spatially segregated, and we show that the interplay between cooperative and inhibitory interactions in space can lead to pattern formation. Our strategy can be applied to achieve spatiotemporal control of expression of a gene of interest in S. cerevisiae to perturb both intercellular and interspecies interactions. IMPORTANCE Recent advances in microbial ecology have highlighted the importance of intercellular interactions in controlling the development, composition, and resilience of microbial communities. In order to better understand the role of these interactions in governing community development, it is critical to be able to alter them in a controlled manner. Optogenetically controlled interactions offer advantages over static perturbations or chemically controlled interactions, as light can be manipulated in space and time and does not require the addition of nutrients or antibiotics. Here, we report a system for rapidly achieving light control of a gene of interest in the important model organism Saccharomyces cerevisiae and demonstrate that by controlling expression of the enzyme invertase, we can control cooperative interactions. This approach will be useful for understanding intercellular and interspecies interactions in natural and synthetic microbial consortia containing S. cerevisiae and serves as a proof of principle for implementing this approach in other consortia.

Automated calibration of optoPlate LEDs to reduce light dose variation in optogenetic experiments.

Optogenetic systems use light to precisely control and investigate cellular processes. Until recently, there had been few instruments available for applying controlled light doses to cultures of cells. The optoPlate, a programmable array of 192 LEDs, was developed to meet this need. However, LED performance varies, and without calibration there are substantial brightness differences between LEDs on an optoPlate. Here we present a method for calibrating an optoPlate that uses a programmable microscope stage and optical power meter to automatically measure all 192 LEDs of an optoPlate. The resulting brightness measurements are used to calculate calibration values that tune the electrical current supplied to each optoPlate LED to reduce brightness variation in optogenetic experiments.