dNEMO: a tool for quantification of mRNA and punctate structures in time-lapse images of single cells.

MOTIVATION: Many biological processes are regulated by single molecules and molecular assemblies within cells that are visible by microscopy as punctate features, often diffraction limited. Here, we present detecting-NEMO (dNEMO), a computational tool optimized for accurate and rapid measurement of fluorescent puncta in fixed-cell and time-lapse images. RESULTS: The spot detection algorithm uses the à trous wavelet transform, a computationally inexpensive method that is robust to imaging noise. By combining automated with manual spot curation in the user interface, fluorescent puncta can be carefully selected and measured against their local background to extract high-quality single-cell data. Integrated into the workflow are segmentation and spot-inspection tools that enable almost real-time interaction with images without time consuming pre-processing steps. Although the software is agnostic to the type of puncta imaged, we demonstrate dNEMO using smFISH to measure transcript numbers in single cells in addition to the transient formation of IKK/NEMO puncta from time-lapse images of cells exposed to inflammatory stimuli. We conclude that dNEMO is an ideal user interface for rapid and accurate measurement of fluorescent molecular assemblies in biological imaging data. AVAILABILITY AND IMPLEMENTATION: The data and software are freely available online at https://github.com/recleelab. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Fold change of nuclear NF-κB determines TNF-induced transcription in single cells.

In response to tumor necrosis factor (TNF), NF-κB enters the nucleus and promotes inflammatory and stress-responsive gene transcription. Because NF-κB deregulation is associated with disease, one might expect strict control of NF-κB localization. However, nuclear NF-κB levels exhibit considerable cell-to-cell variability, even in unstimulated cells. To resolve this paradox and determine how transcription-inducing signals are encoded, we quantified single-cell NF-κB translocation dynamics and transcription in the same cells. We show that TNF-induced transcription correlates best with fold change in nuclear NF-κB, not absolute nuclear NF-κB abundance. Using computational modeling, we find that an incoherent feedforward loop, from competition for binding to κB motifs, could provide memory of the preligand state necessary for fold-change detection. Experimentally, we observed three gene-specific transcriptional patterns that our model recapitulates by modulating competition strength alone. Fold-change detection buffers against stochastic variation in signaling molecules and explains how cells tolerate variability in NF-κB abundance and localization.

Parallel Tempering with Lasso for model reduction in systems biology.

Systems Biology models reveal relationships between signaling inputs and observable molecular or cellular behaviors. The complexity of these models, however, often obscures key elements that regulate emergent properties. We use a Bayesian model reduction approach that combines Parallel Tempering with Lasso regularization to identify minimal subsets of reactions in a signaling network that are sufficient to reproduce experimentally observed data. The Bayesian approach finds distinct reduced models that fit data equivalently. A variant of this approach that uses Lasso to perform selection at the level of reaction modules is applied to the NF-κB signaling network to test the necessity of feedback loops for responses to pulsatile and continuous pathway stimulation. Taken together, our results demonstrate that Bayesian parameter estimation combined with regularization can isolate and reveal core motifs sufficient to explain data from complex signaling systems.

Demystifying the cytokine network: Mathematical models point the way.

Cytokines provide the means by which immune cells communicate with each other and with parenchymal cells. There are over one hundred cytokines and many exist in families that share receptor components and signal transduction pathways, creating complex networks. Reductionist approaches to understanding the role of specific cytokines, through the use of gene-targeted mice, have revealed further complexity in the form of redundancy and pleiotropy in cytokine function. Creating an understanding of the complex interactions between cytokines and their target cells is challenging experimentally. Mathematical and computational modeling provides a robust set of tools by which complex interactions between cytokines can be studied and analyzed, in the process creating novel insights that can be further tested experimentally. This review will discuss and provide examples of the different modeling approaches that have been used to increase our understanding of cytokine networks. This includes discussion of knowledge-based and data-driven modeling approaches and the recent advance in single-cell analysis. The use of modeling to optimize cytokine-based therapies will also be discussed.

Metacaspase Yca1 is required for clearance of insoluble protein aggregates.

In complex organisms, caspase proteases mediate a variety of cell behaviors, including proliferation, differentiation, and programmed cell death/apoptosis. Structural homologs to the caspase family (termed metacaspases) engage apoptosis in single-cell eukaryotes, yet the molecular mechanisms that contribute to nondeath roles are currently undefined. Here, we report an unexpected role for the Saccharomyces cerevisiae metacaspase Yca1 in protein quality control. Quantitative proteomic analysis of Deltayca1 cells identified significant alterations to vacuolar catabolism and stress-response proteins in the absence of induced stress. Yca1 protein complexes are enriched for aggregate-remodeling chaperones that colocalize with Yca1-GFP fusions. Finally, deletion and inactivation mutants of Yca1 accrue protein aggregates and autophagic bodies during log-phase growth. Together, our results show that Yca1 contributes to the fitness and adaptability of growing yeast through an aggregate remodeling activity.

Long-term imaging of individual mRNA molecules in living cells.

Single-cell imaging of individual mRNAs has revealed core mechanisms of the central dogma. However, most approaches require cell fixation or have limited sensitivity for live-cell applications. Here, we describe SunRISER (SunTag-based reporter for imaging signal-enriched mRNA), a computationally and experimentally optimized approach for unambiguous detection of single mRNA molecules in living cells. When viewed by epifluorescence microscopy, SunRISER-labeled mRNAs show strong signal to background and resistance to photobleaching, which together enable long-term mRNA imaging studies. SunRISER variants, using 8× and 10× stem-loop arrays, demonstrate effective mRNA detection while significantly reducing alterations to target mRNA sequences. We characterize SunRISER to observe mRNA inheritance during mitosis and find that stressors enhance diversity among post-mitotic sister cells. Taken together, SunRISER enables a glimpse into living cells to observe aspects of the central dogma and the role of mRNAs in rare and dynamical trafficking events.

Label and quantify mRNA molecules in live cell experiments using SunRISER and dNEMO.

Visualization of mRNA molecules in single cells has revealed their core mechanisms as well as sources of cell-to-cell and spatiotemporal heterogeneity. Here, we describe a protocol to label, visualize, and quantify mRNA molecules by time-lapse imaging with the capability of resolving mRNA molecules over durations of hours to days. We provide links to mRNA-labeling plasmids as well as free software for a semi-automated image analysis pipeline. For complete details on the use and execution of this protocol, please refer to Guo and Lee (2022) and Kowalczyk et al. (2021).

Antibacterial and antitubercular activity of fosmidomycin, FR900098, and their lipophilic analogs.

The nonmevalonate pathway (NMP) of isoprene biosynthesis is an exciting new route toward novel antibiotic development. Inhibitors against several enzymes in this pathway are currently under examination. A significant liability of many of these agents is poor cell penetration. To overcome and improve our understanding of this problem, we have synthesized a series of lipophilic, prodrug analogs of fosmidomycin and FR900098, inhibitors of the NMP enzyme Dxr. Several of these compounds show improved antibacterial activity against a panel of organisms relative to the parent compound, including activity against Mycobacterium tuberculosis (Mtb). Our results show that this strategy can be an effective way for improving whole cell activity of NMP inhibitors.

A variable-gain stochastic pooling motif mediates information transfer from receptor assemblies into NF-κB.

A myriad of inflammatory cytokines regulate signaling pathways to maintain cellular homeostasis. The IκB kinase (IKK) complex is an integration hub for cytokines that govern nuclear factor κB (NF-κB) signaling. In response to inflammation, IKK is activated through recruitment to receptor-associated protein assemblies. How and what information IKK complexes transmit about the milieu are open questions. Here, we track dynamics of IKK complexes and nuclear NF-κB to identify upstream signaling features that determine same-cell responses. Experiments and modeling of single complexes reveal their size, number, and timing relays cytokine-specific control over shared signaling mechanisms with feedback regulation that is independent of transcription. Our results provide evidence for variable-gain stochastic pooling, a noise-reducing motif that enables cytokine-specific regulation and parsimonious information transfer. We propose that emergent properties of stochastic pooling are general principles of receptor signaling that have evolved for constructive information transmission in noisy molecular environments.

Reconstructing regulatory kinase pathways from phosphopeptide data: a bioinformatics approach.

Protein phosphorylation is a widespread cellular process, and simplistic linear pathway models of kinase signaling likely under-represent the complexity of in vivo pathways. The recent massive increase in information available through protein interaction databases now allows construction of in silico models of protein networks that are underpinned by evidence from real biological systems. By combining protein phosphorylation data with current databases of protein-protein and kinase-substrate interactions, sophisticated models of intracellular protein phosphorylation signaling can be constructed for a system of interest. The kinase interaction network can be visualized, analyzed by graph theory, and investigated for hypotheses that are not otherwise obvious.

A System for Analog Control of Cell Culture Dynamics to Reveal Capabilities of Signaling Networks.

Cellular microenvironments are dynamic. When exposed to extracellular cues, such as changing concentrations of inflammatory cytokines, cells activate signaling networks that mediate fate decisions. Exploring responses broadly to time-varying microenvironments is essential to understand the information transmission capabilities of signaling networks and how dynamic milieus influence cell fate decisions. Here, we present a gravity-driven cell culture and demonstrate that the system accurately produces user-defined concentration profiles for one or more dynamic stimuli. As proof of principle, we monitor nuclear factor-κB activation in single cells exposed to dynamic cytokine stimulation and reveal context-dependent sensitivity and uncharacterized single-cell response classes distinct from persistent stimulation. Using computational modeling, we find that cell-to-cell variability in feedback rates within the signaling network contributes to different response classes. Models are validated using inhibitors to predictably modulate response classes in live cells exposed to dynamic stimuli. These hidden capabilities, uncovered through dynamic stimulation, provide opportunities to discover and manipulate signaling mechanisms.

A network-centric approach to drugging TNF-induced NF-κB signaling.

Target-centric drug development strategies prioritize single-target potency in vitro and do not account for connectivity and multi-target effects within a signal transduction network. Here, we present a systems biology approach that combines transcriptomic and structural analyses with live-cell imaging to predict small molecule inhibitors of TNF-induced NF-κB signaling and elucidate the network response. We identify two first-in-class small molecules that inhibit the NF-κB signaling pathway by preventing the maturation of a rate-limiting multiprotein complex necessary for IKK activation. Our findings suggest that a network-centric drug discovery approach is a promising strategy to evaluate the impact of pharmacologic intervention in signaling.

Dopamine D2 receptor modulates Wnt expression and control of cell proliferation.

The Wnt/ß-catenin pathway is one of the most conserved signaling pathways across species with essential roles in development, cell proliferation, and disease. Wnt signaling occurs at the protein level and via ß-catenin-mediated transcription of target genes. However, little is known about the underlying mechanisms regulating the expression of the key Wnt ligand Wnt3a or the modulation of its activity. Here, we provide evidence that there is significant cross-talk between the dopamine D2 receptor (D2R) and Wnt/ß-catenin signaling pathways. Our data suggest that D2R-dependent cross-talk modulates Wnt3a expression via an evolutionarily-conserved TCF/LEF site within the WNT3A promoter. Moreover, D2R signaling also modulates cell proliferation and modifies the pathology in a renal ischemia/reperfusion-injury disease model, via its effects on Wnt/ß-catenin signaling. Together, our results suggest that D2R is a transcriptional modulator of Wnt/ß-catenin signal transduction with broad implications for health and development of new therapeutics.

Evaluation of Parallel Tempering to Accelerate Bayesian Parameter Estimation in Systems Biology.

Models of biological systems often have many unknown parameters that must be determined in order for model behavior to match experimental observations. Commonly-used methods for parameter estimation that return point estimates of the best-fit parameters are insufficient when models are high dimensional and under-constrained. As a result, Bayesian methods, which treat model parameters as random variables and attempt to estimate their probability distributions given data, have become popular in systems biology. Bayesian parameter estimation often relies on Markov Chain Monte Carlo (MCMC) methods to sample model parameter distributions, but the slow convergence of MCMC sampling can be a major bottleneck. One approach to improving performance is parallel tempering (PT), a physics-based method that uses swapping between multiple Markov chains run in parallel at different temperatures to accelerate sampling. The temperature of a Markov chain determines the probability of accepting an unfavorable move, so swapping with higher temperatures chains enables the sampling chain to escape from local minima. In this work we compared the MCMC performance of PT and the commonly-used Metropolis-Hastings (MH) algorithm on six biological models of varying complexity. We found that for simpler models PT accelerated convergence and sampling, and that for more complex models, PT often converged in cases MH became trapped in non-optimal local minima. We also developed a freely-available MATLAB package for Bayesian parameter estimation called PTEMPEST (http://github.com/RuleWorld/ptempest), which is closely integrated with the popular BioNetGen software for rule-based modeling of biological systems.

Shift from stochastic to spatially-ordered expression of serine-glycine synthesis enzymes in 3D microtumors.

Cell-to-cell differences in protein expression in normal tissues and tumors are a common phenomenon, but the underlying principles that govern this heterogeneity are largely unknown. Here, we show that in monolayer cancer cell-line cultures, the expression of the five metabolic enzymes of serine-glycine synthesis (SGS), including its rate-limiting enzyme, phosphoglycerate dehydrogenase (PHGDH), displays stochastic cell-to-cell variation. By contrast, in cancer cell line-derived three-dimensional (3D) microtumors PHGDH expression is restricted to the outermost part of the microtumors' outer proliferative cell layer, while the four other SGS enzymes display near uniform expression throughout the microtumor. A mathematical model suggests that metabolic stress in the microtumor core activates factors that restrict PHGDH expression. Thus, intracellular enzyme expression in growing cell ecosystems can shift to spatially ordered patterns in 3D structured environments due to emergent cell-cell communication, with potential implications for the design of effective anti-metabolic cancer therapies.

NF-κB-Chromatin Interactions Drive Diverse Phenotypes by Modulating Transcriptional Noise.

Noisy gene expression generates diverse phenotypes, but little is known about mechanisms that modulate noise. Combining experiments and modeling, we studied how tumor necrosis factor (TNF) initiates noisy expression of latent HIV via the transcription factor nuclear factor κB (NF-κB) and how the HIV genomic integration site modulates noise to generate divergent (low-versus-high) phenotypes of viral activation. We show that TNF-induced transcriptional noise varies more than mean transcript number and that amplification of this noise explains low-versus-high viral activation. For a given integration site, live-cell imaging shows that NF-κB activation correlates with viral activation, but across integration sites, NF-κB activation cannot account for differences in transcriptional noise and phenotypes. Instead, differences in transcriptional noise are associated with differences in chromatin state and RNA polymerase II regulation. We conclude that, whereas NF-κB regulates transcript abundance in each cell, the chromatin environment modulates noise in the population to support diverse HIV activation in response to TNF.

Discovery of novel isoxazolines as anti-tuberculosis agents.

Nitrofuranyl isoxazolines with increased proteolytic stability over nitrofuranyl amides were designed and synthesized leading to discovery of several compounds with potent in vitro anti-tuberculosis activity. However, their in vivo activity was limited by high protein binding and poor distribution. Consequently, a series of non-nitrofuran containing isoxazolines were prepared to determine if the core had residual anti-tuberculosis activity. This led to the discovery of novel isoxazoline 12 as anti-tuberculosis agent with a MIC(90) value of 1.56microg/mL.

Solid-phase synthesis and biological evaluation of a uridinyl branched peptide urea library.

A 1000-member uridinyl branched peptide library was synthesized on PS-DES support using IRORI technology. High-throughput screening of this library for anti-tuberculosis activity identified several members with a MIC(90) value of 12.5 microg/mL.

NF-κB Dynamics Discriminate between TNF Doses in Single Cells.

Although cytokine-dependent dynamics of nuclear factor κB (NF-κB) are known to encode information that regulates cell fate decisions, it is unclear whether single-cell responses are switch-like or encode more information about cytokine dose. Here, we measure the dynamic subcellular localization of NF-κB in response to a range of tumor necrosis factor (TNF) stimulation conditions to determine the prevailing mechanism of single-cell dose discrimination. Using an information theory formalism that accounts for signaling dynamics and non-responsive cell subpopulations, we find that the information transmission capacity of single cells exceeds that predicted from a switch-like response. Instead, we observe that NF-κB dynamics within single cells contain sufficient information to encode multiple, TNF-dependent cellular states, and have an activation threshold that varies across the population. By comparing single-cell responses to an internal, experimentally observed reference, we demonstrate that cells can grade responses to TNF across several orders of magnitude in concentration. This suggests that cells contain additional control points to fine-tune their cytokine responses beyond the decision to activate.

NF-κB signalling and cell fate decisions in response to a short pulse of tumour necrosis factor.

In tissues and tumours, cell behaviours are regulated by multiple time-varying signals. While in the laboratory cells are often exposed to a stimulus for the duration of the experiment, in vivo exposures may be much shorter. In this study, we monitored NF-κB and caspase signalling in human cancer cells treated with a short pulse of Tumour Necrosis Factor (TNF). TNF is an inflammatory cytokine that can induce both the pro-survival NF-κB-driven gene transcription pathway and the pro-apoptotic caspase pathway. We find that a few seconds of exposure to TNF is sufficient to activate the NF-κB pathway in HeLa cells and induce apoptotic cell death in both HeLa and Kym-1 cells. Strikingly, a 1-min pulse of TNF can be more effective at killing than a 1-hour pulse, indicating that in addition to TNF concentration, duration of exposure also coordinates cell fate decisions.