A Quantitative Multivariate Model of Human Dendritic Cell-T Helper Cell Communication.

Cell-cell communication involves a large number of molecular signals that function as words of a complex language whose grammar remains mostly unknown. Here, we describe an integrative approach involving (1) protein-level measurement of multiple communication signals coupled to output responses in receiving cells and (2) mathematical modeling to uncover input-output relationships and interactions between signals. Using human dendritic cell (DC)-T helper (Th) cell communication as a model, we measured 36 DC-derived signals and 17 Th cytokines broadly covering Th diversity in 428 observations. We developed a data-driven, computationally validated model capturing 56 already described and 290 potentially novel mechanisms of Th cell specification. By predicting context-dependent behaviors, we demonstrate a new function for IL-12p70 as an inducer of Th17 in an IL-1 signaling context. This work provides a unique resource to decipher the complex combinatorial rules governing DC-Th cell communication and guide their manipulation for vaccine design and immunotherapies.

Signals that control MAIT cell function in healthy and inflamed human tissues.

Mucosal-associated invariant T (MAIT) cells have a semi-invariant T-cell receptor that allows recognition of antigen in the context of the MHC class I-related (MR1) protein. Metabolic intermediates of the riboflavin synthesis pathway have been identified as MR1-restricted antigens with agonist properties. As riboflavin synthesis occurs in many bacterial species, but not human cells, it has been proposed that the main purpose of MAIT cells is antibacterial surveillance and protection. The majority of human MAIT cells secrete interferon-gamma (IFNg) upon activation, while some MAIT cells in tissues can also express IL-17. Given that MAIT cells are present in human barrier tissues colonized by a microbiome, MAIT cells must somehow be able to distinguish colonization from infection to ensure effector functions are only elicited when necessary. Importantly, MAIT cells have additional functional properties, including the potential to contribute to restoring tissue homeostasis by expression of CTLA-4 and secretion of the cytokine IL-22. A recent study provided compelling data indicating that the range of human MAIT cell functional properties is explained by plasticity rather than distinct lineages. This further underscores the necessity to better understand how different signals regulate MAIT cell function. In this review, we highlight what is known in regards to activating and inhibitory signals for MAIT cells with a specific focus on signals relevant to healthy and inflamed tissues. We consider the quantity, quality, and the temporal order of these signals on MAIT cell function and discuss the current limitations of computational tools to extrapolate which signals are received by MAIT cells in human tissues. Using lessons learned from conventional CD8 T cells, we also discuss how TCR signals may integrate with cytokine signals in MAIT cells to elicit distinct functional states.

Signal integration in chemoreceptor complexes.

Motile bacteria use large receptor arrays to detect chemical and physical stimuli in their environment, process this complex information, and accordingly bias their swimming in a direction they deem favorable. The chemoreceptor molecules form tripod-like trimers of receptor dimers through direct contacts between their cytoplasmic tips. A pair of trimers, together with a dedicated kinase enzyme, form a core signaling complex. Hundreds of core complexes network to form extended arrays. While considerable progress has been made in revealing the hierarchical structure of the array, the molecular properties underlying signal processing in these structures remain largely unclear. Here we analyzed the signaling properties of nonnetworked core complexes in live cells by following both conformational and kinase control responses to attractant stimuli and to output-biasing lesions at various locations in the receptor molecule. Contrary to the prevailing view that individual receptors are binary two-state devices, we demonstrate that conformational coupling between the ligand binding and the kinase-control receptor domains is, in fact, only moderate. In addition, we demonstrate communication between neighboring receptors through their trimer-contact domains that biases them to adopt similar signaling states. Taken together, these data suggest a view of signaling in receptor trimers that allows significant signal integration to occur within individual core complexes.

Beyond the marks: reader-effectors as drivers of epigenetics and chromatin engineering.

Chromatin is a system of proteins and DNA that regulates chromosome organization and gene expression in eukaryotes. Essential features that support these processes include biochemical marks on histones and DNA, 'writer' enzymes that generate or remove these marks and proteins that translate the marks into transcriptional regulation: reader-effectors. Here, we review recent studies that reveal how reader-effectors drive chromatin-mediated processes. Advances in proteomics and epigenomics have accelerated the discovery of chromatin marks and their correlation with gene states, outpacing our understanding of the corresponding reader-effectors. Therefore, we summarize the current state of knowledge and open questions about how reader-effectors impact cellular function and human disease and discuss how synthetic biology can deepen our knowledge of reader-effector activity.

Signaling is the pathway to macrophage function.

Tissue and inflammatory contexts are well appreciated to shape macrophage function to promote health or disease. However, there has been minimal progress towards understanding how these contexts modify signaling-to-transcription networks. Integration of mechanistic modeling and data-driven approaches will be crucial for investigating how cell state impacts macrophage decision-making.

Dynamics-driven allosteric stimulation of diguanylate cyclase activity in a red light-regulated phytochrome.

Sensor-effector proteins integrate information from different stimuli and transform this into cellular responses. Some sensory domains, like red-light responsive bacteriophytochromes, show remarkable modularity regulating a variety of effectors. One effector domain is the GGDEF diguanylate cyclase catalyzing the formation of the bacterial second messenger cyclic-dimeric-guanosine monophosphate. While critical signal integration elements have been described for different phytochromes, a generalized understanding of signal processing and communication over large distances, roughly 100 Å in phytochrome diguanylate cyclases, is missing. Here we show that dynamics-driven allostery is key to understanding signal integration on a molecular level. We generated protein variants stabilized in their far-red-absorbing Pfr state and demonstrated by analysis of conformational dynamics using hydrogen-deuterium exchange coupled to mass spectrometry that single amino acid replacements are accompanied by altered dynamics of functional elements throughout the protein. We show that the conformational dynamics correlate with the enzymatic activity of these variants, explaining also the increased activity of a non-photochromic variant. In addition, we demonstrate the functional importance of mixed Pfr/intermediate state dimers using a fast-reverting variant that still enables wild-type-like fold-changes of enzymatic stimulation by red light. This supports the functional role of single protomer activation in phytochromes, a property that might correlate with the non-canonical mixed Pfr/intermediate-state spectra observed for many phytochrome systems. We anticipate our results to stimulate research in the direction of dynamics-driven allosteric regulation of different bacteriophytochrome-based sensor-effectors. This will eventually impact design strategies for the creation of novel sensor-effector systems for enriching the optogenetic toolbox.

Characterization of p38α Signaling Networks in Cancer Cells Using Quantitative Proteomics and Phosphoproteomics.

p38α (encoded by MAPK14) is a protein kinase that regulates cellular responses to almost all types of environmental and intracellular stresses. Upon activation, p38α phosphorylates many substrates both in the cytoplasm and nucleus, allowing this pathway to regulate a wide variety of cellular processes. While the role of p38α in the stress response has been widely investigated, its implication in cell homeostasis is less understood. To investigate the signaling networks regulated by p38α in proliferating cancer cells, we performed quantitative proteomic and phosphoproteomic analyses in breast cancer cells in which this pathway had been either genetically targeted or chemically inhibited. Our study identified with high confidence 35 proteins and 82 phosphoproteins (114 phosphosites) that are modulated by p38α and highlighted the implication of various protein kinases, including MK2 and mTOR, in the p38α-regulated signaling networks. Moreover, functional analyses revealed an important contribution of p38α to the regulation of cell adhesion, DNA replication, and RNA metabolism. Indeed, we provide experimental evidence supporting that p38α facilitates cancer cell adhesion and showed that this p38α function is likely mediated by the modulation of the adaptor protein ArgBP2. Collectively, our results illustrate the complexity of the p38α-regulated signaling networks, provide valuable information on p38α-dependent phosphorylation events in cancer cells, and document a mechanism by which p38α can regulate cell adhesion.

Shining a light on bacterial environmental cue integration and its relation to metabolism.

The ability of a bacterium to successfully colonize its host is dependent on proper adaptation to its local environment. Environmental cues are diverse in nature, ranging from ions to bacterial-produced signals, and to host immune responses that can also be exploited by the bacteria as cues. Simultaneously, bacterial metabolism must be matched to the carbon and nitrogen sources available at a given time and location. While initial characterization of a bacterium's response to a given environmental cue or its ability to utilize a particular carbon/nitrogen source requires study of the signal in question in isolation, actual infection poses a situation where multiple signals are present concurrently. This perspective focuses on the untapped potential in uncovering and understanding how bacteria integrate their response to multiple concurrent environmental cues, and in elucidating the possible intrinsic coordination of bacterial environmental response with its metabolism.

Far-red light increases maize volatile emissions in response to volatile cues from neighbouring plants.

Plants perceive the presence and defence status of their neighbours through light and volatile cues, but how plants integrate both stimuli is poorly understood. We investigated if and how low Red to Far red light (R:FR) ratios, indicative of shading or canopy closure, affect maize (Zea mays) responses to herbivore-induced plant volatiles (HIPVs), including the green leaf volatile (Z)-3-hexenyl acetate. We modulated light signalling and perception by using FR supplementation and a phyB1phyB2 mutant, and we determined volatile release as a response readout. To gain mechanistic insights, we examined expression of volatile biosynthesis genes, hormone accumulation, and photosynthesis. Exposure to a full blend of HIPVs or (Z)-3-hexenyl acetate induced maize volatile release. Short-term FR supplementation increased this response. In contrast, prolonged FR supplementation or constitutive phytochrome B inactivation in phyB1phyB2 plants showed the opposite response. Short-term FR supplementation enhanced photosynthesis and stomatal conductance and (Z)-3-hexenyl acetate-induced JA-Ile levels. We conclude that a FR-enriched light environment can prompt maize plants to respond more strongly to HIPVs emitted by neighbours, which might be explained by changes in photosynthetic processes and phytochrome B signalling. Our findings reveal interactive responses to light and volatile cues with potentially important consequences for plant-plant and plant-herbivore interactions.

Multikinase Networks: Two-Component Signaling Networks Integrating Multiple Stimuli.

Bacteria depend on two-component systems to detect and respond to threats. Simple pathways comprise a single sensor kinase (SK) that detects a signal and activates a response regulator protein to mediate an appropriate output. These simple pathways with only a single SK are not well suited to making complex decisions where multiple different stimuli need to be evaluated. A recently emerging theme is the existence of multikinase networks (MKNs) where multiple SKs collaborate to detect and integrate numerous different signals to regulate a major lifestyle switch, e.g., between virulence, sporulation, biofilm formation, and cell division. In this review, the role of MKNs and the phosphosignaling mechanisms underpinning their signal integration and decision making are explored.

A logical way to reprogram plants.

Living cells constantly monitor their external and internal environments for changing conditions, stresses or developmental cues. Networks of genetically encoded components sense and process these signals following pre-defined rules in such a way that specific combinations of the presence or absence of certain signals activate suitable responses. Many biological signal integration mechanisms approximate Boolean logic operations, whereby presence or absence of signals are computed as variables with values described as either true or false, respectively. Boolean logic gates are commonly used in algebra and in computer sciences, and have long been recognized as useful information processing devices in electronic circuits. In these circuits, logic gates integrate multiple input values and produce an output signal according to pre-defined Boolean logic operations. Recent implementation of these logic operations using genetic components to process information in living cells has allowed genetic circuits to enable novel traits with decision-making capabilities. Although several literature reports describe the design and use of these logic gates to introduce new functions in bacterial, yeast and mammalian cells, similar approaches in plants remain scarce, likely due to challenges posed by the complexity of plants and the lack of some technological advances, e.g., species-independent genetic transformation. In this mini review, we have surveyed recent reports describing synthetic genetic Boolean logic operators in plants and the different gate architectures used. We also briefly discuss the potential of deploying these genetic devices in plants to bring to fruition a new generation of resilient crops and improved biomanufacturing platforms.

Mitotic and pheromone-specific intrinsic polarization cues interfere with gradient sensing in Saccharomyces cerevisiae.

Polarity decisions are central to many processes, including mitosis and chemotropism. In Saccharomyces cerevisiae, budding and mating projection (MP) formation use an overlapping system of cortical landmarks that converges on the small G protein Cdc42. However, pheromone-gradient sensing must override the Rsr1-dependent internal polarity cues used for budding. Using this model system, we asked what happens when intrinsic and extrinsic spatial cues are not aligned. Is there competition, or collaboration? By live-cell microscopy and microfluidics techniques, we uncovered three previously overlooked features of this signaling system. First, the cytokinesis-associated polarization patch serves as a polarity landmark independently of all known cues. Second, the Rax1-Rax2 complex functions as a pheromone-promoted polarity cue in the distal pole of the cells. Third, internal cues remain active during pheromone-gradient tracking and can interfere with this process, biasing the location of MPs. Yeast defective in internal-cue utilization align significantly better than wild type with artificially generated pheromone gradients.

Stimulus Reliability Automatically Biases Temporal Integration of Discrete Perceptual Targets in the Human Brain.

Many decisions, from crossing a busy street to choosing a profession, require integration of discrete sensory events. Previous studies have shown that integrative decision-making favors more reliable stimuli, mimicking statistically optimal integration. It remains unclear, however, whether reliability biases operate even when they lead to suboptimal performance. To address this issue, we asked human observers to reproduce the average motion direction of two suprathreshold coherent motion signals presented successively and with varying levels of reliability, while simultaneously recording whole-brain activity using electroencephalography. By definition, the averaging task should engender equal weighting of the two component motion signals, but instead we found robust behavioral biases in participants' average decisions that favored the more reliable stimulus. Using population-tuning modeling of brain activity we characterized tuning to the average motion direction. In keeping with the behavioral biases, the neural tuning profiles also exhibited reliability biases. A control experiment revealed that observers were able to reproduce motion directions of low and high reliability with equal precision, suggesting that unbiased integration in this task was not only theoretically optimal but demonstrably possible. Our findings reveal that temporal integration of discrete sensory events in the brain is automatically and suboptimally weighted according to stimulus reliability.SIGNIFICANCE STATEMENT Many everyday decisions require integration of several sources of information. To safely cross a busy road, for example, one must consider the movement of vehicles with different speeds and trajectories. Previous research has shown that individual stimuli are weighted according to their reliability. Whereas reliability biases typically yield performance that closely mimics statistically optimal integration, it remains unknown whether such biases arise even when they lead to suboptimal performance. Here we combined a novel integrative decision-making task with concurrent brain recording and modeling to address this question. While unbiased decisions were optimal in the task, observers nevertheless exhibited robust reliability biases in both behavior and brain activity, suggesting that reliability-weighted integration is automatic and dissociable from statistically optimal integration.

Integration of reactive oxygen species and hormone signaling during abiotic stress.

Each year, abiotic stress conditions such as drought, heat, salinity, cold and particularly their different combinations, inflict a heavy toll on crop productivity worldwide. The effects of these adverse conditions on plant productivity are becoming ever more alarming in recent years in light of the increased rate and intensity of global climatic changes. Improving crop tolerance to abiotic stress conditions requires a deep understanding of the response of plants to changes in their environment. This response is dependent on early and late signal transduction events that involve important signaling molecules such as reactive oxygen species (ROS), different plant hormones and other signaling molecules. It is the integration of these signaling events, mediated by an interplay between ROS and different plant hormones that orchestrates the plant response to abiotic stress and drive changes in transcriptomic, metabolic and proteomic networks that lead to plant acclimation and survival. Here we review some of the different studies that address hormone and ROS integration during the response of plants to abiotic stress. We further highlight the integration of ROS and hormone signaling during early and late phases of the plant response to abiotic stress, the key role of respiratory burst oxidase homologs in the integration of ROS and hormone signaling during these phases, and the involvement of hormone and ROS in systemic signaling events that lead to systemic acquired acclimation. Lastly, we underscore the need to understand the complex interactions that occur between ROS and different plant hormones during stress combinations.

Distinct roles and requirements for Ras pathway signaling in visceral versus somatic muscle founder specification.

Pleiotropic signaling pathways must somehow engender specific cellular responses. In the Drosophila mesoderm, Ras pathway signaling specifies muscle founder cells from among the broader population of myoblasts. For somatic muscles, this is an inductive process mediated by the ETS-domain downstream Ras effectors Pointed and Aop (Yan). We demonstrate here that for the circular visceral muscles, despite superficial similarities, a significantly different specification mechanism is at work. Not only is visceral founder cell specification not dependent on Pointed or Aop, but Ras pathway signaling in its entirety can be bypassed. Our results show that de-repression, not activation, is the predominant role of Ras signaling in the visceral mesoderm and that, accordingly, Ras signaling is not required in the absence of repression. The key repressor acts downstream of the transcription factor Lame duck and is likely a member of the ETS transcription factor family. Our findings fit with a growing body of data that point to a complex interplay between the Ras pathway, ETS transcription factors, and enhancer binding as a crucial mechanism for determining unique responses to Ras signaling.

Vasopressin Resets the Central Circadian Clock in a Manner Influenced by Sex and Vasoactive Intestinal Polypeptide Signaling.

BACKGROUND/AIMS: Circadian rhythms in behavior and physiology are programmed by the suprachiasmatic nucleus (SCN) of the hypothalamus. A subset of SCN neurons produce the neuropeptide arginine vasopressin (AVP), but it remains unclear whether AVP signaling influences the SCN clock directly. METHODS: Here, we test that AVP signaling acting through V1A and V1B receptors influences molecular rhythms in SCN neurons. V1 receptor agonists were applied ex vivo to PERIOD2::LUCIFERASE SCN slices, allowing for real-time monitoring of changes in molecular clock function. RESULTS: V1A/B agonists reset the phase of the SCN molecular clock in a time-dependent manner, with larger magnitude responses by the female SCN. Further, we found evidence that both Gαq and Gαs signaling pathways interact with V1A/B-induced SCN resetting, and that this response requires vasoactive intestinal polypeptide (VIP) signaling. CONCLUSIONS: Collectively, this work indicates that AVP signaling resets SCN molecular rhythms in conjunction with VIP signaling and in a manner influenced by sex. This highlights the utility of studying clock function in both sexes and suggests that signal integration in central clock circuits regulates emergent properties important for the control of daily rhythms in behavior and physiology.

Structural basis of molecular logic OR in a dual-sensor histidine kinase.

Signal detection and integration by sensory proteins constitute the critical molecular events as living organisms respond to changes in a complex environment. Many sensory proteins adopt a modular architecture that integrates the perception of distinct chemical or physical signals and the generation of a biological response in the same protein molecule. Currently, how signal perception and integration are achieved in such a modular, often dimeric, framework remains elusive. Here, we report a dynamic crystallography study on the tandem sensor domains of a dual-sensor histidine kinase PPHK (phosphorylation-responsive photosensitive histidine kinase) that operates a molecular logic OR, by which the output kinase activity is modulated by a phosphorylation signal and a light signal. A joint analysis of ∼170 crystallographic datasets probing different signaling states shows remarkable dimer asymmetry as PPHK responds to the input signals and transitions from one state to the other. Supported by mutational data and structural analysis, these direct observations reveal the working mechanics of the molecular logic OR in PPHK, where the light-induced bending of a long signaling helix at the dimer interface is counteracted by the ligand-induced structural changes from a different sensor domain. We propose that the logic OR of PPHK, together with an upstream photoreceptor, implements a "long-pass" red light response distinct from those accomplished by classical phytochromes.

BR-dependent phosphorylation modulates PIF4 transcriptional activity and shapes diurnal hypocotyl growth.

Signaling by the hormones brassinosteroid (BR) and gibberellin (GA) is critical to normal plant growth and development and is required for hypocotyl elongation in response to dark and elevated temperatures. Active BR signaling is essential for GA promotion of hypocotyl growth and suppresses the dwarf phenotype of GA mutants. Cross-talk between these hormones occurs downstream from the DELLAs, as GA-induced destabilization of these GA signaling repressors is not affected by BRs. Here we show that the light-regulated PIF4 (phytochrome-interacting factor 4) factor is a phosphorylation target of the BR signaling kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2), which marks this transcriptional regulator for proteasome degradation. Expression of a mutated PIF41A protein lacking a conserved BIN2 phosphorylation consensus causes a severe elongated phenotype and strongly up-regulated expression of the gene targets. However, PIF41A is not able to suppress the dwarf phenotype of the bin2-1 mutant with constitutive activation of this kinase. PIFs were shown to be required for the constitutive BR response of bes1-D and bzr1-1D mutants, these factors acting in an interdependent manner to promote cell elongation. Here, we show that bes1-D seedlings are still repressed by the inhibitor BRZ in the light and that expression of the nonphosphorylatable PIF41A protein makes this mutant fully insensitive to brassinazole (BRZ). PIF41A is preferentially stabilized at dawn, coinciding with the diurnal time of maximal growth. These results uncover a main role of BRs in antagonizing light signaling by inhibiting BIN2-mediated destabilization of the PIF4 factor. This regulation plays a prevalent role in timing hypocotyl elongation to late night, before light activation of phytochrome B (PHYB) and accumulation of DELLAs restricts PIF4 transcriptional activity.

Plant photoreceptors: Multi-functional sensory proteins and their signaling networks.

Light is a crucial environmental cue not only for photosynthetic energy production but also for plant growth and development. Plants employ sophisticated methods to detect and interpret information from incoming light. Five classes of photoreceptors have been discovered in the model plant Arabidopsis thaliana. These photoreceptors act either distinctly and/or redundantly in fine-tuning many aspects of plant life cycle. Unlike mobile animals, sessile plants have developed an enormous plasticity to adapt and survive in changing environment. By monitoring different information arising from ambient light, plants precisely regulate downstream signaling pathways to adapt accordingly. Given that changes in the light environment is typically synchronized with other environmental cues such as temperature, abiotic stresses, and seasonal changes, it is not surprising that light signaling pathways are interconnected with multiple pathways to regulate plant physiology and development. Indeed, recent advances in plant photobiology revealed a large network of co-regulation among different photoreceptor signaling pathways as well as other internal signaling pathways (e.g., hormone signaling). In addition, some photoreceptors are directly involved in perception of non-light stimuli (e.g., temperature). Therefore, understanding highly inter-connected signaling networks is essential to explore the photoreceptor functions in plants. Here, we summarize how plants co-ordinate multiple photoreceptors and their internal signaling pathways to regulate a myriad of downstream responses at molecular and physiological levels.

COLD REGULATED 27 and 28 are targets of CONSTITUTIVELY PHOTOMORPHOGENIC 1 and negatively affect phytochrome B signalling.

Phytochromes are red/far-red light receptors in plants involved in the regulation of growth and development. Phytochromes can sense the light environment and contribute to measuring day length; thereby, they allow plants to respond and adapt to changes in the ambient environment. Two well-characterized signalling pathways act downstream of phytochromes and link light perception to the regulation of gene expression. The CONSTITUTIVELY PHOTOMORPHOGENIC 1/SUPPRESSOR OF PHYA-105 (COP1/SPA) E3 ubiquitin ligase complex and the PHYTOCHROME INTERACTING FACTORs (PIFs) are key components of these pathways and repress light responses in the dark. In light-grown seedlings, phytochromes inhibit COP1/SPA and PIF activity and thereby promote light signalling. In a yeast-two-hybrid screen for proteins binding to light-activated phytochromes, we identified COLD-REGULATED GENE 27 (COR27). COR27 and its homologue COR28 bind to phyA and phyB, the two primary phytochromes in seed plants. COR27 and COR28 have been described previously with regard to a function in the regulation of freezing tolerance, flowering and the circadian clock. Here, we show that COR27 and COR28 repress early seedling development in blue, far-red and in particular red light. COR27 and COR28 contain a conserved Val-Pro (VP)-peptide motif, which mediates binding to the COP1/SPA complex. COR27 and COR28 are targeted for degradation by COP1/SPA and mutant versions with a VP to AA amino acid substitution in the VP-peptide motif are stabilized. Overall, our data suggest that COR27 and COR28 accumulate in light but act as negative regulators of light signalling during early seedling development, thereby preventing an exaggerated response to light.