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1. Inferring the structures of signaling motifs from paired dynamic traces of single cells

   https://doi.org/10.1371/journal.pcbi.1008657  

   Haggerty, Raymond A. ; Purvis, Jeremy E. ( February 2021 , PLOS Computational Biology)

   Asthagiri, Anand R. (Ed.)

   Individual cells show variability in their signaling dynamics that often correlates with phenotypic responses, indicating that cell-to-cell variability is not merely noise but can have functional consequences. Based on this observation, we reasoned that cell-to-cell variability under the same treatment condition could be explained in part by a single signaling motif that maps different upstream signals into a corresponding set of downstream responses. If this assumption holds, then repeated measurements of upstream and downstream signaling dynamics in a population of cells could provide information about the underlying signaling motif for a given pathway, even when no prior knowledge of that motif exists. To test these two hypotheses, we developed a computer algorithm called MISC (Motif Inference from Single Cells) that infers the underlying signaling motif from paired time-series measurements from individual cells. When applied to measurements of transcription factor and reporter gene expression in the yeast stress response, MISC predicted signaling motifs that were consistent with previous mechanistic models of transcription. The ability to detect the underlying mechanism became less certain when a cell’s upstream signal was randomly paired with another cell’s downstream response, demonstrating how averaging time-series measurements across a population obscures information about the underlying signaling mechanism. In some cases, motif predictions improved as more cells were added to the analysis. These results provide evidence that mechanistic information about cellular signaling networks can be systematically extracted from the dynamical patterns of single cells. 

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2. Regulation of biotic interactions and responses to abiotic stresses by MAP kinase pathways in plant pathogenic fungi

   https://doi.org/10.1007/s44154-021-00004-3  

   Zhang, Xue ; Wang, Zeyi ; Jiang, Cong ; Xu, Jin-Rong ( December 2021 , Stress Biology)

   Abstract Like other eukaryotes, fungi use MAP kinase (MAPK) pathways to mediate cellular changes responding to external stimuli. In the past two decades, three well-conserved MAP kinase pathways have been characterized in various plant pathogenic fungi for regulating responses and adaptations to a variety of biotic and abiotic stresses encountered during plant infection or survival in nature. The invasive growth (IG) pathway is homologous to the yeast pheromone response and filamentation pathways. In plant pathogens, the IG pathway often is essential for pathogenesis by regulating infection-related morphogenesis, such as appressorium formation, penetration, and invasive growth. The cell wall integrity (CWI) pathway also is important for plant infection although the infection processes it regulates vary among fungal pathogens. Besides its universal function in cell wall integrity, it often plays a minor role in responses to oxidative and cell wall stresses. Both the IG and CWI pathways are involved in regulating known virulence factors as well as effector genes during plant infection and mediating defenses against mycoviruses, bacteria, and other fungi. In contrast, the high osmolarity growth (HOG) pathway is dispensable for virulence in some fungi although it is essential for plant infection in others. It regulates osmoregulation in hyphae and is dispensable for appressorium turgor generation. The HOG pathway also plays a major role for responding to oxidative, heat, and other environmental stresses and is overstimulated by phenylpyrrole fungicides. Moreover, these three MAPK pathways crosstalk and coordinately regulate responses to various biotic and abiotic stresses. The IG and CWI pathways, particularly the latter, also are involved in responding to abiotic stresses to various degrees in different fungal pathogens, and the HOG pathway also plays a role in interactions with other microbes or fungi. Furthermore, some infection processes or stress responses are co-regulated by MAPK pathways with cAMP or Ca 2+ /CaM signaling. Overall, functions of individual MAP kinase pathways in pathogenesis and stress responses have been well characterized in a number of fungal pathogens, showing the conserved genetic elements with diverged functions, likely by rewiring transcriptional regulatory networks. In the near future, applications of genomics and proteomics approaches will likely lead to better understanding of crosstalk among the MAPKs and with other signaling pathways as well as roles of MAPKs in defense against other microbes (biotic interactions). 

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3. Defining the role of TRPM4 in broadly responsive taste receptor cells

   https://doi.org/10.3389/fncel.2023.1148995  

   Dutta Banik, Debarghya ; Medler, Kathryn F. ( March 2023 , Frontiers in Cellular Neuroscience)

   Peripheral taste receptor cells use multiple signaling pathways to transduce taste stimuli into output signals that are sent to the brain. We have previously identified a subpopulation of Type III taste cells that are broadly responsive (BR) and respond to multiple taste stimuli including bitter, sweet, umami, and sour. These BR cells use a PLCβ3/IP₃R1 signaling pathway to detect bitter, sweet, and umami stimuli and use a separate pathway to detect sour. Currently, the downstream targets of the PLCβ3 signaling pathway are unknown. Here we identify TRPM4, a monovalent selective TRP channel, as an important downstream component in this signaling pathway. Using live cell imaging on isolated taste receptor cells from mice, we show that inhibition of TRPM4 abolished the taste-evoked sodium responses and significantly reduced the taste-evoked calcium responses in BR cells. Since BR cells are a subpopulation of Type III taste cells, they have conventional chemical synapses that require the activation of voltage-gated calcium channels (VGCCs) to cause neurotransmitter release. We found that TRPM4-dependent membrane depolarization selectively activates L-type VGCCs in these cells. The calcium influx through L-type VGCCs also generates a calcium-induced calcium release (CICR) via ryanodine receptors that enhances TRPM4 activity. Together these signaling events amplify the initial taste response to generate an appropriate output signal.

    

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4. Structural determinants driving homoserine lactone ligand selection in the Pseudomonas aeruginosa LasR quorum-sensing receptor

   https://doi.org/10.1073/pnas.1817239116  

   McCready, Amelia R. ; Paczkowski, Jon E. ; Henke, Brad R. ; Bassler, Bonnie L. ( December 2018 , Proceedings of the National Academy of Sciences)

   Quorum sensing is a cell–cell communication process that bacteria use to orchestrate group behaviors. Quorum sensing is mediated by signal molecules called autoinducers. Autoinducers are often structurally similar, raising questions concerning how bacteria distinguish among them. Here, we use thePseudomonas aeruginosaLasR quorum-sensing receptor to explore signal discrimination. The cognate autoinducer, 3OC₁₂homoserine lactone (3OC₁₂HSL), is a more potent activator of LasR than other homoserine lactones. However, other homoserine lactones can elicit LasR-dependent quorum-sensing responses, showing that LasR displays ligand promiscuity. We identify mutants that alter which homoserine lactones LasR detects. Substitution at residue S129 decreases the LasR response to 3OC₁₂HSL, while enhancing discrimination against noncognate autoinducers. Conversely, the LasR L130F mutation increases the potency of 3OC₁₂HSL and other homoserine lactones. We solve crystal structures of LasR ligand-binding domains complexed with noncognate autoinducers. Comparison with existing structures reveals that ligand selectivity/sensitivity is mediated by a flexible loop near the ligand-binding site. We show that LasR variants with modified ligand preferences exhibit altered quorum-sensing responses to autoinducers in vivo. We suggest that possessing some ligand promiscuity endows LasR with the ability to optimally regulate quorum-sensing traits.

    

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5. Antiactivators prevent self-sensing in Pseudomonas aeruginosa quorum sensing

   https://doi.org/10.1073/pnas.2201242119  

   Smith, Parker ; Schuster, Martin ( June 2022 , Proceedings of the National Academy of Sciences)

   Quorum sensing is described as a widespread cell density-dependent signaling mechanism in bacteria. Groups of cells coordinate gene expression by secreting and responding to diffusible signal molecules. Theory, however, predicts that individual cells may short-circuit this mechanism by directly responding to the signals they produce irrespective of cell density. In this study, we characterize this self-sensing effect in the acyl-homoserine lactone quorum sensing system of Pseudomonas aeruginosa . We show that antiactivators, a set of proteins known to affect signal sensitivity, function to prevent self-sensing. Measuring quorum-sensing gene expression in individual cells at very low densities, we find that successive deletion of antiactivator genes qteE and qslA produces a bimodal response pattern, in which increasing proportions of constitutively induced cells coexist with uninduced cells. Comparing responses of signal-proficient and -deficient cells in cocultures, we find that signal-proficient cells show a much higher response in the antiactivator mutant background but not in the wild-type background. Our results experimentally demonstrate the antiactivator-dependent transition from group- to self-sensing in the quorum-sensing circuitry of P. aeruginosa . Taken together, these findings extend our understanding of the functional capacity of quorum sensing. They highlight the functional significance of antiactivators in the maintenance of group-level signaling and experimentally prove long-standing theoretical predictions. 

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