The Nicotinic Acetylcholine Receptor and Its Pentameric Homologs: Toward an Allosteric Mechanism of Signal Transduction at the Atomic Level.

The nicotinic acetylcholine receptor has served, since its biochemical identification in the 1970s, as a model of an allosteric ligand-gated ion channel mediating signal transition at the synapse. In recent years, the application of X-ray crystallography and high-resolution cryo-electron microscopy, together with molecular dynamic simulations of nicotinic receptors and homologs, have opened a new era in the understanding of channel gating by the neurotransmitter. They reveal, at atomic resolution, the diversity and flexibility of the multiple ligand-binding sites, including recently discovered allosteric modulatory sites distinct from the neurotransmitter orthosteric site, and the conformational dynamics of the activation process as a molecular switch linking these multiple sites. The model emerging from these studies paves the way for a new pharmacology based, first, upon the occurrence of an original mode of indirect allosteric modulation, distinct from a steric competition for a single and rigid binding site, and second, the design of drugs that specifically interact with privileged conformations of the receptor such as agonists, antagonists, and desensitizers. Research on nicotinic receptors is still at the forefront of understanding the mode of action of drugs on the nervous system.

Signaling from RAS to RAF: The Molecules and Their Mechanisms.

RAF family protein kinases are a key node in the RAS/RAF/MAP kinase pathway, the signaling cascade that controls cellular proliferation, differentiation, and survival in response to engagement of growth factor receptors on the cell surface. Over the past few years, structural and biochemical studies have provided new understanding of RAF autoregulation, RAF activation by RAS and the SHOC2 phosphatase complex, and RAF engagement with HSP90-CDC37 chaperone complexes. These studies have important implications for pharmacologic targeting of the pathway. They reveal RAF in distinct regulatory states and show that the functional RAF switch is an integrated complex of RAF with its substrate (MEK) and a 14-3-3 dimer. Here we review these advances, placing them in the context of decades of investigation of RAF regulation. We explore the insights they provide into aberrant activation of the pathway in cancer and RASopathies (developmental syndromes caused by germline mutations in components of the pathway).

Light-induced remodeling of phytochrome B enables signal transduction by phytochrome-interacting factor.

Phytochrome B (phyB) and phytochrome-interacting factors (PIFs) constitute a well-established signaling module critical for plants adapting to ambient light. However, mechanisms underlying phyB photoactivation and PIF binding for signal transduction remain elusive. Here, we report the cryo-electron microscopy (cryo-EM) structures of the photoactivated phyB or the constitutively active phyBY276H mutant in complex with PIF6, revealing a similar trimer. The light-induced configuration switch of the chromophore drives a conformational transition of the nearby tongue signature within the phytochrome-specific (PHY) domain of phyB. The resulting α-helical PHY tongue further disrupts the head-to-tail dimer of phyB in the dark-adapted state. These structural remodelings of phyB facilitate the induced-fit recognition of PIF6, consequently stabilizing the N-terminal extension domain and a head-to-head dimer of activated phyB. Interestingly, the phyB dimer exhibits slight asymmetry, resulting in the binding of only one PIF6 molecule. Overall, our findings solve a key question with respect to how light-induced remodeling of phyB enables PIF signaling in phytochrome research.

Gut-liver axis calibrates intestinal stem cell fitness.

The gut and liver are recognized to mutually communicate through the biliary tract, portal vein, and systemic circulation. However, it remains unclear how this gut-liver axis regulates intestinal physiology. Through hepatectomy and transcriptomic and proteomic profiling, we identified pigment epithelium-derived factor (PEDF), a liver-derived soluble Wnt inhibitor, which restrains intestinal stem cell (ISC) hyperproliferation to maintain gut homeostasis by suppressing the Wnt/ß-catenin signaling pathway. Furthermore, we found that microbial danger signals resulting from intestinal inflammation can be sensed by the liver, leading to the repression of PEDF production through peroxisome proliferator-activated receptor-α (PPARα). This repression liberates ISC proliferation to accelerate tissue repair in the gut. Additionally, treating mice with fenofibrate, a clinical PPARα agonist used for hypolipidemia, enhances colitis susceptibility due to PEDF activity. Therefore, we have identified a distinct role for PEDF in calibrating ISC expansion for intestinal homeostasis through reciprocal interactions between the gut and liver.

The phosphorylation landscape of infection-related development by the rice blast fungus.

Many of the world's most devastating crop diseases are caused by fungal pathogens that elaborate specialized infection structures to invade plant tissue. Here, we present a quantitative mass-spectrometry-based phosphoproteomic analysis of infection-related development by the rice blast fungus Magnaporthe oryzae, which threatens global food security. We mapped 8,005 phosphosites on 2,062 fungal proteins following germination on a hydrophobic surface, revealing major re-wiring of phosphorylation-based signaling cascades during appressorium development. Comparing phosphosite conservation across 41 fungal species reveals phosphorylation signatures specifically associated with biotrophic and hemibiotrophic fungal infection. We then used parallel reaction monitoring (PRM) to identify phosphoproteins regulated by the fungal Pmk1 MAPK that controls plant infection by M. oryzae. We define 32 substrates of Pmk1 and show that Pmk1-dependent phosphorylation of regulator Vts1 is required for rice blast disease. Defining the phosphorylation landscape of infection therefore identifies potential therapeutic interventions for the control of plant diseases.

Peptide REF1 is a local wound signal promoting plant regeneration.

Plants frequently encounter wounding and have evolved an extraordinary regenerative capacity to heal the wounds. However, the wound signal that triggers regenerative responses has not been identified. Here, through characterization of a tomato mutant defective in both wound-induced defense and regeneration, we demonstrate that in tomato, a plant elicitor peptide (Pep), REGENERATION FACTOR1 (REF1), acts as a systemin-independent local wound signal that primarily regulates local defense responses and regenerative responses in response to wounding. We further identified PEPR1/2 ORTHOLOG RECEPTOR-LIKE KINASE1 (PORK1) as the receptor perceiving REF1 signal for plant regeneration. REF1-PORK1-mediated signaling promotes regeneration via activating WOUND-INDUCED DEDIFFERENTIATION 1 (WIND1), a master regulator of wound-induced cellular reprogramming in plants. Thus, REF1-PORK1 signaling represents a conserved phytocytokine pathway to initiate, amplify, and stabilize a signaling cascade that orchestrates wound-triggered organ regeneration. Application of REF1 provides a simple method to boost the regeneration and transformation efficiency of recalcitrant crops.

A programmable reaction-diffusion system for spatiotemporal cell signaling circuit design.

Cells self-organize molecules in space and time to generate complex behaviors, but we lack synthetic strategies for engineering spatiotemporal signaling. We present a programmable reaction-diffusion platform for designing protein oscillations, patterns, and circuits in mammalian cells using two bacterial proteins, MinD and MinE (MinDE). MinDE circuits act like "single-cell radios," emitting frequency-barcoded fluorescence signals that can be spectrally isolated and analyzed using digital signal processing tools. We define how to genetically program these signals and connect their spatiotemporal dynamics to cell biology using engineerable protein-protein interactions. This enabled us to construct sensitive reporter circuits that broadcast endogenous cell signaling dynamics on a frequency-barcoded imaging channel and to build control signal circuits that synthetically pattern activities in the cell, such as protein condensate assembly and actin filamentation. Our work establishes a paradigm for visualizing, probing, and engineering cellular activities at length and timescales critical for biological function.

Cytoneme signaling provides essential contributions to mammalian tissue patterning.

During development, morphogens pattern tissues by instructing cell fate across long distances. Directly visualizing morphogen transport in situ has been inaccessible, so the molecular mechanisms ensuring successful morphogen delivery remain unclear. To tackle this longstanding problem, we developed a mouse model for compromised sonic hedgehog (SHH) morphogen delivery and discovered that endocytic recycling promotes SHH loading into signaling filopodia called cytonemes. We optimized methods to preserve in vivo cytonemes for advanced microscopy and show endogenous SHH localized to cytonemes in developing mouse neural tubes. Depletion of SHH from neural tube cytonemes alters neuronal cell fates and compromises neurodevelopment. Mutation of the filopodial motor myosin 10 (MYO10) reduces cytoneme length and density, which corrupts neuronal signaling activity of both SHH and WNT. Combined, these results demonstrate that cytoneme-based signal transport provides essential contributions to morphogen dispersion during mammalian tissue development and suggest MYO10 is a key regulator of cytoneme function.

The FXR1 network acts as a signaling scaffold for actomyosin remodeling.

It is currently not known whether mRNAs fulfill structural roles in the cytoplasm. Here, we report the fragile X-related protein 1 (FXR1) network, an mRNA-protein (mRNP) network present throughout the cytoplasm, formed by FXR1-mediated packaging of exceptionally long mRNAs. These mRNAs serve as an underlying condensate scaffold and concentrate FXR1 molecules. The FXR1 network contains multiple protein binding sites and functions as a signaling scaffold for interacting proteins. We show that it is necessary for RhoA signaling-induced actomyosin reorganization to provide spatial proximity between kinases and their substrates. Point mutations in FXR1, found in its homolog FMR1, where they cause fragile X syndrome, disrupt the network. FXR1 network disruption prevents actomyosin remodeling-an essential and ubiquitous process for the regulation of cell shape, migration, and synaptic function. Our findings uncover a structural role for cytoplasmic mRNA and show how the FXR1 RNA-binding protein as part of the FXR1 network acts as an organizer of signaling reactions.

Protein Kinase C Enzymes in the Hematopoietic and Immune Systems.

The protein kinase C (PKC) family, discovered in the late 1970s, is composed of at least 10 serine/threonine kinases, divided into three groups based on their molecular architecture and cofactor requirements. PKC enzymes have been conserved throughout evolution and are expressed in virtually all cell types; they represent critical signal transducers regulating cell activation, differentiation, proliferation, death, and effector functions. PKC family members play important roles in a diverse array of hematopoietic and immune responses. This review covers the discovery and history of this enzyme family, discusses the roles of PKC enzymes in the development and effector functions of major hematopoietic and immune cell types, and points out gaps in our knowledge, which should ignite interest and further exploration, ultimately leading to better understanding of this enzyme family and, above all, its role in the many facets of the immune system.

Ultrafast distant wound response is essential for whole-body regeneration.

Injury induces systemic responses, but their functions remain elusive. Mechanisms that can rapidly synchronize wound responses through long distances are also mostly unknown. Using planarian flatworms capable of whole-body regeneration, we report that injury induces extracellular signal-regulated kinase (Erk) activity waves to travel at a speed 10-100 times faster than those in other multicellular tissues. This ultrafast propagation requires longitudinal body-wall muscles, elongated cells forming dense parallel tracks running the length of the organism. The morphological properties of muscles allow them to act as superhighways for propagating and disseminating wound signals. Inhibiting Erk propagation prevents tissues distant to the wound from responding and blocks regeneration, which can be rescued by a second injury to distal tissues shortly after the first injury. Our findings provide a mechanism for long-range signal propagation in large, complex tissues to coordinate responses across cell types and highlight the function of feedback between spatially separated tissues during whole-body regeneration.

Sensory circuitry controls cytosolic calcium-mediated phytochrome B phototransduction.

Although Ca2+ has long been recognized as an obligatory intermediate in visual transduction, its role in plant phototransduction remains elusive. Here, we report a Ca2+ signaling that controls photoreceptor phyB nuclear translocation in etiolated seedlings during dark-to-light transition. Red light stimulates acute cytosolic Ca2+ increases via phyB, which are sensed by Ca2+-binding protein kinases, CPK6 and CPK12 (CPK6/12). Upon Ca2+ activation, CPK6/12 in turn directly interact with and phosphorylate photo-activated phyB at Ser80/Ser106 to initiate phyB nuclear import. Non-phosphorylatable mutation, phyBS80A/S106A, abolishes nuclear translocation and fails to complement phyB mutant, which is fully restored by combining phyBS80A/S106A with a nuclear localization signal. We further show that CPK6/12 function specifically in the early phyB-mediated cotyledon expansion, while Ser80/Ser106 phosphorylation generally governs phyB nuclear translocation. Our results uncover a biochemical regulatory loop centered in phyB phototransduction and provide a paradigm for linking ubiquitous Ca2+ increases to specific responses in sensory stimulus processing.

Temporally multiplexed imaging of dynamic signaling networks in living cells.

Molecular signals interact in networks to mediate biological processes. To analyze these networks, it would be useful to image many signals at once, in the same living cell, using standard microscopes and genetically encoded fluorescent reporters. Here, we report temporally multiplexed imaging (TMI), which uses genetically encoded fluorescent proteins with different clocklike properties-such as reversibly photoswitchable fluorescent proteins with different switching kinetics-to represent different cellular signals. We linearly decompose a brief (few-second-long) trace of the fluorescence fluctuations, at each point in a cell, into a weighted sum of the traces exhibited by each fluorophore expressed in the cell. The weights then represent the signal amplitudes. We use TMI to analyze relationships between different kinase activities in individual cells, as well as between different cell-cycle signals, pointing toward broad utility throughout biology in the analysis of signal transduction cascades in living systems.

Hindbrain modules differentially transform activity of single collicular neurons to coordinate movements.

Seemingly simple behaviors such as swatting a mosquito or glancing at a signpost involve the precise coordination of multiple body parts. Neural control of coordinated movements is widely thought to entail transforming a desired overall displacement into displacements for each body part. Here we reveal a different logic implemented in the mouse gaze system. Stimulating superior colliculus (SC) elicits head movements with stereotyped displacements but eye movements with stereotyped endpoints. This is achieved by individual SC neurons whose branched axons innervate modules in medulla and pons that drive head movements with stereotyped displacements and eye movements with stereotyped endpoints, respectively. Thus, single neurons specify a mixture of endpoints and displacements for different body parts, not overall displacement, with displacements for different body parts computed at distinct anatomical stages. Our study establishes an approach for unraveling motor hierarchies and identifies a logic for coordinating movements and the resulting pose.

An oncogenic phenoscape of colonic stem cell polarization.

Cancer cells are regulated by oncogenic mutations and microenvironmental signals, yet these processes are often studied separately. To functionally map how cell-intrinsic and cell-extrinsic cues co-regulate cell fate, we performed a systematic single-cell analysis of 1,107 colonic organoid cultures regulated by (1) colorectal cancer (CRC) oncogenic mutations, (2) microenvironmental fibroblasts and macrophages, (3) stromal ligands, and (4) signaling inhibitors. Multiplexed single-cell analysis revealed a stepwise epithelial differentiation phenoscape dictated by combinations of oncogenes and stromal ligands, spanning from fibroblast-induced Clusterin (CLU)+ revival colonic stem cells (revCSCs) to oncogene-driven LRIG1+ hyper-proliferative CSCs (proCSCs). The transition from revCSCs to proCSCs is regulated by decreasing WNT3A and TGF-ß-driven YAP signaling and increasing KRASG12D or stromal EGF/Epiregulin-activated MAPK/PI3K flux. We find that APC loss and KRASG12D collaboratively limit access to revCSCs and disrupt stromal-epithelial communication-trapping epithelia in the proCSC fate. These results reveal that oncogenic mutations dominate homeostatic differentiation by obstructing cell-extrinsic regulation of cell-fate plasticity.

The Wnt Pathway: From Signaling Mechanisms to Synthetic Modulators.

The Wnt pathway is central to a host of developmental and disease-related processes. The remarkable conservation of this intercellular signaling cascade throughout metazoan lineages indicates that it coevolved with multicellularity to regulate the generation and spatial arrangement of distinct cell types. By regulating cell fate specification, mitotic activity, and cell polarity, Wnt signaling orchestrates development and tissue homeostasis, and its dysregulation is implicated in developmental defects, cancer, and degenerative disorders. We review advances in our understanding of this key pathway, from Wnt protein production and secretion to relay of the signal in the cytoplasm of the receiving cell. We discuss the evolutionary history of this pathway as well as endogenous and synthetic modulators of its activity. Finally, we highlight remaining gaps in our knowledge of Wnt signal transduction and avenues for future research.

Early T cell activation: integrating biochemical, structural, and biophysical cues.

T cells carry out the formidable task of identifying small numbers of foreign antigenic peptides rapidly and specifically against a very noisy environmental background of endogenous self-peptides. Early steps in T cell activation have thus fascinated biologists and are among the best-studied models of cell stimulation. This remarkable process, critical in adaptive immune responses, approaches and even seems to exceed the limitations set by the physical laws ruling molecular behavior. Despite the enormous amount of information concerning the nature of molecules involved in the T cell antigen receptor (TCR) signal transduction network, and the description of the nanoscale organization and real-time analysis of T cell responses, the general principles of information gathering and processing remain incompletely understood. Here we review currently accepted key data on TCR function, discuss the limitations of current research strategies, and suggest a novel model of TCR triggering and a few promising ways of going further into the integration of available data.

Optobiochemistry: Genetically Encoded Control of Protein Activity by Light.

Optobiochemical control of protein activities allows the investigation of protein functions in living cells with high spatiotemporal resolution. Over the last two decades, numerous natural photosensory domains have been characterized and synthetic domains engineered and assembled into photoregulatory systems to control protein function with light. Here, we review the field of optobiochemistry, categorizing photosensory domains by chromophore, describing photoregulatory systems by mechanism of action, and discussing protein classes frequently investigated using optical methods. We also present examples of how spatial or temporal control of proteins in living cells has provided new insights not possible with traditional biochemical or cell biological techniques.

Vertebrate cells differentially interpret ciliary and extraciliary cAMP.

Hedgehog pathway components and select G protein-coupled receptors (GPCRs) localize to the primary cilium, an organelle specialized for signal transduction. We investigated whether cells distinguish between ciliary and extraciliary GPCR signaling. To test whether ciliary and extraciliary cyclic AMP (cAMP) convey different information, we engineered optogenetic and chemogenetic tools to control the subcellular site of cAMP generation. Generating equal amounts of ciliary and cytoplasmic cAMP in zebrafish and mammalian cells revealed that ciliary cAMP, but not cytoplasmic cAMP, inhibited Hedgehog signaling. Modeling suggested that the distinct geometries of the cilium and cell body differentially activate local effectors. The search for effectors identified a ciliary pool of protein kinase A (PKA). Blocking the function of ciliary PKA, but not extraciliary PKA, activated Hedgehog signal transduction and reversed the effects of ciliary cAMP. Therefore, cells distinguish ciliary and extraciliary cAMP using functionally and spatially distinct pools of PKA, and different subcellular pools of cAMP convey different information.

Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling.

During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a "trimer-of-trimers" super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs.