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The multi-subunit translation initiation factor eIF2B is a control node for protein synthesis. eIF2B activity is canonically modulated through stress-responsive phosphorylation of its substrate eIF2. The eIF2B regulatory subcomplex is evolutionarily related to sugar-metabolizing enzymes, but the biological relevance of this relationship was unknown. To identify natural ligands that might regulate eIF2B, we conduct unbiased binding- and activity-based screens followed by structural studies. We find that sugar phosphates occupy the ancestral catalytic site in the eIF2Bα subunit, promote eIF2B holoenzyme formation and enhance enzymatic activity towards eIF2. A mutant in the eIF2Bα ligand pocket that causes Vanishing White Matter disease fails to engage and is not stimulated by sugar phosphates. These data underscore the importance of allosteric metabolite modulation for proper eIF2B function. We propose that eIF2B evolved to couple nutrient status via sugar phosphate sensing with the rate of protein synthesis, one of the most energetically costly cellular processes., The activity of translation initiation factor eIF2B is known to be modulated through stress-responsive phosphorylation of its substrate eIF2. Here, the authors uncover the regulation of eIF2B by the binding of sugar phosphates, suggesting a link between nutrient status and the rate of protein synthesis.

The integrated stress response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity. We show that introduction of a human VWM mutation into mice leads to persistent ISR induction in the central nervous system. ISR activation precedes myelin loss and development of motor deficits. Remarkably, long-term treatment with a small molecule eIF2B activator, 2BAct, prevents all measures of pathology and normalizes the transcriptome and proteome of VWM mice. 2BAct stimulates the remaining activity of mutant eIF2B complex in vivo, abrogating the maladaptive stress response. Thus, 2BAct-like molecules may provide a promising therapeutic approach for VWM and provide relief from chronic ISR induction in a variety of disease contexts., eLife digest Cells must be able to respond to their changing environment in order to survive. When cells encounter particularly unfavorable conditions, they often react by activating a so-called ‘stress’ response. A group of proteins collectively known as eIF2B helps to regulate this response. In a severe neurological condition called Vanishing White Matter (VWM), the genes that produce the eIF2B proteins contain mutations that make eIF2B less active. As a result, certain cells in people with VWM are always stressed. Six years ago, researchers discovered a molecule that boosts the activity of eIF2B. In 2018, they found that it also works on various mutant forms of eIF2B found in VWM. The molecule had so far only been tested in biochemical laboratory experiments. Now, Wong et al. – including some of the researchers involved in the 2018 study – have tested whether an improved version of the molecule treats VWM in mice. The trial treatment successfully halted all signs of the disease in the mice. The molecule blunted the persistent stress response of the cells in the brain and spinal cord, primarily in a cell type that is severely affected by the human form of VWM. Cells in other parts of the body were spared. Overall, the results of the experiments suggest that an eIF2B activator may prove to be an effective treatment for VWM in humans. It could similarly be used to treat other conditions that activate this abnormal cell stress response. The molecule Wong et al. used is not suitable for use in humans, so work is continuing to find a suitable variant.

The Integrated Stress Response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity. We show that introduction of a human VWM mutation into mice leads to persistent ISR induction in the central nervous system. ISR activation precedes myelin loss and development of motor deficits. Remarkably, long-term treatment with a novel eIF2B activator, 2BAct, prevents all measures of pathology and normalizes the transcriptome and proteome of VWM mice. 2BAct stimulates the remaining activity of mutant eIF2B complexin vivo, abrogating the maladaptive stress response. Thus, 2BAct-like molecules may provide a promising therapeutic approach for VWM and provide relief from chronic ISR induction in a variety of other disease contexts.

eIF2B is a dedicated guanine nucleotide exchange factor for eIF2, the GTPase that is essential to initiate mRNA translation. The integrated stress response (ISR) signaling pathway inhibits eIF2B activity, attenuates global protein synthesis and upregulates a set of stress-response proteins. Partial loss-of-function mutations in eIF2B cause a neurodegenerative disorder called Vanishing White Matter Disease (VWMD). Previously, we showed that the small molecule ISRIB is a specific activator of eIF2B (Sidrauski et al., 2015). Here, we report that various VWMD mutations destabilize the decameric eIF2B holoenzyme and impair its enzymatic activity. ISRIB stabilizes VWMD mutant eIF2B in the decameric form and restores the residual catalytic activity to wild-type levels. Moreover, ISRIB blocks activation of the ISR in cells carrying these mutations. As such, ISRIB promises to be an invaluable tool in proof-of-concept studies aiming to ameliorate defects resulting from inappropriate or pathological activation of the ISR.

The Notch–Delta signalling pathway allows communication between neighbouring cells during development^1. It has a critical role in the formation of ‘fine-grained’ patterns, generating distinct cell fates among groups of initially equivalent neighbouring cells and sharply delineating neighbouring regions in developing tissues. The Delta ligand has been shown to have two activities: it transactivates Notch in neighbouring cells and cis-inhibits Notch in its own cell. However, it remains unclear how Notch integrates these two activities and how the resulting system facilitates pattern formation. Here we report the development of a quantitative time-lapse microscopy platform for analysing Notch–Delta signalling dynamics in individual mammalian cells, with the aim of addressing these issues. By controlling both cis- and trans-Delta concentrations, and monitoring the dynamics of a Notch reporter, we measured the combined cis–trans input–output relationship in the Notch–Delta system. The data revealed a striking difference between the responses of Notch to trans- and cis-Delta: whereas the response to trans-Delta is graded, the response to cis-Delta is sharp and occurs at a fixed threshold, independent of trans-Delta. We developed a simple mathematical model that shows how these behaviours emerge from the mutual inactivation of Notch and Delta proteins in the same cell. This interaction generates an ultrasensitive switch between mutually exclusive sending (high Delta/low Notch) and receiving (high Notch/low Delta) signalling states. At the multicellular level, this switch can amplify small differences between neighbouring cells even without transcription-mediated feedback. This Notch–Delta signalling switch facilitates the formation of sharp boundaries and lateral-inhibition patterns in models of development, and provides insight into previously unexplained mutant behaviours.

SUMMARY The Notch signaling pathway comprises multiple ligands that are used in distinct biological contexts. In principle, different ligands could activate distinct target programs in signal-receiving cells, but it is unclear how such ligand discrimination could occur. Here, we show that cells use dynamics to discriminate signaling by the ligands Dll1 and Dll4 through the Notch1 receptor. Quantitative single-cell imaging revealed that Dll1 activates Notch1 in discrete, frequency-modulated pulses that specifically upregulate the Notch target gene Hes1. By contrast, Dll4 activates Notch1 in a sustained, amplitude-modulated manner that predominantly upregulates Hey1 and HeyL. Ectopic expression of Dll1 or Dll4 in chick neural crest produced opposite effects on myogenic differentiation, showing that ligand discrimination can occur in vivo. Finally, analysis of chimeric ligands suggests that ligand-receptor clustering underlies dynamic encoding of ligand identity. The ability of the pathway to utilize ligands as distinct communication channels has implications for diverse Notch-dependent processes., In Brief Notch ligands activate distinct targets through the same Notch receptor by triggering pulsatile or sustained activation dynamics., Graphical Abstract

The Notch signaling pathway consists of multiple types of receptors and ligands, whose interactions can be tuned by Fringe glycosyltransferases. A major challenge is to determine how these components control the specificity and directionality of Notch signaling in developmental contexts. Here, we analyzed same-cell (cis) Notch-ligand interactions for Notch1, Dll1, and Jag1, and their dependence on Fringe protein expression in mammalian cells. We found that Dll1 and Jag1 can cis-inhibit Notch1, and Fringe proteins modulate these interactions in a way that parallels their effects on trans interactions. Fringe similarly modulated Notch-ligand cis interactions during Drosophila development. Based on these and previously identified interactions, we show how the design of the Notch signaling pathway leads to a restricted repertoire of signaling states that promote heterotypic signaling between distinct cell types, providing insight into the design principles of the Notch signaling system, and the specific developmental process of Drosophila dorsal-ventral boundary formation. DOI: http://dx.doi.org/10.7554/eLife.02950.001, eLife digest In animals, cells use a process called Notch signaling to communicate with neighboring cells. During this process, a protein known as a DSL ligand from one cell binds to a protein called a Notch receptor on a neighboring cell. This triggers a series of events in the neighboring cell that change how the genes in this cell are expressed. Notch signaling is involved in many processes including the early growth of embryos, the formation of organs and limbs, and the maintenance of stem cells throughout adult life. Enzymes called Fringe enzymes can control Notch signaling by blocking or promoting the formation of the DSL ligand-Notch receptor pairs. It is also possible for a DSL ligand and a Notch receptor from the same cell to interact. This is thought to be important because it prevents an individual cell from sending and receiving Notch signals at the same time. There are several different DSL ligands, Notch receptors and Fringe enzymes, so it is difficult to determine which configurations of receptors, ligands and Fringe enzymes can enable Notch signals to be sent or received. To address this problem, LeBon et al. investigated how Fringe enzymes acted on several different DSL-Notch receptor pairs in mammalian cells, and also in fruit flies. They focused in particular on the interactions that occurred within the same cell, as the role of Fringe enzymes in this type of interaction has not been examined previously. The experiments revealed that Fringe proteins modify specific same-cell interactions in a way that enables a cell to receive one type of Notch signal from a neighboring cell and send a different type of Notch signal to another cell at the same time. More generally, these results show how an unconventional, ‘bottom-up’ approach can reveal the design principles of cell signaling systems, and suggest that it should now be possible to use these principles to try to understand which cell types send signals to which other cell types in many different contexts. DOI: http://dx.doi.org/10.7554/eLife.02950.002

Developmental patterning requires juxtacrine signaling in order to tightly coordinate the fates of neighboring cells. Recent work has shown that Notch and Delta, the canonical metazoan juxtacrine signaling receptor and ligand, mutually inactivate each other in the same cell. This cis-interaction generates mutually exclusive sending and receiving states in individual cells. It generally remains unclear, however, how this mutual inactivation and the resulting switching behavior can impact developmental patterning circuits. Here we address this question using mathematical modeling in the context of two canonical pattern formation processes: boundary formation and lateral inhibition. For boundary formation, in a model motivated by Drosophila wing vein patterning, we find that mutual inactivation allows sharp boundary formation across a broader range of parameters than models lacking mutual inactivation. This model with mutual inactivation also exhibits robustness to correlated gene expression perturbations. For lateral inhibition, we find that mutual inactivation speeds up patterning dynamics, relieves the need for cooperative regulatory interactions, and expands the range of parameter values that permit pattern formation, compared to canonical models. Furthermore, mutual inactivation enables a simple lateral inhibition circuit architecture which requires only a single downstream regulatory step. Both model systems show how mutual inactivation can facilitate robust fine-grained patterning processes that would be difficult to implement without it, by encoding a difference-promoting feedback within the signaling system itself. Together, these results provide a framework for analysis of more complex Notch-dependent developmental systems., Author Summary Multicellular development requires tightly regulated spatial pattern formation, frequently including the generation of sharp differences over short length scales. Classic examples include boundary formation in the Drosophila wing veins and lateral inhibition patterning in the differentiation of sensory cells. These processes and a diverse variety of others are mediated by the Notch signaling system which allows neighboring cells to exchange information, via interaction between the Notch receptor on one cell and its ligands such as Delta, on another. Interestingly, recent evidence has shown that Notch and Delta within the same cell (in cis) also interact, mutually inactivating each other. However, the significance of this interaction for pattern formation has remained unclear. Here we show, by analytical and computational modeling, how this cis interaction intrinsically generates a difference-promoting logic that optimizes the system for use in fine-grained pattern formation. Specifically, boundary formation and lateral inhibition patterning both operate more effectively and with simpler circuit architectures than they could without this interaction. Our results provide a foundation for understanding these and other Notch-dependent pattern formation processes.