EMBO Journal最新文献

The prevailing view on post-translational modifications (PTMs) is that a single amino acid is modified with a single PTM at any given time. However, recent work has demonstrated crosstalk between different PTMs, some occurring on the same residue. Such interplay is seen with ADP-ribosylation and ubiquitylation. For example, DELTEX E3 ligases were reported to ubiquitylate a hydroxyl group on free NAD⁺ and ADP-ribose in vitro, generating a noncanonical ubiquitin ester-linked species. In this report, we show, for the first time, that this dual PTM occurs in cells on mono-ADP-ribosylated (MARylated) PARP10 on Glu/Asp sites to form a MAR ubiquitin ester. We call this process mono-ADP-ribosyl ubiquitylation or MARUbylation. Using chemical and enzymatic treatments, including a newly characterized bacterial deubiquitinase with esterase-specific activity, we discovered that multiple PARPs are MARUbylated and extended with K11-linked polyubiquitin chains when exogenously expressed. Finally, we show that in response to type I interferon stimulation, MARUbylation can occur endogenously on PARP targets. Thus, MARUbylation represents a new dual PTM that broadens our understanding of the function of PARP-mediated ADP-ribosylation in cells.

The cilium is a microtubule-based eukaryotic organelle critical for many cellular functions. Its assembly initiates at a basal body and continues as an axoneme that projects out of the cell to form a functional cilium. This assembly process is tightly regulated. However, our knowledge of the molecular architecture and the mechanism of assembly is limited. By applying cryo-electron tomography, we obtained structures of the inner junction in three regions of the cilium from Tetrahymena: the proximal, the central core of the basal body, and the axoneme. We identified several protein components in the basal body. While a few proteins are distributed throughout the entire length of the organelle, many are restricted to specific regions, forming intricate local interaction networks in the inner junction and bolstering local structural stability. By examining the inner junction in a POC1 knockout mutant, we found the triplet microtubule was destabilized, resulting in a defective structure. Surprisingly, several axoneme-specific components were found to "infiltrate" into the mutant basal body. Our findings provide molecular insight into cilium assembly at the inner junctions, underscoring its precise spatial regulation.

Our understanding of the functional requirements underpinning biomolecular condensation in vivo is still relatively poor. The Arabidopsis RNA binding protein FLOWERING CONTROL LOCUS A (FCA) is found in liquid-like nuclear condensates that function in transcription termination, promoting proximal polyadenylation at many target genes in the Arabidopsis genome. To further understand the properties of these condensates in vivo, we used single-particle tracking experiments on FCA reporters stably expressed at endogenous levels in plant nuclei. SEC-MALS analyses suggested that FCA forms a core oligomer consistent with a size of four molecules; in vivo particle tracking indicated that this core molecule multimerizes into higher-order particles. The ensuing assemblies coalesce into macromolecular condensates via the coiled-coil protein FLL2, which is genetically required for FCA function. Accordingly, FLL2 predominately co-localizes with FCA in larger-sized condensates. A missense mutation in the FCA RRM domain, also genetically required for FCA function, reduced average size of both FCA particles and condensates, but did not perturb the core oligomer. Our work points to a modular structure for FCA condensates, involving multimerization of core oligomers assembled into functional macromolecular condensates via associated RNA and FLL2 interactions.

The small GTPase Ras is an intracellular signaling hub required for long-term potentiation (LTP) in the hippocampus and for memory formation. Genetic alterations in Ras signaling (i.e., RASopathies) are linked to cognitive disorders in humans. However, it remains unclear how Ras controls synaptic plasticity, and whether different Ras isoforms play overlapping or distinct roles in neurons. Using genetically modified mice, we show here that H-Ras (the most abundant isoform in the brain) does not promote LTP, but instead long-term depression mediated by metabotropic glutamate receptors (mGluR-LTD). Mechanistically, H-Ras is activated locally in spines during mGluR-LTD via c-Src, and is required to trigger Erk activation and de novo protein synthesis. Furthermore, H-Ras deletion impairs object recognition as well as social and spatial memory. Conversely, K-Ras is the isoform specifically required for LTP. This functional specialization correlates with a differential synaptic distribution of the two isoforms H-Ras and K-Ras, which may have important implications for RASopathies and cognitive function.

The cGAS-STING signalling pathway has a central role in the innate immune response to extrinsic and intrinsic sources of cytoplasmic dsDNA. At the core of this pathway is cGAS-dependent production of the intra- and extra-cellular messenger cGAMP, which activates STING and leads to IRF3-dependent expression of cytokines and interferons. Despite its relevance to viral and bacterial infections, cell death, and genome instability, the lack of specific live-cell reporters has precluded spatiotemporal analyses of cGAS-STING signalling. Here, we generate a fluorescent biosensor termed SIRF (STING-IRF3), which reports on the functional interaction between activated STING and IRF3 at the Golgi. We show that cells harbouring SIRF react in a time- and concentration-dependent manner both to STING agonists and to microenvironmental cGAMP. We demonstrate that the new biosensor is suitable for single-cell characterisation of immune responses to HSV-1 infection, mtDNA release upon apoptosis, or other sources of cytoplasmic dsDNA. Furthermore, our results indicate that STING signalling is not activated by ruptured micronuclei, suggesting that other cytosolic pattern recognition receptors underlie the interferon responses to chromosomal instability.

Cell fate instructive genes tend to be regulated by large clusters of enhancers. Whether and how individual enhancers within such clusters cooperate in regulating gene expression is poorly understood. We have previously developed a computational method, SEGCOND, which identifies hubs that we termed Putative Transcriptional Condensates (PTCs), consisting of enhancer clusters and associated target genes. Here, we use SEGCOND to identify PTCs in a CEBPA-induced B-cell-to-macrophage transdifferentiation system. We find that PTCs are enriched for highly expressed, lineage-restricted genes and associate with BRD4, a component of transcriptional condensates. Further, we performed single and combinatorial deletions of enhancers within two PTCs active during induced transdifferentiation, harboring IRF8 and FOS. Two enhancers within the IRF8 PTC were found to provide a backup mechanism when combined, safeguarding IRF8 expression and efficient transdifferentiation. Unexpectedly, two individual enhancers within the FOS PTC antagonize each other on day 1 of transdifferentiation, delaying the conversion of B-cells into macrophages and reducing FOS expression, while on day 7, they cooperate to increase FOS levels induced cells. Our results reveal complex, differentiation-stage-specific interactions between individual enhancers within enhancer clusters.

MAST-like, or Greatwall (Gwl), an atypical protein kinase related to the evolutionarily conserved MAST kinase family, is crucial for cell cycle control during mitotic entry. Mechanistically, Greatwall is activated by Cyclin B-Cdk1 phosphorylation of a 550 amino acids-long insertion in its atypical activation segment. Subsequently, Gwl phosphorylates Endosulfine and Arpp19 to convert them into inhibitors of PP2A-B55 phosphatase, thereby preventing early dephosphorylation of M-phase targets of Cyclin B-Cdk1. Here, searching for an elusive Gwl-like activity in C. elegans, we show that the single worm MAST kinase, KIN-4, fulfills this function in worms and can functionally replace Greatwall in the heterologous Xenopus system. Compared to Greatwall, the short activation segment of KIN-4 lacks a phosphorylation site, and KIN-4 is active even when produced in E. coli. We also show that a balance between Cyclin B-Cdk1 and PP2A-B55 activity, regulated by KIN-4, is essential to ensure asynchronous cell divisions in the early worm embryo. These findings resolve a long-standing puzzle related to the supposed absence of a Greatwall pathway in C. elegans, and highlight a novel aspect of PP2A-B55 regulation by MAST kinases.

During development, epithelial sheets sculpt organs by folding, either apically or basally, into complex 3D structures. Given the presence of actomyosin networks and cell adhesion sites on both sides of cells, a common machinery mediating apical and basal epithelial tissue folding has been proposed. However, unlike for apical folding, little is known about the mechanisms that regulate epithelial folding towards the basal side. Here, using the Drosophila wing imaginal disc and combining genetic perturbations and computational modeling, we demonstrate opposing roles for cell-cell and cell-extracellular matrix (ECM) adhesion systems during epithelial folding. While cadherin-mediated adhesion, linked to actomyosin network, regulates apical folding, a localized reduction on integrin-dependent adhesion, followed by changes in cell shape and reorganization of the basal actomyosin cytoskeleton and E-Cadherin (E-Cad) levels, is necessary and sufficient to trigger basal folding. These results suggest that modulation of the cell mechanical landscape through the crosstalk between integrins and cadherins is essential for correct epithelial folding.

Co-evolution between cereals and pathogenic grass powdery mildew fungi is exemplified by sequence diversification of an allelic series of barley resistance genes encoding Mildew Locus A (MLA) nucleotide-binding leucine-rich repeat (NLR) immunoreceptors with an N-terminal coiled-coil domain (CNLs). Each immunoreceptor recognises a matching, strain-specific powdery mildew effector encoded by an avirulence gene (AVR_a). We present here the cryo-EM structure of barley MLA13 in complex with its cognate effector AVR_A13-1. The effector adopts an RNase-like fold when bound to MLA13 in planta, similar to crystal structures of other RNase-like AVR_A effectors unbound to receptors. AVR_A13-1 interacts via its basal loops with MLA13 C-terminal leucine-rich repeats (LRRs) and the central winged helix domain (WHD). Co-expression of structure-guided MLA13 and AVR_A13-1 substitution variants show that the receptor-effector interface plays an essential role in mediating immunity-associated plant cell death. Furthermore, by combining structural information from the MLA13-AVR_A13-1 heterocomplex with sequence alignments of other MLA receptors, we engineered a single amino acid substitution in MLA7 that enables expanded effector detection of AVR_A13-1 and the virulent variant AVR_A13-V2. In contrast to the pentameric conformation of previously reported effector-activated CNL resistosomes, MLA13 was purified and resolved as a stable heterodimer from an in planta expression system. Our study suggests a common structural principle for RNase-like effector binding to MLAs and highlights the utility of structure-guided engineering of plant immune receptors for broadening their pathogen effector recognition capabilities.