Despite loss of responsiveness to exogenous leptin in obesity, endogenous leptin continues to restrain feeding, although the neural substrates that remain sensitive and mediate this effect remain unknown. Combining spatial transcriptomics with single-nucleus RNA sequencing in mice with diet-induced obesity (DIO), we here show that while most hypothalamic leptin receptor (Lepr)-positive neurons minimally respond to elevated leptin, a single subpopulation defined by glucagon-like peptide-1 receptor (Glp1r) co-expression retains robust leptin sensitivity. These Lepr[+]/Glp1r[+] neurons project onto and inhibit orexigenic Agrp neurons. Lepr deletion from Lepr[+]/Glp1r[+] neurons blocks the anorectic effect of exogenous leptin, reinstates hyperphagic responses normally suppressed in DIO, amplifies the obesogenic response to palatable diet, and unexpectedly attenuates hypothalamic microglial activation - a hallmark of DIO previously attributed to diet rather than leptin signaling. Hence, preserved leptin responses by a single neuronal population in the hypothalamus limit hyperphagia and weight gain, among other responses, during obesity.

Cellular senescence has undergone a remarkable conceptual evolution since its discovery in 1961. Initially described by Hayflick and Moorhead as the finite proliferative lifespan of cultured human cells, senescence was first viewed as a consequence of cellular aging. The identification of telomere shortening, senescence biomarkers, and oncogene- and damage-induced senescence subsequently established its molecular basis and role as a tumor-suppressive stress response. The discovery of the senescence-associated secretory phenotype (SASP) further transformed the field by revealing that senescent cells actively communicate with and remodel their tissue environment. Genetic mouse models later demonstrated causal roles for senescent cells in tissue repair, aging, and age-related disease, while senotherapeutics established senescence as a therapeutic target. Here, we trace the major conceptual transitions that shaped the field, from replicative endpoint to stress-response program, regulator of tissue homeostasis, driver of chronic pathology, and clinically actionable process. We discuss the discoveries, controversies, and technological advances underlying these transitions and how emerging single-cell technologies, precision biomarkers, and targeted interventions are shaping the next era of senescence research.

To fully harness phage therapy against escalating antimicrobial resistance, it is imperative to elucidate the fundamental mechanisms by which viruses systematically dismantle host physiology. In this study, we identify Ped1, a protein from Pseudomonas aeruginosa phage PaoP5, which suppresses bacterial growth through two mechanisms: degradation of genomic DNA and direct inhibition of the replication machinery. Our 2.3-Å crystal structure and biochemical profiling reveal that Ped1 adopts a previously uncharacterized α/β-nuclease architecture, enabling the efficient cleavage of both double-stranded and single-stranded DNA. Furthermore, we demonstrate that Ped1 physically interacts with the host replicative helicase, DnaB, to suppress its activity during DNA replication. Reciprocally, DnaB binds Ped1 to neutralize its nuclease function, acting as an internal immunity partner. Evolutionary analysis indicates that Ped1 is a lineage-specific toxin strictly conserved among Pseudomonas phages. Collectively, this work identifies a novel, dual-function phage effector that coordinately disrupts genomic integrity and replication fidelity, thus advancing our understanding of the phage-host molecular arms race, and describing a promising scaffold for the development of next-generation antimicrobials against multidrug-resistant P. aeruginosa.

Ribosome hibernation helps cells survive stress by reversibly silencing translation and preserving ribosomal complexes. Although well characterized in bacteria and eukaryotes, archaeal hibernation remains poorly understood. Using cryoEM of archaeal lysates, we identified AHA (AMPKγ-HPF from Archaea), a broadly conserved ribosome-associated factor composed of two modules. AHA bound across ribosomal subunits, occluding the mRNA channel and tRNA binding sites, supporting its role in ribosome hibernation. ΔAHA cells displayed reduced viability, loss of ribosomal proteins in the stationary phase, and impaired growth reentry in rich media. Phylogenetic analyses revealed that AHA's C-terminal domain is homologous to the bacterial Hibernation Promoting Factor (HPF), consistent with inheritance from the last universal common ancestor and thereby identifying HPF as a universal hibernation module in prokaryotes. Strikingly, we observed two AMP molecules bound to AHA's N-terminal CBS-tetrad, which showed both sequence and structural similarity to the eukaryotic energy sensor AMPKγ, supporting a shared evolutionary origin of the archaeal CBS-tetrad and the AMPKγ family. Together, these findings uncover a widespread archaeal ribosome hibernation factor and reveal an evolutionary connection between prokaryotic ribosomal hibernation and eukaryotic energy sensing.

Eukaryotic cells regulate the assembly and activation of the essential DNA helicase at the heart of the chromosome replication machinery, to ensure that the chromosomes are copied just once per cell cycle. The Mcm10 protein is essential for helicase activation in budding yeast, but an equivalent role for MCM10 orthologues in animal cells has not been explored. Moreover, complete deletion of the mcm-10 gene is viable in the nematode Caenorhabditis elegans, suggesting the involvement of additional factors. Here we show that MCM-10 and a second factor called SLD-2 are recruited to chromatin after helicase assembly in the C. elegans early embryo and are partially redundant for helicase activation. Moreover, deletion of the Mcm10 gene is viable in mouse embryonic stem cells, but causes synthetic lethality in the absence of RECQL4, which is the orthologue of SLD-2 in vertebrate species. Helicase activation is blocked in the combined absence of MCM10 and RECQL4, mirroring the situation in C. elegans. These findings indicate that metazoan helicase activation requires two conserved factors that are mutated in human disease.

Bacterial pathogens have adapted secreted effector proteins that hijack host ubiquitination to gain control over host responses. These effectors can exhibit, for example, E3 ligase or deubiquitinase activities, often without any homology to eukaryotic regulators. Such convergence in function poses a challenge to the discovery of additional ubiquitin-targeted virulence factors. Pseudomonas aeruginosa, for example, is a pervasive opportunistic pathogen with numerous mechanisms of virulence, yet no described E3 ligases or deubiquitinases. We have developed a workflow to functionally screen natively secreted bacterial effectors for ubiquitin regulatory activities. After benchmarking this approach on Salmonella Typhimurium, Enteropathogenic Escherichia coli, and Shigella flexneri, we identified a cryptic E3 ligase secreted by P. aeruginosa, which we have termed Pseudomonas Ub ligase 1 (PUL-1). PUL-1 resembles none of the other E3 ligases previously established in or outside of the eukaryotic system. Importantly, in an animal model of P. aeruginosa infection, PUL-1 ligase activity plays an important role in regulating virulence. Thus, functional identification of ubiquitin-targeted effectors can expand our appreciation of ubiquitin regulation during bacterial pathogenesis.

The ubiquitin ligase HOIL-1 regulates the formation of Met1-linked (linear) ubiquitin chains through its coordination with the E3 ligase HOIP within the Linear Ubiquitin Chain Assembly Complex (LUBAC). While HOIP-dependent Met1-linked ubiquitination is well established in inflammation and immunity, the physiological importance of its quantitative control remains unclear. Here, we show that cells expressing catalytically inactive HOIL-1 accumulate increased α-synuclein, tau, and amyloid-β aggregates. This is associated with defective late-stage autophagic flux, characterized by impaired delivery of p62-positive aggregates to lysosomes. In parallel, p62 bodies undergo a biophysical transition from dynamic, liquid-like condensates to rigid, solid-like structures. Elevation of Met1-linked ubiquitin chains, either through HOIL-1 inactivation or depletion of the Met1-specific deubiquitinase OTULIN, phenocopies these defects. Together, our findings identify HOIL-1 as a key regulator of aggregate clearance and proteostasis through quantitative control of Met1-linked ubiquitination.

To initiate the process of invasion, apicomplexan parasites must discharge the contents of specialized organelles called rhoptries into host cells. This process requires prior recognition and binding of the host cell by proteins released from another set of parasite organelles, the micronemes. However, the host-parasite interactions required for rhoptry discharge are largely unknown. Here, we performed a host cell-directed genome-wide screen for factors that mediate rhoptry discharge from Toxoplasma gondii, the causative agent of toxoplasmosis. The screen identified host N-glycosylation and cholesterol biosynthesis as pathways required for normal rhoptry discharge. A trimeric microneme complex, MIC1/4/6, interfaces with both pathways by binding host N-glycans to cluster proteins in a process dependent on host plasma membrane cholesterol. The process can be inhibited by depletion of host cholesterol or competition with exogenous glycans. This clustering of host factors by MIC1/4/6 likely prepares the host membrane for rhoptry discharge, delineating a new step in the Toxoplasma invasion process.

Whole-genome duplication (WGD), leading to polyploidy can arise in physiological and pathological contexts. WGD can occur via non-canonical cell cycles such as mitotic slippage, cytokinesis failure or endoreplication. Whether the routes to WGD influence the behaviour of the resulting polyploid cells remains unclear. Here, we compared these routes under both physiological and non-physiological conditions. Remarkably, only mitotic slippage led to widespread nuclear abnormalities defined by highly variable nuclear deformations that we termed nuclear instability. Mechanistically, we found that these nuclei were softer and thus more vulnerable to microtubule-driven deformations. The resulting nuclear instability leads to local nuclear reorganisation and changes in 3D genome organisation. Importantly, we observed similar nuclear instability in megakaryocytes, which are physiological polyploid cells generated by mitotic slippage, providing a molecular mechanism for their atypical nuclear architecture. In striking contrast, nuclear shape was stable in different physiological polyploid cells generated by cytokinesis failure and endoreplication. Overall, our findings highlight that the route towards WGD matters and that mitotic slippage uniquely destabilizes nuclear architecture, with implications for both physiology and disease.

BRCA1-linked cancers contain abundant ~10 kb 'Group 1' tandem duplications (TDs). Group 1 TDs form at a Tus/Ter replication-fork barrier in DNA-end resection-defective mouse embryonic stem (mES) cells lacking Brca1 exon 11. To elucidate how BRCA1 suppresses Group 1 TDs, we analyzed Brca1 coiled-coil (CC)-domain mutants-separation-of-function alleles impaired for homologous recombination (HR) through loss of PALB2-binding and RAD51-loading functions but competent for DNA-end resection. Notably, Brca1 CC mutants retain the ability to suppress Group 1 TDs in the Tus/Ter system and in a mouse model of Brca1-linked mammary tumorigenesis. These data suggest that Brca1 CC domain-mutant cancers follow a path of tumorigenesis distinct from that of other Brca1-linked cancers. FANCM is a TD co-suppressor, loss of which is synthetic lethal/sick in Brca1 exon 11-deleted cells. In contrast, Fancm deletion unexpectedly improves the growth of Brca1 CC mutant and Brca2 mutant mES cells. Thus, Group 1 tandem duplication formation and Fancm synthetic lethality are linked phenotypes, potentially related to defective BRCA1-mediated DNA end resection but genetically separable from BRCA1/PALB2-mediated RAD51 loading.

Epigenetic inheritance provides mutation-free mechanisms for phenotypic adaptation, including modifications to DNA or DNA-associated proteins that create patterns of heritable gene regulation. However, the limits and regulation of this inheritance remain incompletely understood. Here, we developed a C. elegans system to study the transgenerational epigenetic inheritance of H3K27me3, a conserved histone posttranslational modification associated with gene repression. We find that transiently induced alterations of the genome-wide H3K27me3 landscape and the associated fertility defects persist for many generations in genetically wildtype descendants under selective pressure. We uncover that the inheritance of the altered H3K27me3 landscape involves an initiation phase that relies on SET-32/H3K23me3 and a maintenance phase that requires MES-4/H3K36me3. Our results indicate that epigenetic inheritance can act as a mutation-independent, heritable mechanism of adaptation.

Meiotic crossovers (COs) are tightly regulated to ensure chromosome segregation while limiting aberrant recombination. Transverse filament (TF) proteins of the synaptonemal complex (SC) regulate class I crossoverts, yet how the SC coordinates homologous recombination (HR) to maintain faithful recombination remains unclear. Here, we show that loss of rice TF protein ZEP1 does not uniformly enhance HEI10-marked class I COs; instead, ZEP1 null mutants display asynapsis, multivalent formation, and chromosome fragmentation. These defects depend on double-strand breaks (DSBs) and genetically place ZEP1 function after strand invasion. ZEP1 interacts with anti-crossover factors MEICA1, FIGNL1, and RMI1, and is required for their enrichment at the synaptonemal complex; its loss causes persistent DMC1/RAD51 signals, indicating dysregulated strand invasion. Genetic interactions further support impaired recombination intermediate homeostasis, with ZMM removal partially alleviating chromosome abnormalities, while MUS81 becomes increasingly essential. We propose that the synaptonemal complex functions as a structural hub, where ZEP1 concentrates anti-crossover modules that restrain invasion and coordinate recombination intermediate processing to promote HR repair. Furthermore, ZEP1 dosage may provide a constrained lever to tune CO outcomes in a background-dependent manner.

Neural crest cells (NCCs) are multipotent migratory cells essential for cardiac development, yet the lineage trajectories and gene regulatory networks underlying their differentiation in the cardiopharyngeal region remain unclear. Here, we integrate single-cell RNA-seq, spatial transcriptomics, and multiomic analyses to construct a comprehensive map of NCC lineages in developing mouse cardiopharyngeal tissues. We identify a transition from Hox-positive pharyngeal NCCs to Hox-negative intracardiac populations associated with the outflow tract cushion, accompanied by a shift in Meis transcription factor binding and gene-regulatory network architecture. By contrast, NCCs forming the aorticopulmonary septum and great vessel smooth muscle retain distinct Hox-codes. A Meis2-Sox9-Scx gene-regulatory network defines a skeletogenic progenitor-like intermediate state that gives rise to coronary artery smooth muscle and semilunar valves. Our findings suggest that the loss of Hox-dependent regional identity enables pharyngeal NCCs to acquire new fates upon entering the cardiac cushion, providing insight into the developmental origins of coronary and valvular calcification.

Microtubules are key components of the eukaryotic cytoskeleton involved in vital functions in virtually every cell. Among the emerging molecular mechanisms to adapt microtubules to their diverse functions is the biochemical diversification of tubulin molecules by posttranslational modifications (PTMs) and differential gene expression, a concept known as the 'tubulin code'. A key question remains whether the tubulin code has the potential to selectively control microtubule interactions of different microtubule-associated proteins (MAPs) to act as a specific signalling system. To answer this question, we used a medium-throughput in vitro approach to screen 46 proteins for their binding preferences to microtubules with altered PTM or isotype composition. We demonstrate that subsets of these MAPs have unique sensitivities to PTMs, while other proteins are not affected. As a result, PTMs, or combinations of them, differentially attract or repulse individual MAPs to microtubules. Our findings offer mechanistic proof for a key hypothesis of the tubulin code-the capacity to selectively and differentially regulate MAP-microtubule interactions.

Morphogenesis, the process by which cells, tissues, and organs acquire and maintain their form, is essential for embryogenesis, regeneration, and disease progression. During morphogenesis, cells within tissues dynamically interact with biophysical and biochemical signals from their environment by modifying their shape, gene expression, and function. These modifications often lead to transitions between cell states, and with that, to the establishment of functional programs that will in turn define embryonic stages or tissue states. Here, we discuss how the interplay of gene expression programs, chromatin modifications, and biophysical factors from the microenvironment contributes to the robust coordination of morphogenesis by allowing cells within tissues to dynamically transit across functional stages.