Nature Cell Biology

To ensure survival, cells need to buffer the effects of environmental stress on their plasma membrane, yet the structural mechanisms by which this is acutely achieved remain largely unknown. Here, we propose atonosomes as a unifying identity for a class of previously observed but enigmatic, tension-responsive, plasma membrane-derived compartments that arise across contexts of acute and chronic membrane tension loss. Leveraging unprecedented high resolution cryo-FIB-ET imaging in yeast, we show that atonosomes are complex, organelle-containing structures bounded by membranes and cell wall material, spanning hundreds of nanometers, and displaying a remarkable morphological diversity. Atonosomes form within seconds in response to reduced plasma membrane tension, and their emergence appears to require no dedicated molecular machinery, arising instead as a direct consequence of membrane biophysics. Upon formation, they recruit key membrane-associated proteins, including TORC2, Slm1, and septins. Under conditions of chronic disruption of PM homeostasis, atonosomes become constitutively present. Their stability and reversibility are further modulated by the cell wall, whose polymerization state influences atonosome dynamics. Structural conservation in fungi and ichthyosporea, demonstrates that atonosomes are a conserved stress-triggered response of cell-wall enclosed organisms. Together, these findings establish atonosomes as a novel compartment that mediates cellular responses to plasma membrane tension variation, coupling membrane remodeling and lipid homeostasis to preserve cellular integrity under stress.

A central dilemma of epithelial cell turnover is eliminating and replacing cells while simultaneously preserving tissue architecture and barrier function. Conventionally, apoptotic or non-apoptotic cell extrusion has been implicated in the intestinal epithelial turnover. Here, we identify a non-lytic membrane permeabilization program that drives physiological enterocyte turnover in vivo. In the Drosophila intestine, enterocytes undergo erebosis, a non-apoptotic form of cell death characterized by depletion of cytoplasmic proteins. We discover that this process is mediated by transient plasma membrane pores with estimated diameters of 16-50 nm, permitting extracellular protein influx and loss of cytoplasmic contents. The pore-forming protein Ninjurin A (NijA) accumulates as puncta during erebosis, and is necessary and sufficient for driving this process. NijA-mediated transient permeabilization preserves the membrane framework of dying cells, enabling their replacement without disrupting epithelial barrier architecture.

The liver is a metabolic hub with a high protein turnover that renders it uniquely susceptible to proteotoxic stress. Perturbation of proteostasis, either by proteasomal inhibitors or in chronic liver diseases, could adversely impact liver physiology. Here, we show that proteasomal inhibition unexpectedly suppresses basal type I interferon (IFN-I) signaling in the murine liver. Proteasomal inhibition by bortezomib selectively downregulates a subset of interferon-stimulated genes (ISGs), among which USP18 and ISG15 emerge as critical determinants of hepatocellular survival. We identify USP18 as a central cytoprotective factor that prevents proteotoxic apoptosis independently of its deubiquitinase activity, but strictly requires its scaffolding function mediated by isoleucine-60 and interaction with STAT2. Mechanistically, proteotoxic stress disrupts IRF9 nuclear translocation, attenuating USP18 transcription, and drives USP18 and other ISGs into insoluble aggregates with kinetics distinct from canonical IFN-I-induced insolubility. Strikingly, IFN-I priming preserves ISG solubility, restores USP18 abundance, and confers resistance to proteotoxic cell death. Together, these findings uncover an unanticipated link between proteostasis and innate immune signaling, and establish the USP18-STAT2 axis to enhance hepatic resilience under proteotoxic stress.

Intracellular compartmentalization is fundamental to cellular organization, yet mechanobiology has been largely understood through membrane-delimited structures and associated signaling pathways. Whether mechanical forces directly regulate biomolecular condensates, which organize many core cellular functions, remains largely unknown. This question is particularly relevant for the nucleolus, a prominent nuclear condensate that coordinates ribosome biogenesis and is known to remodel in response to diverse biochemical perturbations, placing it at the interface between cellular state and biosynthetic control. Here, we show that mechanical compression remodels nucleolar organization and reduces (ribosomal DNA) rDNA transcription, and identify nucleolin as a key mediator of this adaptive response. Compression induces rapid and reversible redistribution of nucleolin from the nucleolus to the nucleoplasm, accompanied by reduced occupancy at rDNA promoter regions and changes in rDNA transcription and precursor rRNA processing. The nucleolar response occurs independently of classical post-translational regulation of nucleolin and instead depends on the rate of nuclear deformation, with nucleolar organization and function scaling with nuclear volume loss, supporting a mechanism of biophysical regulation. Together, our findings establish the nucleolus as a mechanosensitive condensate and reveal dual regulation of ribosome biogenesis by mechanical compression, through rapid nucleolin-based biophysical adaptation followed by slower epigenetic remodeling.

Severe proteinopathies--such as retinitis pigmentosa, a form of inherited blindness--are driven by genetic mutations that overwhelm the quality control of the post-endoplasmic reticulum (post-ER) secretory pathway, causing toxic protein accumulation. Here, we identify a therapeutic node defined by a hetero-oligomeric cargo receptor complex consisting of TMED7, 2, 9, and 10. This "entrapment complex" anchors structurally and functionally diverse mutant clients within the early secretory pathway via TMED7 binding to the integral Golgi protein GRASP55. Disruption of the entrapment complex results in the clearance of accumulated protein cargoes. In vivo ablation of the entrapment node via inducible genetic deletion or via the small molecule BRD7635 reverses histopathological hallmarks and rescues functional deficits in clinically distinct proteinopathies of the kidney and the eye, including mitigating vision loss in a mouse model of retinitis pigmentosa.

Developmental competence allows tissues to respond to inductive cues before committing to specialized forms, but how this potential is encoded at clustered gene-family loci is poorly understood. We use vertebrate skin to address this problem. Epidermis responds to regional dermal signals before committing to feather, scale, or differentiated programs, and -keratin loci provide a stringent genomic test: separated type-I/type-II clusters show coordinated transcriptional pairing, yet individual keratin genes are selectively deployed across appendage, differentiation, and disease states. Using chicken developmental genomics with comparative mouse and human epidermal datasets, we show that -keratin clusters are organized before commitment as scaffolded chromatin domains. Within these domains, regulatory elements remain broadly accessible but acquire state-specific activity during commitment and differentiation. Inter-cluster contacts and chromatin-factor perturbation link this architecture to keratin output and morphology. These findings reveal a locus-level chromatin basis for developmental competence, enabling domain-level coordination with gene-level selectivity during epidermal diversification.

Cells enable specialized metabolism by compartmentalizing metabolic pathways into distinct organelles, which requires the membrane transport of metabolites. In melanocytes, the amino acid tyrosine is imported into developing melanosomes for the synthesis of the UV-protective pigment melanin1,2. In spite of extensive biochemical characterization, the identity of the melanosomal tyrosine transporter remains unknown. Here, we identify SLC16A6 as an orphan melanosome-localized metabolite transporter. Genetic screens reveal that SLC16A6 expression is driven by the SOX10-MITF axis, the well-characterized master regulatory program governing melanogenesis and melanosomal homeostasis3,4. By redirecting SLC16A6 to the plasma membrane with an S240A mutation5, we demonstrate that SLC16A6 transports tyrosine, a process competitively inhibited by other bulky amino acids. We further determine that SLC16A6 is sufficient for in vitro melanosomal tyrosine uptake. Genetic depletion of SLC16A6 triggered loss of melanosome biogenesis and function as well as depletion of most melanosomal components. Collectively, these findings establish SLC16A6 as a melanosomal tyrosine transporter that is essential for melanosome biogenesis.

Angstrom-scale changes in microtubule (MT) lattice spacing regulate the selective recruitment of MT-associated proteins, yet how these structural states operate in cells remains poorly understood. Here, we show that MT lattice expansion, induced by protein-based expanders or microtubule-stabilizing agents such as Taxol and epothilone D, drives the relocalization of compact lattice-binding proteins, including tau, doublecortin (DCX), and the C1 domain-containing signaling protein GEF-H1, into highly curved MT-associated domains, whereas the compaction-inducing agent laulimalide suppresses this response. In contrast, the tumor suppressor RASSF1A preferentially associates with expanded lattice states, revealing differential lattice sensitivity among closely related C1 domain-containing proteins. These short, curved assemblies are enriched at MT intersections and discrete MT segments, revealing spatially heterogeneous lattice states within individual microtubules. At substoichiometric levels, compact lattice-binding proteins behave as both MT compactors and curvature sensors. Changes in osmotic pressure selectively promote dissociation of compact lattice-binding proteins, whereas expanded lattice-binding proteins remain largely unaffected. Using curved filament formation as an in-cellulo readout of compact lattice regions, we identify widespread lattice-state sensitivity across diverse MT-associated and signaling proteins. Finally, we show that these principles extend to neurons, where somatic, but not axonal, tau exhibits sensitivity to lattice expansion despite the expanded lattice architecture of distal axonal microtubules, suggesting additional neuron-specific regulation of lattice accessibility. Together, our findings identify the MT lattice as a dynamic mechanochemical platform whose nanoscale structural states spatially organize protein recruitment and signaling in cells and neurons.

DNA replication forks frequently encounter obstacles and remodel into four-way junctions to actively slow DNA fork progression. Fork slowing can also spread to undamaged forks via an ATR-dependent mechanism that remained elusive. Here, using mild genotoxic stress, we show that fork slowing and reversal require full ATR activity, but no canonical ATR activators and signalling partners, defining a non-canonical ATR pathway distinct from origin firing control. Phospho-proteomics in S-phase cells under checkpoint-blind replication stress identified a subset of ATR-dependent phospho-sites, such as S343 on NBS1, the regulatory subunit of the MRN complex. This residue is essential to stimulate MRN exonuclease activity in vitro and required in cells for global fork slowing upon mild DNA damage. Ultimately, local UV-C micro-irradiation reveals that ATR-dependent MRN-stimulated resection dampens DNA synthesis at lesions and propagates fork slowing to undamaged chromatin, supporting MRN-mediated ssDNA exposure as a mean to coordinate replication slowdown across the nucleus.

HighlightsCERAMIC enables continuous and high-capacity lineage tracing of liver tumor initiation Fatty liver-associated hepatocytes acquire regenerative and premalignant cell states before malignant transformation Lineage reconstruction identifies Hep_Bi-zonal cells as the cellular origin of liver tumor initiation Transcriptional and regulatory programs distinguish tumor-fated hepatocytes from failed-to-transform lineages Peroxisomal metabolism is required for progenitor-state formation and liver tumor initiation Spatial remodeling identifies a macrophage niche associated with tumor-fated hepatocytes Dual ontogenies and functional specialization of lipid-associated macrophages shape the tumor-fated hepatocyte niche Fatty liver disease predisposes to primary liver cancer, yet the lineage routes and niche mechanisms that select rare tumor-fated hepatocytes remain unclear. Here we developed CERAMIC, a high-capacity CRISPR-Cas9 lineage recorder that co-recovers editing scars and transcriptomes from single cells, and applied it to an AKT/NRAS-driven model of MASLD-associated liver tumor initiation. Longitudinal lineage, single-cell and spatial analyses revealed a hierarchical trajectory in which Hep_Bi-zonal cells, rather than Hep_CVlike cells, generated regenerative and neoplastic hepatocyte progenitor states that progressed toward both hepatocellular carcinoma and intrahepatic cholangiocarcinoma lineages. Tumor-fated cells preferentially expanded along a remodeled midlobular-periportal axis and depended on ACOX1-mediated peroxisomal beta-oxidation to withstand lipotoxic and oxidative stress. Spatial and lineage analyses further identified a sequential lipid-associated macrophage niche, in which monocyte-derived LAMs engaged tumor-fated hepatocytes through an LGALS9-P4HB axis, and P4HB inhibition suppressed tumor expansion. These findings define liver tumor initiation as a lineage-restricted process licensed by peroxisomal metabolic adaptation and macrophage-derived niche signals.

Telomerase RNA (TERC) is known as the essential template for telomere elongation. Here, we report an unexpected role for TERC in regulating chromatin accessibility, which is primed for determining the cell fate of human embryonic stem cells (hESCs). TERC-deficient hESCs retain critical markers for pluripotency but fail to undergo lineage differentiation as shown by standard in vivo teratoma formation as well as in vitro differentiation assays, which is consistent with repressed transcription during differentiation into the three germ lineages. Notably, transient re-introduction of TERC into TERC-deficient hESCs rescued lineage differentiation capacity without restoring telomere length. TERC binds to the promoters and enhancers of developmental genes marked by H3K27ac to maintain an open chromatin state. Loss of TERC reduces H3K27ac deposition and decreases chromatin accessibility through the remodelling of three-dimensional genome organization, including TAD boundary insulation and compartment switching. Collectively, our findings reveal that TERC is a chromatin-associated noncoding RNA that regulates the epigenomic architecture that governs cell fate for lineage commitment during development.

The maternal-to-zygotic transition (MZT) requires coordinated clearance and deadenylation of maternally deposited mRNAs, yet the underlying molecular mechanisms remain poorly understood. N6-methyladenosine (m6A) has emerged as a key regulator of maternal mRNA fate, but prior studies have relied on population-averaged short-read methods that cannot resolve modification state, poly(A) tail length, or isoform identity on the same molecule. Here, we employ nanopore direct RNA sequencing on zebrafish embryos across MZT to resolve the interplay between m6A deposition, mRNA clearance, and poly(A) tail length dynamics at single-molecule resolution. We find that 78% of expressed maternal genes harbor m6A-modified isoforms, significantly exceeding prior bulk estimates. Within-isoform comparisons demonstrate that m6A promotes mRNA decay, with CDS m6A contributing more to maternal mRNA clearance than 3-UTR m6A. The positional context of m6A alone is sufficient to determine the temporal regulation of poly(A) tail lengths. CDS m6A constitutively suppresses tail length throughout MZT, while 3'-UTR m6A acquires shortening activity only after zygotic genome activation (ZGA). Transcriptomic analysis of ythdf2 knockout embryos reveals two unrecognized roles. Ythdf2 stabilizes m6A-marked maternal transcripts to set stoichiometry at MZT onset, and is also responsible for maintaining global poly(A) tail homeostasis prior to ZGA through an m6A-independent mechanism. Together, these findings define the single-molecule logic by which m6A modifications shape transcript fate during vertebrate MZT.

Effective lysosome function is essential for health, and declines with ageing and disease. Upon lysosome damage, cells mount a complex stress response to restore homeostasis. Membrane ATG8ylation plays a central role, orchestrating lysosome repair, replacement and removal, either directly at the damaged membrane, via CASM (conjugation of ATG8s to single membranes), or at nascent autophagosomes, during lysophagy. Here, we identify a novel role for CASM in driving an antioxidant response to lysosome stress. Following damage, CASM regulates both lysosome ubiquitination and p62 recruitment. Membrane-associated p62 undergoes S349-phosphorylation, which permits KEAP1 sequestration, thereby releasing master transcription factor, NRF2. Liberated NRF2 translocates to the nucleus and promotes transcription of antioxidant and detoxifying genes, co-ordinating a cytoprotective response, in a CASM-dependent manner. These findings position CASM as a major upstream response to lysosome stress and uncover the p62/KEAP1/NRF2 axis as a novel effector pathway, termed SOLAR (SQSTM1/p62 Oligomer-mediated lysosome antioxidant response).

Necroptosis is best known as a lytic, proinflammatory cell-death pathway mediated by RIPK3 and MLKL. Effective wound repair requires the rapid resolution of inflammation, and ongoing necroptotic activity would only exacerbate tissue damage, delaying healing. However, damaged skin presents a trigger-rich environment for necroptotic signalling, an apparent paradox that remains unresolved. Using genetic ablation and pharmacological inhibition across multiple wound models, we show that inhibiting necroptosis accelerates wound closure, revealing that necroptotic signalling normally restrains repair. Surprisingly, we found that MLKL activation in wild-type keratinocytes induces differentiation and membrane repair rather than cell lysis. This adaptive, non-lethal mode of necroptotic signalling preserves barrier integrity but slows re-epithelialisation. Our findings redefine epidermal necroptotic signalling as a stress-responsive program that modulates keratinocyte fate in a trigger-rich environment. Temporarily dampening this pathway may enhance regeneration after barrier loss without compromising immune defence, revealing necroptosis as a tunable mechanism balancing tissue repair and inflammation.

Germinal centers (GCs) support physiological B-cell mutagenesis and are considered lymphoma-permissive; nevertheless, lymphoma development is uncommon. Human in situ follicular neoplasia (ISFN) captures this paradox: premalignant B-cells can persist within GCs for prolonged periods without progressing to overt lymphoma. We found that human ISFN, but not normal GCs, are infiltrated by CD8 T-cells, suggesting that premalignant GC B-cells are locally immune-surveilled. Using mouse models that separate early premalignant fitness from lymphoma-associated evolution, we show that fitness-enhanced premalignant GC B-cells expand transiently but are selectively eliminated by infiltrating cytotoxic CD8 T-cells, while normal GC B-cells are spared. By contrast, evolved premalignant GC B-cells retain their fitness but disable productive CD8 T-cell cytotoxic differentiation, allowing persistence and lymphoma-like transcriptional and genomic evolution. These findings establish GCs as active immune-surveillance sites and show that progression from premalignancy to lymphoma requires both enhanced GC fitness and escape from local immune control. Key findingsGCs undergo active immune-surveillance to detect premalignant B-cells. Premalignant GC B-cells trigger cytotoxic CD8 T-cell responses. Lymphoma-associated evolution enables immune-escape within GCs. Fitness and immune-escape drive evolution from premalignancy to lymphoma. BlurbGerminal centers are considered lymphoma-permissive; however, progression from premalignancy is uncommon. Using models of human in situ follicular neoplasia, Zhang et al. demonstrate that infiltrating CD8 T-cells actively eliminate premalignant GC B-cells. Co-occurrence of lymphoma-like alterations blocks this cytotoxic T-cell response, driving immune escape and lymphoma evolution.

Hematopoietic stem cell (HSC) longevity critically depends on maintaining a deep dormant state, yet the molecular mechanisms that preserve this rare and functionally essential population remain poorly understood. Here, we identify the deubiquitinase USP7 as a key regulator of long-term HSC dormancy. Using a Usp7+/- mouse model, we uncover selective depletion of hematopoietic stem and progenitor cells (HSPCs), which is associated with impaired long-term repopulation capacity. Strikingly, H2B-GFP label-retention assays reveal a profound loss of dormant HSCs in Usp7+/- mice, demonstrating a failure to maintain the most quiescent stem cell fraction in vivo. Consistently, single-cell RNA sequencing shows erosion of the transcriptional dormancy program, linking USP7 activity to the preservation of stem cell identity at both functional and molecular levels. Mechanistically, ultra-low-input proteomic profiling and biochemical approaches identify HMGA2 as a novel USP7 substrate, suggesting that ubiquitin-dependent regulation of chromatin architecture contributes to the control of HSC dormancy. Together, our findings establish USP7 as a critical regulator of HSC dormancy, revealing a previously unrecognized post-translational mechanism controlling stem cell longevity, with implications for aging, regeneration, and hematopoietic disorders.

Macrophages are the vanguard of innate immunity, relying on phagocytosis to clear invading pathogens. Podosomes in macrophages have recently emerged as mechanical structures that assemble at phagocytic cups to facilitate pathogen clearance. However, how these unique actin structures are regulated to execute antimicrobial functions remains poorly understood. Here, we reveal that fungal infection drives podosome maturation to strengthen macrophage mechanical immunity. Exposure to the opportunistic fungus Candida albicans upregulates the scaffold protein Tks5 and activates Src kinase, which repurposes dynamin-2 from a membrane fission enzyme into an actin-bundling protein. This functional switch stabilizes the podosome actin core, enhancing the adhesion and mechanical force generation required to efficiently engulf, fold, and trap invading fungi. Ultimately, our findings define a mechanobiological paradigm in which infection-induced molecular repurposing drives macrophage antifungal immunity.

Cholesterol biosynthesis is classically viewed as a tumour supportive metabolic programme, yet cholesterol-associated metabolic states can paradoxically coincide with improved responses to immune checkpoint blockade (ICB)1, highlighting an unresolved context-dependent relationship between cholesterol homeostasis and antitumour immunity. Here we identify PCIF1, a cap-specific RNA methyltransferase2, as a tumour-intrinsic brake on the inflammatory potential of cholesterol biosynthesis. PCIF1 depletion had limited effects on tumour cell proliferation in vitro, but suppressed tumour growth in immunocompetent, not immunodeficient, hosts and sensitized immune-refractory syngeneic tumours to ICB. Immune profiling by single-cell RNA sequencing, flow cytometry and multiplex immunofluorescence revealed increased CD8+ T cell infiltration in PCIF1-deficient tumours, and CD8+ T cell depletion restored tumour growth. Mechanistically, PCIF1 loss selectively enhanced translational engagement of SCAP, activated SREBP2-dependent cholesterol biosynthesis and promoted intracellular cholesterol accumulation. Unexpectedly, activation of this cholesterol programme induced a tumour cell inflammatory cytokine state enriched for T cell-recruiting chemokines, including CXCL10. Pharmacological perturbation of the SCAP-cholesterol axis attenuated this inflammatory programme, placing cholesterol sensing upstream of PCIF1 loss-induced tumour cell inflammatory activation. In ICB-treated patient cohorts, high PCIF1 expression in pre- and on-treatment samples was associated with poor clinical response, and across tumour types, PCIF1 expression inversely correlated with cytolytic immune signatures. These findings reveal PCIF1-mediated SCAP translational control as a tumour cell checkpoint that constrains the antitumour inflammatory activity of a canonical growth-associated cholesterol programme and shapes checkpoint therapy responsiveness.

Nuclear pore complexes (NPCs) control molecular exchange across the nuclear envelope, but how they tailor their selective permeability to meet the needs of specific cell types and/or environments remains poorly understood. We demonstrate that the strength of the NPC diffusion barrier differs across cell types, is particularly stringent in cultured neurons, and correlates with the O-linked N-acetylglucosamine (GlcNAc) modification of nucleoporins. Using conditional tools that specifically control nucleoporin GlcNAcylation, we show that GlcNAc modulates NPC permeability. Interestingly, nucleoporin GlcNAcylation is mechanosensitive, increasing in cells plated on stiff substrates, a condition where nuclear pores dilate. Indeed, we demonstrate that increasing or decreasing GlcNAcylation dilates and constricts NPCs, respectively. Further, O-linked N-acetylglucosamine transferase is recruited to modify NPCs upon their acute constriction during osmotic shock. Thus, cells employ GlcNAc to modulate steady-state NPC permeability in response to mechanical inputs and to counteract critical changes to their osmotic environment.

Nicotinamide adenine dinucleotide (NAD+) and its precursor nicotinamide mononucleotide (NMN) are strictly compartmentalized, yet how individual organelles maintain local metabolic homeostasis remains unresolved. Here, we report FrNADS and FrNMNS1.0, a FRET-based biosensor toolkit that maps NAD+ and NMN dynamics in living cells with subcellular resolution, including the oxidizing lumen of the endoplasmic reticulum. We find that NAD+ recovery in the nucleus following PARP1 activation depends on NAMPT mediated salvage synthesis, while peroxisomes buffer NAD+ via NUDT12 and SLC25A17. In mitochondria, NMNAT3 acts as a NAD+ hydrolase that counterbalances import through SLC25A51; HINT2 functionally enhances this activity. Furthermore, SLC25A48 functions as a critical regulatory node that modulates the compartmental redistribution of the generated NMN. These findings establish a mitochondrial NAD+/NMN regulatory circuit and reveal how organelles independently resolve metabolic stress.