Nature Cell Biology

Cargo receptors are central organizers of the secretory pathway, yet the mechanisms controlling their abundance remain poorly understood. The endoplasmic reticulum (ER)-resident intramembrane protease RHBDL4 promotes substrate turnover via a non-canonical branch of ER-associated degradation and has recently been implicated in regulating secretory pathway components. We previously identified the p24 cargo receptor TMED7 as an RHBDL4 substrate, suggesting that cargo receptor turnover contributes to secretory pathway regulation. Here, quantitative proteomics identify members of the ER-Golgi intermediate compartment (ERGIC) cargo receptor family as endogenous RHBDL4 substrates, demonstrating that RHBDL4 targets multiple cargo receptor families within the early secretory pathway. Accordingly, RHBDL4 modulates multiple ERGIC-dependent transport pathways. In addition, unbiased secretome analysis reveals increased secretion of lysosomal precursor proteins upon RHBDL4 ablation. Mechanistically, we show that this phenotype is mediated, at least in part, by RHBDL4-dependent cleavage of the lysosomal cargo receptor sortilin/SORT1. Together, these findings identify cargo receptors as a major class of RHBDL4 substrates and establish proteolytic remodeling of cargo receptor networks as a mechanism for regulating secretory pathway flux.

The ability to longitudinally track clonal evolution non-invasively would transform cancer interception strategies, long before late-stage disease when most cancer genomes are analysed. Here, we demonstrate that repeated hair sampling from the same individual followed by exome sequencing enables tracking of somatic evolution in vivo over several months after chemically induced skin carcinogenesis. We found that hair follicles accumulate a higher mutation burden than spatially-matched skin and harbour mutations that spread into surrounding epidermis and persist throughout tumour progression. DNA-damaged follicles enter sustained quiescence that delays replication and repair, creating a reservoir for long-lived mutations. During premalignant progression, carcinogen-associated mutations become enriched as follicular clones expand into adjacent skin. Mutation tracking identified genes that may govern tumour predisposition and initiation, many of which are mutated at high incidence in human cutaneous squamous cell carcinoma cohorts. Hair follicles therefore provide a non-invasive readout to forecast the early development of skin cancer, enabling patient risk stratification.

Lineage-restricted transcriptional programs establish cell identity and can create selective dependencies in cancer. Here, we identify POU2AF2, encoding the transcriptional co-activator OCA-T1, as a critical lineage-specific dependency in a subset of diffuse large B-cell lymphoma (DLBCL). Pan-cancer dependency analyses and patient cohorts reveal elevated POU2AF2 expression in genetically aggressive DLBCL, where its depletion markedly suppresses tumor growth in vitro and in vivo. Mechanistically, POU2AF2 cooperates with the B-cell lineage-defining transcription factor POU2F2 (OCT2) to activate lymphocyte activation gene programs through direct chromatin engagement, thereby sustaining malignant transcriptional networks. We further identified a key epigenetic regulatory axis composed of the lineage-specific transcription factor TCF3 and the histone methyltransferase SET1A-COMPASS that drives POU2AF2 expression downstream of B-cell receptor signaling. Single-cell transcriptomic analysis reveals that POU2AF2 marks and sustains an innate-like B1 B-cell population in vivo, a candidate cell of origin for lymphoma. Together, these findings define a lineage-restricted POU2AF2/POU2F2 transcriptional module, controlled by a TCF3/SET1A epigenetic network, that sustains both innate-like B-cell identity and malignant fitness in DLBCL. Our study uncovers a previously unrecognized lineage-specific transcriptional dependency and highlights POU2AF2 and its associated regulatory circuitry as potential therapeutic targets in aggressive B-cell malignancies.

Plant-specific RNA Polymerase V (Pol V) transcribes noncoding RNAs in the RNA-directed DNA methylation pathway, thereby influencing gene expression and genome stability by controlling de novo DNA methylation. However, the mechanisms governing precise chromatin localization and transcriptional activities of Pol V remain elusive. Here we show that Pol V localization is spatially constrained by the chromatin regulators microrchidia (MORC) and MORPHEUS' MOLECULE 1 (MOM1). MORC and MOM1 promote Pol V occupancy at sites near active chromatin, whereas their loss leads to redistribution of Pol V into CMT3-enriched heterochromatin, accompanied by noncoding RNA transcription, small RNA production and DNA methylation. Our findings reveal a combinatorial model in which recruitment, spatial constraint and DNA methylation feedback collectively define Pol V chromatin distribution and epigenetic function.

Single-cell technologies have generated cell censuses of tissues, however, how tissue geometry reflects functional needs remains poorly characterized. The human pilosebaceous unit offers a tractable model, a prenatally-formed complex mini-organ combining hair and sebum production with a stem cell reservoir. Using histomorphology, spatial transcriptomics, and single-cell multiomics on the same human prenatal scalp skin samples (8-19 post-conception weeks), integrated and analyzed using machine learning approaches, we built a spatiotemporal map of pilosebaceous unit development. We demonstrate that epithelial-mesenchymal interactions coordinate cellular fate and organogenesis, using an in vitro hair-bearing skin organoid model to validate this tissue-patterning. In addition, we show sebaceous gland developmental programmes are overcome during tumor formation. Our large-scale multi-modal analysis provides a unique framework for understanding form and function of tissues with applications in tissue engineering and pathology.

Peripheral sensory neurons innervate the skin to detect mechanical, thermal, and noxious stimuli. Within the epidermis, nerve fibers terminate beneath tight junctions, shielding them from environmental exposure. Although epidermal differentiation coordinates tight junction assembly, its role in organizing nerve terminals is poorly understood. Here, we show that activation of Notch, a master regulator of epidermal differentiation, caused near-complete loss of epidermal innervation. This was largely the result of increased contractility rather than impaired differentiation. Inducing epidermal contractility was sufficient to deplete nerve fibers with striking spatial precision, and restoring normal contractility reversed this effect. Actomyosin contractility is highest in the granular layers of the epidermis, where tight junctions form and nerve fibers terminate. Ablation of nonmuscle myosin II allowed nerve fibers to extend beyond their normal termination zone and caused touch hypersensitivity. Together, these findings demonstrate that epidermal contractility positions sensory nerve endings through spatially controlled pruning and defines a mechanical boundary established by epidermal cells that restricts neuronal outgrowth.

Replication-associated errors can cause DNA damage to accumulate on the newly synthesized strand over time. In specific cases such as stem cells, retention of the immortal strand used as template preserves one daughter cell into pluripotency while correlating with terminal differentiation of the damage one. In somatic cells, DNA damage distribution after mitosis remains unclear. Here, we uncovered a mechanism of non-random segregation of the DNA damage marker gH2AX occurring during a single cell division cycle. Replication stress using hydroxyurea (HU) upon release into S phase in RPE-1, BJ, hCEC D29 and fibroblasts showed reproducible Non-Random Segregation (NRS) of gH2AX in the ensuing G1, a phenotype not observed in any of the cancer cell lines analyzed. Notably, removal of R-loops led to a reduction of cells with NRS, whether RNaseH1 was over-expressed globally or exclusively targeted to centromeres, indicating that centromeric DNA-RNA hybrids contribute to NRS of the damage. In line with our previous evidence of centromeric chromatin disruption leading to R-loops, rapid removal of the histone H3 variant CENP-A causes damage and NRS, although to a lower extent than HU alone. This implies that additional mechanisms contribute to centromeric R-loops and NRS of damage in the daughter cells upon mitotic exit. Mechanistically, chemical inhibition of the catalytic activity of Rad51 led to a significant drop in NRS without a change in the total amount of damaged cells, implying involvement of the Homologous Recombination (HR) pathway to accumulation of gH2AX to only one chromatid. In turn, this affects the spindle-kinetochore with a measurable length asymmetry, inducing mechanical and/or epigenetic signals that affect the orientation of the sister chromatids on the metaphase plate to bias segregation. Altogether, we found replication-induced asymmetric segregation of DNA damage during mitosis that is influenced by centromeric R-loops, Rad51 activity and spindle dynamics, with implications on cell fate, chromosome and genome stability in the daughter cells.

Protein synthesis is dynamically regulated to control cell growth, differentiation, and stress responses. Recent single-cell sequencing methods can map ribosome positions on individual transcripts, but cannot capture the global translational states that coordinate protein synthesis across the transcriptome. In contrast, methods that measure the global translational landscape, such as polysome profiling and cryogenic electron tomography, lack either single-cell resolution or throughput. Here we introduce SCISSOR (Single-Cell Inference of Structural States of Ribosomes), a strategy that infers global translation activity in individual cells from the differential protection of ribosomal RNA (rRNA) against nuclease digestion. By integrating these protection signatures with the structure of the ribosome, SCISSOR resolves multiple ribosomal states and quantifies their abundance across thousands of individual cells. Applying SCISSOR reveals systematic variation in global translation across the cell cycle in human cells, as well as during the differentiation of murine intestinal stem cells into distinct epithelial lineages. These findings uncover principles of global translational regulation that are invisible to transcriptomic or ribosome-profiling assays, establishing a framework for studying global translation control at single-cell resolution.

Solid tumors remain refractory to conventional treatments, yet cell surface proteins, by virtue of their extracellular accessibility and critical roles in tumor signaling, represent an attractive class of targets for precision-targeted therapy. Here, we report that TMEM132A is an essential and previously unrecognized pan-cancer target. TMEM132A interacts directly with EGFR and stabilizes its expression, thereby tethering EGFR at the plasma membrane and sustaining constitutive activation of lipid synthesis. Mechanistically, the TMEM132A-EGFR axis promotes lipogenesis by facilitating SREBP nuclear translocation, which in turn upregulates ACLY and ACSS2 expression to drive acetyl-CoA production and downstream lipid biosynthesis, ultimately disrupting lipid droplet homeostasis. To therapeutically target this axis, we developed a nanobody, LFNanoT132A#3, which effectively blocks the TMEM132A-EGFR interaction, abrogates downstream signaling activation, and potently inhibits proliferation across multiple solid tumor types. Notably, LFNanoT132A also exerts robust antitumor activity against H1975 xenografts, a model resistant to first- and second- generation EGFR inhibitors, underscoring its potential to overcome conventional drug resistance. Our findings establish TMEM132A#3 as a critical node in membrane-tethered oncogenic signaling and metabolic rewiring, and position LFNanoT132A#3 as a promising therapeutic candidate for precision cancer therapy.

Cell shape changes during migration, division, or differentiation require the dynamic regulation of actin network mechanics. Actin crosslinkers are central to this regulation, controlling network connectivity and the transmission of contractile forces. A large diversity of crosslinkers exists, differing in length, domain structure, and binding kinetics, yet why cells deploy specific crosslinkers in a physiological context remains unclear. To bridge this gap, we developed a light-controlled actin crosslinker toolbox spanning three physiologically relevant lengths: ~9 nm (fascin-like), ~16 nm (fimbrin-like), and ~56 nm (alpha-actinin-like). Using magnetic pincher experiments and in silico modelling, we show that short and mid-length crosslinkers dynamically tune cortical stiffness and thickness in a density- and myosin-dependent manner, with short crosslinkers also driving pronounced stress-stiffening as the cortex is deformed. Strikingly, minute-scale activation reveals a length-dependent switch in cell behaviour: short crosslinkers cause cortical delamination, while long ones instead drive cell polarization and symmetry breaking. This switch can be overridden by perturbing actin turnover, which unlocks polarization in mid-length crosslinkers that otherwise delaminate. Crosslinker-induced polarization is not merely a local cortical event: it directs subsequent cell spreading, coupling a nanometre-scale molecular choice to a cell-scale decision about movement. Together, these findings establish a versatile optogenetic platform for manipulating actin crosslinking, and show that the cortex can encode a behavioural switch directly in its material architecture.

Hematopoietic stem cell (HSC) aging is associated with epigenetic remodeling, yet the molecular mechanisms driving these changes, their overlap with stress-induced alterations, and whether this course can be durably reset remain incompletely understood. Here, we show that transient induction of the Yamanaka factors OCT4, SOX2, KLF4, and MYC in young mice durably delays and partially reverses physiological and LPS-driven HSC aging in mice. Transient reprogramming improved hematopoietic reconstitution, reduced myeloid bias, and limited DNA damage. Multi-omic analyses revealed reduced chromatin accessibility at AP-1-enriched regulatory regions, attenuated age-associated AP-1 transcriptional programs, and repression of transposable elements (TEs). Pharmacological AP-1 inhibition prevented LPS-induced TE activation and loss of HSC clonogenicity. Reverse transcriptase inhibition in aged mice reduced DNA damage and improved HSC function, demonstrating a functional contribution of TE activity to HSC decline. Together, these findings identify AP-1-associated chromatin remodeling as a candidate mechanism linking inflammatory stress, TE activation and HSC aging.

X-chromosome inactivation involves chromosome-wide gene silencing accompanied by extensive chromatin changes, as well the loss of topologically associating domains. Yet discrete regions of the inactive X chromosome retain activity within localised 3D domains, which contain active genes that variably escape from X inactivation. The transcription factor and architectural protein CTCF has been proposed to be implicated in escape by insulating escape domains or sustaining their topology via cohesin-mediated loop extrusion. Here, we test the role of CTCF and cohesin in escape using acute degron-mediated depletion of CTCF and RAD21 in neural progenitor cells with established escape profiles. Although CTCF occupancy correlates with escape status on the inactive X chromosome, its removal - together with loss of loop extrusion - does not disrupt escapee gene expression, or domain organization, nor does it result in spreading of silencing or activation of genes in cis. Rather, we show that facultative escape regions are self-sustaining compartments of active chromatin enriched in H3K27 acetylation and depleted in H3K27 methylation, with the magnitude of compartment strength scaling up with the degree of transcriptional activity on the inactive X chromosome. These active escapee compartments are propagated independently of CTCF and RAD21-dependent 3D architecture. Our findings identify chromatin compartmentalization as the primary feature of facultative escapee domains.

A comprehensive recording of cell fate transitions and underlying molecular changes remains a fundamental goal in developmental biology. Here, we present DeepTrack, a lineage tracing mouse model that integrates in situ cellular barcoding with high-throughput, single-cell multi-omics to simultaneously profile clonal fates, transcriptomic states, and chromatin accessibility. Using DeepTrack, we profiled clonal behaviors during gastrulation and early organogenesis, uncovered early fate priming within epiblast clones, and revealed clonal architecture within distinct regions of the nervous system. Embryo-wide multi-omic lineage tracing at single-cell resolution revealed transcriptional and epigenetic programs underlying fate commitment in neuromesodermal progenitors (NMPs). Clonal tracing with multi-omic profiles enabled inference of fate-associated gene-regulatory networks and identified the transcription factor Cdx2 as a key regulator of mesodermal specification in NMPs. Genetic perturbation of Cdx2 in chimeric embryos impaired paraxial mesoderm differentiation. Together, DeepTrack provides a versatile framework for decoding multimodal regulation of cell fate across diverse developmental contexts.

Skin, the bodys largest mechanosensitive organ, relies on a tension gradient across epidermal layers to maintain structure and function, but how mechanical force contributes to epidermal development and disease pathogenesis is poorly understood. By anchoring intermediate filaments (IF) to the plasma membrane, desmosomes, the most abundant intercellular junctions in the epidermis, help create a supracellular scaffolding that provides mechanical resilience to the tissue. However, the contribution of the desmosome-IF network to the epidermal response to mechanical strain remains unknown. Here we show that the desmosome-IF connection is not only required to induce a proper cellular mechano-response but is actively strengthened in response to stretch through the PP2A-mediated phospho-regulation of the cytoskeletal linker protein desmoplakin (DP). Additionally, we show in human skin dephosphorylated DP localizes to high tension layers, suggesting this mechano-response mechanism is coordinated with the epidermal tension gradient. Furthermore, in models of Carvajal syndrome, a cardio-cutaneous disorder caused by truncating DP mutations, cells lose mechano-responsive behavior and exhibit abnormal morphology in high-tension epidermal layers. Together, these findings identify the DP-IF network as a key component of the response to mechanical strain and show that its disruption compromises epidermal homeostasis and contributes to disease pathogenesis.

O-linked {beta}-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole writer for intracellular O-GlcNAcylation. It catalyzes O-GlcNAcylation of thousands of protein substrates, but relatively less is known about the post-translational modifications that occur on OGT itself. Herein, we demonstrate that OGT is S-palmitoylated at Cys-472 and Cys-477, which is mediated by the S-acyltransferase Zinc Finger DHHC-Type Palmitoyl transferase 14 (zDHHC14) and removed by acyl protein thioesterase 2 (APT2). S-Palmitoylation stabilizes OGT by shunting it away from the lysosomal chaperone-mediated autophagy (CMA) pathway, as S-palmitoylation decreases the interaction between OGT and heat shock cognate 70 kDa protein (HSC70), the CMA chaperone. Via label-free quantitative mass spectrometry, we find that S- palmitoylation elevates the affinity between OGT and protein phosphatase 1 catalytic subunit gamma (PPP1CC), but not PPP1CB. We further demonstrate that S-palmitoylation of OGT augments binding with Yes-associated protein-1 (YAP), a protein that associates with PPP1CC, and subsequently enhances YAP O-GlcNAcylation. Our work unearths S-palmitoylation of OGT and CMA-mediated degradation of lysosomal OGT, the orchestration of which finetunes the activity of key OGT complexes, such as OGT-PPP1CC, and contributes to OGT substrate selectivity.

Pancreatic ductal adenocarcinoma (PDAC) exhibits profound metabolic rewiring and strong resistance to DNA-damaging therapies, yet how metabolic pathways regulate genome maintenance remains poorly understood. The hexosamine biosynthetic pathway (HBP) integrates nutrient availability with protein glycosylation through production of UDP-GlcNAc, but its role in DNA damage response (DDR) regulation is unclear. Here we show that inhibition of the HBP enzyme phosphoglucomutase-3 (PGM3) reduces DNA repair capacity in pancreatic cancer cells. Transcriptomic and functional analyses reveal that the selective PGM3 inhibitor FR054 amplifies gemcitabine-induced replication stress, disrupts ATR-CHK1 and ATM-CHK2 checkpoint signaling, and selectively impairs homologous recombination. Glycoproteomic profiling identifies the AAA+ ATPase RUVBL2 as a key metabolic-DDR node. Gemcitabine increases RUVBL2 O-GlcNAcylation, with Thr81 identified as a modified residue within the Walker A nucleotide-binding motif. Structural modelling predicts that Thr81 O-GlcNAcylation stabilizes the RUVBL1-RUVBL2 complex without compromising ATP-Mg engagement. PGM3 inhibition and Thr81 mutation similarly reduced ATR and ATM abundance and promoted persistent DNA damage, supporting a role for RUVBL2 Thr81 O-GlcNAcylation in sustaining checkpoint signalling and genome stability. Consequently, PGM3 inhibition induces a BRCAness-like state that sensitizes pancreatic cancer cells to PARP inhibition, both in vitro and in vivo, as well as to ionizing radiation. These findings reveal a nutrient-sensitive mechanism linking protein glycosylation to genome maintenance and identify HBP-dependent DNA repair as a potentially actionable vulnerability in pancreatic cancer.

The X chromosome carries an unusually high density of immune genes and is a major contributor to sex differences in immune function and autoimmune diseases. In females, X-chromosome inactivation (XCI) has two major functional consequences: it shapes X-linked gene dosage through XCI escape and determines the cellular exposure of heterozygous X-linked variants through XCI skew. Yet because XCI creates a mosaic of cells expressing different parental X chromosomes, these properties have remained largely inaccessible in individual women, becoming measurable only where XCI is non-random or after aggregation across large cohorts. Consequently, how X-linked variation contributes to sex-biased immunity and differs between individual women has remained unresolved. Here we present scDaisyChain, a graph-based framework that reconstructs chromosome-scale X haplotypes directly from heterozygous SNPs and single-cell long-read transcriptomes. scDaisyChain achieves near-ground-truth accuracy in highly polymorphic mouse hybrids and shows strong concordance with orthogonal long-read whole-genome phasing in human samples. Applied to peripheral blood immune cells from healthy women, it reveals a lineage-specific escape program in which lymphoid cells escape XCI more broadly than monocytes, with corresponding gains in the inactive X chromatin accessibility and female-biased expression. Lineage-specific skew further alters the proportion of cells expressing each heterozygous X-linked variant, a property we term variant exposure. Predicted deleterious variants are preferentially found in low-exposure states, exemplified by a splice-altering TLR8 variant expressed in few cytotoxic T cells. In rheumatoid arthritis (RA), the monocyte compartment - which has the lowest escape in health - shows reproducible inactive X dysregulation converging on a trained-immunity programme linked to disease flare and synovial macrophage activation, with elevated escape of IL13RA1 and HDAC8. These findings establish lineage-specific escape, skew and variant exposure as quantifiable, patient-resolved determinants of sex-biased immune gene dosage and X-linked variant penetrance in health and autoimmune disease, resolving a dimension of female biology that has been previously inaccessible in individual donors.

Biomolecular condensates (BMCs) organize cellular biochemistry by concentrating selected molecules into dynamic membrane-free compartments. Yet the molecular parameters that determine not only whether condensates form, but also how they behave and what they do, remain poorly defined. Here we show that scaffold binding affinity (Kd) is a quantitative determinant of condensate phase behavior, internal dynamics and biochemical output. Using a modular SUMO-SIM system in which scaffold valency was held constant while binding affinity was systematically varied, we found that affinity governs the phase boundary, resistance to chemical perturbation, and molecular mobility of condensates in vitro and in human cells. In multicomponent mixtures, the highest-affinity scaffold dominated dense-phase composition and dynamics, revealing a hierarchical rule for condensate organization. Finally, affinity-dependent changes in condensate dynamics translated into tunable enzyme activity, establishing binding energetics as an engineerable parameter for programming condensate biochemistry.

The adult endometrium regenerates repeatedly, yet the cells and mechanisms that rebuild its epithelium remain poorly defined. To control the cell types available for regeneration, a genetic model to extensively ablate the uterine epithelium was combined with transplantation of lineage-labeled organoids. Ablation without organoid transplantation triggered re-epithelialization, but resulted in infertility. Transplanted endometrial epithelial organoids engrafted into the ablated uterus, reconstructed both the luminal and glandular epithelia, and restored fertility. Depleting organoids of the glandular lineage before transplantation revealed that luminal epithelial-derived cells acquire glandular identity and function after engraftment. The same luminal-to-glandular epithelial differentiation trajectory emerged during endogenous repair following targeted glandular ablation. Together, these findings establish luminal-to-glandular epithelial conversion as an intrinsic regenerative property of the adult uterine epithelium and establish an endometrial organoid transplantation platform with therapeutic potential.

Tissue regeneration requires careful allocation of metabolic resources, yet how organisms adjust this allocation in response to varying amounts of tissue loss remains poorly understood. Here, we show that the regenerative metabolic response is not fixed: the size of an injury regulates how glucose is allocated at both local and organism-wide levels. We first demonstrate that tail regeneration requires glucose metabolism in the axolotl (Ambystoma mexicanum), a salamander capable of regenerating centimetre-scale tissues. We then mapped glucose uptake in axolotls regenerating from small or large tail injuries using positron emission tomography/magnetic resonance imaging (PET/MRI) and the radiolabelled glucose analogue [18F]FDG. Glucose uptake was elevated in regenerating tails compared to uninjured tails. During early regeneration, larger injuries induced higher glucose uptake than smaller injuries, correlating with faster regenerative outgrowth. Larger injuries also increased glucose uptake in distant organs, indicating a systemic metabolic response. Together, our findings suggest that metabolic responses tuned to injury size underlie faithful tissue regeneration and establish PET/MRI as a powerful approach for studying whole-body metabolic dynamics in large regenerating vertebrates.