Nature Cell Biology

Cells degrade the autophagy receptor p62/sequestosome 1 (SQSTM1) in lysosomes. By knocking the HiBiT peptide into the endogenous SQSTM1 locus, we show that export is a second, quantitatively comparable fate. Specifically, p62 leaves cells continuously inside membrane-enclosed carriers at a rate similar to that of LC3B. Blocking lysosomal degradation amplifies release through an ATG5- and FIP200-dependent route, whereas a basal route persists in autophagy-deficient cells. Thus, p62 exits by at least two parallel pathways. Export requires PB1-mediated self-oligomerization but not LC3 binding, Kelch-like ECH-associated protein 1 (KEAP1) binding, or an intact ubiquitin-associated domain. Hence, oligomerization is the sorting determinant. KEAP1 is also released, and dominant-negative RAB27A retains both proteins inside cells and lowers nuclear factor erythroid 2-related factor 2 transcriptional output. Secretion is therefore a second outlet for the p62-KEAP1 module, and its interruption co-varies with a dampened oxidative stress response.

Cells must prevent incompletely spliced transcripts from entering the cytoplasm, yet how splicing status is recognized and converted into selective nuclear retention remains unclear. Here, we identify a network of RNA-binding proteins that retain pre-mRNPs within nuclear speckles. Proteomic analysis of splicing intermediates accumulated in nuclear speckles identified a set of retention factors including BCLAF1, THRAP3 and SRSF7 that anchor pre-mRNPs to the SON scaffold and are required for pre-mRNP retention in speckles. Distinct RNA-binding patterns and transcript-specific retention factor requirements suggest that multiple mRNP features tune retention combinatorially. Super-resolution imaging revealed that the SON and SRRM2 speckle scaffolds form interdigitated networks with RNA enriched along networks and within interstitial speckle lumens. This architecture remodels in response to pre-mRNP load and is disrupted upon depletion of retention factors. Together, these findings define a pre-mRNP quality-control pathway that links splicing state to nuclear retention and the dynamic organization of nuclear speckles.

The nuclear basket (NB) is a fibrillar scaffold anchored to the nuclear pore complex (NPC), with protein TPR constituting its structural backbone. Recently, ZC3HC1 was identified as an additional structural NB component interconnecting TPR subpopulations. However, it had remained unclear how ZC3HC1 and TPR engage each other and how the resulting TPR:ZC3HC1 ensembles are arranged relative to the NB. Here, we uncover a hierarchy of sequential interactions between distinct ZC3HC1 and TPR domains, resulting in NB-appended ZC3HC1:TPR ensembles that extend beyond the NB dimensions traditionally considered prototypic. Consistent with a modular organisational principle, we furthermore find copy numbers of NPC/NB-associated TPR to be far higher than commonly anticipated. Together with sufficient ZC3HC1 copy numbers, our data define a model in which alternating recruitment of ZC3HC1 and TPR extends the NB's spatial and functional reach into the nuclear interior.

The selective lysosomal turnover of endoplasmic reticulum (ER) portions, termed ERphagy, removes aged, excess or damaged ER domains. The intermediates forming between receptor activation and lysosomal delivery are short-lived and remain poorly resolved. Here we identify a reversible temperature block at 25{degree sign}C that arrests ERphagy before lysosomal delivery. At this temperature, nutrient restriction still drives focal accumulation of the endogenously Halo-tagged ERphagy receptor TEX264 (puncta formation), segregation of ER cargo, remodelling of the ER membrane, whereas delivery of this material to degradative endolysosomes is strongly reduced. Rewarming above 27{degrees} restores lysosomal delivery, showing that the accumulated eTEX264-Halo puncta correspond to pre-lysosomal intermediates of the ERphagy program. Their formation requires ULK1/2 activity, the ATG7-dependent conjugation machinery, ATG9A, ATG13 and FIP200, coupling receptor clustering to autophagosome biogenesis. Consistently, expansion microscopy and electron tomography reveal curved, cradle-like TEX264-positive ER domains containing ER cargo, co-localizing with autophagy markers and lying adjacent to p62-cargo condensates.

During gametogenesis, organelle remodeling is coordinated alongside chromosome segregation to ensure gamete quality. In Saccharomyces cerevisiae, the precursor-cell vacuole permeabilizes following completion of meiotic divisions, releasing hydrolytic enzymes that degrade cellular material excluded from gametes. The regulation and biological significance of this developmentally-programmed process, termed mega-autophagy, remain poorly understood. Here we identify the conserved armadillo repeat-containing protein Vac8 as a key regulator of mega-autophagy. The presence of Vac8 during meiosis is required for timely execution of mega-autophagy. Specifically, during anaphase II, precursor-cell vacuoles undergo Vac8-dependent fusion to form fewer, larger structures. Deleting VAC8 disrupts this remodeling and impairs subsequent vacuolar permeabilization, establishing meiotic vacuolar fusion as a prerequisite for mega-autophagy. Defective mega-autophagy compromises the ability of gametes to exit dormancy and proliferate upon nutrient repletion. Together, these findings reveal that Vac8-mediated vacuolar remodeling during meiosis primes a post-meiotic quality-control mechanism that safeguards gamete developmental competence.

Single-cell transcriptomic analysis predominantly derives cell identity from gene expression analysis, while alternative splicing is processed separately despite its fundamental role for cell homeostasis. To overcome the limits of separate investigations, we developed a probabilistic deep learning framework, Crecerelle, enabling resolution of the contributions of gene expression and alternative splicing in each cell. Crecerelle learns cell embeddings from gene expressions and alternative splicing isoforms, to decipher their mutually dependent impact on the functional characterisation of cells in a data-driven manner, exemplified for the Tabula Muris dataset. This is enabled through a zero-and-N-inflated Dirichlet-Multinomial for a variational autoencoder that learns cell embeddings solely from splicing profiles, as well as a bi-modal variational autoencoder with a relevance-weighted mixture-of-experts variational posterior to consolidate the modality-specific contribution at single-cell level. Crecerelle reveals cell-type-specific isoform markers as well as subpopulations with unique isoforms and uncovers regulatory and disease-associated pathways not detected by gene expression analyses alone. This scalable and interpretable framework thus allows a more holistic study of transcriptomic regulation and will open a route to modality-relevance-weighted investigations across single-cell multiomics datasets and their influence on cellular homeostasis, tissue development and disease phenotypes.

In eutherian females, the choice of which X chromosome gets embryonically inactivated is thought to be random. Somatic tissues are thus assumed to comprise cells with a 50:50 ratio of maternal:paternal X active. However, a phenomenon known as X-skewing has been observed, wherein tissue composition shifts to be dominated by cells expressing one X or the other. On a gross level, such X-skewing is phenotypically silent except in the case where one X-chromosome carries a disease mutation. Here, by leveraging a mouse model of an X-linked disease called Incontinentia Pigmenti, caused by heterozygous mutations in the NF-kB effector, Nemo, we explore how X-linked cellular dynamics shape progenitor biology in the epidermis. Lineage tracing with X-linked reporters shows that at birth, the epidermis is unexpectedly skewed toward mutant cells. Within a week, as inflammation emerges, these dynamics reverse leaving surviving animals with almost entirely wild-type epithelium. The shift is driven by loss rather than growth since mutant cells adopt an aberrant fate and die at higher rates than their wild-type neighbours; proliferation rises in both populations. Notably, wild-type proliferation scales with local tissue composition, suggesting these cells calibrate their response to the extent of the mutant burden to compensate appropriately for tissue loss. Depleting macrophages, the principal TNF- source, attenuates inflammation, mutant cell death and ultimately is expected to preserve the mutant population. Altogether, our results identify inflammation as a selective force capable of resolving deleterious mosaicism, not only in the context of an X-linked disease, but within any dynamic, clonally heterogeneous epithelium.

Chromatin mobility, the exploration of 3D conformations by the chromatin fibre, is implicated in transcription and DNA repair, and is thought to decrease progressively as cells differentiate. Here, combining live-cell 3D single-nucleosome tracking with genomic approaches, we show that chromatin mobility instead changes in a cell-type-specific manner, increasing as pluripotent cells acquire posterior fates. This is accompanied by 3D genome reorganisation, including loss of Polycomb-associated interactions and emergence of CDX2-associated interactions. Withdrawal of posteriorising WNT/{beta}-catenin signalling rapidly reduces chromatin mobility and partially restores Polycomb-associated chromatin interactions, while Cdx1/2/4-null cells fail to acquire these WNT/{beta}-catenin-dependent changes. Together, our findings establish chromatin mobility as a regulated property of cell state, with WNT/{beta}-catenin signalling and CDX factors acting as mediators that fine-tune the sampling of 3D chromatin interactions during cell state transitions.

Mitogens trigger cell-cycle entry by activating E2F at the restriction point, which is followed by B-MYB/FOXM1 activation and progression to mitosis. How mitogens control continued cycling and cell-cycle exit after the restriction point is not well-understood. By developing an E2F and B-MYB/FOXM1 dual transcriptional biosensor system, we show that S/G2 phase duration is set by timed mitogen-regulated B-MYB/FOXM1 activation, while E2F activity gradually declines before mitosis. As a striking consequence, rapid B-MYB/FOXM1 activation shortens S/G2, delivering high mitotic E2F activity to daughter cells which keeps them cycling. Delayed B-MYB/FOXM1 activation prolongs S/G2, depleting mitotic E2F which drives daughters to quiescence. When S/G2 is further prolonged, partially activated B-MYB/FOXM1 frequently reverts, triggering mitotic bypass and polyploid quiescence. Thus, B-MYB/FOXM1 governs a tri-directional (G2 restriction point) where cells commit to continued cycling through early B-MYB/FOXM1 activation; cell-cycle exit through delayed B-MYB/FOXM1 activation; or mitotic bypass by B-MYB/FOXM1 inactivation.

The endoplasmic reticulum (ER) coordinates protein folding and lipid biosynthesis, while peroxisomes govern fatty-acid catabolism and redox balance. However, little is known about the mechanisms by which these organelles communicate. Here we show that activating transcription factor 6 (ATF6), a cytoprotective ER-resident sensor of the unfolded protein response (UPR), forms a molecular tether that zippers the ER and peroxisomes together at a 23-nm intermembrane distance. ATF6 achieves this by inserting a short segment of its unstructured N-terminal cytosolic tail into the solvated transmembrane cavity of the peroxisomal ABC-type lipid transporter ABCD3, trapping it in an off-state. This interaction is reinforced by Ceapin, a previously identified small molecule molecular-glue ligand, which promotes ER-peroxisome contact formation. The junctions serve as a scaffold that can initiate peroxisome turnover by p62-triggered autophagy (pexophagy), as enhanced lipid processing resulting from organelle zippering leads to peroxisome damage due to excessive reactive oxygen species (ROS) accumulation. These findings define ATF6 as a non-transcriptional regulator of organelle quality control and provide a template for engineering Ceapin analogues against ATF6/ABCD3-dependent colorectal and hepatocellular cancers and related diseases.

Oocyte meiosis in animals is essential for producing fertilizable eggs. As transcription becomes largely quiescent, meiotic progression depends on temporally regulated translation of accumulated maternal mRNAs. However, the underlying mechanisms remain unclear. Here, we identified diverse and highly organized mRNA-protein assemblies in mouse oocytes that coordinate cytoplasmic mRNA regulation and enable spatiotemporal translation to drive oocyte meiosis. Twelve RNA-binding proteins (RBPs), including DDX4/VASA, HDLBP/Vigilin, and TDRD3, were assembled into widely distributed aggregates in germinal vesicle (GV)-stage oocytes. We depleted DDX4 and HDLBP using Trim-Away and found that these RBPs regulate the precise assembly of RNA granules and protein aggregates through distinct mechanisms, promoting temporal protein synthesis at cortical translation factories with the endoplasmic reticulum, termed CORTE. All 12 RBPs formed distinct mRNA-protein assemblies, whose size and distribution changed upon meiotic initiation. These findings reveal the molecular basis of spatiotemporal mRNA-protein-organelle networks underlying animal reproduction and development.

Microtubule minus ends are central to non-centrosomal microtubule organization, yet how their recognition, protection and molecular organization are integrated remains unclear. Here we show that Drosophila Patronin forms dynamic condensates at microtubule minus ends through a prion-like domain (PrLD). PrLD-mediated condensation cooperates with the CKK domain and a second CKK-independent recognition region to convert weak or broadly distributed microtubule-binding activities into persistent minus-end recognition and tracking. Replacing the PrLD with a heterologous phase-separating HNRNPA1 IDR restores minus-end targeting, indicating that condensation capacity rather than PrLD primary sequence is critical. Patronin condensates further form a protective cap that resists minus-end depolymerization and recruit the cytoskeletal regulators Sas-4, Katanin and Ena. Disrupting PrLD multivalency impairs Patronin localization at multiple developmental sites and causes early organismal lethality. Thus, Patronin condensation creates a physiologically essential microtubule minus-end compartment coupling end recognition, stabilization and cytoskeletal organization.

The endoplasmic reticulum (ER) chaperone BiP/GRP78 sits at the center of the proteostasis network, coordinating unfolded protein response (UPR) signaling and supporting oncogenic programs of plasticity, metastasis, immune evasion, and chemoresistance. While BiP undergoes liquid-liquid phase separation in vitro, whether mammalian cells dynamically tune this physicochemical behavior to prevent functional exhaustion or pathological condensation under sustained ER stress has remained unclear. Here, we identify the E3 ubiquitin ligase TRIM21 as a phase-state regulator that suppresses BiP's liquid-to-solid phase separation, clearing, and preventing insoluble aggregates-thereby controlling BiP's localization and availability to sustain the adaptive UPR under ER stress. We found that during ER stress, BiP is prone to liquid-to-solid phase separation, a transition that renders the chaperone dysfunctional. TRIM21 is recruited to BiP, where it directly polyubiquitinates BiP at specific K547/553/554/556 sites with K6-, K27-, K33-, and K63-linked chains; VCP recognizes these marks and extracts BiP from the ER lumen via ER-associated degradation (ERAD), coupling ubiquitin tagging to retrotranslocation. This preserves chaperone availability and sustains adaptive UPR-a homeostatic mechanism broadly relevant to UPR-driven diseases. In vivo, genetic ablation of Trim21 in KrasG12D/+; Pdx1Cre/+ mice triggers aberrant BiP solidification while curtailing acinar-to-ductal transdifferentiation and tumorigenesis; notably, TRIM21 co-localizes with BiP condensates in pre-malignant human pancreatic lesions, underscoring the clinical relevance of this regulatory axis. Our findings define the TRIM21-BiP axis as a phase-control system at the interface of proteostasis and stress adaptation, offering a new framework with translational potential.

Nucleolar enlargement is a conserved hallmark of physiological and premature ageing, including Hutchinson-Gilford Progeria Syndrome (HGPS), a disorder caused by LMNA mutations that produce Progerin and profoundly disrupt nuclear architecture. Although both the nuclear lamina and nucleolus organize repressive chromatin, how nucleolar remodelling contributes to genome reorganization during ageing driven by lamina dysfunction remains unclear. Here, we identify the nucleolus as a stress-responsive chromatin-organizing compartment that buffers the initial events of Progerin-induced ageing programs by sequestering lamina-associated domains (LADs). Despite widespread nuclear reorganization and robust transcriptional ageing signatures following Progerin expression, repression of LADs is preserved. This preservation results from an increased nucleolar association of lamina-released LADs, which can retain H3K9me2 and remain transcriptionally silent. Disrupting this compensatory pathway by depleting NPM1, a nucleolar factor required for nucleolar genome association, abolishes nucleolar buffering, leading to loss of H3K9me2 at relocated LADs, derepression of LAD-associated genes, exacerbation of transcriptional ageing programs, and increased DNA damage. Together, these findings establish the nucleolus as a structural fail-safe that preserves heterochromatin integrity and reveal stress-responsive nuclear compartmentalization as a fundamental mechanism safeguarding epigenome stability.

Metastatic cancer cells migrate through physically restrictive environments where nuclear deformation ruptures the nuclear envelope (NE) resulting in genomic instability. Although structural proteins of the NE are established regulators of nuclear mechanics, how nuclear membrane (NM) lipid composition contributes to its biophysical response to mechanical stress remains poorly understood. Here, we show that melanoma progression is associated with broad transcriptional remodeling of cholesterol homeostasis that is established early in disease progression, with altered expression of cholesterol biosynthesis genes associated with reduced patient survival. Building on our previous identification of the inner NM sterol reductase lamin B receptor (LBR) as a regulator of NE fragility, we find that LBR-dependent cholesterol biosynthesis promotes cholesterol enrichment within the NM and alters its spatial organization during cellular confinement. Quantitative fluorescence and lifetime imaging of biosensors of cholesterol distribution, lipid order, and tension reveals that cholesterol remodeling alters NM lipid organization and results in high NM tension in melanoma cells that is selectively reduced by cholesterol depletion. Cellular confinement generates nuclear blebs where cholesterol becomes preferentially enriched at highly curved, rupture-prone regions of the NM. Together, our results establish cholesterol-dependent NM remodeling as a regulator of nuclear mechanics and link metabolic changes acquired during cancer progression to the biophysical response of the NM to mechanical stress.

Nucleosomes regulate chromatin folding, accessibility, and factor recruitment. Current models primarily attribute these functions to histone tail modifications, while the core is largely viewed as a structural scaffold. Yet subtle changes within the nucleosome core can produce profound functional consequences, and the mechanisms underlying these effects remain unclear. Here, we describe nucleosome core allostery as a fundamental principle of chromatin regulation that amplifies the impact of minimal nucleosome variations. Leveraging natural differences between H2A.Z variants, we show that the nucleosome core encodes distinct conformational dynamics that propagate allosterically, thereby controlling nucleosome accessibility and recognition by chromatin factors. As a result, a single buried amino acid substitution alone is sufficient to reprogram nucleosome dynamics and bias cell identity. Our findings establish the nucleosome core as an allosteric regulatory module and provide a generalizable framework for how subtle variation within nucleosomes is amplified into diverse biological outcomes in development and disease.

Adult mammalian wounds typically restore tissue integrity through fibrotic repair rather than regeneration of the original architecture. Here, we compared small non-regenerative and large regenerative skin wounds to investigate how fibroblasts acquire regenerative competence and enter alternative cell-fate programs. Integrated single-cell RNA sequencing identified a transient signaling and extracellular-matrix-remodeling niche enriched in large wounds, together with NCAM1-associated competent fibroblast states. Time-resolved analysis positioned these states within a root-like fibroblast manifold connected to hair dermal-condensate, endothelial, pericyte and glial-associated fate-entry programs. Inferred cell-cell communication networks revealed shared matrix and adhesion signals, superimposed with branch-associated inputs, including VEGF signaling toward NCAM1-positive endothelial-transition cells. Lineage tracing showed that PDGFRA- and NCAM1-expressing lineages contributed to vascular- and nerve/glial-associated regenerative compartments. Functionally, partial NCAM1 perturbation reduced vascular organization while increasing hair follicle neogenesis, whereas stronger inhibition impaired both outcomes. These findings identify NCAM1 as a regulator of regenerative competence and fate specification within a regenerative competence field rather than a marker of a single terminal lineage. We propose that regenerative wounds activate an NCAM1-associated fibroblast competence field whose quantitative modulation biases competent fibroblasts toward alternative outcomes.

The DNA sensor cGAS is largely sequestered on chromatin in an inactive form, but how this state is regulated remains unclear. We show that cGAS preferentially binds nucleosomes containing the histone variant macroH2A1 (mH2A1) marked by H3K27me3 and H3K9me3. Cryo-EM reveals that cGAS selectively engages the mH2A1/H2B acidic patch within heterotypic nucleosomes. Depleting mH2A1.1 or blocking H3K27me3 deposition releases cGAS from chromatin and activates the cGAS-STING-IRF3 pathway. Genome-wide profiling reveals two cGAS-bound chromatin compartments: chromosome-arm sites co-occupied by mH2A1.1, methylated CpG islands, and CTCF, where cGAS and mH2A1.1 binding are interdependent; and centromeric regions (14% of peaks), which lack DNA methylation and mH2A1.1 dependence. These findings establish mH2A1 and H3K27me3 as determinants of cGAS chromatin tethering, linking nuclear cGAS regulation to facultative heterochromatin and opening new avenues for modulating cGAS-STING signaling.

Estrogen receptor (ESR1) fusions are recurrent drivers of endocrine-resistant breast cancer, yet their mechanisms of action remain poorly understood. Here, we show that ESR1-e6 fusion proteins, including ESR1-DAB2, ESR1-SOX9, and ESR1-YAP1, assemble into nuclear condensates across multiple cell types. Sequence analysis reveals that these fusions combine chromatin-targeting domains with increased intrinsic disorder, promoting multivalent interactions and phase separation. Live-cell imaging, fluorescence recovery after photobleaching, and mechanical perturbation demonstrate that condensates exhibit liquid-like properties, but progressively mature into stable, viscoelastic assemblies constrained by chromatin. Disruption of DNA binding abolishes condensate formation, indicating that chromatin engagement is essential for their assembly and organisation. ESR1 fusion condensates localise to chromatin-depleted regions, reduce nuclear stiffness, and promote chromatin decompaction, consistent with active remodelling of nuclear architecture. We propose that ESR1 fusions exploit phase separation to generate chromatin-anchored assemblies that reorganise the nuclear microenvironment, providing a physical framework through which ESR1 fusions may support sustained transcriptional activity, endocrine resistance and tumour progression.

How adult stem cell populations coordinate tissue regeneration across physiological and pathological states remains unclear. Hair follicles contain epithelial and mesenchymal stem cells that codetermine hair regeneration. Here, we integrate a multimodal spatiotemporal atlas with functional validation to define the mesenchymal-epithelial stem cell dynamics governing human hair follicle homeostasis, aging, and androgenetic alopecia. Aging is characterized by passive depletion of hDSC-derived regenerative cues, whereas AGA involves a shift of hDSC toward an inhibitory signaling hub that promotes HFSC dysfunction. We further pinpoint hDSC-secreted PTN as a key regenerative factor that promotes hair growth and counteracts androgen-induced growth suppression. This study provides a holistic framework of adult stem cell crosstalk across physiological and pathological states, offering a mechanistic roadmap to reinstate tissue regeneration.