Nature Cell Biology

The proportional scaling between nuclear and cell size was first described more than 150 years ago and is among the most conserved features of cellular organization. Yet the mechanisms that establish this scaling and its physiological significance have remained unresolved. Here, we address both questions by combining image-enabled cell sorting with genome-wide CRISPR screening, transcriptomics and functional analyses. We identify more than 180 regulators of the nuclear-to-cell ratio and show that distinct classes of genes independently control nuclear and cell size. RNA metabolism predominantly regulates nuclear size, whereas protein synthesis and degradation primarily regulate cell size. This supports a model in which differences in macromolecular partitioning between the nucleus and cytoplasm contribute to osmotic regulation of nuclear size together with mechanical constraints imposed by chromatin, the cytoskeleton, and the nuclear envelope. Changes in nuclear size cause widespread transcriptional remodeling that is independent of changes in cell size. Cells with smaller nuclei exhibit reduced PRC2-dependent H3K27 trimethylation, activation of developmental gene-expression programmes and repression of cell-cycle genes. Consistent with these changes, mouse embryonic stem cells with smaller nuclei show an increased propensity to exit pluripotency and initiate differentiation in response to retinoic acid. Together, our findings provide a mechanistic framework for nuclear size scaling and establish nuclear size as a physical regulator of gene expression and cell-state transitions, linking cellular architecture to cell fate.

Lysosomal damage is increasingly recognized as a hallmark of aging. Understanding lysosomal quality control may open new therapeutic strategies to enhance lysosomal resilience. Here, we identify SOLAR (SQSTM1/p62 Oligomer-mediated Lysosomal Antioxidant Defense And Membrane Repair), a lysosomal quality control pathway that links CASM (conjugation of ATG8 to single membranes) to p62-driven membrane repair and redox signaling. In this pathway, CASM, but not macroautophagy, recruited p62 to damaged lysosomes. Efficient lysosomal recruitment of p62 required its ATG8-binding and self-oligomerizing domains, enabling p62 assemblies to promote membrane repair. p62 mutations associated with neurodegeneration interfered with p62 recruitment and compromised lysosomal repair. Besides membrane repair, the SOLAR pathway also activated the p62-KEAP1-NRF2 redox signaling axis, driving transcriptional upregulation of antioxidative genes and cholesterol biosynthesis. By integrating membrane repair and redox defense, SOLAR establishes a coordinated lysosomal quality control pathway with implications for aging and degenerative disease.

Centriole copy number is tightly regulated, with one procentriole emanating from a torus surrounding each pre-existing centriole. Which proteins are sufficient to generate a full-fledged organelle is unclear. We address this question by engineering a high valency low copy number phase-separated droplet platform to concentrate proteins in an ectopic cellular location. We establish that droplet targeting of the torus protein Cep63 suffices to initiate procentriole assembly. Ectopic procentrioles mature when a limiting interaction involving STIL is alleviated or when pre-existing centrioles are lacking. Ectopic procentrioles disengage from the droplet during mitosis in a PLK1-dependent manner, organize supernumerary spindle poles, and seed procentriole formation at the next cell cycle, demonstrating that synthetic centrioles have been reconstituted. Moreover, we uncover that procentriole number scales with platform surface area. Since the torus surface area is set by pre-existing centriole dimensions, we propose that this constitutes a closed circuit mechanism dictating organelle number homeostasis.

Organoid models have transformed our understanding of intestinal renewal. Fluorescent imaging has been extensively used to identify key cell types and their differentiation pathways, but immunofluorescence provides only static readouts, whereas live imaging requires fluorescent-reporter engineering and is constrained by limited multiplexing and spectral overlap. Here, we introduce NuclearIDTracker, an explainable machine-learning framework that infers cell identity directly from 3D nuclear segmentations. Using a single nuclear marker, NuclearIDTracker accurately classifies intestinal cell types and integrates with single-cell tracking to resolve lineages and reconstruct dynamic state transitions during organoid development. We show that TA-like cells, rather than stem cells, drive early crypt formation and generate enterocyte and Paneth lineages, as well as the stem-cell population, which emerges only later and subsequently replenishes the TA-like compartment. Following stem-cell ablation, crypt regeneration was not driven by a single discrete cell type. Instead, multiple epithelial populations converged on a proliferative regenerative state with a nuclear phenotypic signature that resembled, but remained distinct from, that of homeostatic TA-like cells, and a YAP/TAZ-associated fetal-like transcriptional signature. Thus, nuclear phenotypic signatures resolve cell identity and reveal coordinated epithelial plasticity during crypt regeneration. NuclearIDTracker establishes a non-perturbative tool to quantify cell identity and state dynamics at single-cell resolution, revealing previously inaccessible biological dynamics and expanding the toolkit for studying epithelial homeostasis, regeneration, and disease.

Polyploid cells, which contain more than two copies of the genome, are widely present across plants and animals, where they are often found in tissues with high biosynthetic and metabolic demands, such as the mammalian liver and placenta. While somatic polyploidy is frequently associated with increased cell growth and biosynthetic capacity, unscheduled polyploidization in cell types that are not normally programmed to become polyploid is often linked to reduced cellular fitness and genome instability. To understand whether these divergent outcomes stem from distinct immediate cellular responses to increased ploidy, we systematically compared the early consequences of polyploidization across naturally occurring and experimentally induced systems. Specifically, we examined physiological polyploid cells in the Caenorhabditis elegans intestine and human hepatocyte organoids, alongside unscheduled polyploid human retinal pigment epithelial (RPE1) cells generated through cytokinesis failure. Using quantitative imaging, flow cytometry, and FUCCI based cell-cycle reporters we measured cell size and protein translation dynamics during G1 in diploid and polyploid cells. Across all systems, we observed a strikingly conserved relationship between ploidy, cell size, and biosynthetic capacity: both cell size and protein translation showed similar scaling patterns after polyploidization, regardless of whether polyploidization occurred as part of normal development or by inducing cytokinesis failure. These findings indicate that the immediate cellular response to increased ploidy is broadly similar across contexts. However, in contrast to unscheduled polyploid RPE1 cells, polyploid human hepatocytes extend their G1 phase, leading to a higher accumulation of proteins before cell-cycle progression. Together, our findings suggest that polyploidization elicits similar growth responses across contexts, and that cell-type specific cell-cycle adaptations may determine whether polyploidy becomes advantageous or deleterious.

Quantitative image analysis of subcellular organization requires sufficient spatial resolution to resolve individual organelles and sample size to capture heterogeneity both within cells and between cells. Existing imaging approaches often force a tradeoff between spatial resolution and throughput, limiting the ability to measure organelle-level phenotypes across cell populations. Here, we establish high-throughputs expansion microscopy (HiExM) as a scalable pipeline for single-organelle analysis. As a benchmark, we focus on mapping late endosomes and lysosomes (LELs), a heterogeneous organelle class whose small size, dense intracellular distribution, and functional diversity make it difficult to quantify accurately using conventional light microscopy. HiExM increases effective spatial resolution while preserving compatibility with large-scale image acquisition, enabling robust segmentation and quantitative profiling of individual LELs across large cell populations. Using this pipeline, we identified differences in intracellular trafficking behavior among anti-transferrin receptor antibodies that could not be captured by conventional colocalization analysis alone. We further integrate spatial and morphological features with learned image-based representations that can define relationships between LEL morphology and subcellular position as well as how these relationships respond to perturbations. Together, our work establishes HiExM as a generalizable platform for scalable single-organelle profiling, enabling an analytical framework for quantifying discrete organelles across cells and conditions.

Stress granules are cytoplasmic membraneless organelles assembled during stress to maintain cellular homeostasis. Although fusion is a hallmark of liquid-like behavior of these condensates, whether this process carries functional significance beyond its physical coalescence remains unclear. Here, we show that stress granule fusion is facilitated by mitochondrial dynamics and membrane potential. Intact mitochondria actively associate with stress granules, facilitating fusion and maturation. In contrast, loss of mitochondrial membrane potential, along with disrupted mitochondrial structure or motility, weakens these interactions and reduces fusion frequency. We find that impaired fusion leads to the accumulation of immature granules that retain fewer sequestered components, which correlates with premature cell death. Remarkably, restoring mitochondrial membrane potential rescues granule fusion and enlargement, and is accompanied by increased cell viability and a corresponding increase in granule-associated apoptotic factors. These results demonstrate that stress granule fusion is actively coordinated by mitochondria rather than driven solely by passive coalescence, reshaping how condensate dynamics are understood to integrate with organelle function during cellular stress response.

DNA damage response proteins frequently accumulate in biomolecular condensates, yet how these structures cooperate with classical adaptor-mediated recruitment mechanisms to organize DNA damage signaling remains poorly understood. Here, we identify Treacle and the ATM adaptor NBS1 as prominent components of TOPBP1 condensates and show that these structures activate ATM signaling in the absence of DNA damage. At rDNA breaks, Treacle recruits NBS1 and TOPBP1 through genetically separable interaction modules. Acute protein degradation further revealed that Treacle is required for condensate assembly, whereas TOPBP1 remains continuously required for condensate maintenance. Finally, we show that efficient ATM accumulation at IR-induced DNA double-strand breaks similarly depends on both NBS1 and TOPBP1, indicating that this two-component mechanism is not restricted to nucleolar DNA damage. Together, our findings support a two-component model in which adaptor proteins provide molecular specificity, whereas TOPBP1 condensates create the spatial organization required for robust ATM signaling.

Transcriptional condensates at super-enhancers are thought to concentrate BRD4, Mediator, and RNA polymerase II (Pol II) to promote gene activation, yet their compositional organization and regulation remain poorly understood. We developed a high-throughput live-cell phenomics platform based on endogenous fluorescent tagging of BRD4, MED14 (Mediator), and POLR2A (Pol II) to systematically quantify transcriptional condensate states across >1,000 chemical perturbations. Contrary to prevailing models of largely co-occupied assemblies, we find compositionally heterogenous condensate populations. In particular, BRD4-only spots emerged as a prominent class that is depleted of Mediator and Pol II, enriched at chromatin, and resistant to transcription initiation inhibition. Mechanistically, compound screening coupled to mechanism-of-action analysis identifies histone acetylation as a dominant regulatory axis for BRD4-only spots: Bromodomain and Extra-Terminal motif (BET) and histone acetyltransferase inhibition selectively deplete BRD4-only condensates, while histone deacetylase inhibition expands them. Together, these findings support a model in which acetylation-dependent BRD4 condensates define a distinct chromatin-associated regulatory state that is separable from canonical transcriptionally engaged condensates. More broadly, our work establishes condensate composition as a quantitative phenotype and provides a scalable framework for systematically dissecting the regulation of condensates across perturbations, cell types, and disease contexts.

The nucleolus is the largest nuclear condensate, yet how its assembly is developmentally timed remains poorly understood. In the Caenorhabditis elegans embryo, nucleoli normally appear at the 6- to 8-cell stage. Here we show that the LINC complex and nuclear lamina restrain premature nucleolar assembly through heterochromatin organization. Depletion of the embryonic LINC complex proteins SUN-1 or ZYG-12 induces precocious FIB-1-positive nucleoli at the 4-cell stage, particularly in the EMS and P2 blastomeres. LMN-1 depletion produces a more severe phenotype combining premature assembly with impaired disassembly. Loss of the heterochromatin anchor CEC-4 alone has modest effects but strongly suppresses precocious nucleolar assembly caused by LINC complex depletion. LINC complex and lamin perturbations alter the heterogeneity of HPL-2-marked chromatin, and FIB-1 condensates occupy locally HPL-2-depleted regions. These findings identify nuclear envelope-dependent heterochromatin organization as a developmental gate for nucleolar condensate assembly.

Protein homeostasis relies on protein quality control (PQC) pathways that survey the proteome to eliminate aberrant polypeptides. The BAG6 complex is a central PQC factor that recognizes exposed hydrophobic regions, a feature commonly associated with misfolded, mislocalized, and mistranslated proteins. Whether this surveillance machinery also regulates intact, functional proteins as part of physiological proteostasis has remained unclear. Using unbiased quantitative proteomics, we identify the ribosomal protein RPL22L1 as an endogenous BAG6 substrate whose abundance is controlled by continuous proteasomal degradation. This turnover requires the RNF115 E3 ligase activity but not the canonical BAG6 partner RNF126, defining RPL22L1 as a selective RNF115-dependent substrate. Mechanistically, we map a bipartite hydrophobic degron that distinguishes RPL22L1 from its stable paralog RPL22, and show that BAG6-RNF115-mediated degradation is governed by substrate assembly state. Accordingly, RPL22L1 is protected from degradation upon incorporation into the 60S ribosome, where it substitutes for RPL22. When RPL22 is lost, either genetically or through recurrent inactivating mutations in microsatellite-unstable cancers, the vacant ribosomal binding site permits RPL22L1 incorporation, protecting it from BAG6-mediated degradation. These findings establish unassembly-coupled degradation as a mechanism by which BAG6 regulates the abundance of functional protein components, ensuring that they accumulate only when incorporated into their native macromolecular complexes.

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.

In eukaryotic cells, the function of each organelle depends on its unique protein composition. Protein targeting errors threaten organelle identity and function, yet how mistargeting errors are detected and resolved remains poorly understood. Here, we show that mitochondrial import stress drives widespread rerouting of mitochondrial proteins to the endoplasmic reticulum (ER), with strong enrichment for hydrophobic oxidative phosphorylation (OXPHOS) components. Using proximity proteomics, a split-fluorescence reporter system, and genome-wide CRISPR screening, we find that mistargeted proteins partition into distinct classes with divergent fates, ranging from stable ER residence to rapid degradation by ER-associated degradation (ERAD). The clearance of these mislocalized proteins involves partially redundant ERAD branches, with the ubiquitin ligase MARCHF6 playing a central role. Together, these findings establish the ER as a key organelle for handling mistargeted mitochondrial proteins and reveal how ER quality control maintains proteostasis during mitochondrial dysfunction.

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport through a selective permeability barrier established by nucleoporins (Nups) that contain intrinsically disordered phenylalanine-glycine (FG) repeats. Due to these domains, FG-Nups are prone to condensation, with a potential to transition into insoluble aggregates. How cells keep FG-Nups in a soluble, functional state during NPC assembly and within the native NPC remains poorly understood. Here, we identify the uncharacterized yeast J-domain protein Jjj2 as a Nup chaperone. Disrupting Jjj2 function or its interaction with Hsp70 chaperones triggers the accumulation of newly synthesized Nups in cytoplasmic condensates. Conversely, Jjj2 overexpression suppresses Nup condensation but also disrupts the NPC permeability barrier and is highly toxic. Overall, our data show that Jjj2, in concert with Hsp70, controls Nup phase state. This activity must be tightly regulated; while Jjj2 prevents condensation of newly produced Nups, its overactivity compromises nucleocytoplasmic compartmentalization.

Periodic patterning is fundamental to biological organization. In birds, colour stripes and spots on growing embryonic surface and elongating feather filaments provide a unique system to study continuous periodic patterning in expanding domains. Agouti-signaling protein (ASIP) directly reports epigenomic activity during colour patterning. Using a neural-network-like architecture, we show that ASIPs cis-regulatory landscape functions as hidden layers, integrating morphogen feedback, chromatin topology, and tissue geometry to generate discrete colour outputs. Single-nucleus multiome and Micro-C uncover stage- and context-specific enhancer-silencer coalitions. Epidermal Wnt ligands activate ASIP while inducing Wnt inhibitors in fibroblasts, forming a negative-feedback loop coupling periodic patterning to domain expansion. Comparative cross-tissue and cross-species analyses define cis-regulatory modules comprising an epigenetic grammar; functional assays highlight retrotransposon co-option expanding ASIPs cis-regulatory repertoire, potentially contributing to colour pattern evolution. Together, these findings motivate a Turing-principle-based communication model: ASIP reflects epigenetic coalitions shifting to drive diverse, environmentally tunable colour motifs for adaptation.

The actin cytoskeleton rapidly reorganizes in response to intracellular calcium signals, driving cellular morphogenesis and wound healing. Among actin regulators, the formin INF2 uniquely mediates the "Calcium-mediated Actin Reset" (CaAR) reaction, orchestrating transient and global actin remodeling upon calcium influx. Excessive INF2 activity is linked to kidney and neuronal diseases, underscoring the need for its tight control. Combining live cell imaging with single molecule tracking, biochemistry and structural analysis we discover that INF2 activity is tightly controlled by two interlinked mechanisms: canonical intramolecular autoinhibition and binding of the INF2 N-terminus to the side of actin filaments. Side-binding limits actin elongation and supports re-establishment of autoinhibition. Disruption of this negative feedback prolongs INF2 activity, affecting plasma membrane organization and repair as well as transcriptional control. Our findings uncover a novel product-inhibition mechanism that limits INF2 function and offer important insight into disease mechanisms linked to actin dysregulation.

Nuclear lamina integrity is fundamental to cellular homeostasis across the lifespan 1, and its progressive deterioration is closely linked to human aging 2. Yet, the regulatory mechanism that govern this decline and how they might be counteracted in long-lived individuals remain poorly defined. Here, by combining whole-exome sequencing of Ashkenazi Jewish centenarians with GTEx transcriptomes, we identify ubiquitin E3 ligase UBE3C strongly associated with exceptional longevity and progressively declines with age across human tissues. UBE3C knockdown triggers premature senescence and destabilizes key nuclear lamina components Lamin B1 (LMNB1) and Lamin B receptor (LBR), while the longevity-associated UBE3C variant delays senescence and preserves LMNB1/LBR expression. Mechanistically, UBE3C interacts directly with LMNB1/LBR and modulates their abundance via selective autophagy. Notably, we uncover the ER- resident autophagy trigger CKAP4 3 bridges UBE3C and LMNB1. UBE3C loss enhances LMNB1-CKAP4 binding, linking nuclear lamina turnover to autophagy. Together, our findings establish UBE3C as a central guardian of nuclear lamina maintenance during senescence and offering novel insights into interventions against age-related nuclear lamina deterioration.

Spatial chromatin organization dictates cellular function and resilience, yet scalable imaging methods to quantify chromatin states in situ across aging and interventions are lacking. While ATAC-see can visualize accessible chromatin, its broader application is hindered by protocol variability, low throughput, and incompatibility with complex tissues. Here, we systematically optimize the ATAC-see workflow for robust, high-throughput quantitative imaging in fixed, adherent mammalian cells and fresh frozen tissues. We validate the platforms sensitivity to pharmacologic remodeling and apply it to replicative, chronological, and pathological aging in primary human fibroblasts, revealing progressive age-associated chromatin opening and heterochromatin remodeling. Furthermore, we demonstrate that our optimized ATAC-see captures rapid, reversible chromatin reorganization during OSK(M)-driven partial reprogramming of aged fibroblasts. Finally, we extend a cost-effective and accessible protocol to murine tissue sections, quantifying in situ age-dependent remodeling. This standardized framework establishes chromatin accessibility as a highly scalable, sequencing-compatible imaging biomarker for evaluating aging and rejuvenation. Summary StatementATAC-see was optimized for scalable, quantitative imaging of chromatin remodeling during aging and cellular reprogramming, and extended to characterize age-associated epigenetic changes across organs.

Cell junctions establish and maintain epithelial architecture despite fluctuating environmental and developmental conditions. A central question is how cells respond to challenging conditions to preserve junctional integrity. Here, we report the discovery of a previously unrecognized quality control pathway that monitors epithelial junctions (J-QC). We used the Caenorhabditis elegans epidermis as a model to investigate the DLG-1-AJM-1 complex (DAC), a junctional domain that is critical for embryonic morphogenesis. We identify two mechanisms that sustain junctional integrity: first, localized dlg-1 mRNA ensures appropriate DLG-1 protein levels at the junction; repositioning dlg-1 RNA reduces DLG-1 levels, leading to gaps between epithelial cells. Second, transcription of DAC components responds to perturbations that disrupt the DAC. This response is sequence-independent, distinguishing it from other quality control mechanisms. It is activated by perturbations of the DAC or cytoskeleton and requires the LINC complex component ZYG-12/HOOK1-3 to transduce information about junctional integrity to the nucleus. These findings define a novel junctional QC for epithelial maintenance.

Selective export of membrane proteins from the endoplasmic reticulum (ER) is fundamental for eukaryotic cell biology, yet how trafficking receptors coordinate cargo recognition with membrane adaptation and COPII recruitment remains unknown. Cornichon homolog (CNIH) proteins comprise a conserved family of trafficking receptors that mediate ER export of ion channels, G protein-coupled receptors (GPCRs), ATP-binding cassette (ABC) and solute carrier (SLC) transporters. Here, we determine cryo-electron microscopy structures of the prototypical cornichon receptor Erv14 bound to an SLC transporter in detergent and lipid nanodiscs. We show that cargo recognition is mediated by a dynamic network of interactions, in which structural lipids stabilize the receptor-cargo interface. Nanodisc structures reveal the assembly of a second Erv14 receptor that remodels the receptor-cargo interface in response to membrane architecture, thereby reducing local membrane thickness and providing direct structural evidence that cornichon receptors buffer hydrophobic mismatch during membrane protein biogenesis. Structural and trafficking analyses further show that the second receptor recruits the COPII adaptor Sec24, coupling membrane remodelling to cargo export. Together, our findings establish that cornichon receptors couple lipid-mediated membrane adaptation with cargo selection through sequential receptor assembly, linking membrane protein folding to selective COPII-mediated ER export. One sentence summaryCornichon receptors integrate membrane adaptation with cargo recognition to coordinate membrane protein quality control and selective ER export.