Virological research has traditionally focused on individual viruses or viral families. Advances in DNA synthesis now allow large-scale construction of individual gene products, enabling systematic exploration of the vir...Virological research has traditionally focused on individual viruses or viral families. Advances in DNA synthesis now allow large-scale construction of individual gene products, enabling systematic exploration of the virome. Here, we developed a barcoded library of ∼12,000 viral open reading frames (vORFs) from 513 viral species, which we leveraged to identify hundreds of viral regulators of cellular proliferation, MHC class I antigen presentation, and interferon signaling. Integrating results across these screens revealed unique phenotypic profiles and functional vORF modules, allowing the in-depth characterization of two previously uncharacterized viral proteins, MC162R and Yaba-like disease virus (YLDV) 151R, which impair MHC class I antigen presentation and interferon (IFN)-β signaling, respectively. Together, the viral ORFeome provides a scalable framework for dissecting viral protein function across the breadth of the virome.
Endosymbiosis has spurred the evolution of new organelles across life. Corals and other cnidarians have repeatedly evolved an organelle, called the symbiosome, which houses intracellular algal symbionts. However, the mol...Endosymbiosis has spurred the evolution of new organelles across life. Corals and other cnidarians have repeatedly evolved an organelle, called the symbiosome, which houses intracellular algal symbionts. However, the molecular mechanisms enabling this repeated evolution remain unclear. Using the sea anemone Aiptasia, we generated a high-quality proteome of the symbiosome, revealing protein trafficking mechanisms and the types of biomolecules exchanged during symbiosis. Symbiosomal enrichment of lysosomal proteins, visualization of lysosomal fusion, and reduced symbiosis following knockdown of lysosomal genes indicate that the symbiosome functions through extensive co-option of lysosomal proteins. We identified a symbiosomal bicarbonate/sulfate transporter, SLC26A11, and showed through CRISPR/Cas9 mutagenesis that this lysosomal transporter is required for symbiosis in Aiptasia and a reef-building coral. Together, these findings reveal that corals and anemones have repeatedly co-opted lysosomal proteins to concentrate carbon and shuttle metabolites to support photosymbiosis, providing a relatively simple path for the repeated evolution of new photosymbioses.
Miakicheva S, Hawley KM, Zumbo P
… +15 more, Gearty SV, Grassmann S, Naizir B, Chiu E, Carson S, Kissner M, Owyong M, Zhang Y, Washburn R, Sun JC, Chaligné R, Reiner SL, Maillard I, Betel D, Schietinger A
In settings of persistent (self or foreign) antigen, such as autoimmunity and chronic infection, immune responses are sustained by stem-like T cells. Although TCF1 has emerged as a key transcription factor (TF) associate...In settings of persistent (self or foreign) antigen, such as autoimmunity and chronic infection, immune responses are sustained by stem-like T cells. Although TCF1 has emerged as a key transcription factor (TF) associated with stemness, the TCF1 population is heterogeneous, raising the question of whether TCF1 exclusively defines the stem T cell (T) pool. Using preclinical models of autoimmune type 1 diabetes and chronic infection, we discover that a small subset of TCF1 T cells express the TF LEF1. LEF1 TCF1 T cells define a true self-renewing T pool. T give rise to LEF1 TCF1 progenitor T cells (T), which lack stem functions and generate terminally differentiated TCF1 T cells (T) (T→T→T). We show that LEF1 is essential for T cell stemness. Autoimmune and exhausted LEF1 T share a unique epigenetically encoded core program enriched for genes and pathways characteristic of embryonic and adult stem cells, including WNT/β-catenin and Notch signaling. Spatial positioning, niche signals, and migration regulate stem-cell fate; accordingly, targeting integrins or Notch signaling impairs T cell stemness and prevents disease. Our studies identify LEF1 and niche-derived factors as fundamental regulators of T cell stemness across chronic diseases.
Zhou Z, Luquette LJ, Dong G
… +18 more, Kim J, Ku J, Kim K, Ramesh N, Bae M, Caplin A, Shao DD, Sahile B, Essuman K, Goodman E, Miller MB, Huang AY, Nathan WJ, Nussenzweig A, Park PJ, Lagier-Tourenne C, Lee EA, Walsh CA
Amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease (AD) represent two major categories of neurodegenerative disorders-TAR DNA-binding protein 43 (TDP-43) and tau proteinopathies-f...Amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease (AD) represent two major categories of neurodegenerative disorders-TAR DNA-binding protein 43 (TDP-43) and tau proteinopathies-for which the mechanisms driving neuronal death remain unclear. Single-cell whole-genome sequencing of 469 neurons from C9ORF72 ALS, C9ORF72 FTD, AD, and control brains revealed increased somatic single-nucleotide variants (sSNVs) and insertions/deletions (sIndels) in all three diseases. Mutational signature analysis identified a disease-associated sSNV signature consistent with oxidative damage and an sIndel process affecting 22% of ALS, 76% of FTD, and 61% of AD neurons-but only 2% of control neurons-resembling signature ID4, previously linked to topoisomerase 1 (TOP1)-mediated mutagenesis. Rapid approach to DNA adduct recovery (RADAR) assays confirmed increased TOP1-DNA covalent complexes, and duplex sequencing confirmed the increased sIndels and identified single-strand events as likely precursor lesions. TOP1-associated sIndel mutagenesis and genome instability thus represent a mechanism shared by both TDP-43 and tau neurodegeneration.
Understanding organ formation requires capturing molecular information simultaneously in three-dimensional (3D) space and across developmental time. To this end, we developed 3D DNase-Enhanced Expression Profiling (3DEEP...Understanding organ formation requires capturing molecular information simultaneously in three-dimensional (3D) space and across developmental time. To this end, we developed 3D DNase-Enhanced Expression Profiling (3DEEP), a tissue-clearing approach that removes genomic DNA to extend spatial transcriptomic profiling hundreds of microns into intact tissues. We applied 3DEEP to neonatal mouse skin, capturing hundreds of developing hair follicles across their organogenesis trajectory. Ordering follicles by molecularly inferred developmental age transformed this single spatial snapshot into a four-dimensional (3D + time) molecular map of organogenesis. This map revealed developmental dynamics spanning stem cell compartment stratification, emergence of new cell subtypes within the follicle, and cascading structural transformations leading to hair canal formation. Comparative analysis of Foxn1-deficient nude mice, a hairlessness model, revealed organ-wide changes in developmental dynamics, including delayed molecular progression, reduced coordination, and increased developmental instability, preceding overt structural defects. This work demonstrates how deep-tissue spatial transcriptomics can uncover hidden dynamics of organ formation.
We introduce a whole-cell digital twin framework that integrates four-dimensional (4D) (x, y, z, and t) lattice light-sheet microscopy with particle-based reaction-diffusion simulations in ReaDDy to model mesoscale intra...We introduce a whole-cell digital twin framework that integrates four-dimensional (4D) (x, y, z, and t) lattice light-sheet microscopy with particle-based reaction-diffusion simulations in ReaDDy to model mesoscale intracellular organelle dynamics. Using fluorescence microscopy data from live Cal27 cells, we construct spatially resolved digital twins incorporating mitochondrial networks, microtubule networks, dynein and kinesin motors, the plasma membrane, and the nucleus. Mitochondrial dynamics include fusion/fission remodeling, diffusion, and motor-driven active transport along microtubules. Our simulations reproduce experimental trends in mitochondrial dynamics across control and two microtubule-perturbed conditions, demonstrating predictive capability without reparameterization. We then use stress-mimicking to predict emergent perinuclear mitochondrial clustering. Crucially, these simulations reveal that microtubule topology acts as a structural gate for this reorganization, demonstrating that upregulated retrograde motor kinetics alone are insufficient to drive clustering without permissive filament connectivity. This digital twin framework provides an approach for investigating intracellular dynamics and perturbation effects in an interpretable and biologically grounded manner.
Pathogens deploy effector proteins to exploit host cell biology, and most effector open reading frames (ORFs) are rapidly evolving and lack functional annotation. We developed the effector ORFeome (eORFeome), a scalable...Pathogens deploy effector proteins to exploit host cell biology, and most effector open reading frames (ORFs) are rapidly evolving and lack functional annotation. We developed the effector ORFeome (eORFeome), a scalable functional genomics platform encompassing 3,835 effector ORFs from diverse viruses, bacteria, and parasites. High-throughput barcoded screens across nuclear factor κB (NF-κB), apoptosis, p53, cGAS-STING, and major histocompatibility complex class I (MHC class I) pathways revealed novel pathway-modulating functions for hundreds of uncharacterized eORFs, unexpected activities of known effectors, and distinct pathway-specific functions encoded by single ORFs. Illustrating the power of this approach, we identified HHV6A U14 as a p53 antagonist, HHV7 U21 as a dual-function STING antagonist and MHC-I antigen display inhibitor, and adenoviral 13.6K/i-leader protein as a de novo-evolved TAP inhibitor that suppresses MHC-I display. These results establish a general framework for systematic effector annotation, uncover new mechanisms of host-pathogen interaction across kingdoms, and highlight pathogen effectors as a versatile toolkit for rewiring and probing human cellular pathways.
The Plasmodium moving junction is central to malarial host-cell invasion, and yet its function remains unclear. Here, we determine the endogenous structure of the basic repeating unit of the moving junction, purified dir...The Plasmodium moving junction is central to malarial host-cell invasion, and yet its function remains unclear. Here, we determine the endogenous structure of the basic repeating unit of the moving junction, purified directly from invasion-stalled Plasmodium falciparum parasites, revealing a sailboat-shaped 1:1:1:1 assembly of apical membrane antigen 1 (PfAMA1) and rhoptry neck proteins 2, 4, and 5 (PfRON2, PfRON4, and PfRON5). We observe two PfRON2 transmembrane helices that anchor the complex in the red blood cell (RBC) membrane and display an extracellular handle for PfAMA1 binding. PfAMA1 directly contacts the RBC membrane, strengthening the connection. PfRON2/4/5 form a large, basic platform inside the RBC that electrostatically engages the RBC membrane and wedges seven amphipathic helices deep into the bilayer, suggesting an active role in host-membrane remodeling. We then leverage the native membrane context revealed by our structure, along with recent advances in computational protein design, to enable the rational design of a small protein binder that inhibits invasion.
Schwayer C, Barbiero S, Brückner DB
… +22 more, Oost KC, Baader C, Repina NA, Kim J, Diaz OE, Uccelli I, Capolupo L, Meylan LC, Kalck V, Moos F, Suppinger S, Yang Q, Schnabl J, Kilik U, Bourdon M, Camp JG, Stockinger B, Ferrari A, Bühler M, Stadler MB, Hannezo E, Liberali P
Tissue regeneration requires de novo patterning, which has been proposed to be facilitated by cellular heterogeneity. Yet how such heterogeneities are integrated with the mechanochemical state of the tissue and stabilize...Tissue regeneration requires de novo patterning, which has been proposed to be facilitated by cellular heterogeneity. Yet how such heterogeneities are integrated with the mechanochemical state of the tissue and stabilized at the chromatin level into stable, spatially organized fates remains poorly understood. Using in vivo mouse intestinal regeneration models and organoids, we identify a critical density regime that produces a permissive window for heterogeneity in the mechanosensor Yes-associated protein 1 (YAP1). We show that YAP1 heterogeneity is coupled to lineage-biased chromatin accessibility and is decoded through FOXA1, which integrates the permissive chromatin state to Delta-Notch supracellular feedback and lineage commitment. This circuit generates fate bistability and preserves a memory of transient YAP1 activity, thereby maintaining spatial patterning as tissues return to homeostasis after injury. Together, our findings establish a multiscale framework in which tissue-scale mechanics tune single-cell competence and, through FOXA1-mediated bistability, convert transient heterogeneity into stable and self-organized tissue architecture.
Tau pathology spreads cell to cell, but the mechanisms of intercellular tau transmission remain unclear. We find that the neuronal gene Arc is critical for the release of tau in neuronal extracellular vesicles (EVs) via...Tau pathology spreads cell to cell, but the mechanisms of intercellular tau transmission remain unclear. We find that the neuronal gene Arc is critical for the release of tau in neuronal extracellular vesicles (EVs) via a direct protein-protein interaction. Brain EVs purified from transgenic rTg4510 mutant tau mice (rTg) crossed with Arc knockout mice (rTg) contain less tau and reduced tau seeding potential. Both Arc and tau are co-packaged in mouse and human brain-derived EVs. Moreover, Arc levels in brain-derived EVs isolated from human Alzheimer's disease (AD) brains show a strong positive correlation with phosphorylated EV-tau levels. rTg mice have increased accumulation of intracellular tau and a modest increase in cell toxicity early in disease progression. Strikingly, intercellular tau transmission is almost absent in Arc KO mice. These results show that Arc is critical for the packaging of tau in EVs, which plays a significant role in intercellular tau transmission.
The intricate network of axonal fibers forming the mammalian cortical connectome exhibits a complex topology, being neither completely regular nor random. It also has a characteristic topography in which distinct regions...The intricate network of axonal fibers forming the mammalian cortical connectome exhibits a complex topology, being neither completely regular nor random. It also has a characteristic topography in which distinct regions have specific connectivity profiles. How such properties arise remains a mystery. Here, we formulate a simple analytical model derived from neural field theory that prioritizes physical constraints on connectome architecture, assuming that connectivity is preferentially concentrated between cortical locations that facilitate the excitation of resonant geometric modes of the cortex. Our model outperforms existing approaches in reproducing topological and topographical properties of cortical connectomes mapped via either non-invasive diffusion magnetic resonance imaging (MRI) or invasive viral tract tracing at spatial scales spanning multiple orders of magnitude in humans, chimpanzees, macaques, marmosets, and mice. Our findings point to a fundamental role of geometry in shaping the multiscale architecture of cortical connectomes that has been conserved across 90 million years of evolution.
Chen S, Deng F, Peng B
… +20 more, Liu L, Wang J, Jin F, Xu J, Lin D, Chen W, Zhang D, Yi C, Zhang J, Zhong S, Zhu L, Huang Y, Yang J, Wang R, Sun X, Zhang Y, Ling Z, Ma L, Liu X, Sun B
Gasdermin D (GSDMD)-mediated interleukin (IL)-33 secretion by lung epithelial cells initiates airway inflammation upon allergen challenge. How environmental allergens activate GSDMD remains elusive. Here, we demonstrate...Gasdermin D (GSDMD)-mediated interleukin (IL)-33 secretion by lung epithelial cells initiates airway inflammation upon allergen challenge. How environmental allergens activate GSDMD remains elusive. Here, we demonstrate that exposing epithelial cells to allergens triggers protease-activated receptor 1 (PAR1)-dependent ferritinophagy, elevating intracellular labile iron. This iron pool is essential for noncanonical, protease-independent GSDMD activation. The iron chaperone poly(rC)-binding protein 2 (PCBP2) delivers iron directly to GSDMD, initiating a highly localized Fenton reaction. This generates constrained hydroxyl radicals that cleave GSDMD, releasing the active N-terminal p40 fragment to form pores for IL-33 release. Blocking any step of this iron-GSDMD pathway, via iron chelation or genetic ablation, abolishes IL-33 secretion, prevents group 2 innate lymphoid cell (ILC2) activation, and mitigates allergic airway inflammation and tissue damage in mice. Our findings reveal an unconventional, iron-catalyzed, and protease-independent mechanism for GSDMD activation, offering potential new therapeutic targets for allergic inflammatory diseases.
Kim MJ, Noh JH, Lee HY
… +16 more, Lee B, Park AD, Oh H, Kim GD, Lee HS, Oh Y, Hwang H, Kim C, Lee H, Li X, Oh MM, Lim D, Abraham SN, Cho Y, Chi SW, Choi HW
Epithelial mechanosensation maintains tissue homeostasis by sensing mechanical stimuli. Dysregulated mechanotransduction has been implicated in chronic pain, yet the molecular link between epithelial stress and persisten...Epithelial mechanosensation maintains tissue homeostasis by sensing mechanical stimuli. Dysregulated mechanotransduction has been implicated in chronic pain, yet the molecular link between epithelial stress and persistent sensory dysfunction remains unclear. Here, we report that the mechanosensitive ion channel PIEZO1 is markedly upregulated in bladder epithelial cells under recurrent uropathogenic E. coli infection through interleukin-6 (IL-6)-dependent signaling. PIEZO1 overexpression amplifies mechanotransduction-induced reactive oxygen species (ROS) generation, triggering a homeostatic antioxidant response via the cystine-glutamate antiporter system Xc⁻ (SLC7A11). This protective mechanism inadvertently promotes extracellular glutamate accumulation, driving aberrant sprouting and hyperinnervation of peptidergic nociceptive C fibers into the epithelial layer. This neuroepithelial remodeling sensitizes bladder afferents and induces persistent pain-like states resembling chronic visceral pain and organ dysfunction. Our findings reveal a paradoxical role of epithelial antioxidant defense in promoting pain through the PIEZO1-SLC7A11-glutamate axis and highlight the neuroepithelial interface as a key therapeutic target for infection-induced chronic pain syndromes.
Lin F, Yin S, Zhang Z
… +20 more, Yu Y, Fang H, Liang Z, Zhu R, Zhou H, Li J, Cao K, Guo W, Qin S, Zhang Y, Lu C, Li H, Liu S, Zhang H, Ye B, Lin J, Li Y, Kang X, Xi JJ, Chen PR
The ability to maintain focus wavers over time, impacting performance, yet the neural circuits governing fluctuations in sustained attention remain unclear. The basal forebrain is integral to attention by modulating cort...The ability to maintain focus wavers over time, impacting performance, yet the neural circuits governing fluctuations in sustained attention remain unclear. The basal forebrain is integral to attention by modulating cortical activity, but recent evidence challenges the role of its signature cholinergic neurons in sustaining attention. Here, we demonstrate that another cortex-projecting basal forebrain cell type, parvalbumin-expressing (BF-PV) inhibitory neurons, mediates sustained attention. In mice performing an attention-demanding task, BF-PV activity predicts trial-by-trial fluctuations in attentional performance metrics-reaction time and accuracy. Bidirectional optogenetic manipulations confirm causality: activation enhances, and inhibition impairs, attention. BF-PV neurons also respond to motivationally salient events-predictive cues, outcomes, and surprises-regardless of valence. A computational model bridges these observations and demonstrates how this signal guides attention allocation. Mechanistically, BF-PV neurons target cortical PV+ interneurons, producing disinhibitory cortical amplification to improve detection sensitivity. These findings reveal a disinhibitory BF-to-cortex projection that regulates cortical gain based on motivational salience, thereby promoting sustained attention.
Baker DJ, Frommer LM, Uslu U
… +14 more, Patel KK, Zhu D, Engel NW, George JM, Zhao W, Kim SI, Sun L, Roselle C, Rommel PC, Young RM, Epstein JA, Hayat S, Arany Z, June CH
Chimeric antigen receptor (CAR) T cells have demonstrated curative potential in hematologic cancers and increasing efficacy in solid tumors and non-malignant diseases. However, target identification remains a major bottl...Chimeric antigen receptor (CAR) T cells have demonstrated curative potential in hematologic cancers and increasing efficacy in solid tumors and non-malignant diseases. However, target identification remains a major bottleneck. We developed an artificial intelligence (AI)-driven approach for CAR T cell target discovery by integrating single-cell RNA sequencing datasets from human skin cancer and healthy tissue. Candidates were refined using public datasets to optimize for tumor composition, tissue specificity, and clinical feasibility. Large language models were applied to prioritize and nominate targets with therapeutic promise. Glycoprotein non-metastatic melanoma protein B (GPNMB) was the most frequently nominated target. We validated its expression across hematologic and solid tumors. We engineered a human GPNMB-directed CAR T cell, which showed potent anti-tumor activity in mouse models of monoblastic leukemia, melanoma, and colorectal adenocarcinoma. These findings establish a scalable pipeline for CAR T cell target discovery and support the translation of GPNMB-directed CAR T cells as a multi-cancer therapeutic.
The cGAS-STING pathway is a central mechanism of innate immunity that detects double-stranded DNA and translates it into transcriptional and other cellular effector responses. Beyond its canonical role in antiviral defen...The cGAS-STING pathway is a central mechanism of innate immunity that detects double-stranded DNA and translates it into transcriptional and other cellular effector responses. Beyond its canonical role in antiviral defense, the pathway senses diverse endogenous DNA species generated as byproducts during various contexts of cell stress. In this capacity, cGAS-STING impacts tissue homeostasis and antitumor immunity, but it is also associated with a number of inflammatory disorders. Here, we review the mechanisms, physiological functions, and disease implications of cGAS-STING signaling and discuss how its context-dependent biology informs emerging therapeutic strategies.
Identifying safe and effective targets remains a major bottleneck for CAR T cell therapies. In this issue of Cell, Baker and colleagues developed a large language model (LLM)-assisted scoring framework to streamline this...Identifying safe and effective targets remains a major bottleneck for CAR T cell therapies. In this issue of Cell, Baker and colleagues developed a large language model (LLM)-assisted scoring framework to streamline this process and as a result identified and validated glycoprotein non-metastatic melanoma protein B (GPNMB) as a candidate chimeric antigen receptor (CAR) T target across melanoma, leukemia, and colorectal cancer.
Most cells polarize and migrate in response to electrical fields. In this issue of Cell, Belliveau et al. identify TMEM154/Galvanin, a receptor that serves as a cellular antenna to sense electrical gradients and guide mi...Most cells polarize and migrate in response to electrical fields. In this issue of Cell, Belliveau et al. identify TMEM154/Galvanin, a receptor that serves as a cellular antenna to sense electrical gradients and guide migration toward the cathode.