Biology subjects

Zhang, J.

Publications and source records attributed to Zhang, J..

At least 19 recordsLinked to original sources

bioRxiv2026

ART: A Compositional Physical Theory for the End-to-End Design of Solid-Phase Immunosensors

Predicting complete solid phase immunoassay performance remains challenging because antibody recognition, reactor transport, molecular transduction and physical observation are coupled rather than independent. Here, we formulate ART as a compositional physical theory expressed as Y = Ts[Tm[AR(X)]]. Recognition A and reactor transport R jointly determine the retained molecular state, while molecular transduction Tm and physical observation Ts operate through replaceable interfaces. Finite-time kinetic and reaction diffusion calculations across plate, tubular and bead formats show that antibodies with identical dissociation constants can produce different capture responses because of distinct association and dissociation rates. In fixed-length tubular reactors, mass-conserving transport distinguishes surface capture density from fractional recovery, revising a first-passage-based local density optimum while preserving a multicriterion design window. An AR-only concentration-threshold map identifies when recognition kinetics or reactor redesign offers greater local leverage. The benefits of improved affinity also depend on the kinetic pathway and reactor regime. For enzymatic transduction, we propose a four-parameter, substrate-conserving model in which the saturation-shape parameter is hm = KM/S0 under a one effective substrate approximation. Accumulated TMB color and time-dependent luminol emission represent distinct consequences of reaction progress, while Ts describes measurement limits through a simplified floor slope roof model. ELISA with OD450 readout and tubular chemiluminescence with camera detection illustrate the end-to-end framework. These examples remain conditional model demonstrations, and existing complete chain curves have not yet been recalibrated using the new enzyme model. ART provides a modular quantitative framework for assay design based on explicit physical interfaces and coupled processes.

bioengineering↗

bioRxiv2026

Efficient recovery of transgene-free edited tobacco using the transgene self-elimination system via de novo regeneration

Elimination of integrated editing constructs remains a major challenge for generating transgene-free progeny in plant genome editing based on Agrobacterium-mediated regeneration. Here, we adapted the Transgene Killer CRISPR (TKC) concept to tobacco and established the transgene self-elimination system, in which CRISPR/Cas9 is coupled with developmentally regulated BARNASE cassettes. Three pU3TR-derived vectors carrying DLL-, OsRP3- or PHAS-driven Barnase regenerated normally after leaf-disc transformation. The DLL construct pEB138 showed the highest T editing frequency, with 56 of 69 independent T plants (81.16%) carrying mutations at one or both NtDFR target loci. Under a stringent lineage-level screen in which up to 48 T progeny were tested per T line, 36 of 43 pEB138 lineages (83.72%) yielded uniformly marker-negative progeny for the three assayed T-DNA markers, compared with 0 of 7 lineages transformed with the parental pU3TR vector. The platform also supported recovery of dfr mutants with altered flower pigmentation and NtA622-edited lines with reduced nicotine accumulation. Moreover, introduction of DLL-iBarnase into the independent pDC45 editing backbone did not impair shoot regeneration, and representative NtBBL progeny showed markedly reduced nicotine content together with loss of the selectable cassette. These results demonstrate that BARNASE-assisted transgene elimination can be integrated with Agrobacterium-mediated de novo regeneration in a dicot, enabling one-step recovery of transgene-free edited tobacco progeny.

molecular biology↗

bioRxiv2026

Dynamic merging of enhancer- and promoter-associated assemblies is coupled to transcriptional bursting

Classical models attribute distal enhancer function to chromatin looping, whereas emerging models propose that spatially organized assemblies of transcriptional regulators also facilitate enhancer-promoter communication. To investigate how these processes relate to transcription, we developed an endogenous-tagging-based four-color live-cell imaging platform and an integrated quantitative analysis pipeline in mouse embryonic stem cells (mESCs), enabling simultaneous visualization of regulatory assembly dynamics, chromatin organization and transcriptional bursting at the endogenous Sox2 locus. We find that the pluripotency transcription factor OCT4 and the coactivator BRD4 form numerous spatially distinct but dynamically interacting assemblies. At non-bursting Sox2 alleles, the promoter and the distal Sox2 enhancer SCR occupy distinct assembly environments, with stronger BRD4 association at the SCR than at the promoter. At bursting Sox2 alleles, transcription is coupled to transient merging and partial overlap of enhancer- and promoter-associated assemblies, with the SCR, promoter and nascent RNA frequently sharing OCT4/BRD4-enriched assembly environments across a broad range of SCR-promoter distances. Pharmacological perturbation of bromodomain-dependent BRD4 function preferentially suppresses long-duration, high-amplitude bursts. Together, our findings support a model in which transcriptional bursting is coupled to the dynamic reorganization and merging of enhancer- and promoter-associated regulatory assemblies, linking chromatin-looping and assembly-based views of enhancer-promoter communication.

molecular biology↗

bioRxiv2026

Bayesian in situ point spread function modeling enables high-fidelity three-dimensional nanoscopy in deep tissue

Three-dimensional single-molecule localization microscopy enables nanometer-resolution mapping of biomolecular organization in native cellular environments, but extending this capability to deep tissue requires in situ point spread function (PSF) modeling to account for specimen-induced optical distortions. Existing in situ approaches rely on a deterministic mapping between each single-molecule detection and the underlying PSF model. In deep tissue, however, specimen-induced aberrations, optical scattering and elevated background degrade the information content of single-molecule observations, posing a fundamental barrier to reliable PSF estimation. Here we introduce Bayesian in situ PSF modeling (BIM), a computational framework that explicitly models uncertainty in emitter axial position and propagates it through PSF reconstruction. By using an uncertainty-aware posterior fusion strategy, BIM retrieves accurate specimen-matched PSF models directly from raw blinking data under native imaging conditions. We demonstrate BIM by resolving hollow mitochondrial membranes, amyloid-{beta} fibrils, dendrites and spines with high fidelity. When combined with adaptive optics, BIM improves high-fidelity 3D reconstruction in deep tissue at depths exceeding one hundred micrometers.

biophysics↗

bioRxiv2026

Fine-tuning an intracellular allosteric pocket programs GPCR signaling bias

Biased signaling of G protein-coupled receptors (GPCRs) offers a route for precise therapeutics with fewer adverse effects. Intracellular allosteric ligands can impose such bias, yet how subtle chemical changes within these pockets reshape receptor-transducer coupling and downstream signaling remains unclear. Here, we systematically fine-tuned the intracellular allosteric pocket of neurotensin receptor 1 (NTSR1) through chemical modification of the {beta}-arrestin-biased ligand SBI-553. Minor changes to the ligand scaffold altered transducer-coupling preferences, generating distinct profiles of G-protein and {beta}-arrestin signaling. Cryo-electron microscopy structures of NTSR1-{beta}-arrestin2 complexes bound to designed allosteric probes revealed ligand-dependent remodeling of the intracellular cavity that reshapes the receptor-effector interface. Alanine scanning identified transducer-sensitive residues within and beyond the immediate ligand-contact region. In mice, SBI-553 and N065 produced distinct physiological profiles, with N065 improving survival in a lethal endotoxemia model. Finally, phosphoproteomic profiling revealed partially overlapping but distinct downstream signaling profiles among the three modulators. Together, these findings establish the intracellular allosteric pocket as a tunable determinant of transducer coupling and show that closely related modulators can generate distinct signaling and physiological profiles.

pharmacology and toxicology↗

bioRxiv2026

Foxp1 Coordinates Early Temporal Identity and Neurogenic Progression in Retinal Progenitors

Neural diversity depends on both the temporal competence of progenitors to generate particular cell types and their progression into a neurogenic state. How these distinct features of progenitor development are mechanistically coordinated remains poorly understood. Here, we identify Foxp1 as a dual function transcriptional regulator that couples early progenitor identity to neurogenic progression in the developing retina. By defining its direct transcriptional targets, we show that Foxp1 coordinates complementary regulatory programs: it sustains early competence by activating early-stage retinal progenitor genes and repressing late temporal factors, while promoting neurogenic progression through activation of the proneural factors Atoh7 and Neurog2. We establish a cis-regulatory mechanism linking these functions: Foxp1 activates Atoh7 through its remote shadow enhancer, and deletion of this enhancer prevents Foxp1-dependent induction of Atoh7 and retinal ganglion cell (RGC) production. We further reveal that Neurog2 partially compensates for loss of Atoh7 function. This coordinated early progenitor program is temporally restricted by progressive silencing of Foxp1, involving sequential deposition of H2AK119ub1 followed by H3K27me3 at a distal conserved silencer essential for its repression. Together, these findings establish a mechanistic link between temporal competence and neurogenic progression, revealing how a shared transcriptional regulator coordinates progenitor identity with neuronal production through distinct cis-regulatory elements.

developmental biology↗

bioRxiv2026

Interpreting Short Tandem Repeat Variations with Plant Genomic Foundation Models in Olive

Short tandem repeats (STRs) are highly mutable, multiallelic variants with distinctive mutation dynamics and have been associated with phenotypic variation across diverse species. However, genome-wide STR variation remains poorly characterized in olive, and the large number of naturally occurring alleles creates a challenge for variant interpretation. Genomic language models offer a potential way to assess how STR length changes alter learned sequence representations, but their responses to natural multiallelic STR series have not been systematically evaluated. Here, we identified 117,119 polymorphic STRs across 212 Iranian and Mediterranean olive accessions. STRs recovered the major SNP-defined ancestry axes while resolving additional geographically structured subcomponents. We then quantified STR-induced embedding perturbation using three plant genomic language models: AgroNT, PlantCAD2, and GPN. Across all three models, embedding distance generally increased with repeat-length difference from the reference allele, and 7,417 loci were embedding-sensitive in all three models. These signals were robust to fixed-length input construction and exceeded those of matched nonrepetitive indels at most tested loci. Integration with oil-composition associations prioritized four exploratory loci supported by all three models. Overall, we provide a genome-wide olive STR resource and show that plant genomic language models capture reproducible responses to natural STR length variation. Combining cross-model embedding evidence with phenotype association provides a potential framework for prioritizing candidate STRs for further investigation.

bioinformatics↗

bioRxiv2026

The GA4GH Workflow Execution Service: A Standard API for Workflow Execution and Management in Genomics

Rapid growth in life science data generation has led to large and increasingly distributed collections of sensitive genomic data. To analyse this data, researchers increasingly rely on computational workflows to enable scalable, portable and reproducible analysis. However, these workflows are executed by specialized workflow engines across heterogeneous compute environments, each with its own requirements. As a result, users often need engine- and compute-environment-specific knowledge to submit, monitor and manage workflow execution, increasing the barriers to interoperability. These challenges are compounded in large-scale projects, where workflow runs must be dispatched to local clusters, cloud platforms or data hosting infrastructures with different underlying technologies. We describe the Global Alliance for Genomics and Health (GA4GH) Workflow Execution Service (WES) API, an approved GA4GH standard first released in 2018, together with its data model and the endpoints that define a unified interface for workflow execution. By standardizing workflow invocation, the WES API allows a single client to submit, monitor and cancel workflow runs across diverse workflow engines and compute backends, such as scheduling clusters or cloud platforms. We surveyed thirteen adoptions of the standard, covering standalone services and platforms in four programming languages. In a two-site demonstration, a WES implementation from Germany and one from Japan executed the same federated allele-frequency analysis with site-specific adjustments and only aggregated results leaving either site, demonstrating that it is possible to run the same analysis across independently built and operated WES services in different countries. From the gaps identified, we propose extensions for community discussion.

bioinformatics↗

bioRxiv2026

A polysynaptic hypothalamuspelvic-ganglion circuit regulates prostate tumor growth via WNT3A

Brain-tumor communication is increasingly recognized. Peripheral autonomic nerves regulate prostate cancer progression, yet how the brain influences local nerve-tumor interactions remains unclear. We identify a polysynaptic circuit linking the hypothalamic paraventricular nucleus (PVN), dorsal motor nucleus of the vagus (DMV), major pelvic ganglion (MPG) and prostate tumor in mice. Although both nuclei were activated during tumor growth, PVN activity restrained, whereas DMV activity promoted tumor growth. MPG ablation abolished DMV-induced tumor promotion. Combining LMD with Smart-seq2 revealed transcriptional remodeling and increased Wnt3a expression in tumor-innervating, activated MPG neurons under upstream neural control. MPG explants released WNT3A, whose neutralization suppressed explant-induced cancer-cell proliferation. Together, our findings identify a polysynaptic PVN-DMV-MPG circuit that regulates prostate tumor growth and implicate WNT3A as a mediator of the pro-tumor effects of the MPG, highlighting the therapeutic potential of targeting the neural circuit and downstream molecular pathway.

cancer biology↗

bioRxiv2026

N-Acetylglucosamine ameliorates knee osteoarthritis by enhancing UDP-GlcNAc conversion and chondrocyte DNA repair capacity

Background: Knee osteoarthritis (KOA) is a degenerative joint disease characterized by progressive cartilage degradation and synovial inflammation, while current therapies offer only symptomatic relief. This study aimed to determine whether N-acetylglucosamine (GlcNAc) is superior to glucosamine hydrochloride (GlcN{middle dot}HCl) in slowing the progression of KOA and to elucidate the underlying mechanisms involved. Methods: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to assess serum concentrations of UDP-GlcNAc to conduct a comprehensive evaluation of the pharmacokinetics and pharmacodynamics associated with both GlcNAc and GlcN{middle dot}HCl. Using a destabilization of the medial meniscus (DMM) rat model, we performed micro-computed tomography and histopathological assessments to evaluate structural changes in the joint tissues. Additionally, we conducted bulk RNA sequencing, flow cytometry and ELISA to validate the efficacy of GlcNAc in anti-inflammatory immunoregulation and DNA repair in knee cartilage. Results: GlcNAc achieved higher peak serum concentrations and more efficient conversion to UDP-GlcNAc than GlcN{middle dot}HCl, with UDP-GlcNAc levels 4.57 times higher at 9 hours after administration. GlcNAc significantly attenuated KOA-like pathological changes, downregulated catabolic markers Mmp3 and Adamts4, decreased pain-related neural markers Pgp9.5 and Ngf, and alleviated pain sensitivity. RNA-seq identified 4,395 downregulated DEGs in DMM rats, of which GlcNAc upregulated 2,808 compared to only 391 by GlcN{middle dot}HCl. GlcNAc promoted DNA repair and adaptive immune cell differentiation pathways, and decreased serum IL-1{beta}, IL-6, and IL-17. Four candidate DNA repair genes, Hist1h2an, Poli, Ube2t, and Hells, were identified as significantly upregulated by GlcNAc, with GSEA confirming homologous recombination repair pathway enrichment. Conclusion: This study shows GlcNAc outperforms GlcN{middle dot}HCl in rat DMM knee osteoarthritis via multiple mechanisms, including 4.57-fold higher UDP-GlcNAc conversion, reduced cartilage degeneration, immune rebalancing, and DNA repair activation, supporting GlcNAc as a promising KOA candidate warranting further clinical study.

pharmacology and toxicology↗

bioRxiv2026

BiNVaST: niche boundary-preserving and synthetic data-guided variational autoencoders for concordant inference across high-resolution spatial transcriptomic platforms

High-resolution spatial transcriptomics (ST) enables subcellular spatially resolved transcript profiling but remains difficult to annotate in complex tissues because of gene coverage and super sparseness in expression. These challenges are amplified in cancer research, where neoplastic cell states and rare immune cells in tumor microenvironment are often poorly captured by generic references and/or cell typing tools exclusively built on molecular profile. Here, we present BiNVaST, a niche boundary-preserving and dual-branch variational autoencoder fused with statistical simulator for reliable cell typing across multiple spatial platforms. It generates platform-adjusted in silico training data by integrating query spatial profile and user-defined pre-annotated single-cell reference through Singular Value Decomposition and Optimal Transport, combining molecular similarity and spatial proximity to predict segmented cell labels. BiNVaST was benchmarked against existing methods across Xenium, CosMx, Stereo-seq, Visium HD platforms using six specimens from four tissue types. This tool is sensitive in detecting divergent tumor cell states and improves the cross-platform concordance, distinguishing disease-specific neoplastic and non-neoplastic cellular subpopulations and supporting robust cellular or niche inferences in heterogeneous tissues.

bioinformatics↗

bioRxiv2026

LysM: a bacterial self-encoded peptidoglycan hydrolase as an antimicrobial and anti-biofilm agent for Gram-negative nosocomial pathogens

Agents with both anti-biofilm property and broad-spectrum antimicrobial activity are urgently needed, especially for drug-resistant nosocomial pathogens. Here, we report a bacterial genome-encoded glycoside hydrolase, named LysM, with lytic activity against multiple Gram-negative nosocomial pathogens and little toxicity to human cells. 3D structural modelling and site-directed mutagenesis indicates that LysM is a peptidoglycan-specific glycoside hydrolase. In combination with EDTA, exogenous LysM induces explosive bacterial cell lysis, exhibits potent bactericidal activity, and eliminates 24 h-old biofilms formed by Gram-negative ESKAPE group pathogens with the highest activity on three nosocomial pathogens, namely Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacter cloacae, all of which contain LysM homologs in their genome. More strikingly, LysM supplied in growth media without EDTA, also inhibits the growth of several nosocomial pathogens. Single-cell tracking analysis reveals that LysM completely prevent biofilm formation of P. aeruginosa by reducing bacterial surface-adherence and extending the doubling time. A LysM-based cocktail consisting of LysM, EDTA, and matrix exopolysaccharide-degrading glycosidase PslG, shows potent anti-biofilm activity against P. aeruginosa, including drug-resistant clinical isolates, both in vitro and in human airway organoids (HAOs) biofilm infection model. In addition, LysM enhances the efficacy of many antibiotics from all major classes, including colistin, tobramycin, ciprofloxacin, and tetracycline. Our findings not only provide a clinical candidate for the treatment of nosocomial infections but also open a new path for antimicrobial discovery and anti-biofilm strategies, as homologues of LysM are present in the genomes of many bacteria and phages.

microbiology↗

bioRxiv2026

Imaging biological tissue at extended depth using swept-source visible-light OCT

We present a swept-source visible-light optical coherence tomography (SS-vis-OCT) system with twice the imaging depth of prior SS-vis-OCT and improved sensitivity for imaging biological samples, achieving shot-noise-limited operation. Because second-harmonic generation is limited by the booster optical amplifier's output power, the visible-light SS power is capped at 450 W. Increasing it further would require a more powerful, but costlier, amplifier. Instead, we reduce the receiver noise floor by selecting a balanced detector with NEP approaching the thermal (Johnson-Nyquist) limit. Real-time numerical k-linearization increases the effective number of linear-in-wavenumber samples per sweep, further improving SNR and extending the imaging range. Together, these improvements yield a sensitivity of 89 dB and an imaging depth of 9 mm in air or 6.5 mm in tissue, enabling full anterior-segment imaging of ex vivo porcine eyes.

bioengineering↗

bioRxiv2026

Phylogenomic Characterization of Two Alternaria Lineages Associated with Cotton Leaf Spot in Tennessee

Alternaria leaf spot is an important foliar disease of cotton (Gossypium hirsutum L.) and has historically been associated primarily with Alternaria macrospora in the United States. In November 2025, symptomatic Upland cotton leaves from a research greenhouse at Tennessee Technological University's Oakley Farm in Livingston, Tennessee, yielded two Alternaria isolates, 2-2 and 9-1. Quantitative conidial characterization showed substantial overlap in dimensions, septation, and shape. Concatenated ITS-RPB2 phylogenetic analysis supported different broad affinities, placing isolate 2-2 within an A. alternata- dominated group and isolate 9-1 within a group associated with the A. arborescens species complex (AASC), although their immediate relationships were weakly resolved. Phylogenomic analysis of 1,501 single-copy BUSCO orthologs from 63 genomes placed isolate 2-2 within the A. alternata lineage, sister to BMP2343, and isolate 9-1 within the AASC, sister to MOD1-FUNGI6. In controlled pathogenicity assays, isolate 9-1 reproduced foliar lesions on cotton, whereas mock-inoculated plants remained symptomless, and molecularly confirmed re-isolation fulfilled Koch's postulates. Isolate 2-2 produced insufficient conidia under the culture conditions used to prepare a standardized inoculum, and its pathogenicity was therefore not determined. These results reveal phylogenetically distinct Alternaria lineages associated with symptomatic cotton and establish an AASC isolate as a causal agent of cotton leaf spot in Tennessee.

pathology↗

bioRxiv2026

CucurbitAgent: An AI agent for exploring cucurbit multi-omics resources

Functional genomics in crops increasingly depends on integrating information from an extensive literature and heterogeneous multi-omics datasets. Large language models (LLMs) offer a powerful approach to synthesizing these resources and revealing hidden connections across datasets; however, they frequently hallucinate, producing plausible but unsupported claims, when responding to gene-specific queries. Here, we present CucurbitAgent, an AI research agent for functional genomics in the Cucurbitaceae, a major family of horticultural crops. CucurbitAgent integrates multi-omics resources from nine cultivated cucurbit species and more than 18,000 publications, encompassing approximately 256,000 genes, tissue-resolved expression profiles, over 16 million natural genetic variants, and roughly 242,000 predicted protein structures with more than 14 million predicted binding-site residue annotations. Responses link retrieved evidence to source records, including gene entries, expression measurements, structural predictions, and literature passages. On a curated benchmark of 50 tasks assessed by domain experts under blinded conditions, CucurbitAgent achieved an overall pairwise win rate of 96.0% against nine general-purpose LLMs, based on weighted scores for factual accuracy, completeness, relevance, and traceability. The platform is freely available at https://cucurbitagent.com and supports queries in 37 languages collectively spoken by an estimated 70% or more of the world's population. CucurbitAgent not only unifies evidence retrieval and interpretation but also reveals latent associations across datasets, helping researchers formulate testable hypotheses and substantially accelerating candidate-gene research. Its framework is readily transferable to other crops and, more broadly, provides a blueprint for building specialized agents in any domain.

bioinformatics↗

bioRxiv2026

Efferocytosis Remodels Osteoclast Metabolism and Enhances Antibacterial Defense

Osteoclasts are metabolically specialized for bone resorption, but how apoptotic-cell uptake shapes their immune function remains unclear. During Staphylococcus aureus bone infection, osteoclasts engulfed apoptotic neutrophils. In osteoclast cultures, apoptotic-neutrophil exposure reduced multinucleation and resorption, induced host-defense programs, and enhanced antibacterial activity, whereas macrophages showed no comparable antibacterial response. MERTK was required for efficient osteoclast efferocytosis. Ctsk-Cre-mediated Mertk deletion increased bacterial burden and bone loss, whereas transplantation of MERTK-competent bone marrow restored osteoclast efferocytosis and reduced infection-associated bone damage. Transcriptomic and extracellular flux analyses revealed enhanced glycolytic activity and altered lactate handling in efferocytic osteoclasts. Lactate supplementation reduced osteoclast formation without reproducing the antibacterial phenotype. Inhibiting lactate production or export attenuated antibacterial activity in efferocytic osteoclasts, whereas blocking lactate uptake enhanced it. These findings identify osteoclast efferocytosis as an immune adaptation that links apoptotic-neutrophil clearance to metabolic remodeling and antibacterial defense in bone.

cell biology↗

bioRxiv2026

Co-targeting KRAS and BCL-2 induces pyroptosis and antitumor tertiary lymphoid structures

Mutant KRAS drives lung tumorigenesis through both cell-intrinsic and cell-extrinsic mechanisms, yet mutation subtype-specific differences in metabolic regulation and immune evasion remain incompletely understood. Here we show that the KRAS G12D mutation, particularly in the KRAS/STK11 co-mutant (KL) context, is associated with poor prognosis and a suppressed immune microenvironment characterized by impaired antigen presentation. At the cell-intrinsic level, G12D exhibits weakened interaction with hexokinase 1 (HK1), reduced spare respiratory capacity, and diminished mitochondrial stress resistance compared with G12C. Based on this mitochondrial vulnerability, we screened a panel of clinically available agents that induce mitochondrial stress and identified that co-targeting KRAS and BCL-2 synergistically triggers GSDME-mediated pyroptosis predominantly in G12D tumors. This combination further induces functional tertiary lymphoid structures (TLS) in vivo. Immune cell depletion experiments further demonstrate that the antitumor efficacy is dramatically dependent on B cells. Moreover, this treatment strategy induced membrane attack complex (MAC) deposition in situ and elicited memory antitumor responses. Collectively, these findings establish that combined KRAS and BCL-2 inhibition elicits pyroptosis and TLS formation, leading to B-cell-dependent in situ and memory antitumor immunity, providing a mechanistic rationale for targeting mitochondrial-immune crosstalk in KRAS G12D-mutant lung cancer.

cancer biology↗

bioRxiv2026

Dual role of the RIG-I 2CARD as a signaling module and a brake on dsRNA translocation

RIG-I is an innate immune receptor for viral double-stranded RNA (dsRNA). Upon binding dsRNA, RIG-I nucleates MAVS filaments through its two caspase activation and recruitment domains (2CARD). However, how the 2CARD behaves during activation has remained unclear. Using high-speed atomic force microscopy, we visualized the 2CARD in full-length RIG-I across its apo, dsRNA-bound, translocating, multimeric, and polyubiquitin-bound states. Protrusions consistent with the 2CARD appeared only transiently in the apo state, whereas a mobile 2CARD emerged upon RNA binding. During ATP-driven translocation, wild-type RIG-I moved approximately twofold slower and showed more frequent pauses and multimers than RIG-I {Delta}2CARD. Within multimers, 2CARDs formed a complex that polyubiquitin stabilized into larger, dsRNA-deforming assemblies. These findings reveal the 2CARD as a brake on the RIG-I motor, beyond signaling.

biophysics↗