Biology subjects

Ou, G.

Publications and source records attributed to Ou, G..

At least 19 recordsLinked to original sources

bioRxiv2026

Janusein and EB Cooperate to Enhance Microtubule Plus-end Catastrophe

Microtubule (MT) plus ends are organized by end-binding (EB) proteins, yet how EB-associated factors modulate the transition from growth to catastrophe remains incompletely understood. Using spatially resolved proximity proteomics, we identified Janusein (ZC2HC1A) as a conserved, poorly characterized MT-associated protein (MAP). At low expression levels, Janusein tracked MT plus ends and reduced EB comet velocity; at higher levels, it redistributed to the MT lattice and was associated with MT bundling and widespread MT loss. Loss of Janusein resulted in spindle defects in human cells. In vitro, Janusein alone facilitated MT elongation through its C-terminal fragment, but promoted MT catastrophe through its N-terminal fragment in the presence of EB proteins. Molecular dynamics simulations and mutational analyses suggest that an N-terminal loop may perturb lateral contacts between adjacent MT protofilaments. Together, these findings identify Janusein as an unconventional MAP that functionally cooperates with EB proteins to promote MT catastrophe.

cell biology↗

bioRxiv2026

Spatially Confined Phase Separation Promotes Local Tubulin Availability to Stabilize Ciliary Microtubule Growth

How microtubule (MT) growth is locally sustained within cilia remains a central unanswered question in ciliogenesis. Here we identify RAT-1 as a regulator of ciliary tubulin homeostasis and MT growth. Genetic screens in Caenorhabditis elegans show that loss of RAT-1 suppresses the toxic effects of a mutant -tubulin and restores ciliary structure, while RAT-1 deficiency reduces tubulin turnover at the ciliary tip, indicating its physiological role in tuning ciliary MT dynamics. Mechanistically, RAT-1 forms intra-ciliary condensates selectively at the distal region through an auto-inhibitory -helix and phosphorylation by the distally localized ciliary kinase. RAT-1 enriches tubulin within condensates in vivo/vitro, and cooperates with end-binding proteins to facilitate sustained MT growth in vitro. Notably, heterologous expression of worm RAT-1 reveals conserved condensate behavior within mammalian cilia. Our findings reveal a kinase-regulated mechanism controlling RAT-1 condensation within cilia and establish RAT-1 as a regulator of ciliary MT dynamics.

cell biology↗

bioRxiv2026

Integrating structural and biological evidence to rerank ESMFold2 protein-protein interactions

Large-scale protein structure prediction enables proteome-wide protein-protein interaction (PPI) screening, but distinguishing biologically meaningful interactions from spurious interfaces remains challenging. Here, we develop a scalable framework combining fast, MSA-free ESMFold2 prediction with PAE-guided domain parsing and evidence-based reranking. Three-recycle ESMFold2-Fast achieved 57% acceptable-or-better DockQ scores on FoldBench, comparable to AlphaFold2-Multimer while substantially reducing computation. PAE-guided parsing preserved 98.1% of XL-MS cross-links within parsed domain pairs. We then developed the Structure Prediction and Omics informed Classifier (SPOC)-ESMFold2, which integrates structural features with independent biological evidence to prioritize predicted interactions. SPOC-ESMFold2 achieved an AUROC of 0.93 and AUPR of 0.90, compared with 0.87 and 0.79 for a structural-only classifier. Under a 1:128 positive-to-negative ratio, SPOC-ESMFold2 achieved 17.2% recall at 5% false-discovery rate, substantially outperforming structural confidence metrics alone. This framework enables scalable PPI screening by integrating structural plausibility with orthogonal biological evidence to prioritize candidates for experimental investigation.

bioinformatics↗

bioRxiv2026

A Discrete Language of Protein Words for Functional Discovery and Design

Proteins can preserve conserved functions despite extensive sequence and structural divergence, suggesting that functional organization is governed by distributed constraints not captured by conventional representations. Here we develop a hierarchical sequence-based representation framework that compresses proteins into context-dependent latent states while preserving multiscale organizational information. Using this framework, we identified previously uncharacterized ciliary proteins lacking detectable sequence and structure homology, including ADMAP1, which is required for normal sperm axonemal organization and motility in mice. Discrete latent protein states captured species-level organizational signatures correlated with major evolutionary groups and revealed expansion of intrinsically disordered regulatory environments in eukaryotes. Autoregressive sampling within this latent space further enabled design of synthetic actin-remodeling proteins that maintained robust F-actin severing activity despite extensive sequence rewiring across key functional interfaces. These findings demonstrate that distributed protein organization can be inferred directly from sequence, linking functional discovery, evolutionary analysis, and protein design within a shared representational framework.

bioinformatics↗

bioRxiv2025

Cysteine protease cathepsin B promotes high population density-induced mutagenesis, driving genome evolution and competitive growth in response to the crowding stress

In a wide variety of species from invertebrates to mammals, overpopulation has been shown to induce low fertility and high mortality. Although density-dependent population regulation is widespread in the animal kingdom, the underlying molecular mechanisms remain poorly understood. We show here that C. elegans animals respond to the crowding stress by secreting CPR-4, a homologue of human cathepsin B cysteine protease, to promote chromosomal DNA damage in germ cells, leading to high density-induced deficiencies that include increased embryonic lethality and larval arrest and decreased brood size. CPR-4 mediates these crowding responses through the insulin-like growth factor receptor DAF-2, multiple components in the insulin signaling pathway, and the SKN-1/Nrf transcription factor. Whole genome sequencing analyses of animals from 10 generations of continual growth in the crowded condition reveal that CPR-4-induced DNA damage produces an average of 2.9 more de novo genome mutations per animal per generation and a 75% increase in mutation rate compared with animals grown in the uncrowded condition. CPR-4-induced mutagenesis also facilitates evolution of the genomes through multi-generational crowding selection, leading to biased mutation distributions towards the intergenic regions over the gene bodies and crowd-inducible growth advantage. Our findings suggest that CPR-4 acts as a crucial crowd-responding factor to induce chromosomal DNA damage, leading to density-dependent deficiencies, increased genome mutation rates, and genome evolution and competitive growth of animals in response to the crowding stress.

developmental biology↗

bioRxiv2025

RPRM (Reprimo) triggers SCF(FBXW11)-mediated DNA-PKcs degradation to block non-homologous end joining and radiosensitize tumors

Targeting DNA damage repair pathways represents a promising strategy in cancer radiotherapy, however, the limited insight into the regulation of the non-homologous end joining (NHEJ) repair pathway in cancer cells severely restricts the development of precise radiosensitization approaches. Here, we identify Reprimo (RPRM), a p53-inducible tumor suppressor, is a potent inhibitor of NHEJ in which RPRM promotes proteasomal degradation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylated at the T2609 site. Upon irradiation, RPRM quickly translocates into the nucleus to enhance the transcription of FBXW11, a F-box protein gene that encodes the substrate recognition component of SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complex. In the nucleus, RPRM is also able to scaffold the assembly of the SCFFBXW11 E3 ligase complex by binding to both CUL1 and FBXW11. This complex can specifically ubiquitylate phosphorylated DNA-PKcs (T2609), leading to reduced levels of both phosphorylated DNA-PKcs (T2609) and total DNA-PKcs, thereby suppressing NHEJ-driven repair and enhancing tumor radiosensitivity. In consistence, inhibiting the SCFFBXW11 E3 ligase complex eliminates RPRM-mediated radiosensitization by protecting DNA-PKcs. Clinically, high RPRM expression in non-small cell lung cancer (NSCLC) predicts better patient prognosis. Moreover, RPRM expression correlates positively with FBXW11 and PRKDC (DNA-PKcs) in the tumor tissues of the NSCLC patient cohort. Significantly, high expression of RPRM protein is linked to enhanced radiosensitivity of patient-derived organoids (PDOs) from these NSCLC patients. These results establish RPRM as a key negative regulator in NHEJ repair pathway and the RPRM-SCFFBXW11-DNA-PKcs axis as a potential target to enhance cancer radiosensitivity.

cancer biology↗

bioRxiv2025

TaoChongBao: A Large-Scale Caenorhabditis elegans Mutagenesis and Missense Variant Database Bridging Worm and Human Genomes

We generated and sequenced 12,069 viable Caenorhabditis elegans strains produced by ethyl methanesulfonate mutagenesis, identifying 20,315,536 variants, including 541,102 unique missense mutations across 20,914 genes. To organize and visualize this resource, we developed TaoChongBao, an open-access database and strain repository that integrates C. elegans mutation data with AlphaMissense-predicted pathogenicity scores and ClinVar clinical annotations. TaoChongBao enables users to explore worm missense variants, identify conserved residues corresponding to human pathogenic sites, and access viable strains for experimental validation. Compared with the previous Million Mutation Project in C. elegans, TaoChongBao expands mutation coverage over twenty-fold and emphasizes amino acid-altering variants. This resource provides a scalable platform for functional residuomics, variant interpretation, and comparative analyses between C. elegans and human genomes.

bioinformatics↗

bioRxiv2025

Conserved Gαq-PLCβ-PKC signaling mediates trametinib resistance in BRAFV600E melanoma

Oncogenic BRAFV600E mutations drive constitutive MAPK signaling across various cancers; however, resistance to MEK inhibitors such as trametinib undermines clinical benefits, and the mechanisms sustaining MAPK activity under treatment remain unclear. We developed a lin-45(V627E) Caenorhabditis elegans model to mimic oncogenic BRAF signaling and found that these animals tolerate ultra-high doses of trametinib. This survival is driven by an adaptive resistance bypass, mediated by Gq-PLC{beta}-PKC signaling, which sustains MAPK activity despite MEK inhibition. Transcriptomic analyses of patient tumors and resistant melanoma cells reveal upregulation of the Gq subunits and GPCR PAR2 upon acquisition of resistance. Functional inhibition of PAR2 or downstream Gq signaling suppresses ERK reactivation, selectively impairs the viability of resistant cells. Furthermore, combined inhibition of MEK and Gq-PLC{beta}-PKC signaling synergistically suppresses tumor growth and restores trametinib sensitivity in xenograft models. These findings uncover adaptive GPCR-Gq-driven signaling rewiring as an evolutionarily conserved and therapeutically actionable vulnerability in MAPK-driven cancers.

cancer biology↗

bioRxiv2025

A comprehensive functional landscape of α-tubulin TUBA1A variants illuminates microtubule biology and refines clinical classification

Missense variant interpretation in highly conserved, paralog-rich gene families remains a critical bottleneck for precision medicine. Here, we developed an integrated experimental-computational platform to systematically assess the functional impact of all possible missense mutations in the -tubulin TUBA1A. Combining high-throughput comprehensive mutagenesis, high-content imaging, convolutional neural network- driven phenotyping, and machine learning-guided prediction, we quantified microtubule assembly phenotypes for every coding variant. This approach outperforms conservation-based predictors and enables functional reinterpretation of disease-associated variants. Structural mapping of the TUBA1A mutational landscape reveals distinct domains critical for GTP binding, chaperone-assisted folding or protofilament interaction, illuminating diverse mechanisms of tubulin-related diseases. Integration with ACMG-AMP guidelines demonstrates this mutational landscape improves clinical variant classification in redundant gene families. This framework is broadly applicable to other structurally conserved proteins, linking variant effect prediction to mechanistic insight and clinical translation.

genetics↗

bioRxiv2025

Physiological Reconstitution of Microtubule Doublets

The formation of microtubule doublets (MTDs) is a foundational step in cilia biogenesis, yet how B-tubule nucleation is initiated at the molecular level remains elusive. Here, we identify FAP53 as a crucial factor that promotes B-tubule assembly. Molecular dynamics simulations revealed that FAP53 alleviates steric hindrance from the -tubulin C-terminal tail and stabilizes B-tubule docking at the A-tubule surface. In vitro reconstitution demonstrated that recombinant FAP53 is sufficient to drive MTD formation under physiological tubulin conditions. In cultured HeLa cells, co-expression of CFAP53 and CFAP20--an inner junction protein--induced ectopic MTD-like structures in the cytoplasm. Furthermore, a structurally homologous protein in C. elegans, WFAP-53, localized to sensory cilia; however, when overexpressed, it triggered ectopic MTD formation in neuronal dendrites and concomitantly led to sensory cilia disassembly. These findings uncover a conserved mechanism of B-tubule initiation and underscore the necessity for spatially restricted expression of MTD assembly factors during ciliogenesis.

cell biology↗

bioRxiv2025

Fasting Rescues Locomotion in Neuromodulation-Deficient C. elegans via Octopamine-Gαq Signaling

Nutrient deprivation induces adaptive behavioral and physiological changes that are critical for survival. Here, we demonstrate that fasting ameliorates locomotion defects in Caenorhabditis elegans mutants lacking UNC-31/CAPS, a protein essential for dense-core vesicle (DCV)-mediated neuromodulation. Through forward genetic screening, we identified a gain-of-function mutation in egl-30, which encodes the heterotrimeric G protein subunit Gq, that suppresses the locomotion defects of unc-31 mutants under fed conditions. Transcriptomic analyses revealed that fasting induces upregulation of egl-30 and its downstream effectors in unc-31 mutants. Remarkably, exogenous octopamine treatment, which activates EGL-30/Gq signaling, mimicked the fasting response and restored locomotion in an EGL-30-dependent manner. Our findings uncover a mechanism of neuromodulatory plasticity, in which metabolic stress activates a compensatory octopamine-Gq signaling cascade to bypass impaired DCV-mediated neuromodulation, and suggest potential therapeutic strategies for CAPS-related neuropsychiatric disorders.

neuroscience↗

bioRxiv2025

CRISPR-based shuttle cloning of 1,397 human genes into UAS vectors

The construction of genome-wide DNA libraries from publicly available resources is essential for leveraging functional genomics to investigate complex biological systems. However, all existing high-throughput cloning methods for transferring DNA fragments between vectors require PCR amplification of the DNA fragments, rendering the construction of genome-wide DNA libraries labor-intensive and time-consuming. By introducing a concept of CRISPRshuttle cassette, we herein present a method named CRISPR-based shuttle cloning (CRISPRshuttle cloning). This method enables the high-throughput transfer of numerous DNA fragments from original plasmids with identical backbones to a different vector background without the need for PCR amplification of the DNA fragments. The procedure comprises two-step test tube reactions followed by bacterial transformation. Using CRISPRshuttle we successfully generated a library of GAL4/UAS-based UAS-ORF plasmids covering 1,397 human genes conserved in Drosophila. This library may serve as a valuable resource for gain-of-function screening in cultured cells and for the creation of a transgenic UAS-ORF library in Drosophila.

molecular biology↗

bioRxiv2025

SynSeg: Generating Synthetic Datasets for Accurate Subcellular Segmentation with U-net

Accurate segmentation of subcellular components is crucial for understanding cellular processes, but traditional methods struggle with noise and complex structures. Convolutional neural networks improve accuracy but require large, time-consuming, and biased manually annotated datasets. Here, we developed SynSeg, a pipeline that generates synthetic training data to train a U-net model for subcellular structure segmentation, eliminating the need for manual annotation. SynSeg leverages synthetic datasets with variations in intensity, morphology, and signal distribution to deliver context-aware segmentations, even in challenging imaging conditions. We demonstrate SynSegs superior performance in segmenting vesicles and cytoskeletal filaments from culture cells and live C. elegans, outperforming traditional methods such as Otsus thresholding, ILEE, and FilamentSensor 2.0. Additionally, SynSeg effectively quantified disease-associated microtubule morphology in live cells, uncovering structural defects caused by mutant Tau proteins linked to neurodegenerative diseases. These results highlight the potential of synthetic data-driven approaches to advance biological segmentation and enhance microscopy techniques. Significance StatementThis study introduces a novel approach for accurately segmenting cellular structures, such as microtubules and vesicles, using synthetic datasets and advanced deep learning techniques. By leveraging a U-Net model trained on thousands of artificially generated images, our method eliminates the need for labor-intensive experimental data and simplifies the data creation process. Importantly, it incorporates noise and variability into the training datasets to make the model more robust and biologically relevant. Our findings demonstrate that the model can successfully identify cellular components, paving the way for its application in real-world microscopy images. This innovation has the potential to accelerate discoveries in cell biology by providing an efficient, scalable tool for analyzing complex cellular structures, even in challenging imaging conditions.

bioinformatics↗

bioRxiv2024

Kinesin Autoinhibition Requires Elbow Phosphorylation

Kinesin motor proteins facilitate microtubule-based transport by converting chemical energy into mechanic forces, but this activity is autoinhibited until cargo is loaded. Regulatory mechanisms underlying this autoinhibitory conformation are not well understood. Here, we show that a NEver in mitosis Kinase NEKL-3 directly phosphorylates a flexible elbow region between two coiled coil domains connecting the motor head and tail of an intraflagellar transport kinesin, OSM-3. The phosphor dead (PD) mutation, but not phosphor mimic (PM) mutation, induce constitutive motility of OSM-3 in vitro. Using knock-in animals, we discovered that both PD and PM mutations shorten the C. elegans sensory cilia. The constitutively active OSM-3PD fails to enter cilia and abnormally accumulates in neurites, mimicking another hyperactive mutation, OSM-3G444E. Conversely, OSM-3PM enters cilia but moves at a reduced speed, indicating an inhibitory role of elbow phosphorylation in kinesin motility. These findings highlight the crucial role of elbow phosphorylation in regulating kinesin autoinhibition.

cell biology↗

bioRxiv2024

Artificial Intelligence-Enabled AlphaFold II Pipeline Guides Functional Fluorescence Labeling of Tubulin Across Species

Dynamic properties are essential for microtubule (MT) physiology. Current techniques for in vivo imaging of MTs present intrinsic limitations in elucidating the isotype-specific nuances of tubulins, which contribute to their versatile functions. Harnessing the power of AlphaFold II pipeline, we engineered a strategy for the minimally invasive fluorescence labeling of endogenous tubulin isotypes or those harboring missense mutations. We demonstrated that a specifically designed 16-amino acid linker, coupled with sfGFP11 from the split-sfGFP system and integration into the H1-S2 loop of tubulin, facilitated tubulin labeling without compromising MT dynamics, embryonic development, or ciliogenesis in C. elegans. Extending this technique to human cells and murine oocytes, we visualized MTs with the minimal background fluorescence and a pathogenic tubulin isoform with fidelity. The utility of our approach across biological contexts and species set an additional paradigm for studying tubulin dynamics and functional specificity, with implications for understanding tubulin-related diseases known as tubulinopathies.

cell biology↗

bioRxiv2024

A Machine Learning Enhanced EMS Mutagenesis Probability Map for Efficient Identification of Causal Mutations in Caenorhabditis elegans

Chemical mutagenesis-driven forward genetic screens are pivotal in unveiling gene functions, yet identifying causal mutations behind phenotypes remains laborious, hindering their high-throughput application. Here, we reveal a non-uniform mutation rate caused by Ethyl Methane Sulfonate (EMS) mutagenesis in the C. elegans genome, indicating that mutation frequency is influenced by proximate sequence context and chromatin status. Leveraging these factors, we developed a Machine Learning enhanced pipeline to create a comprehensive EMS mutagenesis probability map for the C. elegans genome. This map operates on the principle that causative mutations are enriched in genetic screens targeting specific phenotypes among random mutations. Applying this map to Whole Genome Sequencing (WGS) data of genetic suppressors that rescue a C. elegans ciliary kinesin mutant, we successfully pinpointed causal mutations without generating recombinant inbred lines. This methodology can be adapted in other species, offering a scalable approach for identifying causal genes and revitalizing the effectiveness of forward genetic screens. Significance statementExploring gene functions through chemical mutagenesis-driven genetic screens is pivotal, yet the cumbersome task of identifying causative mutations remains a bottleneck, limiting their high-throughput potential. In this investigation, we uncovered a non-uniform mutation pattern induced by Ethyl Methane Sulfonate (EMS) mutagenesis in the C. elegans genome, highlighting the influence of proximate sequence context and chromatin status on mutation frequency. Leveraging these insights, we engineered a machine learning enhanced pipeline to construct a comprehensive EMS mutagenesis probability map for the C. elegans genome. This map operates on the principle that causative mutations are selectively enriched in genetic screens targeting specific phenotypes amid the backdrop of random mutations. Applying this mapping tool to Whole Genome Sequencing (WGS) data derived from genetic suppressors rescuing a C. elegans ciliary kinesin mutant, we achieved precise identification of causal mutations without resorting to the conventional generation of recombinant inbred lines. Our work not only advances understanding of mutation dynamics but also revitalizes the efficacy of forward genetic screens, contributing to the refinement of genetic exploration methodologies with implications for various organisms.

genetics↗

bioRxiv2023

Transposase-Assisted Tagmentation: An Economical and Scalable Strategy for Single-Worm Whole-Genome Sequencing

AlphaMissense identifies 23 million human missense variants as likely pathogenic, but only 0.1% have been clinically classified. To experimentally validate these predictions, chemical mutagenesis presents a rapid, cost-effective method to produce billions of mutations in model organisms. However, the prohibitive costs and limitations in the throughput of whole-genome sequencing (WGS) technologies, crucial for variant identification, constrain its widespread application. Here, we introduce a Tn5 transposase-assisted tagmentation technique for conducting WGS in C. elegans, E. coli, S. cervisiae, and C. reinhardtii. This method, demands merely 20 minutes of hands-on time for a single worm or single-cell clones and incurs a cost below 10 US dollars. It effectively pinpoints causal mutations in mutants defective in cilia or neurotransmitter secretion and in mutants synthetically sterile with a variant analogous to the oncogenic BRAF(V600E) mutation. Integrated with chemical mutagenesis, our approach can generate and identify missense variants economically and efficiently, facilitating experimental investigations of missense variants in diverse species.

genomics↗

bioRxiv2023

Utilizing the Scale-Invariant Feature Transform Algorithm to Align Distance Matrices Facilitates Systematic Protein Structure Comparison

Protein structure comparison is pivotal for deriving homological relationships, elucidating protein functions, and understanding evolutionary developments. The burgeoning field of in-silico protein structure prediction now yields billions of models with near-experimental accuracy, necessitating sophisticated tools for discerning structural similarities among proteins, particularly when sequence similarity is limited. Here, we have developed the ADAMS pipeline, which synergizes the distance matrix alignment method with the scale-invariant feature transform (SIFT) algorithm, streamlining protein structure comparison on a proteomic scale. Utilizing a computer vision-centric strategy for contrasting disparate distance matrices, ADAMS adeptly alleviates challenges associated with proteins characterized by a high degree of structural flexibility. Our findings indicate that ADAMS achieves a level of performance and accuracy on par with Foldseek, while maintaining similar speed. Crucially, ADAMS overcomes certain limitations of Foldseek in handling structurally flexible proteins, establishing it as an efficacious tool for in-depth protein structure analysis with heightened accuracy.

bioinformatics↗