Biology subjects

Wu, H.-Y.

Publications and source records attributed to Wu, H.-Y..

17 recordsLinked to original sources

bioRxiv2026

N6-methyladenosine regulates Influenza A virus mRNA stability yet is rarely found on genomic RNA

Previous studies have found widespread N6-methyladenosine (m6A methylation) on all forms of Influenza A virus (IAV) RNA, with m6A found critical for viral replication, pathogenicity as well as viral RNA packaging. Here we applied the latest quantitative technologies to revisit the methylation landscape on the anti-sense genomic RNA of IAV. Unexpectedly, upon Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) analysis of IAV virion -extracted genomic RNA, we detected very little m6A regardless of production from human cells or chicken eggs. Concordantly, Nanopore direct RNA sequencing also detected an overall low occurrence and stoichiometry (generally <5%) of m6A across all viral genomic RNA segments, compared with abundant m6A sites on viral mRNAs at ~20-30% m6A. Cross validation with glyoxal- and nitrite-mediated deamination of unmethylated adenosines (GLORI) confirmed multiple m6A sites on viral mRNA yet very few m6A on the genomic RNA. This paucity of m6A on genomic RNA makes it unlikely that m6A contributes to viral RNA packaging. Knockdown or pharmacological inhibition of the m6A methyltransferase METTL3 as well as the reader protein YTHDF2 both reduced viral mRNA levels and infectious viral particle production, with YTHDF2 promoting viral mRNA stability. Thus, the presence of m6A on IAV transcripts is indeed proviral, yet it is the mRNAs instead of genomic RNAs that are methylated at functionally relevant levels. Lastly, we provide proof of concept that a METTL3 small molecule inhibitor can be antiviral, and propose that m6A-targeted antivirals would mainly impact the intracellular gene expression phase of IAV replication.

microbiology↗

bioRxiv2026

Development and validation of highly selective monoclonal antibodies for the detection of huntingtin neoepitopes

Huntington's disease is caused by a CAG repeat tract expansion in the huntingtin gene, resulting in production of pathogenic N-terminal huntingtin protein fragments associated with disease pathology. Despite their central role, detection of these fragments has relied on a limited antibody repertoire with reproducibility concerns. Here, we describe the generation and characterization of recombinant rabbit monoclonal antibodies targeting two reciprocal neoepitopes flanking the huntingtin exon 1/exon 2 junction corresponding to amino acids P90 and K91. The P90 antibodies (clones 1B12, 11G2) demonstrate fragment-length-selective recognition of the C-terminal HTTexon1 P90 neoepitope with no detectable binding to full length huntingtin. A side-by-side comparison of the widely used monoclonal antibody MW8 from two different sources revealed measurable lot-to-lot drift in its fragment selectivity, whereas the recombinant P90 antibodies, expressed from a defined, sequenced clone, maintained consistent specificity, addressing this source-dependent variability. Whereas P90-positive fragments can arise through alternative splicing of the HTT1a transcript, generation of the reciprocal K91 N-terminal HTTexon2 neoepitope would require site-specific proteolytic cleavage, a mechanism that has not yet been directly tested for lack of a suitable reagent. The K91 antibody (clone 7G10) binds the N-terminal K91 neoepitope with high affinity and specificity over full length huntingtin and provides, for the first time, a tool capable of directly interrogating whether such cleavage occurs. Neoepitope specificity of these antibodies was orthogonally confirmed by protease digestion (Lys-N and Arg-C) coupled with intact mass spectrometry. As an additional outcome of the immunization and selection strategy, we discovered human-mouse cross-reactive antibodies (clones 27F5, 31C10) targeting the proline-rich domain of huntingtin that will facilitate mouse-human translational studies. All antibodies are recombinant, ensuring long-term reproducibility, and are being made available, along with their sequences, to the research community.

neuroscience↗

bioRxiv2026

Geometric optics organizes organelle interactions and positioning

Microtubule asters are central to positioning organelles and organizing intracellular architectures. We show that the multi-stage choreography of microtubule asters which positions pronuclei during the first cell division in Caenorhabditis elegans, is governed by a cellular analog of geometric optics. Large-scale electron tomography reveals that astral microtubules reach cortical and pronuclear surfaces largely along line-of-sight trajectories, leaving complementary regions shadowed and inaccessible. Laser ablation identifies surface-anchored motors pulling on microtubules as the dominant drivers of motion. We develop a biophysical model incorporating dynamic terminator curves that separate microtubule-accessible and -inaccessible surfaces, and quantitatively recapitulate aster separation, pronuclear migration, centering, and rotation in control and genetically perturbed embryos. More broadly, robust positioning emerges from a feedback loop wherein intracellular geometry gates microtubule access, thus sculpting the distribution and magnitude of pulling forces, which in turn reshape that geometry.

cell biology↗

bioRxiv2025

Cell size reduction scales spindle elongation but not chromosome segregation in C. elegans

How embryos adapt their internal cellular machinery to reductions in cell size during development remains a fundamental question in cell biology1-11. Here, we use high-resolution lattice light-sheet fluorescence microscopy and automated image analysis to quantify lineage-resolved mitotic spindle and chromosome segregation dynamics from the 2-to 64-cell stages in Caenorhabditis elegans embryos. While spindle length scales with cell size across both wild-type and size-perturbed embryos, chromosome segregation dynamics remain largely invariant, suggesting that distinct mechanisms govern these mitotic processes. Combining femtosecond laser ablation12,13 with large-scale electron tomography14, we find that central spindle microtubules mediate chromosome segregation dynamics and remain uncoupled from cell size across all stages of early development. In contrast, spindle elongation is driven by cortically anchored motor proteins and astral microtubules, rendering it sensitive to cell size12,13,15-17. Incorporating these experimental results into an extended stoichiometric model for both the spindle and chromosomes, we find that allowing only cell size and microtubule catastrophe rates to vary reproduces elongation dynamics across development. The same model also accounts for centrosome separation and pronuclear positioning in the one-cell C. elegans embryo18, spindle-length scaling across nematode species spanning ~100 million years of divergence17, and spindle rotation in human cells19. Thus, a unified stoichiometric framework provides a predictive, mechanistic account of spindle and nuclear dynamics across scales and species.

cell biology↗

bioRxiv2025

Rediscovery of the Enigmatic Paroxygraphis in Xizang, China: Phylogenetic Evidence for its Reclassification Within Oxygraphis (Ranunculaceae)

The enigmatic monotypic genus Paroxygraphis (Ranunculaceae, trib. Ranunculeae), represented solely by P. sikkimensis, has long posed taxonomic uncertainties due to its rarity. Through field rediscovery in southern Xizang province, China, and comprehensive molecular phylogenetic analyses combining nrITS and four plastid regions, we resolve its phylogenetic position within the Oxygraphis clade, with an estimated divergence time of about 3.73 Ma (95% HPD: 1.98-5.77 Ma) from its sister clade. Morphological and ecological characteristics, including small herb with a rosette of basal simple leaves, longitudinally ribbed achenes, and its alpine adaptations, further support this relationship. Furthermore, the results showed that persistent sepals and unisexual flowers in trib. Ranunculeae reflects multiple independent evolutionary origins. We propose the new combination Oxygraphis sikkimensis and highlight its critically endangered status due to habitat vulnerability. Lectotypification of P. sikkimensis is also made. This study provides new data for the floristic research of China and resolves the long-standing taxonomic controversy of this problematic genus within trib. Ranunculeae.

evolutionary biology↗

bioRxiv2024

Cytosolic Carboxypeptidase 5 maintains mammalian ependymal multicilia to ensure proper homeostasis and functions of the brain

Ependymal multicilia position at one-side on the cell surface and beat synchronously across tissue to propel the flow of cerebrospinal fluid. Loss of ependymal cilia often causes hydrocephalus. However, molecules contributing to their maintenance remain yet fully revealed. Cytosolic carboxypeptidase (CCP) family are erasers of polyglutamylation, a conserved posttranslational modification of ciliary-axoneme microtubules. CCPs possess a unique domain (N-domain) N-terminal to their carboxypeptidase (CP) domain with unclear function. Here, we show that a novel mutant mouse of Agbl5, the gene encoding CCP5, with deletion of its N-terminus and partial CP domain (designated Agbl5M1/M1), developed lethal hydrocephalus due to degeneration of ependymal multicilia. Interestingly, multiciliogenesis was not impaired in Agbl5M1/M1 ependyma, but the initially formed multicilia beat in intercellularly diverse directions, indicative of aberrant tissue-level coordination. Moreover, actin networks are severely disrupted and basal body patches are improperly displaced in mutant cells, suggesting impaired cell level polarity. In contrast, Agbl5 mutants with disruption solely in the CP domain of CCP5 (Agbl5M2/M2) do not develop hydrocephalus despite increased glutamylation levels in ependymal cilia as similarly seen in Agbl5M1/M1. This study revealed an unappreciated role of CCP5, particularly its N-domain, in ependymal multicilia stability associated with their polarization and coordination.

cell biology↗

bioRxiv2023

Clustering of cortical dynein regulates the mechanics of spindle orientation in human mitotic cells

The forces which orient the spindle in human cells remain poorly understood due to a lack of direct mechanical measurements in mammalian systems. We use magnetic tweezers to measure the force on human mitotic spindles. Combining the spindles measured resistance to rotation, the speed it rotates after laser ablating astral microtubules, and estimates of the number of ablated microtubules reveals that each microtubule contacting the cell cortex is subject to [~]1 pN of pulling force, suggesting that each is pulled on by an individual dynein motor. We find that the concentration of dynein at the cell cortex and extent of dynein clustering are key determinants of the spindles resistance to rotation, with little contribution from cytoplasmic viscosity, which we explain using a biophysically based mathematical model. This work reveals how pulling forces on astral microtubules determine the mechanics of spindle orientation and demonstrates the central role of cortical dynein clustering. HighlightsO_LICytoplasmic viscosity does not determine the spindles resistance to rotation C_LIO_LIEach astral microtubule that contacts the cell cortex is pulled on by a single dynein motor C_LIO_LIPulling forces on astral microtubules determine the mechanics of spindle orientation C_LIO_LIThe mechanics of spindle orientation is regulated by clustering of dynein motors at the cell cortex C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=178 HEIGHT=200 SRC="FIGDIR/small/557210v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@56e8daorg.highwire.dtl.DTLVardef@1d16449org.highwire.dtl.DTLVardef@f3006org.highwire.dtl.DTLVardef@119f6aa_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Isolation and whole genome characterization of antagonistic Paenibacillus polymyxa 188 and its biocontrol potential against several fungi

Discovery of new antifungal compounds and biocontrol agents is important due to the emergence of drug-resistant fungi and new fungal pathogens. The aim of this study was to isolate marine bacteria that are able to produce metabolites with antifungal properties. In this study, we isolated a P. polymyxa, named as 188, from a marine sediment sample and evaluated its antifungal ability. The results indicated that the bacterium showed excellent antifungal activity against many pathogenic fungi of plants and humans. The antifungal compounds produced by P. polymyxa 188 were extracted and analyzed using MALDI-TOF/MS. The complete genome sequence and biosynthetic gene clusters were characterized, and further to compare the genomes of P. polymyxa 188 with other strains. Various CAZymes were identified in P. polymyxa 188. Five antibiotic gene clusters including paenilan, paenibacillin, fusaricidin, polymyxin, and tridecaptin can be found in P. polymyxa strains, but gene clusters of paenilan, paenibacillin, and polymyxin were absent in P. polymyxa 188. Our findings provided detail genetic information about P. polymyxa 188 and suggested that P. polymyxa 188 is the potential agent of biocontrol and disease management.

bioengineering↗

bioRxiv2021

Laser Ablation and Fluid Flows Show a Single Force Mechanism Governs Spindle Positioning

Few techniques are available for elucidating the nature of forces that drive subcellular behaviors. Here we develop two complementary ones: 1) femtosecond stereotactic laser ablation (FESLA), which rapidly creates complex cuts of subcellular structures, thereby allowing precise dissection of when, where, and in what direction forces are generated; and 2) assessment of subcellular fluid flows, by comparing direct flow measurements, using microinjected fluorescent nanodiamonds, to large-scale fluid-structure simulations of different models of force transduction. We apply these to study centrosomes in Caenorhabditis elegans early embryos, and use the data to construct a biophysically-based model of centrosome dynamics. Taken together, we demonstrate that cortical pulling forces provide a general explanation for many behaviors mediated by centrosomes, including pronuclear migration/centration and rotation, metaphase spindle positioning, asymmetric spindle elongation and spindle oscillations. In sum, this work establishes new methodologies for disentangling the forces responsible for cell biological phenomena.

biophysics↗

bioRxiv2021

Self-organization of kinetochore-fibers in human mitotic spindles

During eukaryotic cell division, chromosomes are linked to microtubules (MTs) in the spindle by a macromolecular complex called the kinetochore. The bound kinetochore microtubules (KMTs) are crucial to ensuring accurate chromosome segregation. Recent electron tomography reconstructions (Kiewisz et al. 2021) captured the positions and configurations of every MT in human mitotic spindles, revealing that many KMTs in these spindles do not reach the pole. Here, we investigate the processes that give rise to this distribution of KMTs using a combination of analysis of the electron tomography reconstructions, photoconversion experiments, quantitative polarized light microscopy, and biophysical modeling. Our results indicate that in metaphase, KMTs grow away from the kinetochores along well-defined trajectories, continually decreasing in speed as they approach the poles. The locations of KMT minus ends, and the turnover and movements of tubulin in KMTs, are consistent with models in which KMTs predominately nucleate de novo at kinetochores and are inconsistent with substantial numbers of non-KMTs being recruited to the kinetochore in metaphase. Taken together, this work leads to a mathematical model of the self-organization of kinetochore-fibers in human mitotic spindles.

biophysics↗

bioRxiv2021

Community Evaluation of Glycoproteomics Informatics Solutions Reveals High-Performance Search Strategies of Glycopeptide Data

Glycoproteome profiling (glycoproteomics) is a powerful yet analytically challenging research tool. The complex tandem mass spectra generated from glycopeptide mixtures require sophisticated analysis pipelines for structural determination. Diverse software aiding the process have appeared, but their relative performance remains untested. Conducted through the HUPO Human Proteome Project - Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates the performance of informatics solutions for system-wide glycopeptide analysis. Mass spectrometry-based glycoproteomics datasets from human serum were shared with all teams. The relative team performance for N- and O-glycopeptide data analysis was comprehensively established and validated through orthogonal performance tests. Excitingly, several high-performance glycoproteomics informatics solutions were identified. While the study illustrated that significant informatics challenges remain, as indicated by a high discordance between annotated glycopeptides, lists of high-confidence (consensus) glycopeptides were compiled from the standardised team reports. Deep analysis of the performance data revealed key performance-associated search variables and led to recommendations for improved "high coverage" and "high accuracy" glycoproteomics search strategies. This study concludes that diverse software for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies, and specifies key variables that may guide future software developments and assist informatics decision-making in glycoproteomics.

biochemistry↗

bioRxiv2020

Dynamin-2 regulates synaptic podosome maturation to facilitate neuromuscular junction development

Neuromuscular junctions (NMJs) govern rapid and efficient neuronal communication with muscle cells, which relies on the proper architecture of specialized postsynaptic compartments. However, the intrinsic mechanism in muscle cells contributing to elaborate NMJ development has been unclear. In this study, we reveal that the GTPase dynamin-2 (Dyn2), best-known for catalyzing synaptic vesicle endocytosis at the presynaptic membrane, is also involved in postsynaptic morphogenesis. We demonstrate that Dyn2 is enriched in the postsynaptic membrane of muscle cells and is involved in the maturation of neurotransmitter receptor clusters via its actin bundling ability. Dyn2 functions as a molecular girdle to regulate synaptic podosome turnover and promote morphogenesis of the postsynaptic apparatus. In Drosophila NMJs, Dyn2 is required to organize the postsynaptic actin cytoskeleton and to mediate its electrophysiological activities. Mechanistically, the actin binding, self-assembly, GTP hydrolysis ability, and Y597 phosphorylation of Dyn2 all regulate its actin bundling activity. Together, our study uncovers a role for Dyn2 in cytoskeleton remodeling and organization at the postsynaptic membrane of NMJs.

molecular biology↗

bioRxiv2019

Selective protein O-GlcNAcylation in cells by a proximity-directed O-GlcNAc transferase

O-Linked N-acetylglucosamine (O-GlcNAc) is a monosaccharide that plays an essential role in cellular signaling throughout the nucleocytoplasmic proteome of eukaryotic cells. Yet, the study of post-translational modifications like O-GlcNAc has been limited by the lack of strategies to induce O-GlcNAcylation on a target protein in cells. Here, we report a generalizable genetic strategy to induce O-GlcNAc to specific target proteins in cells using a nanobody as a proximity-directing agent fused to O-GlcNAc transferase (OGT). Fusion of a nanobody that recognizes GFP (nGFP) or a nanobody that recognizes the four-amino acid sequence EPEA (nEPEA) to OGT(4), a truncated form of OGT, yielded a nanobody-OGT(4) construct that selectively delivered O-GlcNAc to the target protein (e.g., JunB, cJun, Nup62) and reduced alteration of global O-GlcNAc levels in the cell. Quantitative chemical proteomics confirmed the selective increase in O-GlcNAc to the target protein by nanobody-OGT(4). Glycoproteomics revealed that nanobody-OGT(4) or full-length OGT produced a similar glycosite profile on the target protein. Finally, we demonstrate the ability to selectively target endogenous -synuclein for glycosylation in HEK293T cells. Thus, the use of nanobodies to redirect OGT substrate selection is a versatile strategy to induce glycosylation of desired target proteins in cells that will facilitate discovery of O-GlcNAc functions and provide a mechanism to engineer O-GlcNAc signaling. The proximity-directed OGT approach for protein-selective O-GlcNAcylation is readily translated to additional protein targets and nanobodies that may constitute a generalizable strategy to control post-translational modifications in cells.\n\nSignificance StatementNature uses post-translational modifications (PTMs) like glycosylation as a mechanism to alter protein signaling and function. However, the study of these modified proteins in cells is confined to loss-of-function strategies, such as mutagenic elimination of the modification site. Here, we report a generalizable strategy for induction of O-GlcNAc to a protein target in cells. The O-GlcNAc modification is installed by O-GlcNAc transferase (OGT) to thousands of nucleocytoplasmic proteins. Fusion of a nanobody to OGT enables the selective increase of O-GlcNAc levels on a series of target proteins. The described approach will facilitate direct studies of O-GlcNAc and its regulatory enzymes and drive new approaches to engineer protein signaling via a strategy that may be conceptually translatable to additional PTMs.

biochemistry↗

bioRxiv2019

Discovery of a celecoxib binding site on PTGES with a cleavable chelation-assisted biotin probe

The coxibs are a subset of non-steroidal anti-inflammatory drugs (NSAIDs) that primarily target cyclooxygenase-2 (COX-2) to inhibit prostaglandin signaling and reduce inflammation. However, mechanisms to inhibit other members of the prostaglandin signaling pathway may improve selectivity and reduce off-target toxicity. Here, we report a novel binding site for celecoxib on prostaglandin E synthase (PTGES), an enzyme downstream of COX-2 in the prostaglandin signaling pathway, using a cleavable chelation-assisted biotin probe 6. Evaluation of the multi-functional probe 6 revealed significantly improved tagging efficiencies attributable to the embedded picolyl functional group. Application of the probe 6 within the small molecule interactome mapping by photo-affinity labeling (SIM-PAL) platform using photo-celecoxib as a reporter for celecoxib identified PTGES and other membrane proteins in the top eight enriched proteins from A549 cells. Carbonic anhydrase 12, a known protein target of celecoxib, was also enriched. Four binding sites to photo-celecoxib were additionally mapped by the probe 6, including a binding site with PTGES. The binding interaction with PTGES was validated by competitive displacement with celecoxib and known PTGES inhibitor licofelone. The binding site of photo-celecoxib on PTGES enabled the development of a structural model of the interaction and will inform the design of new selective inhibitors of the prostaglandin signaling pathway.

pharmacology and toxicology↗

bioRxiv2019

Retinoic acid promotes in vitro development of haploid germ cells from pre-pubertal porcine spermatogenic cells

Spermatogonial stem cells (SSCs) self-renew and contribute genetic information to the next generation. Inducing directional differentiation of porcine SSCs may be an important strategy in exploring the mechanisms of spermatogenesis and developing better treatment methods for male sterility. Here, we established an in vitro culture model for porcine small seminiferous tubule segments, to induce SSCs to differentiate into single-tail haploid spermatozoa. The culture model subsequently enabled spermatozoa to express the sperm-specific protein acrosin, and oocytes to develop to blastocyst stage after round spermatid injection. The addition of retinoic acid (RA) to the differentiation media promoted the efficiency of haploid differentiation. RT-PCR analysis indicated that RA stimulated the expression of Stra8 but reduced the expression of NANOS2 in spermatogonia. Genes involved in post-meiotic development, Prm1 and Tnp1, were up-regulated in the presence of RA. The addition of RAR inhibitor, BMS439, showed that RA enhanced the expression of cAMP responsive-element binding protein through RAR, and promoted the formation of round spermatids.

developmental biology↗

bioRxiv2019

Central spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

Spindle microtubules, whose dynamics vary over time and at different locations, cooperatively drive chromosome segregation. Measurements of microtubule dynamics and spindle ultrastructure can provide insight into the behaviors of microtubules, helping elucidate the mechanism of chromosome segregation. Much work has focused on the dynamics and organization of kinetochore microtubules, i.e. on the region between chromosomes and poles. In comparison, microtubules in the central spindle region, between segregating chromosomes, have been less thoroughly characterized. Here, we report measurements of the movement of central spindle microtubules during chromosome segregation in human mitotic spindles, and Caenorhabditis elegans mitotic and female meiotic spindles. We found that these central spindle microtubules slide apart at the same speed as chromosomes, even as chromosomes move towards spindle poles. In these systems, damaging central spindle microtubules by laser ablation caused an immediate and complete cessation of chromosome motion, suggesting a strong coupling between central spindle microtubules and chromosomes. Electron tomographic reconstruction revealed that the analyzed anaphase spindles all contain microtubules with both ends between segregating chromosomes. Our results provide new dynamical, functional, and ultrastructural characterizations of central spindle microtubules during chromosome segregation in diverse spindles, and suggest that central spindle microtubules and chromosomes are strongly coupled in anaphase.

cell biology↗

bioRxiv2017

Dynein Pulling Forces On Ruptured Nuclei Counteract Lamin-Mediated Nuclear Envelope Repair Mechanisms In Vivo

Recent work done exclusively in tissue culture cells revealed that the nuclear envelope (NE) undergoes ruptures leading to transient mixing of nuclear and cytoplasmic components. The duration of transient NE ruptures depends on lamins, however the underlying mechanisms and the relevance to in vivo events is not known. Here, we use C. elegans embryos to show that dynein forces that position nuclei increase the severity of lamin-induced NE ruptures in vivo. In the absence of dynein forces, lamin prevents nuclear-cytoplasmic mixing caused by NE ruptures. By monitoring the dynamics of NE rupture events, we demonstrate that lamin is required for a distinct step in NE recovery that restricts nucleocytoplasmic mixing prior to the full restoration of NE rupture sites. We show that laser-induced puncture of the NE recapitulates phenotypes associated with NE recovery in wild type cells. Surprisingly, we find that embryonic lethality does not correlate with the incidence of NE rupture events suggesting that embryos survive transient losses of NE compartmentalization during early embryogenesis. In addition to presenting the first mechanistic analysis of transient NE ruptures in vivo, this work demonstrates that lamin controls the duration of NE ruptures by opposing dynein forces on ruptured nuclei to allow reestablishment of the NE permeability barrier and subsequent restoration of NE rupture sites.

developmental biology↗