Biology subjects

Wood, A. J.

Publications and source records attributed to Wood, A. J..

18 recordsLinked to original sources

bioRxiv2026

Temporal phosphoproteomic programmes set a redox-gated rheostat on neutrophil phagocytosis

Neutrophils are essential frontline immune cells in the defence against bacterial pathogens such as Staphylococcus aureus, but their killing machinery also causes host tissue damage. How neutrophils regulate these processes remains incompletely understood. Here, we profile the proteome and phosphoproteome of human neutrophils across a two-hour exposure to S. aureus, alongside five simultaneous functional readouts, before investigating how an initial encounter modulates the response to a second. The phosphoproteomic response resolves into qualitatively distinct early and late windows: the early landscape drives particle uptake, while by the late window this machinery is been deactivated, with a broadly supressed kinome, and cells geared towards inflammatory output. Correlating protein phosphorylation and functional kinetics - an approach we term KINETIC - implicates putative mediators of key neutrophil antimicrobial functions. A genome-wide CRISPR knockout screen in neutrophil-like PLB-985 cells, sorted on phagocytic load, recovers canonical engulfment machinery among the strongest facilitators of phagocytosis, additionally identifying the lysosomal Ragulator-mTORC1 nutrient sensing pathway and HOPS complex corresponding to the KINETIC-identified pathways. Conversely, the NRF2 repressor KEAP1 is revealed as the strongest phagocytic brake, positioning oxidative burden as a regulator of phagocytic appetite. Neutrophils in the late window exhibit impaired phagocytosis and phagosomal acidification on S.aureus re-challenge. Underlying these functional deficits, cells in the late window are refractory across multiomic layers: a second S. aureus meal alters no phosphoprotein, protein, or transcript, whereas the chemoattractants PAF and fMLP evoke a largely consistent phosphoproteomic programme, delineating the phenotype as receptor-specific rather than a global loss of signalling capacity. Consistent with de-repressing the antioxidant response via genetic loss of KEAP1 increasing phagocytosis, reducing ROS production via inhibition of the NADPH oxidase results in a partial rescue of phagocytosis of the re-exposed S. aureus. Together, these findings describe a ROS-gated phagocytic rheostat, enabling neutrophils to meter their phagocytic appetite according to their exposure history, likely limiting uptake of pathogens they lack the capacity to effectively digest. Inappropriate activation of this rheostat under conditions of physiological stress or severe illness may underpin the defects in phagocytosis identified in such patients.

immunology↗

bioRxiv2026

Universal donor plasmids for biallelic degron-tagging in mammalian cells and organoids

Degron tags enable rapid, tuneable and reversible control of protein expression, and have become a critical tool for target validation and elucidating biological mechanisms. Tag engineering uses genome editing to generate biallelic gene fusions, though this process remains laborious and requires new donor vectors to be generated for each locus. Here, we have generated a series of universal donor vectors to enable biallelic integration of different degron tags (AID, dTAG, BromoTag, SMASh) using different selection strategies (FACS, drug selection). Using this system, tags can be fused in-frame to endogenous protein C-termini via homology-independent repair following Cas9-induced double strand breaks, as shown here using human lymphoma cells, mouse embryonic stem cells and colonic tumour organoids. This workflow simplifies the genetic engineering of degron tags and is applicable in contexts where homology-dependent repair is challenging.

synthetic biology↗

bioRxiv2026

A mammalian-specific domain of MSH5 drives the transition from crossover licensing to designation during meiotic prophase I

SUMMARYMeiotic recombination initiates with DNA double-strand breaks (DSBs) repaired as either crossovers (COs) or non-crossovers. Across eukaryotes, MSH4/MSH5 (MutS{gamma}) licenses DSB repair intermediates, directing repair into the class I CO pathway via recruitment of MLH1/MLH3 (MutL{gamma}). In mammals, excess MutS{gamma} sites relative to final MutL{gamma} foci suggest additional MutS{gamma} functions, including directing repair through the minor class II CO pathway. We investigated the role of a mammalian-specific 38-amino acid C-terminal domain of MSH5 using mice lacking this domain (Msh5{Delta}C/{Delta}C). Spermatocytes and oocytes load MSH4 normally to achieve CO licensing in zygonema, but these numbers decline precipitously in pachynema, leading to dramatically reduced MutL{gamma} foci and associated pro-CO factors HEI10 and CNTD1. Despite this, licensing factors RNF212B and MutS{gamma}-associated kinase CDK4 remain persistently upregulated in pachynema. Strikingly, the switch from licensing-associated CDK4 to CO-site-associated CDK2 fails to occur in Msh5{Delta}C/{Delta}C mice, even at residual class I CO events. The result is rapid germ cell death prior to prophase I completion in both sexes. Thus, the loss of the MSH5 C-terminus functionally uncouples the regulatory proteins that define the stepwise patterning of class I COs. Our findings reveal novel early roles for the C-terminus of mammalian MSH5 in converting licensed DSB repair intermediates to designated class I COs.

genetics↗

bioRxiv2026

Structure and inhibition of the sperm TMEM95-FIMP complex in mammalian fertilization

TMEM95 is a sperm acrosomal membrane protein essential for mammalian fertilization. How TMEM95 facilitates sperm-egg interaction has largely remained unknown. Analogous sperm fertilization proteins function as complexes, leading us to hypothesize that TMEM95 may have a binding partner on sperm. Here, we surveyed interactions between TMEM95 and individual proteins in a curated library of testis-expressed proteins using AlphaFold3. We identify FIMP, a fertilization-essential acrosomal membrane protein, as a high-confidence interaction partner of TMEM95. These two proteins form a high-affinity complex through their ectodomains. Using single-particle cryo-EM, we determine the structure of the human TMEM95-FIMP ectodomain complex at high resolution. An aromatic motif of FIMP binds to a conserved surface of TMEM95, and amino acid substitutions within this motif ablate the TMEM95-binding activity of FIMP. We isolate an anti-TMEM95 antibody, termed 3A02, that binds to human and murine TMEM95 and disrupts the interaction between TMEM95 and FIMP. By determining the cryo-EM structure of human TMEM95 bound to the 3A02 fragment antigen-binding region, we find that 3A02 recognizes the FIMP-binding site on TMEM95. 3A02 inhibits fusion of murine sperm with eggs, independent of antibody size, suggesting that the TMEM95-FIMP interface is critical for sperm-egg interaction. Together, these results establish the human sperm TMEM95-FIMP complex and suggest that a FIMP-mediated interaction of TMEM95 facilitates membrane fusion during mammalian fertilization.

biochemistry↗

bioRxiv2026

Divergent effects of target protein stabilisation versus overproduction on PROTAC activity

Protein degrader drugs such as PROTACs are being advanced as therapeutics targeted against oncogenic proteins. During tumorigenesis, oncogenic proteins can become constitutively activated via mechanisms including gene amplification, which increases protein production, and point mutations, which can extend protein half-life. Few experimental studies have addressed how disease-associated changes in target protein homeostasis influence PROTAC activity. We developed orthogonal methods to increase production or enhance stability of {beta}-catenin, an important oncoprotein and target for degrader therapeutics, and used the dTAG system to evaluate the consequences for PROTAC activity. Stabilising oncogenic missense mutations increased protein expression up to 5-fold but do not alter the PROTAC-imposed minimal steady-state level. In contrast, transcriptional upregulation increases both pre- and post-treatment target levels, revealing a synthesis-dependent ceiling on achievable depletion. Our results highlight distinct constraints on PROTAC activity arising from different mechanisms of oncogene activation, with potential implications for preclinical modelling, drug resistance and personalised medicine.

cancer biology↗

bioRxiv2025

Chromosome length is not the sole determinant of sexually dimorphic crossover rates during mammalian meiosis: Insights from genetically diverse mouse strains

Meiotic recombination generates crossovers (COs), reciprocal exchanges between homologous chromosomes critical for accurate chromosome segregation. Inappropriate CO frequency and distribution drive aneuploidy in human oocytes, with error rates up to 10-fold higher than in sperm despite females exhibiting higher CO frequencies. COs form in the context of the proteinaceous synaptonemal complex (SC) that tethers homologs during prophase I. SC length strongly correlates with CO number, and sexual dimorphism in recombination has long been attributed to longer SCs in females. However, this model is challenged by wild-derived PWD mice in which males consistently generate more COs despite having shorter SCs. Here, we exploit natural genetic variation among inbred mouse strains to dissect the structural and regulatory basis of sexually dimorphic CO regulation. Using cytological markers of SC assembly (SYCP3), recombination progression (RAD51, MSH4), class I CO designation (HEI10, MLH1/MLH3), and chiasmata, we show that SC length is not the sole predictor of CO number. PWD males exhibit stronger CO interference and higher CO number than females, despite reduced SC length. Notably, females show reduced efficiency in designating recombination intermediate to become COs, whereas PWD males display exceptional proficiency. Unexpectedly, although class II COs are rare, they play a disproportionate role in ensuring that every chromosome pair receives at least one CO, thereby safeguarding against aneuploidy. Together, these findings challenge the prevailing view that SC length is the primary determinant of sexually dimorphic CO rates and instead highlight sex-specific regulation of CO designation and pathway usage as key drivers of recombination outcomes.

genetics↗

bioRxiv2025

Tissue-specific consequences of tag fusions on protein expression in transgenic mice

Genetic fusion of protein tags is widely used to study protein functions in vivo. It is well known that tag fusion can cause unwanted changes in protein stability, but whether this is an inherent property of the tagged protein, or can be influenced by the cell and tissue environment, is unclear. Using a series of genome edited mouse models, we show that tag-dependent changes in protein expression can vary across different primary cell and tissue contexts. In one case (Ncaph2), a C-terminal auxin-inducible degron fusion strongly increased protein stability in some tissues but decreased it in others. Destabilisation resulted from tissue-specific leakage of the auxin-inducible degron, which depended on TIR1 expression, and occurred selectively in the small intestine where basal concentrations of auxin / indole-3-acetic acid can reach levels that are sufficient to trigger protein degradation in cultured cells. Stabilisation occurred in post-mitotic cells via an endogenous degradation signal situated at the Ncaph2 C-terminus, which normally undergoes activation upon cell cycle exit, but is inactivated by C-terminal tag fusion. Our results highlight the underappreciated importance of cell and tissue environment in determining the consequences of tag fusions on protein expression, which may be particularly important in animal models that contain diverse cell types.

molecular biology↗

bioRxiv2024

The potential impacts of vector host species fidelity on zoonotic arbovirus transmission

The interaction between vector host preference and host availability on vector blood feeding behaviour has important implications for the transmission of vector-borne pathogens. In particular in multi-host disease systems the fidelity of the vector biting behaviour has the potential to have important implications to disease outcomes, particularly when there are amplifying and dead-end hosts. Using a mathematical model we showed that vector fidelity to the host species they take a first blood meal from leads to non-homogeneous mixing between hosts and vectors. Taking Japanese encephalitis virus (JEV) as a case study, we investigated how vector preference for amplifying vs dead-end hosts and fidelity can influence JEV transmission. We show that in regions where pigs (amplifying hosts) are scarce compared to cattle (dead-end hosts preferred by common JEV vectors), JEV can still be maintained through vector fidelity. Our findings demonstrate the importance of considering fidelity as a potential driver of transmission, particularly in scenarios such as Bangladesh and India where the composition of the host community might initially suggest that transmission is not possible.

ecology↗

bioRxiv2024

The utility of whole-genome sequencing to identify likely transmission pairs for pathogens with slow and variable evolution

Pathogen whole-genome sequencing (WGS) has been used to track the transmission of infectious diseases in extraordinary detail, especially for pathogens that undergo fast and steady evolution, as is the case with many RNA viruses. However, for other pathogens evolution is less predictable, making interpretation of these data to inform our understanding of their epidemiology more challenging and the value of densely collected pathogen genome data uncertain. Here, we assess the utility of WGS for one such pathogen, in the "who-infected-whom" identification problem. We study samples from hosts (130 cattle, 111 badgers) with confirmed infection of M. bovis (causing bovine Tuberculosis), which has an estimated clock rate as slow as [~]0.1-1 variations per year. For each potential pathway between hosts, we calculate the relative likelihood that such a transmission event occurred. This is informed by an epidemiological model of transmission, and host life history data. By including WGS data, we shrink the number of plausible pathways significantly, relative to those deemed likely on the basis of life history data alone. Despite our uncertainty relating to the evolution of M. bovis, the WGS data are therefore a valuable adjunct to epidemiological investigations, especially for wildlife species whose life history data are sparse.

ecology↗

bioRxiv2023

Mutational scanning reveals oncogenic CTNNB1 mutations have diverse effects on signalling and clinical traits

CTNNB1, the gene encoding {beta}-catenin, is a frequent target for oncogenic mutations activating the canonical Wnt signalling pathway, typically via missense mutations within a degron hotspot motif in exon 3. Here, we combine saturation genome editing with a fluorescent reporter assay to quantify signalling phenotypes for all 342 missense mutations in the mutation hotspot, including 74 recurrent mutations reported in over 6000 tumours. Our data define the genetic requirements for {beta}-catenin degron function and reveal diverse levels of signal activation among known driver mutations. Tumorigenesis in different human tissues involves selection for CTNNB1 mutations spanning distinct ranges of effect size. In hepatocellular carcinoma, mutations that activate {beta}-catenin relatively weakly are associated with worse prognosis compared to stronger activating mutations, despite greater immune cell infiltration in the tumour microenvironment. Our work therefore provides a resource to understand mutational diversity within a pan-cancer mutation hotspot, with potential implications for targeted therapy.

cancer biology↗

bioRxiv2022

The effects of antibiotic combination treatments on Pseudomonas aeruginosa tolerance evolution and coexistence with Stenotrophomonas maltophilia

Pseudomonas aeruginosa bacterium is a common pathogen of Cystic Fibrosis (CF) patients due to its ability to evolve resistance to antibiotics during treatments. While P. aeruginosa resistance evolution is well characterised in monocultures, it is less well understood in polymicrobial CF infections. Here, we investigated how exposure to ciprofloxacin, colistin, or tobramycin antibiotics, administered at sub-MIC doses alone and in combination, shaped the tolerance evolution of P. aeruginosa (PAO1 lab and clinical CF LESB58 strains) in the absence and presence of a commonly co-occurring species, Stenotrophomonas maltophilia. Increases in antibiotic tolerances were primarily driven by the presence of that antibiotic in the treatment. We observed a reciprocal cross-tolerance between ciprofloxacin and tobramycin, and when combined these antibiotics selected increased MICs for all antibiotics. Though the presence of S. maltophilia did not affect the tolerance or the MIC evolution, it drove P. aeruginosa into extinction more frequently in the presence of tobramycin due to its relatively greater innate tobramycin tolerance. In contrast, P. aeruginosa dominated and drove S. maltophilia extinct in most other treatments. Together, our findings suggest that besides driving high-level antibiotic tolerance evolution, sub-MIC antibiotic exposure can alter competitive bacterial interactions, leading to target pathogen extinctions in multi-species communities.

microbiology↗

bioRxiv2022

Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons

Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here, we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. Using mouse genetics, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase subunit Tir1. Rapid degradation of condensin I and condensin II - two essential regulators of mitotic chromosome structure - revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues. HighlightsO_LIAuxin-inducible degradation of endogenously tagged proteins in living mice and a range of primary cells. C_LIO_LIMost but not all cell types are competent for degradation C_LIO_LIDosage of the tagged protein, E3 ligase substrate receptor and ligand can all determine degradation kinetics C_LIO_LIRapid degradation of condensin subunits in lymphocytes reveals stage-specific requirements during cell division C_LI

genetics↗

bioRxiv2020

A commercial antibody to the human condensin II subunit NCAPH2 cross-reacts with a SWI/SNF complex component

Condensin complexes compact and disentangle chromosomes in preparation for cell division. Commercially available antibodies raised against condensin subunits have been widely used to characterise their cellular interactome. Here we have assessed the specificity of a polyclonal antibody (Bethyl A302-276A) that is commonly used as a probe for NCAPH2, the kleisin subunit of condensin II, in mammalian cells. We find that, in addition to its intended target, this antibody cross-reacts with one or more components of the SWI/SNF family of chromatin remodelling complexes in an NCAPH2-independent manner. This cross-reactivity with an abundant chromatin-associated factor is likely to affect the interpretation of protein and chromatin immunoprecipitation experiments that make use of this antibody probe.

cell biology↗

bioRxiv2020

Developmental regulation of mitotic chromosome formation revealed by condensin reporter mice

Withdrawal Note: The authors have withdrawn their manuscript due to the discovery of artifacts that call into question key findings from the study. Both artifacts originate from cell-type differences that arose during sample processing, creating the impression that non-SMC subunits of condensin proteins were present at substantially higher levels in thymic T cells compared to bone marrow B cells or erythroblast cells. Based on data collected since the preprint was submitted, we no longer believe this to be true. The experimental artifacts do not affect the data contributed by the Papadopoulos laboratory (Figure 4). The authors wish to make it clear that all data and experimental protocols in the original manuscript were accurately reported, and that the experimental artifacts which affect data interpretation were discovered during internal review, independently of peer review, and are being reported at the authors own instigation. They do not, to our knowledge, affect the interpretation of data from any other published work. Due to the issues outlined above, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

cell biology↗

bioRxiv2020

Macrophages provide a transient muscle stem cell niche via NAMPT secretion

Skeletal muscle is paradigmatic of a regenerative tissue that repairs itself via the activation of a resident stem cell1. Termed the satellite cell, these normally quiescent cells are induced to proliferate by ill-defined wound-derived signals2. Identifying the source and nature of these pro-regenerative cues has been hampered by an inability to visualise the complex cellular interactions that occur within the wound environment. We therefore developed a zebrafish muscle injury model to systematically capture satellite cell interactions within the injury site, in real time, throughout the repair process. This analysis identified that a specific subset of macrophages dwells within the injury, establishing a transient but obligate stem cell niche required for stem cell proliferation. Single cell profiling identified specific signals secreted from dwelling macrophages that include the cytokine, Nicotinamide phosphoribosyltransferase (NAMPT/Visfatin/PBEF). Here we show that NAMPT secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (CCR5) expressed on muscle stem cells. This analysis reveals that along with their well-described ability to modulate the pro-inflammatory and anti-inflammatory phases of wound repair, specific macrophage populations also provide a transient stem cell-activating niche, directly supplying pro-proliferative cues that govern the timing and rate of muscle stem cell-mediated repair processes.

cell biology↗

bioRxiv2020

Complement C5a impairs phagosomal maturation in the neutrophil through phosphoproteomic remodelling.

Critical illness is accompanied by the release of large amounts of the anaphylotoxin, C5a. C5a suppresses antimicrobial functions of neutrophils which is associated with adverse outcomes. The signalling pathways that mediate C5a-induced neutrophil dysfunction are incompletely understood. Healthy donor neutrophils exposed to purified C5a demonstrated a prolonged defect (7 hours) in phagocytosis of Staphylococcus aureus. Phosphoproteomic profiling of 2712 phosphoproteins identified persistent C5a signalling and selective impairment of phagosomal protein phosphorylation on exposure to S. aureus. Notable proteins included early endosomal marker ZFYVE16 and V-ATPase proton channel component ATPV1G1. A novel assay of phagosomal acidification demonstrated C5a-induced impairment of phagosomal acidification which was recapitulated in neutrophils from critically ill patients. Examination of the C5a-impaired protein phosphorylation indicated a role for the phosphatidylinositol 3-kinase VPS34 in phagosomal maturation. Inhibition of VPS34 impaired neutrophil phagosomal acidification and killing of S. aureus. This study provides a phosphoproteomic assessment of human neutrophil signalling in response to S. aureus and its disruption by C5a, identifying a defect in phagosomal maturation and new mechanisms of immune failure in critical illness.

immunology↗

bioRxiv2019

Simulating the evolutionary trajectories of metabolic pathways for insect symbionts in the Sodalis genus

1Insect-bacterial symbioses are ubiquitous, but there is still much to uncover about how these relationships establish, persist and evolve. The tsetse endosymbiont Sodalis glossinidius displays intriguing metabolic adaptations to its microenvironment, but the process by which this relationship evolved remains to be elucidated. The recent chance discovery of the free-living secies of the Sodalis genus, S. praecaptivus, provides a serendipitous starting point from which to investigate the evolution of this symbiosis. Here, we present a flux balance model for S. praecaptivus. Metabolic modelling is used in combination with a multi-objective evolutionary algorithm to explore the trajectories that S. glossinidius may have undertaken after becoming internalised. The time-dependent loss of key genes is shown to influence the evolved populations, providing possible targets for future in vitro genetic manipulation. This method provides an unusually detailed perspective on possible evolutionary trajectories for S. glossinidius in this fundamental process of evolutionary and ecological change.

evolutionary biology↗

bioRxiv2018

Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence repair outcome

CRISPR-Cas9 genome editing occurs in the context of chromatin, which is heterogeneous in structure and function across the genome. Chromatin heterogeneity is thought to affect genome editing efficiency, but this has been challenging to quantify due to the presence of confounding variables. Here, we develop a method that exploits the allele-specific chromatin status of imprinted genes in order to address this problem. Because maternal and paternal alleles of imprinted genes have identical DNA sequence and are situated in the same nucleus, allele-specific differences in the frequency and spectrum of Cas9-induced mutations can be attributed unequivocally to epigenetic mechanisms. We found that heterochromatin can impede mutagenesis, but to a degree that depends on other key experimental parameters. Mutagenesis was impeded by up to 7-fold when Cas9 exposure was brief and when intracellular Cas9 expression was low. Surprisingly, the outcome of mutagenic DNA repair was independent of chromatin state, with similar efficiencies of homology directed repair and deletion spectra on maternal and paternal chromosomes. Combined, our data show that heterochromatin imposes a permeable barrier that influences the kinetics, but not the endpoint of CRISPR-Cas9 genome editing, and suggest that therapeutic applications involving low-level Cas9 exposure will be particularly affected by chromatin status.

genetics↗