Biology subjects

Bond, A.

Publications and source records attributed to Bond, A..

10 recordsLinked to original sources

bioRxiv2026

Inhibitory Fc Receptor sets a time limit on macrophage response to IgG

Antibodies engage both activating Fc Receptors and the inhibitory receptor Fc{gamma}RIIB. Why macrophages need a dedicated inhibitory receptor rather than simply tuning activating receptor signaling is unclear. Using DNA-based chimeric receptors and in silico modeling, we independently controlled activating and inhibitory Fc Receptors. We found that Fc{gamma}RIIB imposed a time limit on macrophage phagocytosis and ERK signaling. The time limit is due to activating Fc Receptors converting PI(4,5)P2 to PI(3,4,5)P3, which is subsequently converted to PI(3,4)P2 by Fc{gamma}RIIB. This leads to a pulse of active signaling, which is sufficient for phagocytosis of small bacteria-sized targets but not phagocytosis of large targets and TNF secretion. Unlike engaging Fc{gamma}RIIB, reducing activating Fc Receptor signaling decreased initiation of phagocytosis, the speed of PI(3,4,5)P3 generation, and the amplitude of ERK signaling. Our results demonstrate that Fc{gamma}RIIB controls the duration of IgG signaling, while the activating Fc Receptors control sensitivity.

immunology↗

bioRxiv2026

Make it so: Rapid and affordable plasmid sequencing on ONT platforms with PICARD-seq

Plasmid construction underpins molecular biology and synthetic biology, yet validation is often limited to the inserted fragment rather than the whole plasmid, and around a third of laboratory-made plasmids carry sequence errors that can affect function. Sanger sequencing scales poorly across whole plasmids, while short-read approaches cannot resolve the repeated DNA parts, such as promoters, that are common in synthetic constructs. We present PICARD-seq, a rapid nanopore-based protocol that uses off-the-shelf Tn5 rapid barcoding reagents and a MinION to sequence pools of whole plasmids in under a day, and we systematically benchmark the computational pipelines available for analysing the resulting data. Using a curated set of 25 plasmids of known sequence spanning 3.0-20.6 kbp, including various dilution series and repetitive multi-part constructs, we ran five independent replicates of each pipeline. The ONT EPI2ME Clone Validation workflow was fast (13-18 min) but stochastic, varying between replicates for both plasmids assembled and what sequence was returned; Canu outperformed the default Flye assembler, and reducing the minimum coverage parameter from 60x to 20x substantially improved assembly of large, repetitive, and dilute samples. The ensemble assembler Autocycler was slower (81-111 min) but gave the highest and most consistent rate of recovering the expected sequence. Complementary read mapping with minimap2 distinguished genuine sequence differences from assembly artefacts. Applying PICARD-seq to problematic plasmids revealed backbone concatemers, a misincorporated promoter part, and a mixed population of rearranged molecules in a repetitive construct. PICARD-seq makes routine whole-plasmid validation practical and affordable for individual laboratories.

bioinformatics↗

bioRxiv2026

IL-10/ACOD1 axis regulates catabolism of phagocytosed lipids in trained macrophages

Macrophages clear excess host and microbial debris to restore homeostasis in inflamed tissues, yet the regulation and molecular fate of phagocytosed lipids during innate immune training remains largely unexplored. Leveraging stable isotope tracing of 13C-labeled bacteria, we establish an experimental framework to track microbe-to-host lipid transfer and define the fates of microbial lipids in macrophages in vitro and in vivo. While naive macrophages scavenge phagocytosed bacterial fatty acids into the host lipidome, TLR4-trained macrophages direct flux to mitochondria for {beta}-oxidation or lipid droplets in the context of mitochondrial dysfunction. While TLR4 signaling increases ACOD1 expression to produce itaconate that throttles TCA flux, trained macrophages produce IL-10 that reduces ACOD1 to sustain bacterial lipid disposal and promote resolution. These findings reveal an IL-10/ACOD1 regulatory axis in trained macrophages that reprograms lipid metabolism to optimally reestablish tissue homeostasis post-inflammation.

immunology↗

bioRxiv2025

CD47 prevents Rac-mediated phagocytosis through Vav1 dephosphorylation

CD47 is expressed by viable cells to protect against phagocytosis. CD47 is recognized by SIRP, an inhibitory receptor expressed by macrophages and other myeloid cells. Activated SIRP recruits SHP-1 and SHP-2 phosphatases but the inhibitory signaling cascade downstream of these phosphatases is not clear. In this study, we used time lapse imaging to measure how CD47 impacts the kinetics of phagocytosis. We found that targets with IgG antibodies were primarily phagocytosed through a Rac-based reaching mechanism. Targets also containing CD47 were only phagocytosed through a less frequent Rho-based sinking mechanism. Hyperactivating Rac2 eliminated the suppressive effect of CD47, suggesting that CD47 prevents activation of Rac and reaching phagocytosis. During IgG-mediated phagocytosis, the tyrosine kinase Syk phosphorylates the GEF Vav, which then activates the GTPase Rac to drive F-actin rearrangement and target internalization. CD47 inhibited Vav1 phosphorylation without impacting Vav1 recruitment to the phagocytic synapse or Syk phosphorylation. Macrophages expressing a hyperactive Vav1 were no longer sensitive to CD47. Together this data suggests that Vav1 is a key target of the CD47 signaling pathway.

cell biology↗

bioRxiv2024

eIF4ET regulates meiotic proteome levels to enable oocyte formation and storage

Animals store oocytes in a dormant state for weeks to decades before ovulation. The homeostatic programs oocytes use to endure long-term storage are poorly understood. Using female nematodes as a short-lived model, we found that oocyte formation and storage required IFET-1, the conserved eIF4E-Transporter protein (eIF4ET). IFET-1 co-assembled with CAR-1 (Lsm14) to form micron-scale condensates in stored oocytes, which dissipated after oocyte activation. Depletion of IFET-1 destabilized the stored oocyte proteome, leading to lower translation, a decline in microtubule maintenance proteins, and errors in microtubule organization and meiotic spindle assembly. Deleting individual domains within IFET-1 impaired oocyte storage without affecting oocyte formation. Thus, in addition to establishing a healthy oocyte reserve in young mothers, IFET-1 ensures that correct levels of cytoskeletal proteins are maintained as oocytes age. Human eIF4ET also localized to micron-scale puncta in dormant oocytes in a reproductively healthy patient. Our results clarify the role for eIF4ET in maintaining the oocyte reserve and further support eIF4ET dysfunction as an upstream cause of embryonic aneuploidy and age-related infertility.

developmental biology↗

bioRxiv2023

Prior Fc Receptor activation primes macrophage for increased sensitivity to IgG via long term and short term mechanisms

Macrophages measure the eat-me signal IgG to identify targets for phagocytosis. We wondered if prior encounters with IgG influence macrophage appetite. IgG is recognized by the Fc Receptor. To temporally control Fc Receptor activation, we engineered an Fc Receptor that is activated by light-induced oligomerization of Cry2, triggering phagocytosis. Using this tool, we demonstrate that Fc Receptor activation primes macrophages to be more sensitive to IgG in future encounters. Macrophages that have previously experienced Fc Receptor activation eat more IgG-bound cancer cells. Increased phagocytosis occurs by two discrete mechanisms - a short- and long-term priming. Long term priming requires new protein synthesis and Erk activity. Short term priming does not require new protein synthesis and correlates with an increase in Fc Receptor mobility. Our work demonstrates that IgG primes macrophages for increased phagocytosis, suggesting that therapeutic antibodies may become more effective after initial priming doses.

cell biology↗

bioRxiv2022

How woodcocks produce the most brilliant white plumage patches among the birds

Until recently, and when compared with diurnal birds that use contrasting plumage patches and complex feather structures to convey visual information, communication in nocturnal species was considered to follow acoustic and chemical channels. However, many nocturnal birds have evolved intensely white plumage patches within otherwise inconspicuous plumages. We used spectrophotometry, electron microscopy, and optical modelling to explain the mechanisms producing bright white tail feather tips of the Eurasian woodcock Scolopax rusticola. Their diffuse reflectance was [~]30% higher than any previously measured feather. This intense reflectance is the result of incoherent light scattering from a disordered nanostructure composed of keratin and air within the barb rami. In addition, the flattening, thickening, and arrangement of those barbs creates a Venetian-blind-like macrostructure that enhances the surface area for light reflection. We suggest that the woodcocks have evolved these bright white feather patches for long-range visual communication in dimly lit environments.

ecology↗

bioRxiv2021

Prolactin and prolactin receptor expression in the HPG axis and crop sac during parental care in both sexes of a biparental bird (Columba livia)

During breeding, multiple circulating hormones, including prolactin, facilitate reproductive transitions in species that exhibit parental care. Prolactin underlies parental behaviors and related physiological changes across many vertebrates, including birds and mammals. While circulating prolactin levels often fluctuate across breeding, less is known about how relevant target tissues vary in their prolactin responsiveness via prolactin receptor (PRLR) expression. Recent studies have also investigated prolactin (PRL) gene expression outside of the pituitary (i.e., extra-pituitary PRL), but how PRL gene expression varies during parental care in non-pituitary tissue (e.g., hypothalamus, gonads) remains largely unknown. Further, it is unclear if and how tissue-specific PRL and PRLR vary between the sexes during biparental care. To address this, we measured PRL and PRLR gene expression in tissues relevant to parental care, the endocrine reproductive hypothalamic-pituitary-gonadal (HPG) axis and the crop (a tissue with a similar function as the mammalian mammary gland), across various reproductive stages in both sexes of a biparental bird, the rock dove (Columba livia). We also assessed how these genes responded to changes in offspring presence by adding chicks mid-incubation, simulating an early hatch when prolactin levels were still moderately low. We found that pituitary PRL expression showed similar increases as plasma prolactin levels, and detected extra-pituitary PRL in the hypothalamus, gonads and crop. Hypothalamic and gonadal PRLR expression also changed as birds began incubation. Crop PRLR expression correlated with plasma prolactin, peaking when chicks hatched. In response to replacing eggs with a novel chick mid-incubation, hypothalamic and gonadal PRL and PRLR gene expression differed significantly compared to mid-incubation controls, even when plasma prolactin levels did not differ. We also found sex differences in PRL and PRLR that suggest gene expression may allow males to compensate for lower levels in prolactin by upregulating PRLR in all tissues. Overall, this study advances our understanding of how tissue-specific changes in responsiveness to parental hormones may differ across key reproductive transitions, in response to offspring cues, and between the sexes.

animal behavior and cognition↗

bioRxiv2021

Sex-specific life extension in tauopathy mice by CSF1R inhibition causing selective microglial depletion and suppressed pathogenesis

Microglia are central to pathogenesis in many neurological conditions. Drugs targeting colony- stimulating factor-1 receptor (CSF1R) to block microglial proliferation in preclinical disease models have shown mixed outcomes, thus the therapeutic potential of this approach remains unclear. Here, CSF1R inhibitors were evaluated in tauopathy mice using multiple dosing schemes, drug analogs, and longitudinal measurements in the brain and plasma. A sex- independent reduction in pathogenic tau was seen in several models and non-microglial gene expression patterns reverted toward a normal wild type signature. Surprisingly, despite greater drug exposure in male mice, functional rescue and extended survival was only observed in female mice. A dose-dependent upregulation of immediate early genes and neurotransmitter dysregulation were observed in the brains of male mice only, indicating that excitotoxicity may have precluded functional benefits. Drug-resilient microglia in male mice exhibited morphological and gene expression patterns consistent with increased neuroinflammatory signaling, suggesting a mechanistic basis for sex-specific excitotoxicity. These data argue that complete microglial ablation is neither required nor desirable for neuroprotection and that therapeutics targeting microglia must consider sex-dependent effects on functional outcomes.

neuroscience↗

bioRxiv2019

Material aging causes centrosome weakening and disassembly during mitotic exit

Centrosomes must resist microtubule-mediated forces for mitotic chromosome segregation. During mitotic exit, however, centrosomes are deformed and fractured by those same forces, which is a key step in centrosome disassembly. How the functional material properties of centrosomes change throughout the cell cycle, and how they are molecularly tuned remain unknown. Here, we used optically-induced flow perturbations to determine the molecular basis of centrosome strength and ductility in C. elegans embryos. We found that both properties declined sharply at anaphase onset, long before natural disassembly. This mechanical transition required PP2A phosphatase and correlated with inactivation of PLK-1 (Polo Kinase) and SPD-2 (Cep192). In vitro, PLK-1 and SPD-2 directly protected centrosome scaffolds from force-induced disassembly. Our results suggest that, prior to anaphase, PLK-1 and SPD-2 confer strength and ductility to the centrosome scaffold so that it can resist microtubule-pulling forces. In anaphase, centrosomes lose PLK-1 and SPD-2 and transition to a weak, brittle state that enables force-mediated centrosome disassembly.

cell biology↗