Biology subjects

Ambrose, B.

Publications and source records attributed to Ambrose, B..

10 recordsLinked to original sources

bioRxiv2026

INO80 rapidly shuttles nucleosomes between chromatin barriers

ATP-dependent chromatin remodelers establish nucleosome organization, but how they move nucleosomes over extended distances and respond to neighboring chromatin remains unclear. Here, we combine correlative optical tweezers and fluorescence microscopy, and single-molecule FRET to visualize human INO80 activity on naked and chromatinized DNA. hINO80 undergoes free one-dimensional diffusion along DNA with multiple, nucleotide-regulated, diffusive states. Upon engaging a nucleosome, hINO80 drives rapid, processive nucleosome sliding over thousands of base pairs in bidirectional bursts. Neighboring nucleosomes prevent passage and redirect translocation, confining mobile nucleosomes to repeated movement between chromatin boundaries. Single-molecule FRET reveals transitions consistent with INO80 switching between opposing nucleosome-binding orientations, while fluorescence stoichiometry and mass photometry show that two hINO80 complexes can simultaneously occupy one nucleosome. These findings identify non-exclusive mechanisms for directional reversal and reveal how long-range hINO80 translocation is converted into boundary-constrained nucleosome repositioning, providing a dynamic framework for understanding nucleosome organization within chromatin.

biophysics↗

bioRxiv2026

DeepDiffusion: a Physics-Informed Neural Network for Heterogeneous Facilitated 1D Diffusion

Single-molecule fluorescence microscopy combined with optical tweezers has enabled the direct observation of diffusing proteins on tethered DNA. These and complementary techniques reveal facilitated one-dimensional diffusion as a common functional mechanism for numerous DNA-binding proteins, with a wide range of heterogeneous diffusive behaviours arising from different DNA-binding modes. However, detailed investigations have been limited by the lack of methods to detect such heterogeneous diffusion. We have developed DeepDiffusion, a physics-informed neural network model for estimating the instantaneous diffusion at each point along a single-molecule trajectory. We show, using synthetic trajectories, that DeepDiffusion can accurately detect subtle changes in diffusion even when challenged with large underlying errors. DeepDiffusion can recapitulate previously characterised heterogeneity in experimental data and reveal mechanistic details of facilitated diffusion that are inaccessible to current methods. We expect DeepDiffusion to become a powerful tool for single-molecule researchers, allowing them to investigate the diffusion of their proteins of interest in unprecedented detail.

biophysics↗

bioRxiv2025

Multi-omics Analysis of Human Blood Cells Reveals Unique Features of Age-associated Type2 CD8 Memory T cells

Aging impacts immune function, but the mechanisms driving age-related changes in immune cell subsets remain unclear. To explore age-dependent changes in immune cell populations, we analyzed human peripheral blood mononuclear cells (PBMCs) from a cohort of healthy donors aged 20-82 years using a 36-color spectral flow cytometry panel focused on T cells. We identified a unique population of memory CD8 T cells, which lack CXCR3 and produce a Th2-like cytokine response, accumulate with age. We discovered an age-dependent bias in naive CD8 T cells toward Th2 cytokine production, accompanied by transcriptional and epigenetic changes supporting this phenotype. Moreover, health outcome association analysis linked the accumulation of these unique CXCR3- central memory CD8 T cells to asthma, chronic liver conditions, and type 2 diabetes. Together, our results support the model that an age-dependent drift in epigenetic regulation towards a Th2-like phenotype drives a pathogenic Th2-like immune population.

immunology↗

bioRxiv2025

Brassinosteroid treatment reveals the importance of xyloglucan transglucosylase/hydrolase (XTH) genes in growth habit determination of twining common bean vines

O_LIBrassinosteroids impact the development of G-fibers --specialized cells that generate tension in plants. To explore the functional and genetic relationships between G-fibers and twining stems of common bean, we applied an active brassinosteroid and a brassinosteroid inhibitor to perturb G-fiber development and probed these phenotypes through gene expression and anatomical analyses. C_LIO_LIBrassinosteroid treatment generated phenotypes that aKected the three key features of twining: elongation, circumnutation, and G-fiber development. We examined anatomical and biochemical changes in the G-fibers through cross-sections, macerations, and immunohistochemistry. RNA sequencing and differential gene expression analysis allowed us to identify unique gene expression patterns for each treatment. C_LIO_LIBrassinosteroid treatment led to significantly elongated internodes with disrupted circumnutation and long, thin-walled G-fibers. In contrast, inhibitor treatment produced short internodes with thick G-fibers. These phenotypes corresponded with significant differential expression of XTH genes, both at the onset of elongation and later, during G-layer deposition. Detection of xyloglucan epitopes in the G-layer, along with in situ hybridization, confirmed active xyloglucan remodeling after twining. C_LIO_LIOur results confirm the presence of xyloglucan in the G-layer of common bean, underscoring its importance in G-fiber function, and suggests a regulatory role for XTH genes in shaping the twining growth habit through modulation of cell wall properties. C_LI

plant biology↗

bioRxiv2024

Herpes simplex virus type 1 origin binding protein UL9 tethers and loops origin- and non-origin-DNA intra- and intermolecularly

Herpesviruses are ubiquitous human pathogens, which are the causative agent of mild to severe symptoms ranging from cold sore to nasopharyngeal carcinoma. Even though replication of the linear dsDNA genome has been studied for decades, we still lack a complete molecular understanding of its mechanism. It has been proposed, but never shown directly, that the HSV-1 origin binding protein UL9 binds two closely located binding sites within the oriS origin sequence, thereby mediating origin looping, which in turn facilitates replication initiation. Here, we used an array of single-molecule approaches to test this long-standing hypothesis directly. Surprisingly, the data show that UL9 does not loop oriS efficiently. However, we demonstrate that UL9 can form large DNA loops at non-origin sequences very efficiently, as well as tether two oriS DNA molecules intermolecularly. Contrary to the origin bending hypothesis, our findings indicate that UL9 does not loop oriS DNA, but rather may play an alternative role in replication initiation, such as tethering two separate molecules to facilitate recombination.

microbiology↗

bioRxiv2024

Rep structures can be tuned by ionicity via metastable intermediates in the absence of DNA

DNA helicases undergo conformational changes; however, their structural dynamics are poorly understood. Here, we study single molecules of superfamily 1A DNA helicase Rep, which undergo conformational transitions during bacterial DNA replication, repair and recombination. We use time-correlated single-photon counting (TCSPC), fluorescence correlation spectroscopy (FCS), rapid single-molecule Forster resonance energy transfer (smFRET), Anti-Brownian ELectrokinetic (ABEL) trapping and molecular dynamics simulations (MDS) to provide unparalleled temporal and spatial resolution of Reps domain movements. We detect four states revealing two hitherto hidden intermediates (S2, S3), between the open (S1) and closed (S4) structures, whose stability is salt dependent. Reps open-to-closed switch involves multiple changes to all four subdomains 1A, 1B, 2A and 2B along the S1[->]S2[->]S3[->]S4 transitional pathway comprising an initial truncated swing of 2B which then rolls across the 1B surface, following by combined rotations of 1B, 2A and 2B. High forward and reverse rates for S1[->]S2 suggest that 1B may act to frustrate 2B movement to prevent premature Rep closure in the absence of DNA. These observations support a more general binding model for accessory DNA helicases that utilises conformational plasticity to explore a multiplicity of structures whose landscape can be tuned by salt prior to locking-in upon DNA binding.

biophysics↗

bioRxiv2023

The genome of the Wollemi pine, a critically endangered living fossil unchanged since the Cretaceous, reveals extensive ancient transposon activity.

We present the genome of the living fossil, Wollemia nobilis, a southern hemisphere conifer morphologically unchanged since the Cretaceous. Presumed extinct until rediscovery in 1994, the Wollemi pine is critically endangered with less than 60 wild adults threatened by intensifying bushfires in the Blue Mountains of Australia. The 12 Gb genome is among the most contiguous large plant genomes assembled, with extremely low heterozygosity and unusual abundance of DNA transposons. Reduced representation and genome re-sequencing of individuals confirms a relictual population since the last major glacial/drying period in Australia, 120 ky BP. Small RNA and methylome sequencing reveal conservation of ancient silencing mechanisms despite the presence of thousands of active and abundant transposons, including some transferred horizontally to conifers from arthropods in the Jurassic. A retrotransposon burst 8-6 my BP coincided with population decline, possibly as an adaptation enhancing epigenetic diversity. Wollemia, like other conifers, is susceptible to Phytophthora, and a suite of defense genes, similar to those in loblolly pine, are targeted for silencing by sRNAs in leaves. The genome provides insight into the earliest seed plants, while enabling conservation efforts.

genomics↗

bioRxiv2022

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than {+/-} 0.06, corresponding to an interdye distance precision of [≤] 0.2 nm and accuracy of [≤] 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to [lsim] 10% of the Forster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.

biophysics↗

bioRxiv2022

An Open-source, Cost-efficient Excitation Module for Single-molecule Microscopy

In fluorescence microscopy, scientific-grade laser diodes are key components and contribute a considerable expense to the total cost of the microscope setup. Existing open-source laser excitation modules are mainly designed for super-resolution imaging and have not been fully characterised to meet the requirements of single-molecule spectroscopy measurements. In this paper we introduce an open-source, cost-efficient excitation module that can be used for confocal, accurate single-molecule FRET, fluorescence correlation spectroscopy (FCS) and low-power super-resolution techniques. The module delivers two diode lasers (520 nm and 638 nm) via a single-mode fibre, with stable power output (<1% deviation), which can be modulated on the microsecond timescale. Here, we benchmark these lasers against smFRET standards, and recover consistent diffusion coefficients from FCS measurements, thereby demonstrating their suitability for a range of single-molecule spectroscopic experiments, whilst reducing the overall costs of our open-source smFRET instrument (the smfBox) by ~50%, making these types of experiments even more accessible to the widest possible userbase.

biophysics↗

bioRxiv2019

The smfBox: an open-source platform for single-molecule FRET

Single-molecule Forster Resonance Energy Transfer (smFRET) is a powerful technique capable of resolving both relative and absolute distances within and between structurally dynamic biomolecules. High instrument costs, and a lack of open-source hardware and acquisition software have limited smFRETs broad application by non-specialists. Here, we present the smfBox, a cost-effective confocal smFRET platform, providing detailed build instructions, open-source acquisition software, and full validation, thereby democratising smFRET for the wider scientific community.

biophysics↗