Biology subjects

O'Toole, P.

Publications and source records attributed to O'Toole, P..

9 recordsLinked to original sources

bioRxiv2026

Dynamic diffusion analysis of the yeast plasma membrane using Airyscan based microscopic techniques

The yeast plasma membrane (PM) is highly compartmentalised into distinct nanoscale domains. The mechanisms by which this organisation regulates surface proteins are not fully understood, and it remains unclear how different biophysical modalities capture diffusion kinetics across varying spatial scales. Using confocal microscopy and an Airyscan2 detector, we benchmarked two prominent techniques: Fluorescence Correlation Spectroscopy (FCS) via the Zeiss Dynamics Profiler and Fluorescence Recovery After Photobleaching (FRAP). We quantified the lateral diffusion of three functionally diverse GFP-tagged model proteins: the exocytic t-SNARE Sso2, the lipid-binding protein Pmp3, and the eisosome-associated protein Ycp4. While diffusion coefficients aligned tightly between both modalities for Pmp3 and Ycp4, Sso2 exhibited a stark 14-fold discrepancy, displaying drastically faster local mobility by FCS compared to macroscopic recovery by FRAP. High-resolution 3D Structured Illumination Microscopy (3D-SIM) shows that Sso2 is partitioned into regional subdomains, that occupy less PM area than the network-like localisation of Pmp3. Our findings suggest that FCS captures rapid, localised diffusion within these microenvironments, whereas FRAP measures highly restricted transit across domain boundaries. Ultimately, this work demonstrates that membrane diffusion coefficients cannot be interpreted in isolation and capturing true lateral mobility requires pairing kinetic measurements with super-resolution spatial mapping to decode complex membrane compartmentalisation.

cell biology↗

bioRxiv2026

NMR reveals specific remodelling of protein folding landscapes in ionic liquids

Ionic liquids (ILs) are designer solvents with tunable properties that have emerged as powerful cosolutes in protein science and biotechnology. While ILs are known to stabilize or destabilize proteins, their molecular mechanisms remain poorly defined, particularly regarding effects on the unfolded ensemble. Here, we use the metastable SH3 domain of the Drosophila adaptor protein Drk, which coexists in a slow two-state equilibrium between folded and unfolded states, as a model to dissect how ILs modulate protein conformational equilibria. Using nuclear magnetic resonance (NMR) spectroscopy, we show that cholinium glutamate ([Ch][Glu]) stabilizes the folded state by preferential exclusion from the protein surface, raising the barrier to unfolding in a manner reminiscent of osmolytes and crowding agents. In contrast, 1-butyl-3-methylimidazolium dicyanamide ([Bmim][dca]) shifts the equilibrium toward the unfolded state, not by destabilizing the native fold, but by stabilizing a compact, non-native -helical conformation within the unfolded ensemble. Notably, this concentration-dependent mechanism differs from traditional denaturants such as urea or guanidinium chloride, which promote random-coil unfolded states. Kinetic measurements further reveal that [Ch][Glu] slows unfolding, whereas [Bmim][dca] slows folding, indicating that the two ILs reshape the folding landscape in opposite directions. These findings challenge the prevailing view that cosolutes act primarily on the folded state and establish unfolded-state stabilization as a critical determinant of protein stability. More broadly, this work provides a molecular framework for understanding how ILs reshape protein folding landscapes and offers design principles for tailoring ILs as stabilizers or destabilizers in biotechnology and therapeutic applications. Significance StatementProteins exist in a delicate balance between folded and unfolded states, and cosolutes such as salts are generally viewed as acting only on the folded state. Here, we show that ionic liquids (ILs) are able to reshape this balance and uniquely modulate both folded and unfolded protein states through distinct molecular mechanisms. These results challenge the conventional view of protein stabilization/destabilization and demonstrate that unfolded-state modulation is a critical determinant of protein behavior. By establishing principles for how ILs alter protein folding landscapes, this work provides a framework for designing ILs with tailored effects, with implications for biocatalysis, protein engineering, and therapeutic formulation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/700775v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@372d3borg.highwire.dtl.DTLVardef@7a7fadorg.highwire.dtl.DTLVardef@543f4eorg.highwire.dtl.DTLVardef@1084836_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Identifying the non-Bifidobacterial hallmarks of a Bifidobacterium-receptive gut microbiome

Bifidobacteria are key health-associated members of the human gut and widely used as probiotics, but their colonization success varies across individuals, partly due to baseline microbiome composition. In this analysis encompassing 51,058 gut microbiomes (148 cohorts spanning 46 countries across six continents), we investigated non-Bifidobacterial features associated with the overall detection and abundance of eight major Bifidobacterial species. While non-Bifidobacterial community composition significantly correlated with Bifidobacterial detection in 74% of analyzed cohorts, the associations for the individual species showed distinct age-/lifestyle-/disease-specific patterns (greater similarity within similar cohorts). We quantified these relationships as Association-Scores, further stratified by age group, lifestyle, profiling strategy (16S/WGS) and nine diseases, and reasonably reproduced in an external validation-cohort-set of 8 Bifidobacterium intervention studies (1,063 gut microbiomes). We subsequently utilized these scores to build microbiome-level receptive-scores corresponding to the 8 major Bifidobacterium species and their overall detection. Both robust linear regression and cross-study validation showed that combining baseline abundance of the administered Bifidobacterium with the corresponding baseline receptive-scores significantly predicted post-treatment persistence/increase in 75% trial-probiotic pairs. We identify 129 species-specific-genome-level functional-groups in non-Bifidobacterial taxa correlating with Bifidobacterial Association-Scores. This global analysis highlights non-Bifidobacterial microbiome features influencing Bifidobacterial colonization and provides tools to predict personalized responses to Bifidobacterial-probiotic interventions.

microbiology↗

bioRxiv2025

Ontogeny-independent expression of LPCAT2 in granuloma macrophages during experimental visceral leishmaniasis

Granulomas are organized inflammatory lesions that form in response to persistent stimuli such as infections. Murine infection with Leishmania donovani results in the formation of granulomas around infected Kupffer cells in the liver and serves as a well-defined model of immune granuloma formation. The formation and resolution of granulomatous inflammation requires dynamic shifts in immune cell activation states, imposing significant metabolic demands. As mediators of energy homeostasis and cell signaling, lipids and lipid metabolism play a key role in regulating immune cell function during inflammation and the response to infection. However, the extent to which alterations in lipids are spatially linked to altered immune cell transcription has yet to be resolved. In this study, we performed a multimodal imaging analysis combining MALDI mass spectrometry, spatial and single cell transcriptomics, proteomics of flow-sorted macrophages and histopathology of L. donovani induced hepatic granulomas. Using this spatially-integrated approach, we identified LPCAT2-mediated membrane re-modelling of myeloid cells as a novel feature of these granulomas. Our study provides new insights into local immunometabolic changes associated with granuloma formation and macrophage activation.

immunology↗

bioRxiv2025

Pathological mutation in SMN impairs modulation of GAR1 phase separation linking condensate dysfunction to Spinal Muscular Atrophy

Pathological mutations in liquid-liquid phase separation (LLPS) scaffold proteins have been linked to biomolecular condensate dysfunction in neurodegenerative diseases, while the possible impact of client protein mutations remains unclear. In spinal muscular atrophy (SMA), the disease-associated E134K mutation in the Tudor domain (TD) of the survival motor neuron (SMN) protein disrupts its interaction with GAR1, an RGG-rich protein within the H/ACA small nucleolar ribonucleoprotein complex. The consequences of this impaired interaction have not been elucidated. Here, we identified GAR1 as an LLPS scaffold protein that is phase separated in nuclear compartments and forms gel-like condensates via complex coacervation with RNA in vitro. In cells, we reveal that SMN co-localizes with GAR1 in Cajal bodies and modulates its dynamics. Using confocal microscopy and NMR spectroscopy, we further show that SMN TD is a client protein that regulates the architecture and dynamics of GAR1 condensates through competitive RNA interactions, implicating GAR1 phase separation in RNA accumulation and release processes regulated by SMN. Notably, the SMA-associated E134K variant of SMN exhibits a reduced affinity for GAR1, impairing the modulation of GAR1 condensates and displacement RNA. Our findings suggest a mechanistic link between phase separation dysregulation and SMA, driven by disrupted scaffold-client interactions, that highlights the therapeutic potential of targeting SMN-dependent condensate regulation in SMA.

biochemistry↗

bioRxiv2024

Towards a Health-Associated Core Keystone (HACK) index for the human gut microbiome

Variations in the normal gut microbiome and the existence of context-dependent disease associations have confounded the identification of microbiome markers of health. A reliable indexing of taxa based on their association with host health and microbiome resilience could accelerate development of microbiome-based therapeutics including selection of live biotherapeutics and facilitate microbiome comparisons in diverse study populations. Here we first investigated 196 taxa for their association with three hallmark properties of health and microbiome-resilience, namely prevalence/community-influence in non-diseased subjects, longitudinal stability and host health, using a discovery cohort of 39,926 publicly available adult (> 18 years) gut microbiomes from 127 studies spanning 42 countries and 28 disease conditions (including 9,434 longitudinal samples). We identified 18 species-level-taxa, referred to as Health-Associated Core Keystones (HACKs), with association-strengths in the top 30 percentile for all three properties. We integrated these association-strengths into a single value, the HACK-index, that ranks taxa based on their estimated contribution to both microbiome stability and host health. We then demonstrate the reproducibility of these indices for the taxon associations with the three properties, using a validation cohort of 4,500 gut microbiomes (from 11 studies with various demographics and diseases). Specific consortia of high HACK-index taxa are also associated with positive response to Mediterranean Diet interventions and Immuno-Checkpoint-Inhibitor therapies. We finally identify the distinguishing microbial genomic functions associated with high HACK-indices/HACK-taxa that can be investigated using targeted mechanistic studies to identify microbiome effectors of improved health. The availability of HACK-indices provides a rational basis for microbiome comparisons and will facilitate the selection and design of microbiome-based therapeutics.

microbiology↗

bioRxiv2020

p53-dependent polyploidisation after DNA damage in G2 phase

Cell cycle progression in the presence of damaged DNA can lead to accumulation of mutations and pose a risk for tumour development. In response to DNA damage in G2 phase, human cells can be forced to exit the cell cycle in a p53-p21- and APC/CCdh1-dependent manner. Cells that exit the cell cycle in G2 phase become senescent, but it is unclear what determines this commitment and whether other cell fates occur. We find that a subset of immortalised RPE-1 cells and primary human fibroblasts spontaneously initiate DNA re-replication several days after forced cell cycle exit in G2 phase. By combining single cell tracking for more than a week with quantitative immunofluorescence, we find that the resulting polyploid cells contain increased levels of damaged DNA and frequently exit the cell cycle again in the next G2 phase. Subsequently, these cells either enter senescence or commit to another round of DNA re-replication, further increasing the ploidy. At least a subset of the polyploid cells show abnormal centrosome numbers or localisation. In conclusion, cells that are forced to exit the cell cycle in G2 phase face multiple choices that lead to various phenotypes, including propagation of cells with different ploidies. Our findings suggest a mechanism by which p53-positive cells can evade senescence that risks genome integrity. Main points-Cell cycle exit from G2 phase does not necessarily lead to senescence -Resumption of proliferation after G2 phase cell cycle exit starts with DNA replication -Successive cell cycle exits lead to propagation of cells with different ploidies -A p53-dependent mechanism allows eventual proliferation after DNA damage

cell biology↗

bioRxiv2019

Arrhythmic gut microbiome signatures for risk profiling of Type-2 Diabetes

To combat the epidemic increase in Type-2-Diabetes (T2D), risk factors need to be identified. Diet, lifestyle and the gut microbiome are among the most important factors affecting metabolic health. We demonstrate in 1,976 subjects of a prospective population cohort that specific gut microbiota members show diurnal oscillations in their relative abundance and we identified 13 taxa with disrupted rhythmicity in T2D. Prediction models based on this signature classified T2D with an area under the curve of 73%. BMI as microbiota-independent risk marker further improved diagnostic classification of T2D. The validity of this arrhythmic risk signature to predict T2D was confirmed in 699 KORA subjects five years after initial sampling. Shotgun metagenomic analysis linked 26 pathways associated with xenobiotic, amino acid, fatty acid, and taurine metabolism to the diurnal oscillation of gut bacteria. In summary, we determined a cohort-specific risk pattern of arrhythmic taxa which significantly contributes to the classification and prediction of T2D, highlighting the importance of circadian rhythmicity of the microbiome in targeting metabolic human diseases.

microbiology↗

bioRxiv2019

Voltage-dependent activation of Rac1 by Nav1.5 channels promotes cell migration

Ion channels can regulate the plasma membrane potential (Vm) and cell migration as a result of altered ion flux. However, the mechanism by which Vm regulates motility remains unclear. Here, we show that the Nav1.5 sodium channel carries persistent inward Na+ current which depolarizes the resting Vm at the timescale of minutes. This Nav1.5-dependent Vm depolarization increases Rac1 colocalization with phosphatidylserine, to which it is anchored at the leading edge of migrating cells, promoting Rac1 activation. A genetically-encoded FRET biosensor of Rac1 activation shows that depolarization-induced Rac1 activation results in acquisition of a motile phenotype. By identifying Nav1.5-mediated Vm depolarization as a regulator of Rac1 activation, we link ionic and electrical signaling at the plasma membrane to small GTPase-dependent cytoskeletal reorganization and cellular migration. We uncover a novel and unexpected mechanism for Rac1 activation, which fine tunes cell migration in response to ionic and/or electric field changes in the local microenvironment.

cell biology↗