Biology subjects

O'Neill, J. S.

Publications and source records attributed to O'Neill, J. S..

12 recordsLinked to original sources

bioRxiv2026

High sensitivity imaging of intracellular Cl- reveals the spatiotemporal evolution of GABAergic chloride fluxes in vitro and in vivo

Fast GABAergic inhibition in the brain is mediated by chloride currents whose driving force depends on intracellular Chloride concentration ([Cl-]i). Resolving the spatiotemporal dynamics of [Cl-]i in neurons requires a sensor combining high Cl- affinity, pH independence, and photostability - properties no existing tool achieves simultaneously. Here we introduce iClima (improved Chloride Imaging), a ratiometric genetically encoded sensor with high Cl- affinity (Kd=3.5 mM at pH 7.2), pH-insensitive under physiological conditions, and markedly improved photostability compared to existing sensors. These properties enable dynamic Cl- imaging without simultaneous pH correction. We demonstrate that iClima resolves GABAA-driven Cl- transients in cultured neurons, detects discrete Cl- hotspots along dendrites, and captures sensory-evoked Cl- transients in vivo. Finally, exploiting the spectral compatibility of iClima with GCaMP6f, we measure in interleaved trials Cl- and Ca2+ transients in the same neurons in vivo, revealing a divergence between the somatic inhibitory drive and the neuronal excitation state as reported by Ca2+ dynamics.

neuroscience↗

bioRxiv2026

An unrecognized host response to microbial exposure resets circadian timing

As ubiquitous features of every natural environment, microbes have profoundly shaped eukaryotic biology throughout evolution. Circadian clocks evolved in all domains of life as central regulators that align physiology with environmental cycles, yet whether they respond directly to microbial signals remains unknown. Here, we demonstrate that evolutionarily diverse microbes potently reset mammalian cellular clocks and can drive phase shifts in plants and algae, indicating cross-kingdom effects of microbes on circadian rhythms. In mammals, exposure to soluble bacterial components distinct from canonical innate immune ligands induces acute PER2 upregulation independently of Bmal1 or nascent transcription. A targeted inhibitor screen and biochemical assays implicate p38 MAPK as a modulator of this response. Taken together, this positions bacterial exposure as a previously unrecognized circadian clock input, revealing a new axis of host-microbe interaction that modulates biological timing at the cellular level.

cell biology↗

bioRxiv2024

Potassium rhythms couple the circadian clock to the cell cycle

Circadian ([~]24 h) rhythms are a fundamental feature of life, and their disruption increases the risk of infectious diseases, metabolic disorders, and cancer1-6. Circadian rhythms couple to the cell cycle across eukaryotes7,8 but the underlying mechanism is unknown. We previously identified an evolutionarily conserved circadian oscillation in intracellular potassium concentration, [K+]i9,10. As critical events in the cell cycle are regulated by intracellular potassium11,12, an enticing hypothesis is that circadian rhythms in [K+]i form the basis of this coupling. We used a minimal model cell, the alga Ostreococcus tauri, to uncover the role of potassium in linking these two cycles. We found direct reciprocal feedback between [K+]i and circadian gene expression. Inhibition of proliferation by manipulating potassium rhythms was dependent on the phase of the circadian cycle. Furthermore, we observed a total inhibition of cell proliferation when circadian gene expression is inhibited. Strikingly, under these conditions a sudden enforced gradient of extracellular potassium was sufficient to induce a round of cell division. Finally, we provide evidence that interactions between potassium and circadian rhythms also influence proliferation in mammalian cells. These results establish circadian regulation of intracellular potassium levels as a primary factor coupling the cell- and circadian cycles across diverse organisms.

cell biology↗

bioRxiv2023

Thermosensitivity of translation underlies the mammalian nocturnal-diurnal switch

Early mammals were nocturnal until the Cretaceous-Paleogene extinction enabled diurnal niche expansion. Diurnality evolved multiple times independently, but the mechanisms driving this shift remain unclear. We identify a conserved cell-intrinsic signal inversion that facilitates the transition from nocturnality to diurnality. Diurnal and nocturnal mammalian cells respond oppositely to temperature and osmotic cycles, mirroring species activity patterns. Cells exhibit differential global responses to temperature changes, including the phosphoproteome and protein synthesis. mTOR signaling is identified as a central mediator of this inversion, with diurnal mammals converging on modifications to mTOR and WNK pathways during evolution. Reducing mTOR activity induces nocturnal-to-diurnal shifting at cellular, tissue, and organismal levels. Therefore, the mTOR pathway is a cellular nexus that integrates energetic state and environmental signals to determine activity niche.

cell biology↗

bioRxiv2023

Propylene glycol inactivates respiratory viruses and prevents airborne transmission

Viruses are vulnerable as they transmit between hosts and we aimed to exploit this critical window. We found that the ubiquitous, safe, inexpensive and biodegradable small molecule propylene glycol (PG) has robust virucidal activity. Propylene glycol rapidly inactivates influenza, SARS-CoV-2 and a broad range of other enveloped viruses, and reduces disease burden in mice when administered intranasally at concentrations commonly found in nasal sprays. Most critically, aerosolized PG efficiently abolishes influenza and SARS-CoV-2 infectivity within airborne droplets, potently preventing infection at levels significantly below those well-tolerated by mammals. We present PG vapor as a first-in-class non-toxic airborne virucide, to prevent transmission of existing and emergent viral pathogens, with clear and immediate implications for public health. One-sentence summaryPropylene glycol is a potent and safe virucidal compound that could be used to limit and control infections.

microbiology↗

bioRxiv2022

Circadian regulation of protein turnover and proteome renewal

Although costly to maintain, protein homeostasis is indispensable for normal cellular function and long-term health. In mammalian cells and tissues, daily variation in global protein synthesis has been observed, but its utility and consequences for proteome integrity are not fully understood. Using several different pulse-labelling strategies, here we gain direct insight into the relationship between protein synthesis and abundance proteome-wide. We show that protein degradation varies in-phase with protein synthesis, facilitating rhythms in turnover rather than abundance. This results in daily consolidation of proteome renewal whilst minimising changes in composition. Coupled rhythms in synthesis and turnover are especially salient to the assembly of macromolecular protein complexes, particularly the ribosome, the most abundant species of complex in the cell. Daily turnover and proteasomal degradation rhythms render cells and mice more sensitive to proteotoxic stress at specific times of day, potentially contributing to daily rhythms in the efficacy of proteasomal inhibitors against cancer. Our findings suggest that circadian rhythms function to minimise the bioenergetic cost of protein homeostasis through temporal consolidation of protein turnover.

cell biology↗

bioRxiv2021

Clock gene-independent daily regulation of haemoglobin oxidation in red blood cells

Cellular circadian rhythms confer temporal organisation upon physiology that is fundamental to human health. Rhythms are present in red blood cells (RBCs), the most abundant cell type in the body, but their physiological function is poorly understood. Here, we present a novel biochemical assay for haemoglobin (Hb) oxidation status which relies on a redox-sensitive covalent haem-Hb linkage that forms during SDS-mediated cell lysis. Formation of this linkage is lowest when ferrous Hb is oxidised, in the form of ferric metHb. Daily haemoglobin oxidation rhythms are observed in RBCs cultured in vitro or taken from freely behaving mice or humans exhibit and are unaffected by mutations that affect circadian rhythms in nucleated cells. These rhythms correlate with daily rhythms in core body temperature, with temperature lowest when metHb levels are highest. Raising metHb levels with dietary sodium nitrite can further decrease daytime core body temperature in mice via NO signaling. These results extend our molecular understanding of RBC circadian rhythms and suggest they contribute to the regulation of body temperature.

cell biology↗

bioRxiv2021

Differential effects of environmental and endogenous 24h rhythms within a deep-coverage spatiotemporal proteome

The cellular landscape of most eukaryotic cells changes dramatically over the course of a 24h day. Whilst the proteome responds directly to daily environmental cycles, it is also regulated by a cellular circadian clock that anticipates the differing demands of day and night. To quantify the relative contribution of diurnal versus circadian regulation, we mapped spatiotemporal proteome dynamics under 12h:12h light:dark cycles compared with constant light. Using Ostreococcus tauri, a prototypical eukaryotic cell, we achieved 85% coverage of the theoretical proteome which provided an unprecedented insight into the identity of proteins that drive and facilitate rhythmic cellular functions. Surprisingly, the overlap between diurnally- and circadian-regulated proteins was quite modest (11%). These proteins exhibited different phases of oscillation between the two conditions, consistent with an interaction between intrinsic and extrinsic regulatory factors. The relative amplitude of rhythmic protein abundance was much lower than would be expected from daily variations in transcript abundance. Transcript rhythmicity was poorly predictive of daily variation in abundance of the encoded protein. We observed coordination between the rhythmic regulation of organelle-encoded proteins with the nuclear-encoded proteins that are targeted to organelles. Rhythmic transmembrane proteins showed a remarkably different phase distribution compared with rhythmic soluble proteins, indicating the existence of a novel circadian regulatory process specific to the biogenesis and/or degradation of membrane proteins. Taken together, our observations argue that the daily spatiotemporal regulation of cellular proteome composition is not dictated solely by clock-regulated gene expression. Instead, it also involves extensive rhythmic post-transcriptional, translational, and post-translational regulation that is further modulated by environmental timing cues.

cell biology↗

bioRxiv2020

Compensatory ion transport buffers daily protein rhythms to regulate osmotic balance and cellular physiology

Between 6-20% of the cellular proteome is under circadian control to tune cell function with cycles of environmental change. For cell viability, and to maintain volume within narrow limits, the osmotic pressure exerted by changes in the soluble proteome must be compensated. The mechanisms and consequences underlying compensation are not known. Here, we show in cultured mammalian cells and in vivo that compensation requires electroneutral active transport of Na+, K+, and Cl- through differential activity of SLC12A family cotransporters. In cardiomyocytes ex vivo and in vivo, compensatory ion fluxes alter their electrical activity at different times of the day. Perturbation of soluble protein abundance has commensurate effects on ion composition and cellular function across the circadian cycle. Thus, circadian regulation of the proteome impacts ion homeostasis with substantial consequences for the physiology of electrically active cells such as cardiomyocytes.

cell biology↗

bioRxiv2020

CRYPTOCHROME suppresses the circadian proteome and promotes protein homeostasis

The daily organisation of most mammalian cellular functions is attributed to circadian regulation of clock-controlled protein expression, driven by daily cycles of CRYPTOCHROME-dependent transcriptional feedback repression. To test this, we compared the circadian proteome and phosphoproteome of wild type and CRY-deficient fibroblast cells. Strikingly, CRY-deficient cells showed a two-fold increase in circadian-regulated proteins, phosphopeptides, and K+ transport. This was accompanied by extensive remodelling of the cellular proteome overall, including reduced phosphatase and proteasome subunit expression. These adaptations rendered CRY-deficient cells more sensitive to stress, which may account for their reduced circadian robustness and contribute to the wide-ranging phenotypes of CRY-deficient mice. We suggest that CRY ultimately functions to suppress, rather than generate, daily rhythms in cellular protein abundance, thereby maintaining protein and osmotic homeostasis.

cell biology↗

bioRxiv2020

CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping

SummaryCircadian (approximately daily) rhythms are a pervasive property of mammalian cells, tissues, and behaviour, ensuring physiological and metabolic adaptation to solar time. Models of daily cellular timekeeping revolve around transcriptional feedback repression, whereby CLOCK and BMAL1 activate the expression of clock proteins PERIOD (PER) and CRYPTOCHROME (CRY), which in turn repress CLOCK/BMAL1 activity. CRY proteins are thus considered essential negative regulators of the oscillation; a function supported by behavioural arrhythmicity of CRY-deficient mice when kept under constant conditions. Challenging this interpretation, however, we find evidence for persistent circadian rhythms in mouse behaviour and cellular PER2 levels when CRY is absent. CRY-less oscillations are variable in their expression and have a shorter period than wild type controls. Importantly, we find classic circadian hallmarks such as temperature compensation and determination of period by casein kinase 1{delta}/{varepsilon} activity to be maintained. In the absence of CRY-mediated transcriptional feedback repression and rhythmic Per2 transcription, PER2 protein rhythms are sustained for several cycles, accompanied by circadian variation in protein stability. We suggest that, whereas circadian transcriptional feedback imparts robustness and functionality onto biological clocks, the core timekeeping mechanism is post-translational. Our findings suggest that PER proteins normally act as signalling hubs that transduce timing information to the nucleus, imparting daily rhythms upon the activity of transcriptional effectors. Highlights[tpltrtarr] PER/CRY-mediated negative feedback is dispensable for mammalian circadian timekeeping [tpltrtarr]Circadian variation in PER2 levels persists in the absence of rhythmic Per2 transcription [tpltrtarr]CK1 and GSK3 are plausible mechanistic components of a cytoscillator mechanism [tpltrtarr]CRY-mediated feedback repression imparts robustness to biological timekeeping In briefCircadian turnover of mammalian clock protein PERIOD2 persists in the absence of canonical transcriptional feedback repression and rhythmic clock gene activity, demanding a re-evaluation of cellular clock function and evolution.

cell biology↗

bioRxiv2018

Energetic substrate availability regulates synchronous activity in an excitatory neural network

Neural networks are required to meet significant metabolic demands associated with performing sophisticated computational tasks in the brain. The necessity for efficient transmission of information imposes stringent constraints on the metabolic pathways that can be used for energy generation at the synapse, and thus low availability of energetic substrates can reduce the efficacy of synaptic function. Here we study the effects of energetic substrate availability on global neural network behavior and find that glucose alone can sustain excitatory neurotransmission required to generate high-frequency synchronous bursting that emerges in culture. In contrast, obligatory oxidative energetic substrates such as lactate and pyruvate are unable to substitute for glucose, indicating that processes involving glucose metabolism form the primary energy-generating pathways supporting coordinated network activity. Our experimental results are discussed in the context of the role that metabolism plays in supporting the performance of individual synapses, including the relative contributions from postsynaptic responses, astrocytes, and presynaptic vesicle cycling. We propose a simple computational model for our excitatory cultures that accurately captures the inability of metabolically compromised synapses to sustain synchronous bursting when extracellular glucose is depleted.

neuroscience↗