Circadian regulation of macromolecular complex 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.

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.

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 a broad range of viruses including influenza A, SARS-CoV-2 and rotavirus and reduces disease burden in mice when administered intranasally at concentrations commonly found in nasal sprays. Most critically, vaporised PG efficiently abolishes influenza A virus and SARS-CoV-2 infectivity within airborne droplets, potently preventing infection at levels well below those tolerated by mammals. We present PG vapour as a first-in-class non-toxic airborne virucide that can prevent transmission of existing and emergent viral pathogens, with clear and immediate implications for public health.

Macromolecular condensation buffers intracellular water potential.

Optimum protein function and biochemical activity critically depends on water availability because solvent thermodynamics drive protein folding and macromolecular interactions1. Reciprocally, macromolecules restrict the movement of 'structured' water molecules within their hydration layers, reducing the available 'free' bulk solvent and therefore the total thermodynamic potential energy of water, or water potential. Here, within concentrated macromolecular solutions such as the cytosol, we found that modest changes in temperature greatly affect the water potential, and are counteracted by opposing changes in osmotic strength. This duality of temperature and osmotic strength enables simple manipulations of solvent thermodynamics to prevent cell death after extreme cold or heat shock. Physiologically, cells must sustain their activity against fluctuating temperature, pressure and osmotic strength, which impact water availability within seconds. Yet, established mechanisms of water homeostasis act over much slower timescales2,3; we therefore postulated the existence of a rapid compensatory response. We find that this function is performed by water potential-driven changes in macromolecular assembly, particularly biomolecular condensation of intrinsically disordered proteins. The formation and dissolution of biomolecular condensates liberates and captures free water, respectively, quickly counteracting thermal or osmotic perturbations of water potential, which is consequently robustly buffered in the cytoplasm. Our results indicate that biomolecular condensation constitutes an intrinsic biophysical feedback response that rapidly compensates for intracellular osmotic and thermal fluctuations. We suggest that preserving water availability within the concentrated cytosol is an overlooked evolutionary driver of protein (dis)order and function.

Mechanisms and physiological function of daily haemoglobin oxidation rhythms 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 mouse and human RBCs cultured in vitro, or taken from humans in vivo, 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 nitric oxide (NO) signalling. These results extend our molecular understanding of RBC circadian rhythms and suggest they contribute to the regulation of body temperature.

A daily temperature rhythm in the human brain predicts survival after brain injury.

Patients undergo interventions to achieve a 'normal' brain temperature; a parameter that remains undefined for humans. The profound sensitivity of neuronal function to temperature implies the brain should be isothermal, but observations from patients and non-human primates suggest significant spatiotemporal variation. We aimed to determine the clinical relevance of brain temperature in patients by establishing how much it varies in healthy adults. We retrospectively screened data for all patients recruited to the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) High Resolution Intensive Care Unit Sub-Study. Only patients with direct brain temperature measurements and without targeted temperature management were included. To interpret patient analyses, we prospectively recruited 40 healthy adults (20 males, 20 females, 20-40 years) for brain thermometry using magnetic resonance spectroscopy. Participants were scanned in the morning, afternoon, and late evening of a single day. In patients (n = 114), brain temperature ranged from 32.6 to 42.3°C and mean brain temperature (38.5 ± 0.8°C) exceeded body temperature (37.5 ± 0.5°C, P < 0.0001). Of 100 patients eligible for brain temperature rhythm analysis, 25 displayed a daily rhythm, and the brain temperature range decreased in older patients (P = 0.018). In healthy participants, brain temperature ranged from 36.1 to 40.9°C; mean brain temperature (38.5 ± 0.4°C) exceeded oral temperature (36.0 ± 0.5°C) and was 0.36°C higher in luteal females relative to follicular females and males (P = 0.0006 and P < 0.0001, respectively). Temperature increased with age, most notably in deep brain regions (0.6°C over 20 years, P = 0.0002), and varied spatially by 2.41 ± 0.46°C with highest temperatures in the thalamus. Brain temperature varied by time of day, especially in deep regions (0.86°C, P = 0.0001), and was lowest at night. From the healthy data we built HEATWAVE-a 4D map of human brain temperature. Testing the clinical relevance of HEATWAVE in patients, we found that lack of a daily brain temperature rhythm increased the odds of death in intensive care 21-fold (P = 0.016), whilst absolute temperature maxima or minima did not predict outcome. A warmer mean brain temperature was associated with survival (P = 0.035), however, and ageing by 10 years increased the odds of death 11-fold (P = 0.0002). Human brain temperature is higher and varies more than previously assumed-by age, sex, menstrual cycle, brain region, and time of day. This has major implications for temperature monitoring and management, with daily brain temperature rhythmicity emerging as one of the strongest single predictors of survival after brain injury. We conclude that daily rhythmic brain temperature variation-not absolute brain temperature-is one way in which human brain physiology may be distinguished from pathophysiology.

Using ALLIGATORs to Capture Circadian Bioluminescence.

Luciferases are a popular tool in circadian biology research as longitudinal reporters of gene expression. Here, we describe a short updated protocol for the use of an Automated Longitudinal Luciferase Imaging Gas and Temperature-Optimized Recorder (ALLIGATOR) to record cellular bioluminescence over many days. The ALLIGATOR has superior capacity and flexibility compared with traditional luminometers that employ photomultiplier tubes (PMTs), with high-throughput capability and spatial resolution. It can be readily adapted to a wide variety of applications, such as different sample types and plate sizes, under a wide range of physiologically relevant conditions.

Detecting Circadian Rhythms in Human Red Blood Cells by Dielectrophoresis.

Dielectrophoresis (DEP) enables the measurement of population-level electrophysiology in many cell types by examining their interaction with an externally applied electric field. Here we describe the application of DEP to the measurement of circadian rhythms in a non-nucleated cell type, the human red blood cell. Using DEP, population-level electrophysiology of ~20,000 red blood cells can be measured from start to finish in less than 3 min, and can be repeated over several days to reveal cell-autonomous daily regulation of membrane electrophysiology. This method is amenable to the characterization of circadian rhythms by altering entrainment and free-run conditions or through pharmacological perturbation.

CRYPTOCHROMES promote daily 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 used quantitative mass spectrometry to compare wild-type and CRY-deficient fibroblasts under constant conditions. In CRY-deficient cells, we found that temporal variation in protein, phosphopeptide, and K+ abundance was at least as great as wild-type controls. Most strikingly, the extent of temporal variation within either genotype was much smaller than overall differences in proteome composition between WT and CRY-deficient cells. This proteome imbalance in CRY-deficient cells and tissues was associated with increased susceptibility to proteotoxic stress, which impairs circadian robustness, and may contribute to the wide-ranging phenotypes of CRY-deficient mice. Rather than generating large-scale daily variation in proteome composition, we suggest it is plausible that the various transcriptional and post-translational functions of CRY proteins ultimately act to maintain protein and osmotic homeostasis against daily perturbation.

Understanding circadian regulation of mammalian cell function, protein homeostasis, and metabolism.

Circadian rhythms are ∼24 h cycles of organismal and cellular activity ubiquitous to mammalian physiology. A prevailing paradigm suggests that timing information flows linearly from rhythmic transcription via protein abundance changes to drive circadian regulation of cellular function. Challenging this view, recent evidence indicates daily variation in many cellular functions arises through rhythmic post-translational regulation of protein activity. We suggest cellular circadian timing primarily functions to maintain proteome homeostasis rather than perturb it. Indeed, although relevant to timekeeping mechanism, daily rhythms of clock protein abundance may be the exception, not the rule. Informed by insights from yeast and mammalian models, we propose that optimal bioenergetic efficiency results from coupled rhythms in mammalian target of rapamycin complex activity, protein synthesis/turnover, ion transport and protein sequestration, which drive facilitatory rhythms in metabolic flux and substrate utilisation. Such daily consolidation of proteome renewal would account for many aspects of circadian cell biology whilst maintaining osmotic homeostasis.

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 and tunes mammalian cell function with daily environmental cycles. For cell viability, and to maintain volume within narrow limits, the daily variation in osmotic potential exerted by changes in the soluble proteome must be counterbalanced. The mechanisms and consequences of this osmotic compensation have not been investigated before. In cultured cells and in tissue we find that compensation involves 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 confer daily variation in electrical activity. 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.

Deep-coverage spatiotemporal proteome of the picoeukaryote Ostreococcus tauri reveals differential effects of environmental and endogenous 24-hour rhythms.

The cellular landscape changes dramatically over the course of a 24 h day. The proteome responds directly to daily environmental cycles and is additionally regulated by the circadian clock. To quantify the relative contribution of diurnal versus circadian regulation, we mapped proteome dynamics under light:dark cycles compared with constant light. Using Ostreococcus tauri, a prototypical eukaryotic cell, we achieved 85% coverage, which allowed an unprecedented insight into the identity of proteins that facilitate rhythmic cellular functions. The overlap between diurnally- and circadian-regulated proteins was modest and these proteins exhibited different phases of oscillation between the two conditions. Transcript oscillations were generally poorly predictive of protein oscillations, in which a far lower relative amplitude was observed. 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 different phase distribution compared with rhythmic soluble proteins, indicating the existence of a circadian regulatory process specific to the biogenesis and/or degradation of membrane proteins. Our observations argue that the cellular spatiotemporal proteome is shaped by a complex interaction between intrinsic and extrinsic regulatory factors through rhythmic regulation at the transcriptional as well as post-transcriptional, translational, and post-translational levels.

Targeted modification of the Per2 clock gene alters circadian function in mPer2luciferase (mPer2Luc) mice.

Modification of the Per2 clock gene in mPer2Luc reporter mice significantly alters circadian function. Behavioral period in constant dark is lengthened, and dissociates into two distinct components in constant light. Rhythms exhibit increased bimodality, enhanced phase resetting to light pulses, and altered entrainment to scheduled feeding. Mechanistic mathematical modelling predicts that enhanced protein interactions with the modified mPER2 C-terminus, combined with differential clock regulation among SCN subregions, can account for effects on circadian behavior via increased Per2 transcript and protein stability. PER2::LUC produces greater suppression of CLOCK:BMAL1 E-box activity than PER2. mPer2Luc carries a 72 bp deletion in exon 23 of Per2, and retains a neomycin resistance cassette that affects rhythm amplitude but not period. The results show that mPer2Luc acts as a circadian clock mutation illustrating a need for detailed assessment of potential impacts of c-terminal tags in genetically modified animal models.

Distinct circadian mechanisms govern cardiac rhythms and susceptibility to arrhythmia.

Electrical activity in the heart exhibits 24-hour rhythmicity, and potentially fatal arrhythmias are more likely to occur at specific times of day. Here, we demonstrate that circadian clocks within the brain and heart set daily rhythms in sinoatrial (SA) and atrioventricular (AV) node activity, and impose a time-of-day dependent susceptibility to ventricular arrhythmia. Critically, the balance of circadian inputs from the autonomic nervous system and cardiomyocyte clock to the SA and AV nodes differ, and this renders the cardiac conduction system sensitive to decoupling during abrupt shifts in behavioural routine and sleep-wake timing. Our findings reveal a functional segregation of circadian control across the heart's conduction system and inherent susceptibility to arrhythmia.