RESUMO
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.
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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.
Assuntos
Substâncias Macromoleculares , Proteínas , Solventes , Termodinâmica , Água , Morte Celular , Citosol/química , Citosol/metabolismo , Homeostase , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Concentração Osmolar , Pressão , Proteínas/química , Proteínas/metabolismo , Solventes/química , Solventes/metabolismo , Temperatura , Fatores de Tempo , Água/química , Água/metabolismoRESUMO
Circadian rhythms in physiology and behavior allow organisms to anticipate the daily environmental changes imposed by the rotation of our planet around its axis. Although these rhythms eventually manifest at the organismal level, a cellular basis for circadian rhythms has been demonstrated. Significant contributors to these cell-autonomous rhythms are daily cycles in gene expression and protein translation. However, recent data revealed cellular rhythms in other biological processes, including ionic currents, ion transport, and cytosolic ion abundance. Circadian rhythms in ion currents sustain circadian variation in action potential firing rate, which coordinates neuronal behavior and activity. Circadian regulation of metal ions abundance and dynamics is implicated in distinct cellular processes, from protein translation to membrane activity and osmotic homeostasis. In turn, studies showed that manipulating ion abundance affects the expression of core clock genes and proteins, suggestive of a close interplay. However, the relationship between gene expression cycles, ion dynamics, and cellular function is still poorly characterized. In this review, I will discuss the mechanisms that generate ion rhythms, the cellular functions they govern, and how they feed back to regulate the core clock machinery.
Assuntos
Relógios Circadianos , Ritmo Circadiano , Ritmo Circadiano/fisiologia , Neurônios/metabolismo , Fenômenos Fisiológicos Celulares , Homeostase , Relógios Circadianos/genéticaRESUMO
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.
Assuntos
Ritmo Circadiano/fisiologia , Criptocromos/metabolismo , Proteostase , Animais , Criptocromos/deficiência , Transporte de Íons , Camundongos , Fosfoproteínas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteoma/metabolismo , Proteômica , Reprodutibilidade dos Testes , Estresse Fisiológico , Fatores de TempoRESUMO
Circadian rhythms are â¼24 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.
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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.
Assuntos
Proteínas de Algas/genética , Clorófitas/fisiologia , Meio Ambiente , Periodicidade , Proteoma/genética , Proteínas de Algas/metabolismo , Clorófitas/genética , Proteoma/metabolismo , Análise Espaço-TemporalRESUMO
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.
Assuntos
Fenômenos Fisiológicos Celulares , Ritmo Circadiano/fisiologia , Transporte de Íons/fisiologia , Osmose , Animais , Sistema Cardiovascular/patologia , Células Cultivadas , Cloretos/metabolismo , Fibroblastos , Homeostase , Pulmão , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Potássio/metabolismo , Proteoma , Sódio/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/genéticaRESUMO
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.
Assuntos
Arritmias Cardíacas/fisiopatologia , Nó Atrioventricular/fisiologia , Ritmo Circadiano/fisiologia , Frequência Cardíaca/fisiologia , Miócitos Cardíacos/fisiologia , Nó Sinoatrial/fisiologia , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Adulto , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Nó Atrioventricular/metabolismo , Sistema Nervoso Autônomo/fisiologia , Relógios Circadianos/fisiologia , Eletrocardiografia , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismo , Nó Sinoatrial/metabolismo , Sono/fisiologiaRESUMO
Ness-Cohn et al claim that our observations of transcriptional circadian rhythms in the absence of the core clock gene Bmal1 in mouse skin fibroblast cells are supported by inadequate evidence. They claim that they were unable to reproduce some of the original ï¬ndings with their reanalysis. We disagree with their analyses and outlook.
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Fatores de Transcrição ARNTL , Ritmo Circadiano , Fatores de Transcrição ARNTL/genética , Animais , Ritmo Circadiano/genética , CamundongosRESUMO
Abruzzi et al argue that transcriptome oscillations found in our study in the absence of Bmal1 are of low amplitude, statistical significance, and consistency. However, their conclusions rely solely on a different statistical algorithm than we used. We provide statistical measures and additional analyses showing that our original analyses and observations are accurate. Further, we highlight independent lines of evidence indicating Bmal1-independent 24-hour molecular oscillations.
Assuntos
Fatores de Transcrição ARNTL , Ritmo Circadiano , Fatores de Transcrição ARNTL/genética , Ritmo Circadiano/genética , TranscriptomaRESUMO
Inductively coupled plasma mass spectrometry (ICP-MS) is a sensitive instrumental analysis technique used for multielemental and isotopic determination. Here we provide a sample preparation and circadian ICP-MS analysis protocol for use with mammalian tissues and cells, using mouse fibroblasts as a case study.
Assuntos
Relógios Circadianos , Espectrometria de Massas/métodos , Animais , Células Cultivadas , Fibroblastos/metabolismo , Espectrometria de Massas/instrumentação , CamundongosRESUMO
The ability to image single molecules in living cells has been impaired by the absence of bright, photostable fluorophores. Quantum dots (QDs) offer an attractive solution to this problem due to their exceptional photostability and brightness. Here, we describe in detail a protocol to chemically deliver functionalized QDs into the cytosol of living cells, based on cell-penetrating poly(disulfide)s (CPDs). This protocol is highly efficient and delivers hundreds of QDs per cell after incubation of cells with functionalized QDs at nanomolar concentrations. We also detail a pipeline for automated detection and tracking of diffusive QDs in living cells, which may provide a useful means to study the biophysical properties of the cytosol and their dynamics. Last, we describe a protocol for conjugating streptavidin fusion proteins to QDs, in order to permit the codelivery of QDs with functional proteins of interest into cells. The protocol has been successfully applied to a broad range of different cell types, thus offering a flexible and generalizable means to image single molecules in living cells.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Pontos Quânticos/química , Imagem Individual de Molécula/métodos , Citosol/metabolismo , Difusão , Corantes Fluorescentes/metabolismo , Nanotecnologia , Proteínas/metabolismo , Coloração e Rotulagem/métodosRESUMO
Circadian (~24 hour) clocks have a fundamental role in regulating daily physiology. The transcription factor BMAL1 is a principal driver of a molecular clock in mammals. Bmal1 deletion abolishes 24-hour activity patterning, one measure of clock output. We determined whether Bmal1 function is necessary for daily molecular oscillations in skin fibroblasts and liver slices. Unexpectedly, in Bmal1 knockout mice, both tissues exhibited 24-hour oscillations of the transcriptome, proteome, and phosphoproteome over 2 to 3 days in the absence of any exogenous drivers such as daily light or temperature cycles. This demonstrates a competent 24-hour molecular pacemaker in Bmal1 knockouts. We suggest that such oscillations might be underpinned by transcriptional regulation by the recruitment of ETS family transcription factors, and nontranscriptionally by co-opting redox oscillations.
Assuntos
Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/fisiologia , Relógios Circadianos/genética , Ritmo Circadiano/genética , Fígado/fisiologia , Fenômenos Fisiológicos da Pele , Animais , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Deleção de Genes , Regulação da Expressão Gênica , Fígado/metabolismo , Camundongos , Camundongos Knockout , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteoma/fisiologia , Transcrição Gênica , Transcriptoma/fisiologiaRESUMO
In mammals, endogenous circadian clocks sense and respond to daily feeding and lighting cues, adjusting internal â¼24 h rhythms to resonate with, and anticipate, external cycles of day and night. The mechanism underlying circadian entrainment to feeding time is critical for understanding why mistimed feeding, as occurs during shift work, disrupts circadian physiology, a state that is associated with increased incidence of chronic diseases such as type 2 (T2) diabetes. We show that feeding-regulated hormones insulin and insulin-like growth factor 1 (IGF-1) reset circadian clocks in vivo and in vitro by induction of PERIOD proteins, and mistimed insulin signaling disrupts circadian organization of mouse behavior and clock gene expression. Insulin and IGF-1 receptor signaling is sufficient to determine essential circadian parameters, principally via increased PERIOD protein synthesis. This requires coincident mechanistic target of rapamycin (mTOR) activation, increased phosphoinositide signaling, and microRNA downregulation. Besides its well-known homeostatic functions, we propose insulin and IGF-1 are primary signals of feeding time to cellular clocks throughout the body.
Assuntos
Relógios Circadianos/fisiologia , Comportamento Alimentar/fisiologia , Proteínas Circadianas Period/metabolismo , Animais , Ritmo Circadiano/fisiologia , Feminino , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Mamíferos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptor IGF Tipo 1/metabolismo , Transdução de SinaisRESUMO
Mg2+ is an essential ion for the cell but whether it can act as a bona fide second messenger has long been questioned. A recent study supports this hypothesis and shows a signalling role for Mg2+ in GABA-mediated neuronal maturation.
Assuntos
Magnésio , Sistemas do Segundo Mensageiro , Citoplasma , Transdução de Sinais , Ácido gama-AminobutíricoRESUMO
cAMP/PKA signalling is compartmentalised with tight spatial and temporal control of signal propagation underpinning specificity of response. The cAMP-degrading enzymes, phosphodiesterases (PDEs), localise to specific subcellular domains within which they control local cAMP levels and are key regulators of signal compartmentalisation. Several components of the cAMP/PKA cascade are located to different mitochondrial sub-compartments, suggesting the presence of multiple cAMP/PKA signalling domains within the organelle. The function and regulation of these domains remain largely unknown. Here, we describe a novel cAMP/PKA signalling domain localised at mitochondrial membranes and regulated by PDE2A2. Using pharmacological and genetic approaches combined with real-time FRET imaging and high resolution microscopy, we demonstrate that in rat cardiac myocytes and other cell types mitochondrial PDE2A2 regulates local cAMP levels and PKA-dependent phosphorylation of Drp1. We further demonstrate that inhibition of PDE2A, by enhancing the hormone-dependent cAMP response locally, affects mitochondria dynamics and protects from apoptotic cell death.
Assuntos
Apoptose , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Dinaminas/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Animais , Linhagem Celular , Humanos , Camundongos , Fosforilação , Processamento de Proteína Pós-Traducional , RatosRESUMO
The circadian clock is a ubiquitous timekeeping system that organizes the behavior and physiology of organisms over the day and night. Current models rely on transcriptional networks that coordinate circadian gene expression of thousands of transcripts. However, recent studies have uncovered phylogenetically conserved redox rhythms that can occur independently of transcriptional cycles. Here we identify the pentose phosphate pathway (PPP), a critical source of the redox cofactor NADPH, as an important regulator of redox and transcriptional oscillations. Our results show that genetic and pharmacological inhibition of the PPP prolongs the period of circadian rhythms in human cells, mouse tissues, and fruit flies. These metabolic manipulations also cause a remodeling of circadian gene expression programs that involves the circadian transcription factors BMAL1 and CLOCK, and the redox-sensitive transcription factor NRF2. Thus, the PPP regulates circadian rhythms via NADPH metabolism, suggesting a pivotal role for NADPH availability in circadian timekeeping.