CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping.

Circadian rhythms are a pervasive property of mammalian cells, tissues and behaviour, ensuring physiological adaptation to solar time. Models of cellular timekeeping revolve around transcriptional feedback repression, whereby CLOCK and BMAL1 activate the expression of PERIOD (PER) and CRYPTOCHROME (CRY), which in turn repress CLOCK/BMAL1 activity. CRY proteins are therefore considered essential components of the cellular clock mechanism, supported by behavioural arrhythmicity of CRY-deficient (CKO) mice under constant conditions. Challenging this interpretation, we find locomotor rhythms in adult CKO mice under specific environmental conditions and circadian rhythms in cellular PER2 levels when CRY is absent. CRY-less oscillations are variable in their expression and have shorter periods than wild-type controls. Importantly, we find classic circadian hallmarks such as temperature compensation and period determination by CK1δ/ε activity to be maintained. In the absence of CRY-mediated 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.

Daily magnesium fluxes regulate cellular timekeeping and energy balance.

Circadian clocks are fundamental to the biology of most eukaryotes, coordinating behaviour and physiology to resonate with the environmental cycle of day and night through complex networks of clock-controlled genes. A fundamental knowledge gap exists, however, between circadian gene expression cycles and the biochemical mechanisms that ultimately facilitate circadian regulation of cell biology. Here we report circadian rhythms in the intracellular concentration of magnesium ions, [Mg(2+)]i, which act as a cell-autonomous timekeeping component to determine key clock properties both in a human cell line and in a unicellular alga that diverged from each other more than 1 billion years ago. Given the essential role of Mg(2+) as a cofactor for ATP, a functional consequence of [Mg(2+)]i oscillations is dynamic regulation of cellular energy expenditure over the daily cycle. Mechanistically, we find that these rhythms provide bilateral feedback linking rhythmic metabolism to clock-controlled gene expression. The global regulation of nucleotide triphosphate turnover by intracellular Mg(2+) availability has potential to impact upon many of the cell's more than 600 MgATP-dependent enzymes and every cellular system where MgNTP hydrolysis becomes rate limiting. Indeed, we find that circadian control of translation by mTOR is regulated through [Mg(2+)]i oscillations. It will now be important to identify which additional biological processes are subject to this form of regulation in tissues of multicellular organisms such as plants and humans, in the context of health and disease.

Peroxiredoxins are conserved markers of circadian rhythms.

Cellular life emerged ∼3.7 billion years ago. With scant exception, terrestrial organisms have evolved under predictable daily cycles owing to the Earth's rotation. The advantage conferred on organisms that anticipate such environmental cycles has driven the evolution of endogenous circadian rhythms that tune internal physiology to external conditions. The molecular phylogeny of mechanisms driving these rhythms has been difficult to dissect because identified clock genes and proteins are not conserved across the domains of life: Bacteria, Archaea and Eukaryota. Here we show that oxidation-reduction cycles of peroxiredoxin proteins constitute a universal marker for circadian rhythms in all domains of life, by characterizing their oscillations in a variety of model organisms. Furthermore, we explore the interconnectivity between these metabolic cycles and transcription-translation feedback loops of the clockwork in each system. Our results suggest an intimate co-evolution of cellular timekeeping with redox homeostatic mechanisms after the Great Oxidation Event ∼2.5 billion years ago.

Domain swapping to assess the mechanistic basis of Arabidopsis phototropin 1 receptor kinase activation and endocytosis by blue light.

Phototropins (phot1 and phot2) are plasma membrane-associated receptor kinases that respond specifically to blue and UV wavelengths. In addition to a C-terminal Ser/Thr kinase domain, phototropins contain two N-terminal chromophore binding LOV domains that function as photoswitches to regulate a wide range of enzymatic activities in prokaryotes and eukaryotes. Through domain swapping, we show that the photochemical properties of Arabidopsis thaliana phot1 rely on interactions between LOV1 and LOV2, which are facilitated by their intervening linker sequence. Functional analysis of domain-swap proteins supports a mechanism whereby LOV2 acts as a dark-state repressor of phot1 activity both in vitro and in vivo. Moreover, we find a photoactive role for LOV1 in arresting chloroplast accumulation at high light intensities. Unlike LOV2, LOV1 cannot operate as a dark-state repressor, resulting in constitutive receptor autophosphorylation and accelerated internalization from the plasma membrane. Coexpression of active and inactive forms of phot1 demonstrates that autophosphorylation can occur intermolecularly, independent of LOV1, via light-dependent receptor dimerization in vivo. Indeed, transphosphorylation is sufficient to promote phot1 internalization through a clathrin-dependent endocytic pathway triggered primarily by phosphorylation of Ser-851 within the kinase activation loop. The mechanistic implications of these findings in regard to light-driven receptor activation and trafficking are discussed.

Mammalian Circadian Period, But Not Phase and Amplitude, Is Robust Against Redox and Metabolic Perturbations.

AIMS: Circadian rhythms permeate all levels of biology to temporally regulate cell and whole-body physiology, although the cell-autonomous mechanism that confers ∼24-h periodicity is incompletely understood. Reports describing circadian oscillations of over-oxidized peroxiredoxin abundance have suggested that redox signaling plays an important role in the timekeeping mechanism. Here, we tested the functional contribution that redox state and primary metabolism make to mammalian cellular timekeeping. RESULTS: We found a circadian rhythm in flux through primary glucose metabolic pathways, indicating rhythmic NAD(P)H production. Using pharmacological and genetic perturbations, however, we found that timekeeping was insensitive to changes in glycolytic flux, whereas oxidative pentose phosphate pathway (PPP) inhibition and other chronic redox stressors primarily affected circadian gene expression amplitude, not periodicity. Finally, acute changes in redox state decreased PER2 protein stability, phase dependently, to alter the subsequent phase of oscillation. INNOVATION: Circadian rhythms in primary cellular metabolism and redox state have been proposed to play a role in the cellular timekeeping mechanism. We present experimental data testing that hypothesis. CONCLUSION: Circadian flux through primary metabolism is cell autonomous, driving rhythmic NAD(P)+ redox cofactor turnover and maintaining a redox balance that is permissive for circadian gene expression cycles. Redox homeostasis and PPP flux, but not glycolysis, are necessary to maintain clock amplitude, but neither redox nor glucose metabolism determines circadian period. Furthermore, cellular rhythms are sensitive to acute changes in redox balance, at least partly through regulation of PER protein. Redox and metabolic state are, thus, both inputs and outputs, but not state variables, of cellular circadian timekeeping. Antioxid. Redox Signal. 28, 507-520.

Circadian actin dynamics drive rhythmic fibroblast mobilization during wound healing.

Fibroblasts are primary cellular protagonists of wound healing. They also exhibit circadian timekeeping, which imparts an approximately 24-hour rhythm to their biological function. We interrogated the functional consequences of the cell-autonomous clockwork in fibroblasts using a proteome-wide screen for rhythmically expressed proteins. We observed temporal coordination of actin regulators that drives cell-intrinsic rhythms in actin dynamics. In consequence, the cellular clock modulates the efficiency of actin-dependent processes such as cell migration and adhesion, which ultimately affect the efficacy of wound healing. Accordingly, skin wounds incurred during a mouse's active phase exhibited increased fibroblast invasion in vivo and ex vivo, as well as in cultured fibroblasts and keratinocytes. Our experimental results correlate with the observation that the time of injury significantly affects healing after burns in humans, with daytime wounds healing ~60% faster than nighttime wounds. We suggest that circadian regulation of the cytoskeleton influences wound-healing efficacy from the cellular to the organismal scale.

In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells.

Firefly luciferase (Fluc) is frequently used to report circadian gene expression rhythms in mammalian cells and tissues. During longitudinal assays it is generally assumed that enzymatic substrates are in saturating excess, such that total bioluminescence is directly proportional to Fluc protein level. To test this assumption, we compared the enzyme kinetics of purified luciferase with its activity in mammalian cells. We found that Fluc activity in solution has a lower Michaelis constant (Km) for luciferin, lower temperature dependence, and lower catalytic half-life than Fluc in cells. In consequence, extracellular luciferin concentration significantly affects the apparent circadian amplitude and phase of the widely used PER2::LUC reporter in cultured fibroblasts, but not in SCN, and we suggest that this arises from differences in plasma membrane luciferin transporter activity. We found that at very high concentrations (>1 mM), luciferin lengthens circadian period, in both fibroblasts and organotypic SCN slices. We conclude that the amplitude and phase of circadian gene expression inferred from bioluminescence recordings should be treated with some caution, and we suggest that optimal luciferin concentration should be determined empirically for each luciferase reporter and cell type.

The Pentose Phosphate Pathway Regulates the Circadian Clock.

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.

Metabolic Cycles in Yeast Share Features Conserved among Circadian Rhythms.

Cell-autonomous circadian rhythms allow organisms to temporally orchestrate their internal state to anticipate and/or resonate with the external environment. Although ∼24-hr periodicity is observed across aerobic eukaryotes, the central mechanism has been hard to dissect because few simple models exist, and known clock proteins are not conserved across phylogenetic kingdoms. In contrast, contributions to circadian rhythmicity made by a handful of post-translational mechanisms, such as phosphorylation of clock proteins by casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3), appear conserved among phyla. These kinases have many other essential cellular functions and are better conserved in their contribution to timekeeping than any of the clock proteins they phosphorylate. Rhythmic oscillations in cellular redox state are another universal feature of circadian timekeeping, e.g., over-oxidation cycles of abundant peroxiredoxin proteins. Here, we use comparative chronobiology to distinguish fundamental clock mechanisms from species and/or tissue-specific adaptations and thereby identify features shared between circadian rhythms in mammalian cells and non-circadian temperature-compensated respiratory oscillations in budding yeast. We find that both types of oscillations are coupled with the cell division cycle, exhibit period determination by CK1 and GSK3, and have peroxiredoxin over-oxidation cycles. We also explore how peroxiredoxins contribute to YROs. Our data point to common mechanisms underlying both YROs and circadian rhythms and suggest two interpretations: either certain biochemical systems are simply permissive for cellular oscillations (with frequencies from hours to days) or this commonality arose via divergence from an ancestral cellular clock.

Circadian redox and metabolic oscillations in mammalian systems.

SIGNIFICANCE: A substantial proportion of mammalian physiology is organized around the day/night cycle, being regulated by the co-ordinated action of numerous cell-autonomous circadian oscillators throughout the body. Disruption of internal timekeeping, by genetic or environmental perturbation, leads to metabolic dysregulation, whereas changes in metabolism affect timekeeping. RECENT ADVANCES: While gene expression cycles are essential for the temporal coordination of normal physiology, it has become clear that rhythms in metabolism and redox balance are cell-intrinsic phenomena, which may regulate gene expression cycles reciprocally, but persist in their absence. For example, a circadian rhythm in peroxiredoxin oxidation was recently observed in isolated human erythrocytes, fibroblast cell lines in vitro, and mouse liver in vivo. CRITICAL ISSUES: Mammalian timekeeping is a cellular phenomenon. While we understand many of the cellular systems that contribute to this biological oscillation's fidelity and robustness, a comprehensive mechanistic understanding remains elusive. Moreover, the formerly clear distinction between "core clock components" and rhythmic cellular outputs is blurred since several outputs, for example, redox balance, can feed back to regulate timekeeping. As with any cyclical system, establishing causality becomes problematic. FUTURE DIRECTIONS: A detailed molecular understanding of the temporal crosstalk between cellular systems, and the coincidence detection mechanisms that allow a cell to discriminate clock-relevant from irrelevant stimuli, will be essential as we move toward an integrated model of how this daily biological oscillation works. Such knowledge will highlight new avenues by which the functional consequences of circadian timekeeping can be explored in the context of human health and disease.

Mutational analysis of phototropin 1 provides insights into the mechanism underlying LOV2 signal transmission.

Phototropins (phot1 and phot2) are blue light-activated serine/threonine protein kinases that elicit a variety of photoresponses in plants. Light sensing by the phototropins is mediated by two flavin mononucleotide (FMN)-binding domains, designated LOV1 and LOV2, located in the N-terminal region of the protein. Exposure to light results in the formation of a covalent adduct between the FMN chromophore and a conserved cysteine residue within the LOV domain. LOV2 photoexcitation is essential for phot1 function in Arabidopsis and is necessary to activate phot1 kinase activity through light-induced structural changes within a conserved alpha-helix situated C-terminal to LOV2. Here we have used site-directed mutagenesis to identify further amino acid residues that are important for phot1 activation by light. Mutagenesis of bacterially expressed LOV2 and full-length phot1 expressed in insect cells indicates that perturbation of the conserved salt bridge on the surface of LOV2 does not play a role in receptor activation. However, mutation of a conserved glutamine residue (Gln(575)) within LOV2, reported previously to be required to propagate structural changes at the LOV2 surface, attenuates light-induced autophosphorylation of phot1 expressed in insect cells without compromising FMN binding. These findings, in combination with double mutant analyses, indicate that Gln(575) plays an important role in coupling light-driven cysteinyl adduct formation from within LOV2 to structural changes at the LOV2 surface that lead to activation of the C-terminal kinase domain.

Determination of the elemental composition of mature wheat grain using a modified secondary ion mass spectrometer (SIMS).

An imaging secondary ion mass spectrometry system has been developed that allows the distribution of elements or ions to be superimposed on an image of the plant cell or tissue generated by ion-induced secondary electrons. This system has been evaluated by analysing the aleurone and sub-aleurone cells of mature wheat grain, showing high spatial resolution (100-200 nm) images of O-, PO(2)-, Mg+, Ca+, Na+ and K+ within the phytate granules of the aleurone, with CN- being diagnostic for proteins and C(2)- being diagnostic for starch in the starchy endosperm cells. This system should provide improved localization of elements in a range of other plant systems.

A role for HKT1 in sodium uptake by wheat roots.

The high affinity potassium transporter, HKT1 from wheat was introduced into Florida wheat in sense and antisense orientation under control of a ubiquitin promoter. Ten transgenic lines expressing the transgene were identified and two of these showed strong down-regulation of the native HKT1 transcript. One line (271) was expressing the antisense construct and the other (223) was expressing a truncated sense construct. The two lines were examined further for phenotype relating to cation transport. Membrane depolarisations were measured in low (0.1 mm) K+ and high (100 mm) NaCl. Under these conditions there was no difference between line 271 and the control at low K+, but at high Na+ there was a rapid depolarisation that was significantly larger in control plants. 22Na uptake was measured in this line and there was a significant decrease in uptake at 100 mm NaCl in the transgenic line when compared with the control. The two transgenic lines were grown at high NaCl (200 mm) and analysed for growth and root sodium content. Lines 271 and 223 showed enhanced growth under salinity when compared with the control and had lower sodium in the root. Secondary ion mass spectrometry (SIMS) analysis of transverse sections of the root showed that Na+ and K+ were strongly localised to stelar regions when compared with other ions, and that the Na+ : K+ ratios were reduced in salt-stressed transgenic tissue when compared with the control.