Lost in clocks: non‐canonical circadian oscillation discovered in Drosophila cells (original) (raw)
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Understand the Timing Mechanism of Circadian Clock Proteins
Based on the cycles of day/night change, life on Earth evolved molecular mechanism to keep track of time. This timing mechanism arises from generating stable oscillations with a 24 hour period known as the circadian rhythm. Major physiologies such as metabolism, gene expression and cell division are tightly regulated by the circadian oscillatory machinery. To understand how the circadian oscillation is generated, our lab focuses on the circadian oscillator of cyanobacteria: a simple mixture of clock proteins KaiA, KaiB, and KaiC, and ATP produces a self- sustained ~24 h rhythm of KaiC phosphorylation. There are two distinct phases to this oscillator: in Phosphorylation Phase, KaiA binds to the C-terminal residues of KaiC known as the A-loop and stimulate KaiC autophosphorylation; in Dephosphorylation Phase, KaiA is inhibited from binding to A-loop by KaiB, then KaiC autodephosphorylates. Our goal here is to elucidate the transient and time-dependent interactions that are central to ...
Design Principles of Phosphorylation-Dependent Timekeeping in Eukaryotic Circadian Clocks
Cold Spring Harbor Perspectives in Biology, 2017
The circadian clock in cyanobacteria employs a posttranslational oscillator composed of a sequential phosphorylation-dephosphorylation cycle of KaiC protein, in which the dynamics of protein structural changes driven by temperature-compensated KaiC's ATPase activity are critical for determining the period. On the other hand, circadian clocks in eukaryotes employ transcriptional feedback loops as a core mechanism. In this system, the dynamics of protein accumulation and degradation affect the circadian period. However, recent studies of eukaryotic circadian clocks reveal that the mechanism controlling the circadian period can be independent of the regulation of protein abundance. Instead, the circadian substrate is often phosphorylated at multiple sites at flexible protein regions to induce structural changes. The phosphorylation is catalyzed by kinases that induce sequential multisite phosphorylation such as casein kinase 1 (CK1) with temperature-compensated activity. We propose that the design principles of phosphorylation-dependent circadian-period determination in eukaryotes may share characteristics with the posttranslational oscillator in cyanobacteria.
Molecular biological approach to the circadian clock mechanism
Neuroscience Research the Official Journal of the Japan Neuroscience Society, 1995
Many circadian phenomena have been described in a diverse range of species, from single cellular organisms to higher species of plants and animals. From several lines of evidence from Drosophila and Neurospora, the oscillation of the circadian clock seems to involve cycling gene expression. Although a great deal of information concerning the anatomy, neurophysiology and neurochemistry of circadian pacemakers has been obtained over the last decade, molecular and cellular approaches to this problem have only just begun. I will summarize recent progress of the molecular biological approach to the circadian clock mechanism. Finally, the importance of' transcription factors to envision the common mechanism of circadian clock in the diverged species will be discussed considering with the existence of a hypothetical 'Time Box'.
Neuron, 2001
fluh et al., 2000c). Strong hypomorphic mutations of dbt also influence cell proliferation and cell survival (Kloss et al., 1998; Zilian et al., 1999). dbt encodes a protein similar to human casein kinase I⑀ (CKI⑀; Kloss et al., 1998). Because PER and DBT form a complex both in The Rockefeller University vitro and in cultured Drosophila S2 cells (Kloss et al., 1998), PER may be a direct substrate of DBT. Such a 1230 York Avenue New York, New York 10021 role for DBT is also suggested by recent findings in mammals. The tau mutation, which causes short-period (20 hr) circadian behavior in the hamster, involves an amino acid change in CKI⑀ (Lowrey et al., 2000). The Summary wild-type hamster kinase binds and phosphorylates mammalian PER proteins in vitro and in cultured cells, The clock gene double-time (dbt) encodes an ortholog but PER phosphorylation is defective when the tau muof casein kinase I⑀ that promotes phosphorylation and tant kinase is employed in such assays (Lowrey et al., turnover of the PERIOD protein. Whereas the period 2000). In humans, Familial Advance Sleep-Phase Syn-(per), timeless (tim), and dClock (dClk) genes of Drodrome (FASPS) is caused by a missense mutation in sophila each contribute cycling mRNA and protein to hPer2. The mutation maps to a region of hPER2 that a circadian clock, dbt RNA and DBT protein are constibinds to CKI⑀, and changes a consensus CKI⑀ target site. tutively expressed. Robust circadian changes in DBT This mutation shortens the period of human circadian subcellular localization are nevertheless observed in rhythms and causes hypophosphorylation of hPER2 by clock-containing cells of the fly head. These localiza-CKI⑀ in vitro (Toh et al., 2001). tion rhythms accompany formation of protein com-In contrast to its mammalian orthologs, bacterially plexes that include PER, TIM, and DBT, and reflect produced, recombinant DBT has shown no enzymatic periodic redistribution between the nucleus and the activity in vitro, suggesting a requirement for additional cytoplasm. Nuclear phosphorylation of PER is strongly factors, or presence of an activating modification of DBT enhanced when TIM is removed from PER/TIM/DBT or its substrate(s) in vivo (Suri et al., 2000; S. Kivimä e, complexes. The varying associations of PER, DBT and L.S., and M.W.Y., unpublished observation). Neverthe-TIM appear to determine the onset and duration of less, it has been demonstrated that PER phosphorylanuclear PER function within the Drosophila clock. tion is dependent on DBT in pacemaker cells of the organism, properties not yet demonstrated for mamma-Introduction lian PER and casein kinase I⑀ (Price et al., 1998; Kloss et al., 1998; Lowrey et al., 2000; Vielhaber et al., 2000; In Drosophila, circadian behavioral rhythms are con-Toh et al., 2001). trolled by "clock" genes that interact to form autoregula-In this report, we show that the level of DBT protein tory loops (Dunlap, 1999; Scully and Kay, 2000; Young, in flies is not under circadian control. However, DBT 2000). Activation of the period (per) and timeless (tim) shows striking circadian changes in subcellular localizagenes depends on two transcription factors, dCLOCK tion in photoreceptor cells and in the brain's pacemaker (CLK) and CYCLE (CYC), which bind to the per and tim cells, the lateral neurons. The localization rhythms coinpromoters. PER and TIM proteins suppress the activities cide with changes in the subcellular distribution of PER of dCLK and CYC, but only after PER and TIM heterodiproteins. We demonstrate that DBT is found in commerize and translocate to nuclei. The PER and TIM proplexes with PER at all times in vivo. Association of DBT teins also positively regulate levels of dClk RNA (Dunlap, with TIM occurs only in the presence of PER. We also 1999; Scully and Kay, 2000; Young, 2000). Formation of show that dissociation of PER from TIM proteins coin-PER/TIM complexes is delayed by the action of DOUcides with increasing phosphorylation of nuclear PER. BLE-TIME (DBT), a kinase that promotes PER phosphor-Because elimination of TIM does not stimulate PER ylation and turnover in the absence of high levels of TIM phosphorylation in the absence of DBT (Price et al., (further reviewed later). These regulatory interactions 1998), we suggest that TIM excludes an activity of DBT lead to cycling expression of per, tim, and dClk (Scully when the latter protein is associated with a PER/TIM and Kay, 2000; Young, 2000). complex. These protein interactions should help deter-Mutant alleles of dbt have been identified that shorten, mine the timing of PER's phosphorylation and turnover lengthen, or abolish circadian behavioral rhythms. Such in the nucleus. mutations similarly affect per and tim mRNA and protein oscillations (Price et al., 1998; Suri et al., 2000; Rothen-Results
Circadian clocks drive the daily rhythms in our physiology and behaviour that adapt us to the 24-h solar and social worlds. Because they impinge upon every facet of metabolism, their acute or chronic disruption compromises performance (both physical and mental) and systemic health, respectively. Equally, the presence of such rhythms has significant implications for pharmacological dynamics and efficacy, because the fate of a drug and the state of its therapeutic target will vary as a function of time of day. Improved understanding of the cellular and molecular biology of circadian clocks therefore offers novel approaches for therapeutic development, for both clock-related and other conditions. At the cellular level, circadian clocks are pivoted around a transcriptional/post-translational delayed feedback loop (TTFL) in which the activation of Period and Cryptochrome genes is negatively regulated by their cognate protein products. Synchrony between these, literally countless, cellular clocks across the organism is maintained by the principal circadian pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus. Notwithstanding the success of the TTFL model, a diverse range of experimental studies has shown that it is insufficient to account for all properties of cellular pacemaking. Most strikingly, circadian cycles of metabolic status can continue in human red blood cells, devoid of nuclei and thus incompetent to sustain a TTFL. Recent interest has therefore focused on the role of oscillatory cytosolic mechanisms as partners to the TTFL. In particular, cAMP-and Ca 2+ -dependent signalling are important components of the clock, whilst timekeeping activity is also sensitive to a series of highly conserved kinases and phosphatases. This has led , # Springer-Verlag Berlin Heidelberg 2013 67 to the view that the 'proto-clock' may have been a cytosolic, metabolic oscillation onto which evolution has bolted TTFLs to provide robustness and amplify circadian outputs in the form of rhythmic gene expression. This evolutionary ascent of the clock has culminated in the SCN, a true pacemaker to the innumerable clock cells distributed across the body. On the basis of findings from our own and other laboratories, we propose a model of the SCN pacemaker that synthesises the themes of TTFLs, intracellular signalling, metabolic flux and interneuronal coupling that can account for its unique circadian properties and pre-eminence.
Circadian rhythms from multiple oscillators: lessons from diverse organisms
Nature Reviews Genetics, 2005
| The organization of biological activities into daily cycles is universal in organisms as diverse as cyanobacteria, fungi, algae, plants, flies, birds and man. Comparisons of circadian clocks in unicellular and multicellular organisms using molecular genetics and genomics have provided new insights into the mechanisms and complexity of clock systems. Whereas unicellular organisms require stand-alone clocks that can generate 24-hour rhythms for diverse processes, organisms with differentiated tissues can partition clock function to generate and coordinate different rhythms. In both cases, the temporal coordination of a multi-oscillator system is essential for producing robust circadian rhythms of gene expression and biological activity.