Organisms are adapted to the relentless cycles of day and night, because they evolved timekeeping systems called circadian clocks, which regulate biological activities with ~24-h rhythms. The clock of cyanobacteria is driven by a three-protein oscillator comprised of KaiA, KaiB, and KaiC, which together generate a circadian rhythm of KaiC phosphorylation. We show that KaiB flips between two distinct three-dimensional folds, and its rare transition to an active state provides a time delay that is required to match the timing of the oscillator to that of earth’s rotation. Once KaiB switches folds, it binds phosphorylated KaiC and captures KaiA, initiating a phase transition of the circadian cycle, and regulates components of the clock-output pathway, providing the link that joins the timekeeping and signaling functions of the oscillator.

Circadian clocks are ubiquitous biological oscillators that coordinate an organism's behavior with the daily cycling of the external environment. To ensure synchronization with the environment, the period of the clock must be maintained near 24 h even as amplitude and phase are altered by input signaling. We show that, in a reconstituted circadian system from cyanobacteria, these conflicting requirements are satisfied by distinct functions for two domains of the central clock protein KaiC: the C-terminal autokinase domain integrates input signals through the ATP/ADP ratio, and the slow N-terminal ATPase acts as an input-independent timer. We find that phosphorylation in the C-terminal domain followed by an ATPase cycle in the N-terminal domain is required to form the inhibitory KaiB•KaiC complexes that drive the dynamics of the clock. We present a mathematical model in which this ATPase-mediated delay in negative feedback gives rise to a compensatory mechanism that allows a tunable phase and amplitude while ensuring a robust circadian period.biological clock | in vitro | robustness | metabolic input processing C ircadian clocks are endogenous oscillatory systems that allow an organism to anticipate daily rhythmic variations in the external environment. Despite having apparently diverse molecular origins, the functional properties of circadian systems are remarkably conserved across most organisms that have been studied. Even when deprived of a rhythmic input, circadian clocks continue to generate self-sustaining oscillations, and these oscillations have a period that is close to 24 h over a wide range of conditions, including varying light levels, nutrient abundance, and temperature (1). The robust period of the oscillator seems to be critical for its physiological role: in multiple organisms, mutants with clocks whose free-running periods are markedly different from 24 h are associated with decreased lifespan and fitness defects (2-4). In contrast, the phase and amplitude of circadian rhythms are generally tunable, and input signals can reset the oscillator to efficiently bring the clock into synchrony with a rhythmic environment.The competing demands for both a sensitive response to input signaling and a robustly invariant oscillator period place strong constraints on the possible mechanisms underlying the circadian clock. It is currently unclear how the biochemical circuitry that generates circadian rhythms satisfies these constraints in any organism. To study the molecular origins of robust periodicity, we analyzed the biochemically tractable circadian oscillator from the cyanobacterium Synechococcus elongatus.The Synechococcus core oscillator can be reconstituted in vitro using three purified proteins, KaiA, KaiB, and KaiC, and ATP (Fig. 1A). KaiC is a multifunctional enzyme with slow ATPase, autokinase, and autophosphatase activities. The effector proteins KaiA and KaiB work together to modulate KaiC's enzymatic activity in a phosphorylation-dependent manner, switching the system between kinase-and phospha...

Summary Circadian clocks are oscillatory systems that schedule daily rhythms of organismal behavior. The ability of the clock to reset its phase in response to external signals is critical for proper synchronization with the environment. In the model clock from cyanobacteria, the KaiABC proteins that comprise the core oscillator [1, 2] are directly sensitive to metabolites. Reduced ATP/ADP ratio and the oxidized quinones cause clock phase shifts in vitro [3, 4]. But it is unclear what determine the metabolic response of the cell to darkness and thus the magnitude of clock resetting. We show that the cyanobacterial circadian clock generates a rhythm in metabolism that causes cells to accumulate glycogen in anticipation of nightfall. Mutation of the histidine kinase CikA creates an insensitive clock input phenotype by misregulating clock output genome-wide, leading to over-accumulation of glycogen and subsequently high ATP in the dark. Conversely, we show that disrupting glycogen metabolism results in low ATP in the dark and makes the clock hypersensitive to dark pulses. The observed changes in cellular energy are sufficient to recapitulate phase shifting phenotypes in an in vitro model of the clock. Our results show that clock input phenotypes can arise from metabolic dysregulation and illustrate a framework for circadian biology where clock outputs feed back through metabolism to control input mechanisms.

Microglia with increased expression of the macrophage colony-stimulating factor receptor (M-CSFR; c-fms) are found surrounding plaques in Alzheimer's disease (AD) and in mouse models for AD and after ischemic or traumatic brain injury. Increased expression of M-CSFR causes microglia to adopt an activated state that results in proliferation, release of cytokines, and enhanced phagocytosis. To determine whether M-CSFR-induced microglial activation affects neuronal survival, we assembled a coculture system consisting of BV-2 microglia transfected to overexpress the M-CSFR and hippocampal organotypic slices treated with NMDA. Twenty-four hours after assembly of the coculture, microglia overexpressing M-CSFR proliferated at a higher rate than nontransfected control cells and exhibited enhanced migration toward NMDA-injured hippocampal cultures. Surprisingly, coculture with c-fms-transfected microglia resulted in a dramatic reduction in NMDA-induced neurotoxicity. Similar results were observed when cocultures were treated with the teratogen cyclophosphamide. Biolistic overexpression of M-CSFR on microglia endogenous to the organotypic culture also rescued neurons from excitotoxicity. Furthermore, c-fms-transfected microglia increased neuronal expression of macrophage colony-stimulating factor (M-CSF), the M-CSFR, and neurotrophin receptors in the NMDA-treated slices, as determined with laser capture microdissection. In the coculture system, direct contact between the exogenous microglia and the slice was necessary for neuroprotection. Finally, blocking expression of the M-CSF ligand by exogenous c-fms-transfected microglia with a hammerhead ribozyme compromised their neuroprotective properties. These results demonstrate a protective role for microglia overexpressing M-CSFR in our coculture system and suggest under certain circumstances, activated microglia can help rather than harm neurons subjected to excitotoxic and teratogen-induced injury.

It has been proposed that the concentration of proteins in the cytoplasm maximizes the speed of important biochemical reactions. Here we have used the Xenopus extract system, which can be diluted or concentrated to yield a range of cytoplasmic protein concentrations, to test the effect of cytoplasmic concentration on mRNA translation and protein degradation. We found that protein synthesis rates are maximal in ~1x cytoplasm, whereas protein degradation continues to rise to an optimal concentration of ~1.8x. This can be attributed to the greater sensitivity of translation to cytoplasmic viscosity, perhaps because it involves unusually large macromolecular complexes like polyribosomes. The different concentration optima sets up a negative feedback homeostatic system, where increasing the cytoplasmic protein concentration above the 1x physiological level increases the viscosity of the cytoplasm, which selectively inhibits translation and drives the system back toward the 1x set point.

No abstract

Supplementary Table 2 from Dose-Dependent Effects of Focal Fractionated Irradiation on Secondary Malignant Neoplasms in <i>Nf1</i> Mutant Mice

Supplementary Table 1 from Dose-Dependent Effects of Focal Fractionated Irradiation on Secondary Malignant Neoplasms in <i>Nf1</i> Mutant Mice