Robust and tunable circadian rhythms from differentially sensitive catalytic domains.

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.

Viscosity-dependent control of protein synthesis and degradation.

It has been proposed that the concentration of proteins in the cytoplasm maximizes the speed of important biochemical reactions. Here we have used Xenopus egg extracts, 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 find that protein synthesis rates are maximal in ~1x cytoplasm, whereas protein degradation continues to rise to a higher optimal concentration of ~1.8x. We show that this difference in optima can be attributed to a greater sensitivity of translation to cytoplasmic viscosity. The different concentration optima could produce 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.

Protein homeostasis from diffusion-dependent control of protein synthesis and degradation.

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.

Microglia overexpressing the macrophage colony-stimulating factor receptor are neuroprotective in a microglial-hippocampal organotypic coculture system.

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.

Circadian rhythms. A protein fold switch joins the circadian oscillator to clock output in cyanobacteria.

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-hour rhythms. The clock of cyanobacteria is driven by a three-protein oscillator composed 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, which initiates a phase transition of the circadian cycle, and it regulates components of the clock-output pathway, which provides the link that joins the timekeeping and signaling functions of the oscillator.

Rhythms in energy storage control the ability of the cyanobacterial circadian clock to reset.

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 are directly sensitive to metabolites. Reduced ATP/ADP ratio and oxidized quinones cause clock phase shifts in vitro. However, it is unclear what determines 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 overaccumulation of glycogen and subsequently high ATP in the dark. Conversely, we show that disruption of 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.

Dose-dependent effects of focal fractionated irradiation on secondary malignant neoplasms in Nf1 mutant mice.

Secondary malignant neoplasms (SMN) are increasingly common complications of cancer therapy that have proven difficult to model in mice. Clinical observations suggest that the development of SMN correlates with radiation dose; however, this relationship has not been investigated systematically. We developed a novel procedure for administering fractionated cranial irradiation (CI) and investigated the incidence and spectrum of cancer in control and heterozygous Nf1 mutant mice irradiated to a moderate (15 Gy) or high dose (30 Gy). Heterozygous Nf1 inactivation cooperated with CI to induce solid tumors and myeloid malignancies, with mice developing many of the most common SMNs found in human patients. CI-induced malignancies segregated according to radiation dose as Nf1(+/-) mice developed predominately hematologic abnormalities after 15 Gy, whereas solid tumors predominated at 30 Gy, suggesting that radiation dose thresholds exist for hematologic and nonhematologic cancers. Genetic and biochemical studies revealed discrete patterns of somatic Nf1 and Trp53 inactivation and we observed hyperactive Ras signaling in many radiation-induced solid tumors. This technique for administering focal fractionated irradiation will facilitate mechanistic and translational studies of SMNs.