SUMMARY Studies in mammals have indicated a connection between circadian clocks and feeding behavior, but the nature of the interaction, and its relationship to nutrient metabolism, are not understood. In Drosophila clock proteins are expressed in many metabolically important tissues, but have not been linked to metabolic processes. Here we demonstrate that Drosophila feeding behavior displays a 24-hour circadian rhythm which is regulated by clocks in digestive/metabolic tissues. Flies lacking clocks in these tissues, in particular in the fat body, also display increased food consumption, but have decreased levels of glycogen and a higher sensitivity to starvation. Interestingly, glycogen levels and starvation sensitivity are also affected by clocks in neuronal cells but the effects of neuronal clocks generally oppose those of the fat body. We propose that the input of neuronal clocks and clocks in metabolic tissues is coordinated to provide effective energy homeostasis.

Organisms ranging from bacteria to humans synchronize their internal clocks to daily cycles of light and dark. Photic entrainment of the Drosophila clock is mediated by proteasomal degradation of the clock protein TIMELESS (TIM). We have identified mutations in jetlag-a gene coding for an Fbox protein with leucine-rich repeats-that result in reduced light sensitivity of the circadian clock. Mutant flies show rhythmic behavior in constant light, reduced phase shifts in response to light pulses, and reduced light-dependent degradation of TIM. Expression of JET along with the circadian photoreceptor cryptochrome (CRY) in cultured S2R+ cells confers light-dependent degradation onto TIM, thereby reconstituting the acute response of the circadian clock to light in a cell culture system. Our results suggest that JET is essential for resetting the clock by transmitting light signals from CRY to TIM.

SUMMARY Though much is known about the cellular and molecular components of the circadian clock, output pathways that couple clock cells to overt behaviors have not been identified. We conducted a screen for circadian-relevant neurons in the Drosophila brain, and report here that cells of the pars intercerebralis (PI), a functional homologue of the mammalian hypothalamus, comprise an important component of the circadian output pathway for rest:activity rhythms. GRASP analysis demonstrates that PI cells are connected to the clock through a polysynaptic circuit extending from pacemaker cells to PI neurons. Molecular profiling of relevant PI cells identified the corticotropin releasing factor (CRF) homologue, DH44, as a circadian output molecule that is specifically expressed by PI neurons and required for normal rest:activity rhythms. Notably, selective activation or ablation of just 6 DH44+ PI cells causes arrhythmicity. These findings delineate a circuit through which clock cells can modulate locomotor rhythms.

The posttranscriptional mechanisms that control the cycling of circadian clock protein levels are not known. Here we demonstrate a role for protein phosphatase 2A (PP2A) in the cyclic expression of the PER protein. PP2A regulatory subunits TWS and WDB target PER and stabilize it in S2 cells. In adult fly heads, expression of tws cycles robustly under control of the circadian clock. Hypomorphic tws mutants show delayed accumulation of PER, while overexpression of tws in clock neurons produces shorter, weaker rhythms. Reduction of PP2A activity reduces PER expression in central clock neurons and results in long periods and arrhythmia. In addition, overexpression of the PP2A catalytic subunit results in loss of behavioral rhythms and constitutive nuclear expression of PER. PP2A also affects PER phosphorylation in vitro and in vivo. We propose that the posttranslational mechanisms that drive cycling of PER require the rhythmic expression of PP2A.