A novel period mutation implicating nuclear export in temperature compensation of the Drosophila circadian clock.

Circadian clocks are self-sustained molecular oscillators controlling daily changes of behavioral activity and physiology. For functional reliability and precision, the frequency of these molecular oscillations must be stable at different environmental temperatures, known as "temperature compensation." Despite being an intrinsic property of all circadian clocks, this phenomenon is not well understood at the molecular level. Here, we use behavioral and molecular approaches to characterize a novel mutation in the period (per) clock gene of Drosophila melanogaster, which alters a predicted nuclear export signal (NES) of the PER protein and affects temperature compensation. We show that this new perI530A allele leads to progressively longer behavioral periods and clock oscillations with increasing temperature in both clock neurons and peripheral clock cells. While the mutant PERI530A protein shows normal circadian fluctuations and post-translational modifications at cool temperatures, increasing temperatures lead to both severe amplitude dampening and hypophosphorylation of PERI530A. We further show that PERI530A displays reduced repressor activity at warmer temperatures, presumably because it cannot inactivate the transcription factor CLOCK (CLK), indicated by temperature-dependent altered CLK post-translational modification in perI530A flies. With increasing temperatures, nuclear accumulation of PERI530A within clock neurons is increased, suggesting that wild-type PER is exported out of the nucleus at warm temperatures. Downregulating the nuclear export factor CRM1 also leads to temperature-dependent changes of behavioral rhythms, suggesting that the PER NES and the nuclear export of clock proteins play an important role in temperature compensation of the Drosophila circadian clock.

Real time, in vivo measurement of neuronal and peripheral clocks in Drosophila melanogaster.

Circadian clocks are highly conserved transcriptional regulators that control ~24 hr oscillations in gene expression, physiological function, and behavior. Circadian clocks exist in almost every tissue and are thought to control tissue-specific gene expression and function, synchronized by the brain clock. Many disease states are associated with loss of circadian regulation. How and when circadian clocks fail during pathogenesis remains largely unknown because it is currently difficult to monitor tissue-specific clock function in intact organisms. Here, we developed a method to directly measure the transcriptional oscillation of distinct neuronal and peripheral clocks in live, intact Drosophila, which we term Locally Activatable BioLuminescence, or LABL. Using this method, we observed that specific neuronal and peripheral clocks exhibit distinct transcriptional properties. Loss of the receptor for PDF, a circadian neurotransmitter critical for the function of the brain clock, disrupts circadian locomotor activity but not all tissue-specific circadian clocks. We found that, while peripheral clocks in non-neuronal tissues were less stable after the loss of PDF signaling, they continued to oscillate. We also demonstrate that distinct clocks exhibit differences in their loss of oscillatory amplitude or their change in period, depending on their anatomical location, mutation, or fly age. Our results demonstrate that LABL is an effective tool that allows rapid, affordable, and direct real-time monitoring of individual clocks in vivo.

The daily rhythms in our lives are driven by biological mechanisms called circadian clocks. These biological clocks are protein machines found in almost every cell and organ of the body, in nearly all living things, from fungi and plants to fruit flies and humans. These clocks control 24-hour cycles of gene activity and behaviour, and are kept in-time by so-called 'master clocks' in the brain. Ideally, scientists would be able to observe how circadian clocks work in different parts of the brain in a living animal and track changes throughout the day, as the animal performs different behaviours. However, the tools that are currently available to study circadian clocks do not allow this. To overcome this difficulty, Johnstone et al. used fruit flies to develop a new method that allows scientists to measure the oscillations of the circadian clocks in the brain in real time. Circadian clocks are composed of proteins called 'transcription factors' that activate different genes throughout the day, producing different proteins at different times. Transcription factors control the activity of genes by binding to DNA sequences called 'promoters' and switching the genes regulated by these promoters on or off. Knowing this, Johnstone et al. engineered fruit flies to carry the gene that codes for a protein called luciferase, which emits light, and placed it under the control of the promoter for the period gene, a gene that is regulated by the circadian clock. To prevent all of the cells in the fly from producing luciferase any time the period promoter was active, Johnstone et al. placed a second gene between the promoter and the luciferase gene. This second gene contains 'stop' sequences that prevent luciferase from being produced as long as the second gene is present. Importantly, this gene can be genetically removed from specific cells in live flies, so only these cells will produce luciferase. When Johnstone et al. removed the second gene from specific cells in the fly brain that are involved in controlling behaviours related to the circadian clocks, these cells started emitting light in cycles that reproduced the activity of the circadian clocks. Thus, by monitoring how the brightness of luciferase changed throughout the day in these flies, Johnstone et al. were able to reveal how the circadian clocks work in different parts of the fly brain. They found that each clock had slightly different cycling lengths, suggesting that the clocks work differently in different parts of the brain to control behaviour. Interestingly, Johnstone et al. found that if a key gene responsible for communication between cells was mutated, the effects of the mutation also varied in different parts of the brain. This suggests that different clocks respond differently to communication cues. Additionally, the results showed that circadian clock activity also changed with age: older flies had weaker circadian behaviours ­ fewer changes in both behavioural and genetic activity levels between the day and night ­ than younger animals. Johnstone et al.'s approach makes it possible to track a living animal's circadian clocks in different parts of the brain and in different organs in real time without the need to dissect the animal. In the future, this method will help scientists understand the links between different circadian clocks, the genes associated with them, and the behaviours they control.

The yeast 2-micron plasmid Rep2 protein has Rep1-independent partitioning function.

Equal partitioning of the multi-copy 2-micron plasmid of the budding yeast Saccharomyces cerevisiae requires association of the plasmid Rep1 and Rep2 proteins with the plasmid STB partitioning locus. Determining how the Rep proteins contribute has been complicated by interactions between the components. Here, each Rep protein was expressed fused to the DNA-binding domain of the bacterial repressor protein LexA in yeast harboring a replication-competent plasmid that had LexA-binding sites but lacked STB. Plasmid transmission to daughter cells was increased only by Rep2 fusion expression. Neither Rep1 nor a functional RSC2 complex (a chromatin remodeler required for 2-micron plasmid partitioning) were needed for the improvement. Deletion analysis showed the carboxy-terminal 65 residues of Rep2 were required and sufficient for this Rep1-independent inheritance. Mutation of a conserved basic motif in this domain impaired Rep1-independent and Rep protein/STB-dependent plasmid partitioning. Our findings suggest Rep2, which requires Rep1 and the RSC2 complex for functional association with STB, directly participates in 2-micron plasmid partitioning by linking the plasmid to a host component that is efficiently partitioned during cell division. Further investigation is needed to reveal the host factor targeted by Rep2 that contributes to the survival of these plasmids in their budding yeast hosts.