Perturbing Dynamin Reveals Potent Effects on the Drosophila Circadian Clock

Abstract

BackgroundTranscriptional feedback loops are central to circadian clock function. However, the role of neural activity and membrane events in molecular rhythms in the fruit fly Drosophila is unclear. To address this question, we expressed a temperature-sensitive, dominant negative allele of the fly homolog of dynamin called shibirets1 (shits1), an active component in membrane vesicle scission.Principal FindingsBroad expression in clock cells resulted in unexpectedly long, robust periods (>28 hours) comparable to perturbation of core clock components, suggesting an unappreciated role of membrane dynamics in setting period. Expression in the pacemaker lateral ventral neurons (LNv) was necessary and sufficient for this effect. Manipulation of other endocytic components exacerbated shits1's behavioral effects, suggesting its mechanism is specific to endocytic regulation. PKA overexpression rescued period effects suggesting shits1 may downregulate PKA pathways. Levels of the clock component PERIOD were reduced in the shits1-expressing pacemaker small LNv of flies held at a fully restrictive temperature (29°C). Less restrictive conditions (25°C) delayed cycling proportional to observed behavioral changes. Levels of the neuropeptide PIGMENT-DISPERSING FACTOR (PDF), the only known LNv neurotransmitter, were also reduced, but PERIOD cycling was still delayed in flies lacking PDF, implicating a PDF-independent process. Further, shits1 expression in the eye also results in reduced PER protein and per and vri transcript levels, suggesting that shibire-dependent signaling extends to peripheral clocks. The level of nuclear CLK, transcriptional activator of many core clock genes, is also reduced in shits1 flies, and Clk overexpression suppresses the period-altering effects of shits1.ConclusionsWe propose that membrane protein turnover through endocytic regulation of PKA pathways modulates the core clock by altering CLK levels and/or activity. These results suggest an important role for membrane scission in setting circadian period.