Molecular mechanisms and novel genes mediating temperature compensation in circadian clock neurons of Drosophila melanogaster

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Circadian clocks are endogenous oscillators, able to control biological rhythms in a constant environment with a period of ca. 24 h. These biological timers are exquisitely sensitive to temperature, because small temperature changes between day and night result in robust clock synchronization. In contrast, their self-sustained 24 h period is independent of its surrounding temperatures, i.e., circadian clocks are temperature compensated. This is a remarkable feature, because all other biological processes speed up with increasing temperatures. It is also essential, because a clock that changes its speed with temperature is no accurate timer. Most organisms are poikilothermic, and therefore heavily depend on proper temperature compensation of their circadian clocks. However, the buffering of the oscillator against temperature changes (temperature compensation) is molecularly not well understood. In the first funding period, we showed that nuclear export of two key Drosophila circadian clock proteins is important for temperature compensation. Our results support the Hastings and Sweeney model for temperature compensation (1957). It assumes that two reactions showing the normal temperature dependent rate increase can nevertheless produce oscillations with constant period length, as long as the second reaction inhibits the first. In Drosophila, this implies that higher rates of nuclear import at warm temperatures would be ‘compensated’ by equally increased rates of nuclear export, keeping the period length constant across temperatures. In the next funding period, we aim to confirm this model using live imaging approaches, which allow to distinguish between nuclear and cytoplasmic localization of clock proteins at different temperatures. We could also show that Casein kinase 1ε (CK1ε, or DBT), which phosphorylates PERIOD and regulates its stability, plays an important role in temperature compensation. Interestingly, we found that a PERIOD mutation interfering with nuclear export shows a phosphorylation defect, specifically at warm temperatures, and a CK1ε mutation that affects temperature compensation in mammals and flies strongly enhances the temperature compensation phenotype of this mutation. We will therefore analyse the function of CK1ε and its interaction with PERIOD for temperature compensation in the next funding period. We could also show that other, so far unknown proteins that function in temperature compensation are subject to nuclear export. Moreover, it is expected that other kinases and proteins are important for temperature compensation. We will therefore perform a genetic screen of ~ 200 isogenic lines generated from natural variants caught in the wild, as well as a candidate screen targeting the known Drosophila kinases. Combined, these approaches will significantly increase our understanding of one of the most fundamental characteristics of circadian clocks.