The higher the temperature, the faster the physiological process. But there is one exception: the so-called biological clock, which regulates the sleep-wake cycle of an organism. A fascinating question for scientists is why internal clocks operate in an almost constant manner despite temperature fluctuations. This is a phenomenon known as temperature compensation. Studies have shown that different molecular mechanisms contribute to this. A team of biologists led by Prof. Ralf Stanewsky of the University of Münster (Germany), in collaboration with teams at Dalhousie University in Canada and the University of Mainz in Germany, has now found an important piece of the puzzle, providing an answer to this question . The results of their work have been published in the journal “current biology“.
The team discovered a point mutation in fruit flies Drosophila This results in a temperature-dependent prolongation of the circadian clock cycle. It is located in what is called the “period“(Every). Flies with this perI530A The mutants displayed a normal sleep-wake rhythm for 24 hours at 18 degrees Celsius. In contrast, at 29 degrees Celsius, the internal clock is about 5 hours behind, or 29 hours. The prolongation of the period also affects the expression of period genes in the brain’s clock neurons, in other words, the activity of period genes.
Typically, the associated protein (PERIOD) undergoes gradual chemical changes over a 24-hour period—specifically, it becomes phosphorylated. After maximal phosphorylation, it is degraded. Here, too, the process is generally the same at temperatures between 18 and 29 degrees Celsius, when the flies are active.As the researchers have shown, phosphorylation occurs in a normal manner at perI530A Mutated at 18°C, but decreased with increasing temperature. This results in “PERIOD” proteins being stable at warmer temperatures.
The mutation studied by the team affects the so-called nuclear export signal (NES), which also occurs in this form in period genes in mammals and plays a role in transporting the PERIOD protein out of the nucleus. The biological function of this export from the nucleus was previously unknown. The current study shows that the mutation leads to prolonged retention of the PERIOD protein in the nucleus of central clock neurons — again, only at higher temperatures. “We therefore hypothesized,” says Ralf Stanewsky, “that the export of proteins from the nucleus plays an important role in temperature compensation—at least in Drosophila.”
In their study, the scientists used Drosophila mutants, and in period Gene(perI530A), which they generated using modern molecular genetic methods (CRISPR/Cas9 mutagenesis and homologous recombination). The animals were then tested to see if their sleep-wake cycles — and thus their running activity — differed according to the ambient temperature. The researchers used a variety of methods to visualize clock genes and their activity in neurons in the brain. One of the methods they used was a new method called locally activatable bioluminescence (LABL) that the Münster team developed in collaboration with Canadian researchers. The method, which involves bioluminescence, made it possible to measure rhythmic gene expression in clock neurons — which make up only a small fraction of all brain neurons — in living fruit flies.
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