When temperatures are higher, bodily functions speed up. However, the so-called circadian clock that controls the sleep-wake cycle in living things is an exception to this rule. It’s a fascinating mystery why the internal clock keeps time almost consistently regardless of temperature changes. The term “temperature compensation” describes this occurrence. Several studies have pointed to distinct molecular processes that contribute to this phenomenon.
An international group of biologists led by Professor Ralf Stanewsky of Germany’s University of Münster and including researchers from Dalhousie University in Canada and the University of Mainz in Germany, have recently solved this mystery. Current Biology features the team’s findings.
The group identified a single mutation in Drosophila melanogaster that causes circadian clock durations to prolong in response to changes in environmental temperature. It is situated in the “clock gene” (sometimes called a “period”). The perI530A mutation results in a 24-hour sleep-wake cycle at 18 degrees Celsius in flies. At 29 degrees Celsius, on the other hand, the internal clock operates around five hours slower, so that it runs for 29 hours. The expression, or activity, of the period gene in the brain’s clock neurons is similarly modified by this lengthening of the cycle.
Over the course of a day, the protein in question (PERIOD) undergoes a slow but steady chemical transformation, called phosphorylation. The protein is broken down after it has been phosphorylated to its maximum. Also, at temperatures between 18 and 29 degrees Celsius, when fruit flies are active, this process is typically the same. Researchers revealed that in the perI530A mutant, phosphorylation is normal at 18 degrees Celsius but diminishes with increasing temperature. A consequence of this is that the “PERIOD” protein becomes more stable in hotter environments.
The team’s research focused on a mutation that affects a so-called nuclear export signal (NES), which is present in animals’ period genes and is involved in exporting the PERIOD proteins from the nucleus. Until recently, the nuclear export was thought to serve no biological purpose. The present research confirms that the mutation causes the PERIOD protein to remain in the nucleus of central clock neurons for a longer period of time—and once again, only at higher temperatures. Consequently, the researchers assume that the export of the protein from the cell nucleus plays a significant role in temperature compensation—at least in the fruit fly.
The researchers used fruit fly mutants that they had created using cutting-edge molecular genetics techniques (CRISPR/Cas9 mutagenesis and homologous recombination) and carried out their experiments with these mutants. In order to determine whether the animals’ sleep-wake cycle and, by extension, their running activity, varied as a function of environmental temperature, they were put through a series of tests.
The researchers were able to visualize the clock genes and the activity of the neurons in the brain using a number of different techniques. They made advantage of a recently discovered technique termed locally activatable bioluminescence (LABL), which had its origins in a joint effort by scientists from Canada and Münster. Using bioluminescence, researchers can monitor the rhythmic gene expression of clock neurons—a small subset of all brain neurons—in living flies.
Astrid Giesecke et al. (2022). A novel period mutation implicating nuclear export in temperature compensation of the Drosophila circadian clock, Current Biology. DOI: 10.1016/j.cub.2022.12.011