Timeless and diapause in Drosophila

Hibernation is a protective state of suspended animation that comes around every season in plants and animals that live in the temperate zones of the world. In insects, this seasonal cycle is called diapause, and is stimulated by the reduction in daylength as winter approaches. The other great rhythm on this planet is the circadian cycle which is the daily 24 hour cycle that we all recognise as our 'body clock'. Recently we have found a connection between a gene called timeless that controls the 24 hour rhythm of the geneticists favourite pet, the fruitfly, and the seasonal diapause cycle. A new mutation in the timeless gene, ls-tim, occurred in south-eastern Italy about 8000 years ago, a few thousand years after the last glaciation when flies invaded Europe from Africa This mutation allows the fly to make two types of TIM protein, L-TIM and S-TIM, whereas the ancient gene, s-tim can only make S-TIM. The new mutation, ls-tim, is spreading under natural selection, because it adapts the fly to the seasonal environment of Europe. It does this by reducing the light sensitivity of the ls-tim fly's diapause mechanism. Thus, even in relatively long days, ls-tim flies 'see' shorter days, and this stimulates them diapause earlier than in s-tim flies. The earlier the fly goes into diapause in Europe, the better its chances of surviving the oncoming winter. In addition, the 24 hour circadian clock of ls-tim is also less light-sensitive and this too is adaptive in Europe, which has very exotic photoperiods in summer which causes the fly's biorhythms to creak. Consequently, L-TIM attenuates the fly's photosensitivity for both circadian and seasonal behaviours, and that is perhaps why this new mutation has spread in Europe. Biochemically, the L-TIM protein is less light sensitive because of its weak physical interaction with a circadian photoreceptor called Cryptochrome. The ls-tim mutation creates a site in the L-TIM protein that is not shared in S-TIM. This new site may be phosphorylated, a protein modification that may alter the shape and stability of L-TIM. This may provide the biochemical clue for why ls-tim flies behave differently. We shall study this site to see whether it is indeed phosphorylated. We shall also investigate whether ls-tim flies change the ratios of the L-TIM and S-TIM they produce under the short days and cold temperature that produce diapause. In addition we will study under these conditions how strongly L-TIM and S-TIM 'touch' the photoreceptor Cryptochrome in the fly's brain and eyes, as this physical interaction provides the light input into the circadian mechanism. There has been a long running debate about whether the circadian clock contributes to diapause. Using the timeless gene, we shall genetically dissect the fly's nervous system in order to investigate which groups of neurons are important for diapause. Because the 24 hour clock neurons are well known and easily identified, we should be able to see whether these cells are relevant for the fly's hibernation. We can then visualise L-TIM and S-TIM protein in the relevant cells of ls-tim flies under diapause conditions, to see whether the two proteins behave differently. Insect diapause is a potential target for the control of medical and agricultural pests in temperate regions, and so our work may have practical spin-offs in future.

A new mutation in the timeless clock gene of Drosophila melanogaster, ls-tim, has spread throughout Europe in the last few thousand years. This variant shows an attenuated photoresponsiveness, both in its circadian clock, and in diapause, the protective response to oncoming winters compared to the ancestral variant s-tim. Both phenotypes are adaptive in seasonal environments. The new variant generates both an L-TIM isoform from an upstream methionine, that is 23 residues longer than S-TIM, which is translated from the downstream methionine. s-tim flies produce only the truncated S-TIM protein. L-TIM shows a reduced physical interaction with the circadian photoreceptor Cryptochrome, which may explain its reduced photosensitivity. The ls-tim mutation generates a de novo phosphorylation site between residues 17-25. We will mutagenise the relevant Serine to an Alanine to see whether this reverts the ls-tim phenotypes to s-tim. If not, we shall randomise the order of the 23 N-terminal residues of L-TIM to examine whether this maintains the ls-tim phenotypes. We will also investigate in ls-tim fly heads the relative levels of L- and S-TIM under temperatures and photoperiods that induce diapause, as well as the dimerization efficiencies of L- and S-TIM with CRY. The tim-null mutant shows a temperature-sensitive diapause which, unlike wild-type, is not suppressed at longer summer photoperiods. We shall restore TIM function to various subsets of neuronal tissues as well as some peripheral tissues using the gal4/gal80/UAS system. We shall determine whether M and E neuronal clocks contribute to the diapause phenotype. Those tissue that our studies deem to be contributing to diapause will then be investigated by ICC in ls-tim flies to examine the dynamics of L- and S-TIM expression, using some of the tools we have generated.