Like most organisms, Drosophila melanogaster can synchronize its physiological and behavioural processes by possessing internal circadian clock that regulates. Naturally fluctuating timing cues, like light and temperature (also known as Zeitgebers), synchronize these endogenous and self‐sustained clocks with external time. In Drosophila, synchronization of the circadian clock by light has been studied in detail, but much less is known about the molecular mechanisms underlying temperature entrainment. Previous data from our lab shows that Nocte, a Chordotonal organ (Ch organ) located protein, is required for normal temperature entrainment in Drosophila. However, neither the function of Nocte in temperature entrainment nor the molecular underlying mechanisms are clear. To address these issues, a proteomics strategy of combing co‐immunoprecipitation and MS/MS sequencing was applied to isolate potential interactors of Nocte. IR25a was one of the most promising candidates, which was later confirmed by behavioural tests using RNA interference: Reducing IR25a expression in Chorgan resulted in abnormal behaviour during temperature cycles, similar to what had been described for Nocte mutant. To further confirm the interaction between Nocte and IR25a, I showed that IR25a physically interacts with Nocte in vivo. Moreover, using an IR25a‐gal4 line, I was able to show that IR25a is expressed in subsets of chordotonal organs (Ch organ) including Johnston's Organs (JO), where Nocte is also highly expressed. These results, along with the behavioural data mentioned above are consistent with the proteomics results and suggest that Nocte and IR25a physically and functionally interact. IR25a mutants were employed to further investigate the function of IR25a in temperature entrainment. First of all, I found that both central and peripheral clocks in wild type flies can be synchronized to temperature cycles with only two degree differences (12h: 12h, 27 °C: 25 °C). In contrast, synchronization of locomotor activity rhythms in the IR25a null mutants to the same temperature cycles and other TC's with 2°C amplitude was eliminated. Under the same conditions, the oscillations of the core clock proteins TIMLESS (TIM) and PERIOD (PER) that normally occur in fly heads were completely abolished inIR25a null mutants, suggesting that IR25a is required for temperature entrainment of peripheral clocks. In the central brain pacemaker neurons, the oscillations of TIM in dorsal and lateral neurons were also affected by the IR25a mutants. On the contrary, IR25a is not required for light entrainment and temperature compensation, suggesting that IR25a is specifically involved in temperature synchronization. Moreover, temperature entrainment of the IR25a null mutants can be partially restored by applying larger temperature intervals (29°C: 25°C) indicating that IR25amay function as amplitude detector independent of absolute temperature values. Finally, neuronal activity in IR25a+ neurons is crucial for the synchronization of circadian clocks to low amplitude temperature cycles. Re‐constitution of functional olfactory receptors required the assembly of IR25a with IR76a and IR76b. Interestingly, IR76a and IR76b are neither required for temperature entrainment at the behavioural level nor expressed in the Ch organs. To check if other potential IRs interacting with IR25a exist, I screened the expression pattern of most divergent IRs using IR‐gal4/UAS‐GFP flies. IR56a was isolated as a potential partner of IR25a because it is also expressed in the femur chordotonal organs. To investigate the function of IR56a in temperature entrainment, I generated a null mutant of IR56a. Surprisingly, this gene is not required for synchronizing clocks to a temperature cycle (27°C: 25°C) at the behavioural level. However, the behavioural and molecular phenotypes of IR56a mutant under different temperature cycles need to be further characterized.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:765754 |
| Date | January 2014 |
| Creators | Chen, Chenghao |
| Publisher | Queen Mary, University of London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://qmro.qmul.ac.uk/xmlui/handle/123456789/26967 |
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