Agrawal, Parul (2016-08). Characterizing Novel Circadian Clock Functions for Drosophila Phosphatases and Non-clock Functions for Circadian Photoreceptors. Doctoral Dissertation. | Thesis individual record

Circadian (~24 h) clocks regulate daily cycles in gene expression to control overt rhythms in physiology, metabolism and behavior. In Drosophila, a transcriptional feedback loop activated by CLOCK-CYCLE (CLK-CYC) complexes, repressed by PERIOD-TIMELESS (PER-TIM) complexes, and synchronized to light: dark cycles primarily by CRYPTOCHROME (CRY), keeps circadian time. The timing of activation and repression is regulated post-translationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Although kinases that control CLK, PER, and TIM levels, activity, and/or subcellular localization have been identified, less is known about phosphatases that control clock protein dephosphorylation. Using genetic, behavioral and molecular analyses, I identified protein phosphatases that function within the Drosophila circadian clock. Moreover, I took advantage of behavioral, molecular, and electrophysiological analyses to characterize CRY expression and function in peripheral tissues of Drosophila melanogaster.

To identify phosphatases, clock cell-specific RNAi knockdowns of all annotated phosphatases in Drosophila were screened for altered activity rhythms. I identified 22 such phosphatases that either lengthened or shortened circadian period by >= 1 h or were significantly arrhythmic. The efficacy and specificity of RNAis was validated by testing RNAis that targeted other regions of the mRNA, transposon inserts, and either existing or CRISPR-generated loss of function mutants for defects in activity rhythms. One phosphatase identified, Leukocyte-Antigen-Related (LAR), regulates the development of neuronal circuit underlying the communication between clock neurons in the fly brain. This work, along with the analysis of another 15 candidates that remain after validation, will reveal novel features of the circadian timekeeping mechanism in Drosophila that may be conserved in all animals including humans.

Furthermore, using a GFP-tagged-cry transgene, I show that CRY is expressed in Drosophila peripheral tissues and promotes light-dependent TIM degradation. Electrophysiological recordings from larval salivary glands which lack a circadian clock and are non-excitable, demonstrated that CRY regulates cell membrane physiology in collaboration with K+ channels. These findings for the first time define the expression profile of CRY in Drosophila peripheral tissues, and reveal that CRY functions in a light-independent and K+ channel-dependent manner to alter membrane function in peripheral tissues devoid of a canonical circadian clock.

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