The long-term goals are to understand the molecular basis of circadian (approximately 24 hour) rhythms. These rhythms are controlled by clocks endogenous to most organisms and are manifest in many different physiological processes. In humans, for instance, a circadian periodicity is discernible in sleep-wake cycles, body temperature, hormonal secretions, blood pressure and liver metabolism. Disrupted functioning of clocks has been associated with sleep disorders as well as other pathologies such as tumor growth. The molecular nature of the endogenous circadian clock was determined largely through studies done in the fruit fly, Drosophila melanogaster. These studies showed that the clock is composed of one of more feedback loops in which protein products of specific genes (so-called """"""""clock genes"""""""") regulate the synthesis of their own mRNAs in a circadian fashion. As a result, cyclic expression of these mRNAs and proteins, and thereby of downstream physiological components, is maintained. We found that cyclic expression of the Drosophila clock proteins period (PER) and timeless (TIM), occur even when the mRNAs encoding them do not cycle and that rhythmic protein expression is sufficient to drive rhythmic behavior. Studies in mammals corroborate the relative RNA-independence of clock protein cycling which is most likely achieved through rhythmic phosphorylation events that lead to periodic turnover. However, the kinases that phosphorylate PER and TIM do not appear to cycle. We have recently found that the PP2A phosphatase family affects stability and nuclear expression of PER and that two of the regulatory subunits of this family, wdb and tws, cycle with a circadian rhythm. Manipulations of either of these subunits or of the catalytic subunit of PP2A results in defects in the molecular clock and in behavioral rhythms. In addition, PP2A directly dephosphorylates PER in vitro. We propose to determine the role of PP2A in the circadian clock. We will (1) Determine when and where PP2A subunits are expressed in the fly head and also determine their subcellular distribution at different times of day, given that the nuclear localization of PER-TIM displays a circadian rhythm. (2) Determine which PP2A subunit mediates each of the different effects of the phosphatase complex on PER. (3) Generate and characterize additional mutants of wdb and tws. (4) Determine how the clock controls the cycling of tws and address the relevance of this cycling for behavioral rhythms.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS048471-02
Application #
7009557
Study Section
Biological Rhythms and Sleep Study Section (BRS)
Program Officer
Mitler, Merrill
Project Start
2005-02-01
Project End
2010-01-31
Budget Start
2006-02-01
Budget End
2007-01-31
Support Year
2
Fiscal Year
2006
Total Cost
$302,533
Indirect Cost
Name
University of Pennsylvania
Department
Neurosciences
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Sehgal, Amita (2015) Methods in Enzymology. Circadian rhythms and biological clocks, part A. Preface. Methods Enzymol 551:xvii-xviii
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Jang, A Reum; Moravcevic, Katarina; Saez, Lino et al. (2015) Drosophila TIM binds importin ?1, and acts as an adapter to transport PER to the nucleus. PLoS Genet 11:e1004974
Maguire, Sarah E; Sehgal, Amita (2015) Heating and cooling theDrosophila melanogasterclock. Curr Opin Insect Sci 7:71-75
Kumar, Shailesh; Chen, Dechun; Jang, Christopher et al. (2014) An ecdysone-responsive nuclear receptor regulates circadian rhythms in Drosophila. Nat Commun 5:5697
Maguire, Sarah E; Schmidt, Paul S; Sehgal, Amita (2014) Natural Populations of Drosophila melanogaster Reveal Features of an Uncharacterized Circadian Property: The Lower Temperature Limit of Rhythmicity. J Biol Rhythms 29:167-180

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