Abstract
Robust circadian oscillations of the proteins PERIOD (PER) and TIMELESS (TIM) are hallmarks of a functional clock in the fruit fly Drosophila melanogaster. Early morning phosphorylation of PER by the kinase Doubletime (DBT) and subsequent PER turnover is an essential step in the functioning of the Drosophila circadian clock. Here using time-lapse fluorescence microscopy we study PER stability in the presence of DBT and its short, long, arrhythmic, and inactive mutants in S2 cells. We observe robust PER degradation in a DBT allele-specific manner. With the exception of doubletime-short (DBT(S)), all mutants produce differential PER degradation profiles that show direct correspondence with their respective Drosophila behavioral phenotypes. The kinetics of PER degradation with DBT(S) in cell culture resembles that with wild-type DBT and posits that, in flies DBT(S) likely does not modulate the clock by simply affecting PER degradation kinetics. For all the other tested DBT alleles, the study provides a simple model in which the changes in Drosophila behavioral rhythms can be explained solely by changes in the rate of PER degradation.
Highlights
Several mutations of the dbt locus have been isolated and the short-period and the long-period alleles are among the best characterized dbt mutants in the literature [1,2,3, 6, 9]. dbtL is a Met-80 3 Ile mutation that causes period lengthening of PER and TIM oscillations and animal behavioral activity, to ϳ27 h
Our initial hypothesis involves the best studied alleles DBT, DBTS, and DBTL and argues that the shortening of fly rhythm from dbtL to dbtS can be explained in terms of (a) earlier onset of PER turnover, (b) shorter half-life of PER, or (c) higher fraction of PER that is degraded in the process
Our studies following the temporal pattern of fluorescently labeled PER and DBT indicate that the onset of PER degradation is very tightly regulated in Schneider 2 (S2) cells and PER abundance starts to diminish typically within 3–5 h after protein induction with only ϳ10% of the substrate remaining at the end of the degradation process (Fig. 1)
Summary
Several mutations of the dbt locus have been isolated and the short-period (dbtS) and the long-period (dbtL) alleles are among the best characterized dbt mutants in the literature [1,2,3, 6, 9]. dbtL is a Met-80 3 Ile mutation that causes period lengthening of PER and TIM oscillations and animal behavioral activity, to ϳ27 h. The protein translocation data, indicate that DBT may enter the nucleus separately from PER and overexpression of the kinase in S2 cells does not significantly alter the average timing of nuclear entry of the PER/TIM complex.
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