1. Posttranslational Regulation of Drosophila PERIOD Protein by Protein Phosphatase 2A 
Sriram Sathyanarayanan, Xiangzhong Zheng, Rui Xiao, Amita Sehgal 
Cell 
Volume 116, Issue 4, Pages (February 2004)
DOI: /S (04)00128-X

2. Figure 1 PP2A Regulates PER Stability in S2 Cells
(A) The phosphatase inhibitor Calyculin A destabilizes PER in S2 cells. The Western blots show levels of PER and β-GAL in cells transfected with pAct-per alone, or along with pAct-tim (indicated in parentheses), and β-gal-V5 expression vectors. The cells were treated with medium containing 30 nM Calyculin A (+) or with vehicle control (−). Cell lysates were separated on an 8% SDS-PAGE gel and membranes were probed sequentially with anti-PER and anti-V5 (β-Gal) antibodies. PER levels were quantified from three experiments and plotted (Figure S1).
(B) Calyculin A-mediated destabilization of the PER-TIM complex can be reversed by the proteasome inhibitor MG132. S2 cells were transfected with pAct-per or pAct-tim or both expression vectors and treated with 30 nM Calyculin A alone or along with 100 μM MG132 or vehicle control for 60 min. Cell lysates were run on 6% SDS-PAGE gels and probed with anti-PER and anti-TIM antibodies. Note that the 6% gel resolves hypo- and hyperphosphorylated forms of PER and it is the hyperphosphorylated (low mobility) form that is increased in the presence of Calyculin A. A nonspecific band (NS) that appeared while probing with the anti-TIM antibody is shown as a loading control. The blots shown are representative of two independent experiments.
(C) RNAi-mediated knockdown of regulatory subunits of PP2A, WDB, and TWS affects PER levels in S2 cells. S2 cells were transfected with per and tim expression vectors along with double-stranded RNA (dsRNA) against the indicated proteins. The extent of knockdown of MTS, TWS, and WDB was estimated using antibodies against each of these three proteins. The specificity of the knockdown was confirmed by probing for the heat shock protein, Hsp-70. The blots shown here are representative of three experiments.
(D) Overexpression of wdb and tws in S2 cells increases PER levels. A pAc-tws-V5 or pAc-wdb-V5 expression vector (200 ng) or vector control was cotransfected into S2 cells with 300 ng of pAct-per alone or along with pAct-tim as indicated. Protein lysates were subjected to Western blot analysis and PER and TIM bands were visualized. Equal protein loading was ensured by probing with an anti-MAP kinase (MAPK) antibody. Similar results were obtained in three independent experiments with TWS-P1 and also with TWS-P2 (see Figure 2A).
Cell  , DOI: ( /S (04)00128-X)

3. Figure 2
Circadian Regulation of wdb and tws Expression in the Adult Fly Head
(A) A simplified, schematic representation of the tws gene, showing exons, introns, and start codons of the P1 and P2 isoforms. The first two exons are specific for P1 transcripts. The location of the tws probe used for RNase protection assays (RPAs) is indicated by the dark line. The twsP insertion that was excised to generate the tws60 mutant is also indicated.
(B) Autoradiographs showing the levels of tws-P1, -P2, wdb, and tubulin transcripts in total RNA isolated from adult fly heads collected at different times of day. Adult fly heads were collected at indicated zeitgeber times (ZT) from wild-type (WT) and cyc0 flies entrained to 12:12 hr light dark cycles; ZT-0 is lights on and ZT-12 is lights off. tws or wdb expression was determined by RNase protection analysis. In each experiment, a tubulin probe was included to control for RNA concentration. The tws probe protects a larger band corresponding to the P1 transcript and shorter bands corresponding to P2. The WDB probe protects a single band.
(C) Quantification of the levels of tws-P1 and wdb mRNA at different times of day. The bands were quantified using a phosphorimager and expressed as ratios relative to tubulin. Relative levels were then normalized to the wild-type peak level, which was set as 1. The levels in cyc0 flies were also normalized relative to the control peak value (controls were run along with cyc0 for absolute comparison). The wild-type results are an average of four independent experiments and error bars depict standard error of the mean (SEM). The cyc0 results represent an average of two independent experiments. The tws-P1 mRNA levels showed a significant circadian oscillation (p = 1 × 10−5) in wild-type flies, and this oscillation was abolished in cyc0 mutants (p = 0.28). Similarly, wdb mRNA levels showed a significant oscillation (p = 0.95 × 10−4) in wild-type flies; this oscillation was blunted (p = 0.06) in cyc0 mutants.
Cell  , DOI: ( /S (04)00128-X)

4. Figure 3
Overexpression of WDB in tim Neurons Lengthens Behavioral Period, Increases PER Levels, and Advances PER Nuclear Entry
(A) wdb mRNA levels are increased in EP3559 flies carrying the tim-Gal4 driver (TG). wdb mRNA levels were determined by RNase protection analysis using a wdb-specific radioactive cRNA probe. A tubulin probe was included to control for RNA concentration. The autoradiogram shows the wdb- and tubulin-specific protected fragments.
(B) Overexpression of WDB in clock neurons lengthens the period of rest:activity rhythms. Representative locomotor activity records (actograms) of individual flies kept in constant darkness (DD) for 10 days after light:dark entrainment are shown. The subjective light:dark phases, genotype, and the circadian period (τ), as determined by chi-square periodogram analysis, are indicated. All the flies in the parental EP3559 line were rhythmic, while 5% of the flies carrying EP3559 along with the TG driver were arrhythmic after ∼4 days in constant conditions.
(C) WDB overexpression increases PER levels in the adult fly head. Flies were collected on the first day in DD after three days of entrainment to a light:dark cycle (under these conditions, CT0 or circadian time 0 refers to subjective “lights on” and CT12 is subjective “lights off”) . Equivalent amounts of total protein from wild-type and WDB overexpressing (TG/cy0; EP3559) flies were blotted and sequentially probed with anti-PER and anti-TIM antibodies. Equal loading was ensured by Ponceau S staining. The asterisk (*) denotes a breakdown product of PER that is usually present in samples with high levels of PER.
(D) PER levels are increased in flies overexpressing WDB. Three sets of extracts were assayed on 6% acrylamide gels and levels of PER and TIM were determined through densitometry using a Kodak image station. Levels were normalized relative to the wild-type peak levels (set as 1) and plotted. Although peak PER levels were relatively unchanged, levels were significantly higher at other points (p < 0.02). However, such an increase in TIM levels was not seen.
(E) WDB overexpression advances PER nuclear entry. LNvs were visualized in larval brains through whole-mount immunofluoresence. The upper panel shows PDF staining (green), which specifically labels cell bodies and axons of the larval lateral neurons (lvLNs); the middle panel shows PER staining (red) and the lower panel represents the merge of both signals (yellow indicates colocalization). Similar localization was seen in 16 larval brain hemispheres examined for each time point.
Cell  , DOI: ( /S (04)00128-X)

5. Figure 4 Nuclear Accumulation of PER Is Affected in tws Mutants
(A) Analysis of PER expression in tws60 larvae. Larvae were entrained to a light:dark cycle for 3 days then shifted to constant darkness (DD). Third instar larval brains (n = 8 to10 for each point) were dissected at the indicated circadian times (CT) on the first day in DD and processed for whole-mount staining. PER protein staining (red) is shown in pacemaker cells that were co-stained with antibodies to PDF. PER accumulated to high levels at CT-18 in control siblings (distinguished by the Tubby (Tb) marker on the TM6B balancer), while in the tws60 mutants PER immunoreactivity was minimal at this time point.
(B) Quantification of PER immunoreactivity in larval ventro-lateral neurons. PER immunoreactivity, relative to background, was blind scored on a scale of 1–10. Relative PER intensity scores at various circadian times were plotted. PER staining was significantly lower (p = , two-sample unpaired t test) than the control at CT18.
(C) Delayed PER nuclear entry in tws60 mutants. PER staining was visualized essentially as described above in control (Tb) and tws60 mutants. Confocal images of larval lateral neurons are shown. Nuclear expression of PER was seen at CT18:30 in controls and yellow, white (y w) flies (data not shown), while in the tws60 mutants PER immunoreactivity was very weak (strongest levels in the 20 hemispheres of larval brain examined are shown) and predominantly cytoplasmic. Note that the timing of nuclear entry varies somewhat in different genetic backgrounds (compare the control here with the one in Figure 3); we have compared with the appropriate control.
(D) tws is expressed in pacemaker cells. Third instar larval brains were stained for PDF (green) and nuclear β-GAL (red). In addition to other cells in the central brain, β-GAL expression was seen in ventro-lateral neurons of the tws enhancer trap line. No specific staining could be seen in y w controls (data not shown).
Cell  , DOI: ( /S (04)00128-X)

6. Figure 5
Modulation of PP2A Levels Affects Overt Behavioral Rhythms in Flies
(A) Expression of tws in Pdf neurons shortens and weakens locomotor rhythms. Representative locomotor activity records (actograms) of individual flies kept in constant darkness (DD) for 14 days after light:dark entrainment are shown. The subjective light:dark cycle, genotype, and the circadian period (τ), as determined by chi-square periodogram analysis, are indicated. Locomotor rhythms got weaker (Table 1) and were lost after 4 to 7 days in constant conditions in most flies tested.
(B) Overexpression of the catalytic subunit of PP2A (mts) in tim neurons causes arrhythmia. Representative actograms of individual flies kept in constant darkness (DD) for 10 days are shown.
(C) Expression of a dominant-negative version of mts in Pdf neurons lengthens period and reduces rhythm strength (Table 1). Note the instability of the rhythm, demonstrated by the occurrence of prolonged activity on some days.
Cell  , DOI: ( /S (04)00128-X)

7. Figure 6 Effect of PP2A on PER Expression and Phosphorylation
(A) Changes in levels of PP2A in clock neurons affect PER stability and nuclear localization. Representative confocal images of PER (red) and PDF (green) immunoreactivity in lateral neurons of third instar larvae are shown. 10–18 hemispheres were examined for each time point.
(B) Overexpression of the catalytic subunit of PP2A (mts) in clock neurons affects PER phosphorylation. Total protein from adult fly heads, collected at the indicated circadian time points, was analyzed on a 6% SDS-polyacryamide gel and probed with PER antibody.
(C) Representative autoradiograph showing direct dephosphorylation of PER by PP2A. Purified PER was in vitro phosphorylated with CKIδ in the presence of γ32p-ATP (see Supplemental Figure S4 online). Equal amounts of phosphorylated PER [PER-(P)] were incubated with varying amounts (0.01 to 0.1 units) of purified PP2A AC dimer in the presence of kinase inhibitor (5-IT). Okadaic acid (OA), Calyculin A (Cal-A), and PP1 specific inhibitor (PPI-2) were used to assay the specificity of the effect.
(D) Autoradiograph showing that PP1 does not dephosphorylate PER in the in vitro assay. Varying amounts of purified PP1 (0.01 to 0.5 units) were used and compared with PP2A for their ability to dephosphorylate PER.
Cell  , DOI: ( /S (04)00128-X)

8. Figure 7
A Model of the Drosophila Circadian Clock Showing Dual Control of PER Cycling
A schematic representation of the Drosophila feedback loop, incorporating the proposed function of PP2A. PER is stabilized by the PP2A regulatory subunits WDB and TWS. We propose that PER protein cycling is regulated at both RNA and protein levels by its own rhythmic transcription and by rhythmic phosphatase expression, respectively. Since wdb and tws are regulated by cycle (cyc) in the adult fly head (Figure 2), both mechanisms that drive PER cycling are shown to depend upon CLK-CYC. Also a CACGTG E box is present in the first intron of tws (see Discussion), suggesting that it could be a direct target of the CLK-CYC complex.
Cell  , DOI: ( /S (04)00128-X)