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Dominant-negative CK2alpha induces potent effects on circadian rhythmicity.

Smith EM, Lin JM, Meissner RA, Allada R - PLoS Genet. (2007)

Bottom Line: CK2alpha(Tik), when targeted to a subset of pacemaker neurons, generates period splitting, resulting in flies exhibiting both long and near 24-h periods.These behavioral effects are evident even when CK2alpha(Tik) expression is induced only during adulthood, implicating an acute role for CK2alpha function in circadian rhythms.CK2alpha(Tik) expression results in reduced PER phosphorylation, delayed nuclear entry, and dampened cycling with elevated trough levels of PER.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois, United States of America.

ABSTRACT
Circadian clocks organize the precise timing of cellular and behavioral events. In Drosophila, circadian clocks consist of negative feedback loops in which the clock component PERIOD (PER) represses its own transcription. PER phosphorylation is a critical step in timing the onset and termination of this feedback. The protein kinase CK2 has been linked to circadian timing, but the importance of this contribution is unclear; it is not certain where and when CK2 acts to regulate circadian rhythms. To determine its temporal and spatial functions, a dominant negative mutant of the catalytic alpha subunit, CK2alpha(Tik), was targeted to circadian neurons. Behaviorally, CK2alpha(Tik) induces severe period lengthening (approximately 33 h), greater than nearly all known circadian mutant alleles, and abolishes detectable free-running behavioral rhythmicity at high levels of expression. CK2alpha(Tik), when targeted to a subset of pacemaker neurons, generates period splitting, resulting in flies exhibiting both long and near 24-h periods. These behavioral effects are evident even when CK2alpha(Tik) expression is induced only during adulthood, implicating an acute role for CK2alpha function in circadian rhythms. CK2alpha(Tik) expression results in reduced PER phosphorylation, delayed nuclear entry, and dampened cycling with elevated trough levels of PER. Heightened trough levels of per transcript accompany increased protein levels, suggesting that CK2alpha(Tik) disturbs negative feedback of PER on its own transcription. Taken together, these in vivo data implicate a central role of CK2alpha function in timing PER negative feedback in adult circadian neurons.

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Expression of CK2αTik in PDF-positive Pacemaker Neurons Increases PER Levels and Delays Nuclear Accumulation(A) Average behavior activity profile for the first day of constant darkness reveals a delayed evening peak for both long (pdfTikL) and split (pdfTikS) flies expressing UASTik in PDF+ cells relative to the UAS control alone.(B) Representative images of smLNv cells labeled for PDF (green) and PER (red) in control (pdfGal4/+) and pdfTik (pdfGal4/+; UASTikT1/+).(C) Quantification of PER levels indicates a delay in the decline of PER protein during middle of the subjective day.(D) Ratio of nuclear:cytoplasmic PER signal in smLNv from control and UASTik-expressing brains shows a diminished relative nuclear accumulation of PER in pdfTik cells.(E) Average pixel intensity of nuclear PER levels in control and pdfTik smLNv nuclei shows delayed nuclear accumulation with CK2α loss of function.(F) Cytoplasmic pixel intensity demonstrates elevated levels of PER in pdfTik smLNv during early subjective day, suggesting that reduced CK2α activity causes excess PER to be sequestered in the cytoplasm. n = 50–76 cells from two independent experiments.
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pgen-0040012-g004: Expression of CK2αTik in PDF-positive Pacemaker Neurons Increases PER Levels and Delays Nuclear Accumulation(A) Average behavior activity profile for the first day of constant darkness reveals a delayed evening peak for both long (pdfTikL) and split (pdfTikS) flies expressing UASTik in PDF+ cells relative to the UAS control alone.(B) Representative images of smLNv cells labeled for PDF (green) and PER (red) in control (pdfGal4/+) and pdfTik (pdfGal4/+; UASTikT1/+).(C) Quantification of PER levels indicates a delay in the decline of PER protein during middle of the subjective day.(D) Ratio of nuclear:cytoplasmic PER signal in smLNv from control and UASTik-expressing brains shows a diminished relative nuclear accumulation of PER in pdfTik cells.(E) Average pixel intensity of nuclear PER levels in control and pdfTik smLNv nuclei shows delayed nuclear accumulation with CK2α loss of function.(F) Cytoplasmic pixel intensity demonstrates elevated levels of PER in pdfTik smLNv during early subjective day, suggesting that reduced CK2α activity causes excess PER to be sequestered in the cytoplasm. n = 50–76 cells from two independent experiments.

Mentions: To determine the effects of CK2α loss of function on core molecular clock rhythms, we tested whether expression of UASTik in PDF-positive LNv altered cycling of the core clock protein PER. Levels and cellular distribution of PER protein in smLNv were examined quantitatively on the first day of DD in pdfTik or control Gal4 flies. Although we do observe splitting in these flies, behavior remains largely synchronous on the first day of DD (Figure 4A). Control flies show the typical evening peak of activity at ∼CT12 while the long-period pdfTik flies have a delayed evening activity peak, regardless of whether they exhibit split periods or not (Figure 4A, pdfTikL v. pdfTikS). Measurements of pixel intensity indirectly report the amount of PER protein in smLNv [44]; as seen in Figure 4B and 4C, PER levels are elevated in smLNv of pdfTik during the subjective day relative to controls. Wild type PER levels wane from CT4–8 and begin accumulating again in the subjective evening (CT16–20); in contrast, a prolonged decline in PER throughout the day (CT4–12) is evident in pdfTik flies, and levels only disappear during subjective evening (CT12–20), consistent with a long period phenotype. Peak and trough PER levels are also elevated in pdfTik flies relative to controls (p < 0.001 comparing pdfGal4/+ CT0 to pdfTik CT4 for peak and pdfGal4/+ CT12 to pdfTik CT16 for trough, Figure 4C). PER typically transitions from the cytoplasm to a predominantly nuclear distribution during the middle of the night, and such a pattern is observed in pdfGal4/+ control flies (Figure 4B and 4D). However, the amplitude of the localization rhythm (as quantified by the nuclear:cytoplasmic ratio) is seriously reduced in pdfTik flies (Figure 4D, p < 0.001 at CT0, CT4, CT8, and CT20 pdfGal4/+ v. pdfTik). The timing of nuclear localization is also delayed in pdfTik flies; while PER never becomes predominantly nuclear, the time at which the most PER is localized to the nucleus occurs later from CT4–12 in pdfTik smLNv, rather than CT0–4 for the GAL4 control (Figure 4D and 4E). This finding is supported by analysis of nuclear PER levels in pdfGal4/+ and pdfTik smLNv. Nuclear PER levels accumulate to a similar degree in pdfTik as in the Gal4 control; however, nuclear levels do not rise until later in the subjective day relative to control (Figure 4E). The overall fraction of nuclear PER is lower (Figure 4D), as more of the PER protein in pdfTik neurons remains sequestered in the cytoplasm (Figure 4F). Indeed, the reduced nuclear PER levels in the face of elevated cytoplasmic PER levels at CT0 provide the most compelling evidence that CK2α is important for nuclear PER localization independent of regulating its cytoplasmic abundance. These results are consistent with prior reports that reduction of CK2 activity inhibits nuclear entry [16].


Dominant-negative CK2alpha induces potent effects on circadian rhythmicity.

Smith EM, Lin JM, Meissner RA, Allada R - PLoS Genet. (2007)

Expression of CK2αTik in PDF-positive Pacemaker Neurons Increases PER Levels and Delays Nuclear Accumulation(A) Average behavior activity profile for the first day of constant darkness reveals a delayed evening peak for both long (pdfTikL) and split (pdfTikS) flies expressing UASTik in PDF+ cells relative to the UAS control alone.(B) Representative images of smLNv cells labeled for PDF (green) and PER (red) in control (pdfGal4/+) and pdfTik (pdfGal4/+; UASTikT1/+).(C) Quantification of PER levels indicates a delay in the decline of PER protein during middle of the subjective day.(D) Ratio of nuclear:cytoplasmic PER signal in smLNv from control and UASTik-expressing brains shows a diminished relative nuclear accumulation of PER in pdfTik cells.(E) Average pixel intensity of nuclear PER levels in control and pdfTik smLNv nuclei shows delayed nuclear accumulation with CK2α loss of function.(F) Cytoplasmic pixel intensity demonstrates elevated levels of PER in pdfTik smLNv during early subjective day, suggesting that reduced CK2α activity causes excess PER to be sequestered in the cytoplasm. n = 50–76 cells from two independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211540&req=5

pgen-0040012-g004: Expression of CK2αTik in PDF-positive Pacemaker Neurons Increases PER Levels and Delays Nuclear Accumulation(A) Average behavior activity profile for the first day of constant darkness reveals a delayed evening peak for both long (pdfTikL) and split (pdfTikS) flies expressing UASTik in PDF+ cells relative to the UAS control alone.(B) Representative images of smLNv cells labeled for PDF (green) and PER (red) in control (pdfGal4/+) and pdfTik (pdfGal4/+; UASTikT1/+).(C) Quantification of PER levels indicates a delay in the decline of PER protein during middle of the subjective day.(D) Ratio of nuclear:cytoplasmic PER signal in smLNv from control and UASTik-expressing brains shows a diminished relative nuclear accumulation of PER in pdfTik cells.(E) Average pixel intensity of nuclear PER levels in control and pdfTik smLNv nuclei shows delayed nuclear accumulation with CK2α loss of function.(F) Cytoplasmic pixel intensity demonstrates elevated levels of PER in pdfTik smLNv during early subjective day, suggesting that reduced CK2α activity causes excess PER to be sequestered in the cytoplasm. n = 50–76 cells from two independent experiments.
Mentions: To determine the effects of CK2α loss of function on core molecular clock rhythms, we tested whether expression of UASTik in PDF-positive LNv altered cycling of the core clock protein PER. Levels and cellular distribution of PER protein in smLNv were examined quantitatively on the first day of DD in pdfTik or control Gal4 flies. Although we do observe splitting in these flies, behavior remains largely synchronous on the first day of DD (Figure 4A). Control flies show the typical evening peak of activity at ∼CT12 while the long-period pdfTik flies have a delayed evening activity peak, regardless of whether they exhibit split periods or not (Figure 4A, pdfTikL v. pdfTikS). Measurements of pixel intensity indirectly report the amount of PER protein in smLNv [44]; as seen in Figure 4B and 4C, PER levels are elevated in smLNv of pdfTik during the subjective day relative to controls. Wild type PER levels wane from CT4–8 and begin accumulating again in the subjective evening (CT16–20); in contrast, a prolonged decline in PER throughout the day (CT4–12) is evident in pdfTik flies, and levels only disappear during subjective evening (CT12–20), consistent with a long period phenotype. Peak and trough PER levels are also elevated in pdfTik flies relative to controls (p < 0.001 comparing pdfGal4/+ CT0 to pdfTik CT4 for peak and pdfGal4/+ CT12 to pdfTik CT16 for trough, Figure 4C). PER typically transitions from the cytoplasm to a predominantly nuclear distribution during the middle of the night, and such a pattern is observed in pdfGal4/+ control flies (Figure 4B and 4D). However, the amplitude of the localization rhythm (as quantified by the nuclear:cytoplasmic ratio) is seriously reduced in pdfTik flies (Figure 4D, p < 0.001 at CT0, CT4, CT8, and CT20 pdfGal4/+ v. pdfTik). The timing of nuclear localization is also delayed in pdfTik flies; while PER never becomes predominantly nuclear, the time at which the most PER is localized to the nucleus occurs later from CT4–12 in pdfTik smLNv, rather than CT0–4 for the GAL4 control (Figure 4D and 4E). This finding is supported by analysis of nuclear PER levels in pdfGal4/+ and pdfTik smLNv. Nuclear PER levels accumulate to a similar degree in pdfTik as in the Gal4 control; however, nuclear levels do not rise until later in the subjective day relative to control (Figure 4E). The overall fraction of nuclear PER is lower (Figure 4D), as more of the PER protein in pdfTik neurons remains sequestered in the cytoplasm (Figure 4F). Indeed, the reduced nuclear PER levels in the face of elevated cytoplasmic PER levels at CT0 provide the most compelling evidence that CK2α is important for nuclear PER localization independent of regulating its cytoplasmic abundance. These results are consistent with prior reports that reduction of CK2 activity inhibits nuclear entry [16].

Bottom Line: CK2alpha(Tik), when targeted to a subset of pacemaker neurons, generates period splitting, resulting in flies exhibiting both long and near 24-h periods.These behavioral effects are evident even when CK2alpha(Tik) expression is induced only during adulthood, implicating an acute role for CK2alpha function in circadian rhythms.CK2alpha(Tik) expression results in reduced PER phosphorylation, delayed nuclear entry, and dampened cycling with elevated trough levels of PER.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois, United States of America.

ABSTRACT
Circadian clocks organize the precise timing of cellular and behavioral events. In Drosophila, circadian clocks consist of negative feedback loops in which the clock component PERIOD (PER) represses its own transcription. PER phosphorylation is a critical step in timing the onset and termination of this feedback. The protein kinase CK2 has been linked to circadian timing, but the importance of this contribution is unclear; it is not certain where and when CK2 acts to regulate circadian rhythms. To determine its temporal and spatial functions, a dominant negative mutant of the catalytic alpha subunit, CK2alpha(Tik), was targeted to circadian neurons. Behaviorally, CK2alpha(Tik) induces severe period lengthening (approximately 33 h), greater than nearly all known circadian mutant alleles, and abolishes detectable free-running behavioral rhythmicity at high levels of expression. CK2alpha(Tik), when targeted to a subset of pacemaker neurons, generates period splitting, resulting in flies exhibiting both long and near 24-h periods. These behavioral effects are evident even when CK2alpha(Tik) expression is induced only during adulthood, implicating an acute role for CK2alpha function in circadian rhythms. CK2alpha(Tik) expression results in reduced PER phosphorylation, delayed nuclear entry, and dampened cycling with elevated trough levels of PER. Heightened trough levels of per transcript accompany increased protein levels, suggesting that CK2alpha(Tik) disturbs negative feedback of PER on its own transcription. Taken together, these in vivo data implicate a central role of CK2alpha function in timing PER negative feedback in adult circadian neurons.

Show MeSH
Related in: MedlinePlus