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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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Overexpression of Dominant-Negative CK2α in LNv, but Not All Circadian Neurons, Induces Behavioral Splitting(A) Percentages of flies in each genotype that show a single ∼24-h period (%R-24, white), a single long ∼36-h period (%R-long, light gray), two split peaks of ∼23 h and ∼35 h, (%S, dark gray), and arrhythmicity (%AR, black). UASTik/+: control, pdfTik: pdfGal4/+; UASTikT1/+, timTik: timGal4/+; UASTikT1/+. Overexpression of UASTik in LNv only causes behavioral splitting in 45% of flies; such splitting is not observed when all circadian neurons are subjected to UASTik expression.(B) Representative Lomb-Scargle periodograms depicting pdfGal4/+; UASTikT1/+ flies with a single long period (left) or split periods (right).
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pgen-0040012-g002: Overexpression of Dominant-Negative CK2α in LNv, but Not All Circadian Neurons, Induces Behavioral Splitting(A) Percentages of flies in each genotype that show a single ∼24-h period (%R-24, white), a single long ∼36-h period (%R-long, light gray), two split peaks of ∼23 h and ∼35 h, (%S, dark gray), and arrhythmicity (%AR, black). UASTik/+: control, pdfTik: pdfGal4/+; UASTikT1/+, timTik: timGal4/+; UASTikT1/+. Overexpression of UASTik in LNv only causes behavioral splitting in 45% of flies; such splitting is not observed when all circadian neurons are subjected to UASTik expression.(B) Representative Lomb-Scargle periodograms depicting pdfGal4/+; UASTikT1/+ flies with a single long period (left) or split periods (right).

Mentions: While pdfTik flies show a long period phenotype, we also noted variability in the period measurement and reduction of the strength of the rhythm in these flies (Table 1). It was hypothesized that “wild-type” non-PDF clock neurons were unable to entrain to the long period program in PDF+ cells, and were expressing a secondary rhythm. To see if flies were exhibiting more than one period, we performed periodogram analysis using the Lomb-Scargle method [37,38]. This approach eliminates misidentification of periods that are simply multiples of a true period. This analysis reveals that approximately 45% of pdfTik animals display two significant periods (Figures 1E and 2A). The dominant period is 35.3 (+/− 0.3) h while a secondary peak indicates an average period of 23.2 (+/− 0.1) h (Figures 2B and S2). When UASTik is expressed as a heterozygote with the broader expressing timGal4 driver, reduced rhythm strength and splitting is not detectable (Figure 2A; Table 1), suggesting that hyper-elongating period only in PDF-positive LNv causes uncoupling of clock cell groups. We propose that non-PDF neurons are unable to maintain synchrony with PDF clocks with extreme periods. To our knowledge, this is the first example of complex rhythmicity due to altering period length in a subset of pacemaker neurons.


Dominant-negative CK2alpha induces potent effects on circadian rhythmicity.

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

Overexpression of Dominant-Negative CK2α in LNv, but Not All Circadian Neurons, Induces Behavioral Splitting(A) Percentages of flies in each genotype that show a single ∼24-h period (%R-24, white), a single long ∼36-h period (%R-long, light gray), two split peaks of ∼23 h and ∼35 h, (%S, dark gray), and arrhythmicity (%AR, black). UASTik/+: control, pdfTik: pdfGal4/+; UASTikT1/+, timTik: timGal4/+; UASTikT1/+. Overexpression of UASTik in LNv only causes behavioral splitting in 45% of flies; such splitting is not observed when all circadian neurons are subjected to UASTik expression.(B) Representative Lomb-Scargle periodograms depicting pdfGal4/+; UASTikT1/+ flies with a single long period (left) or split periods (right).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211540&req=5

pgen-0040012-g002: Overexpression of Dominant-Negative CK2α in LNv, but Not All Circadian Neurons, Induces Behavioral Splitting(A) Percentages of flies in each genotype that show a single ∼24-h period (%R-24, white), a single long ∼36-h period (%R-long, light gray), two split peaks of ∼23 h and ∼35 h, (%S, dark gray), and arrhythmicity (%AR, black). UASTik/+: control, pdfTik: pdfGal4/+; UASTikT1/+, timTik: timGal4/+; UASTikT1/+. Overexpression of UASTik in LNv only causes behavioral splitting in 45% of flies; such splitting is not observed when all circadian neurons are subjected to UASTik expression.(B) Representative Lomb-Scargle periodograms depicting pdfGal4/+; UASTikT1/+ flies with a single long period (left) or split periods (right).
Mentions: While pdfTik flies show a long period phenotype, we also noted variability in the period measurement and reduction of the strength of the rhythm in these flies (Table 1). It was hypothesized that “wild-type” non-PDF clock neurons were unable to entrain to the long period program in PDF+ cells, and were expressing a secondary rhythm. To see if flies were exhibiting more than one period, we performed periodogram analysis using the Lomb-Scargle method [37,38]. This approach eliminates misidentification of periods that are simply multiples of a true period. This analysis reveals that approximately 45% of pdfTik animals display two significant periods (Figures 1E and 2A). The dominant period is 35.3 (+/− 0.3) h while a secondary peak indicates an average period of 23.2 (+/− 0.1) h (Figures 2B and S2). When UASTik is expressed as a heterozygote with the broader expressing timGal4 driver, reduced rhythm strength and splitting is not detectable (Figure 2A; Table 1), suggesting that hyper-elongating period only in PDF-positive LNv causes uncoupling of clock cell groups. We propose that non-PDF neurons are unable to maintain synchrony with PDF clocks with extreme periods. To our knowledge, this is the first example of complex rhythmicity due to altering period length in a subset of pacemaker neurons.

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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