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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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Circadian CK2α Loss of Function Alters Period and Rhythmicity(A–E) Representative double-plotted actograms of indicated genotypes. The x-axis symbolizes two consecutive 24-h time frames; gray bars, subjective day; black bars, subjective night. The y-axis represents consecutive days and vertical black bars represent activity of a single fly.(A) The UASTik alone control shows a normal 24-h period.(B) Expression of a single copy of UASTik with timGal4, a broad circadian driver, lengthens period while (C) increased transgene dosage induces behavioral arrhythmicity.(D) Overexpression of dominant-negative CK2α in PDF-positive clock neurons causes long periods or (E) period splitting.
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pgen-0040012-g001: Circadian CK2α Loss of Function Alters Period and Rhythmicity(A–E) Representative double-plotted actograms of indicated genotypes. The x-axis symbolizes two consecutive 24-h time frames; gray bars, subjective day; black bars, subjective night. The y-axis represents consecutive days and vertical black bars represent activity of a single fly.(A) The UASTik alone control shows a normal 24-h period.(B) Expression of a single copy of UASTik with timGal4, a broad circadian driver, lengthens period while (C) increased transgene dosage induces behavioral arrhythmicity.(D) Overexpression of dominant-negative CK2α in PDF-positive clock neurons causes long periods or (E) period splitting.

Mentions: To examine the behavioral consequences of CK2αTik expression, we crossed flies bearing UAS-driven CK2αTik (UASTik) with timGal4–62 driver flies [27] and assayed circadian behavior in the progeny (timGal4/+; UASTikT1/+, “timTik”). The Drosophila circadian network consists of six bilateral groups of cells: large and small ventral lateral neurons (lg- and sm- LNv), dorsal lateral neurons (LNd), and three clusters of dorsal neurons (DN1–3) [28]. The tim promoter induces GAL4 expression in all of these key neuronal clusters that coordinate circadian behavior [29]. To our surprise, these timTik flies display extraordinarily long periods averaging ∼33 h relative to control periods of ∼24 h (Figure 1, compare Figure 1A and Figure 1B; Table 1). Moreover, the influence on period is dose-dependent; by increasing Gal4 dosage in timTik flies with a second circadian driver, cry16Gal4 [30], the period is further lengthened to ∼37 h (Table 1). Confirming the circadian specificity of this result, expression of UASTik only in photoreceptor neurons with the GMRGal4 driver [31] does not result in period lengthening (data not shown). Heterozygous Tik/+ mutant flies display periods 2–3 h longer than wild-type controls with a reduction of ∼50% in CK2 activity [16]. The magnitude of the period effects strongly argues that CK2 activity is more gravely inhibited in timTik flies. The fact that the magnitude of period effects exceeds that of nearly all circadian mutant alleles suggests that CK2 activity is critically important for setting circadian period.


Dominant-negative CK2alpha induces potent effects on circadian rhythmicity.

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

Circadian CK2α Loss of Function Alters Period and Rhythmicity(A–E) Representative double-plotted actograms of indicated genotypes. The x-axis symbolizes two consecutive 24-h time frames; gray bars, subjective day; black bars, subjective night. The y-axis represents consecutive days and vertical black bars represent activity of a single fly.(A) The UASTik alone control shows a normal 24-h period.(B) Expression of a single copy of UASTik with timGal4, a broad circadian driver, lengthens period while (C) increased transgene dosage induces behavioral arrhythmicity.(D) Overexpression of dominant-negative CK2α in PDF-positive clock neurons causes long periods or (E) period splitting.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211540&req=5

pgen-0040012-g001: Circadian CK2α Loss of Function Alters Period and Rhythmicity(A–E) Representative double-plotted actograms of indicated genotypes. The x-axis symbolizes two consecutive 24-h time frames; gray bars, subjective day; black bars, subjective night. The y-axis represents consecutive days and vertical black bars represent activity of a single fly.(A) The UASTik alone control shows a normal 24-h period.(B) Expression of a single copy of UASTik with timGal4, a broad circadian driver, lengthens period while (C) increased transgene dosage induces behavioral arrhythmicity.(D) Overexpression of dominant-negative CK2α in PDF-positive clock neurons causes long periods or (E) period splitting.
Mentions: To examine the behavioral consequences of CK2αTik expression, we crossed flies bearing UAS-driven CK2αTik (UASTik) with timGal4–62 driver flies [27] and assayed circadian behavior in the progeny (timGal4/+; UASTikT1/+, “timTik”). The Drosophila circadian network consists of six bilateral groups of cells: large and small ventral lateral neurons (lg- and sm- LNv), dorsal lateral neurons (LNd), and three clusters of dorsal neurons (DN1–3) [28]. The tim promoter induces GAL4 expression in all of these key neuronal clusters that coordinate circadian behavior [29]. To our surprise, these timTik flies display extraordinarily long periods averaging ∼33 h relative to control periods of ∼24 h (Figure 1, compare Figure 1A and Figure 1B; Table 1). Moreover, the influence on period is dose-dependent; by increasing Gal4 dosage in timTik flies with a second circadian driver, cry16Gal4 [30], the period is further lengthened to ∼37 h (Table 1). Confirming the circadian specificity of this result, expression of UASTik only in photoreceptor neurons with the GMRGal4 driver [31] does not result in period lengthening (data not shown). Heterozygous Tik/+ mutant flies display periods 2–3 h longer than wild-type controls with a reduction of ∼50% in CK2 activity [16]. The magnitude of the period effects strongly argues that CK2 activity is more gravely inhibited in timTik flies. The fact that the magnitude of period effects exceeds that of nearly all circadian mutant alleles suggests that CK2 activity is critically important for setting circadian period.

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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