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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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Conditional Induction of CK2αTik Induces Period Lengthening during AdulthoodFlies were raised at 18 °C to allow Gal80 inhibition of Gal4-mediated transcription and then tested at either the Gal80-permissive (18 °C) or -restrictive (29 °C) temperatures.(A) Average period values. Period lengthening is observed when UASTik is induced in adult LNv acutely during the testing period.(B) Sample actograms for the indicated genotypes at Gal80-permissive or -restrictive temperatures.
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pgen-0040012-g003: Conditional Induction of CK2αTik Induces Period Lengthening during AdulthoodFlies were raised at 18 °C to allow Gal80 inhibition of Gal4-mediated transcription and then tested at either the Gal80-permissive (18 °C) or -restrictive (29 °C) temperatures.(A) Average period values. Period lengthening is observed when UASTik is induced in adult LNv acutely during the testing period.(B) Sample actograms for the indicated genotypes at Gal80-permissive or -restrictive temperatures.

Mentions: Given that CK2 acts in multiple pathways throughout the life cycle of the fly, we queried if the CK2αTik phenotype is due to developmental/compensatory effects or whether loss of CK2α function during adulthood would still result in lengthening of period. In order to address this concern, we utilized a temperature sensitive Gal80 inhibitor of Gal4 expressed in all cells under the tubulin promoter (tubGal80ts, [39]). This conditional, temporal and regional gene expression targeting (TARGET) system has been previously used to examine the genetic basis of complex behaviors such as memory in Drosophila [39]. tubGal80ts represses GAL4 at the permissive temperature of 18 °C, but is inactivated and fails to repress GAL4 at the restrictive 29 °C temperature. We generated pdfGal4/tubGal80ts; UASTikT1/+ flies and raised them at the permissive temperature (18 °C) to prevent expression of UASTik so that CK2α function would be largely intact during development. Flies were then tested at either permissive (18 °C) or restrictive (29 °C) temperatures and period was calculated during constant conditions. A cardinal feature of circadian clocks is their temperature compensation, i.e., period is roughly invariant over a broad temperature range [40,41]. Consistent with this idea, the control strain here (tubGal80ts/+; UASTikT1/+) shows little period change between 18 °C and 29 °C (Figure 3A and 3B, top panels). Constitutively inhibiting CK2α in pdfTik flies again demonstrates the severe period lengthening effect at both temperatures (Figure 3A and 3B, middle panels); splitting of rhythms in these flies is observed at levels similar to those described above, but only at 29 °C (unpublished data). Interestingly, when dominant-negative UASTik is selectively activated at 29 °C during testing of adult flies, the extreme long period phenotype (>30 h) is still manifested (Figure 3A and 3B, bottom panels). Slight period lengthening is detectable at 18 °C; however, this effect is much smaller than observed at 29 °C, and is likely due to incomplete Gal80 inhibition of Gal4. Additionally, two split periods are again observed at 29 °C in conditionally inhibited circadian CK2α flies, but not at 18 °C (data not shown). These results indicate that CK2α plays a direct role in adult circadian rhythms, and its loss of function in Tik and UASTik animals is not likely due to some developmental artifact. Consistent with this idea, inspection of LNv structure and PDF labeling in UASTik-expressing brains reveals no gross abnormalities of circadian pacemaker anatomy (unpublished data). To our knowledge, this is one of the few temporal investigations of clock gene function demonstrating an acute role of a circadian gene during adulthood [42,43].


Dominant-negative CK2alpha induces potent effects on circadian rhythmicity.

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

Conditional Induction of CK2αTik Induces Period Lengthening during AdulthoodFlies were raised at 18 °C to allow Gal80 inhibition of Gal4-mediated transcription and then tested at either the Gal80-permissive (18 °C) or -restrictive (29 °C) temperatures.(A) Average period values. Period lengthening is observed when UASTik is induced in adult LNv acutely during the testing period.(B) Sample actograms for the indicated genotypes at Gal80-permissive or -restrictive temperatures.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211540&req=5

pgen-0040012-g003: Conditional Induction of CK2αTik Induces Period Lengthening during AdulthoodFlies were raised at 18 °C to allow Gal80 inhibition of Gal4-mediated transcription and then tested at either the Gal80-permissive (18 °C) or -restrictive (29 °C) temperatures.(A) Average period values. Period lengthening is observed when UASTik is induced in adult LNv acutely during the testing period.(B) Sample actograms for the indicated genotypes at Gal80-permissive or -restrictive temperatures.
Mentions: Given that CK2 acts in multiple pathways throughout the life cycle of the fly, we queried if the CK2αTik phenotype is due to developmental/compensatory effects or whether loss of CK2α function during adulthood would still result in lengthening of period. In order to address this concern, we utilized a temperature sensitive Gal80 inhibitor of Gal4 expressed in all cells under the tubulin promoter (tubGal80ts, [39]). This conditional, temporal and regional gene expression targeting (TARGET) system has been previously used to examine the genetic basis of complex behaviors such as memory in Drosophila [39]. tubGal80ts represses GAL4 at the permissive temperature of 18 °C, but is inactivated and fails to repress GAL4 at the restrictive 29 °C temperature. We generated pdfGal4/tubGal80ts; UASTikT1/+ flies and raised them at the permissive temperature (18 °C) to prevent expression of UASTik so that CK2α function would be largely intact during development. Flies were then tested at either permissive (18 °C) or restrictive (29 °C) temperatures and period was calculated during constant conditions. A cardinal feature of circadian clocks is their temperature compensation, i.e., period is roughly invariant over a broad temperature range [40,41]. Consistent with this idea, the control strain here (tubGal80ts/+; UASTikT1/+) shows little period change between 18 °C and 29 °C (Figure 3A and 3B, top panels). Constitutively inhibiting CK2α in pdfTik flies again demonstrates the severe period lengthening effect at both temperatures (Figure 3A and 3B, middle panels); splitting of rhythms in these flies is observed at levels similar to those described above, but only at 29 °C (unpublished data). Interestingly, when dominant-negative UASTik is selectively activated at 29 °C during testing of adult flies, the extreme long period phenotype (>30 h) is still manifested (Figure 3A and 3B, bottom panels). Slight period lengthening is detectable at 18 °C; however, this effect is much smaller than observed at 29 °C, and is likely due to incomplete Gal80 inhibition of Gal4. Additionally, two split periods are again observed at 29 °C in conditionally inhibited circadian CK2α flies, but not at 18 °C (data not shown). These results indicate that CK2α plays a direct role in adult circadian rhythms, and its loss of function in Tik and UASTik animals is not likely due to some developmental artifact. Consistent with this idea, inspection of LNv structure and PDF labeling in UASTik-expressing brains reveals no gross abnormalities of circadian pacemaker anatomy (unpublished data). To our knowledge, this is one of the few temporal investigations of clock gene function demonstrating an acute role of a circadian gene during adulthood [42,43].

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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