A recent study shows that cycling of cryptochrome proteins is dispensable for circadian clock function in mammalian cells. Is it time for a paradigm shift in how we think about the circadian clock mechanism?

Merrow, Martha

Roenneberg, Till

English

A recent study shows that cycling of cryptochrome proteins is dispensable for circadian clock function in mammalian cells. Is it time for a paradigm shift in how we think about the circadian clock mechanism

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Circadian Clock: Time for a Phase Shift of Ideas? 

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   University of GroningenCircadian clockMerrow, Martha; Roenneberg, TillPublished in:Current BiologyDOI:10.1016/j.cub.2007.06.041IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2007Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Merrow, M., & Roenneberg, T. (2007). Circadian clock: Time for a phase shift of ideas? Current Biology,17(16), R636-R638. https://doi.org/10.1016/j.cub.2007.06.041CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 31-10-2023treadmill and to be implicated ina mitotic-like process.References1. Michie, K.A., and Löwe, J. (2006).Dynamic filaments of the bacterialcytoskeleton. Annu. Rev. Biochem. 75,467–492.2. Gitai, Z. (2007). Diversification andspecialization of the bacterialcytoskeleton. Curr. Opin. Cell. Biol. 19,5–12.3. Margolin, W. (2005). FtsZ and the divisionof prokaryotic cells and organelles. Nat.Rev. Mol. Cell. Biol. 6, 862–871.4. Schlieper, D., Oliva, M.A., Andreu, J.M.,and Löwe, J. (2005). Structure of bacterialtubulin BtubA/B: evidence for horizontalgene transfer. Proc. Natl. Acad. Sci. USA102, 9170–9175.5. Vaughan, S., Wickstead, B., Gull, K., andAddinall, S.G. (2004). Molecular evolutionof FtsZ protein sequences encoded withinthe genomes of archaea, bacteria, andeukaryota. J. Mol. Evol. 58, 19–29.6. Koehler, T.M. (2002). Bacillus anthracisgenetics and virulence gene regulation.Curr. Top. Microbiol. Immunol. 271,143–164.7. Tinsley, E., and Khan, S.A. (2006). A novelFtsZ-like protein is involved in replicationof the anthrax toxin-encoding pXO1plasmid in Bacillus anthracis. J. Bacteriol.188, 2829–2835.8. Berry, C., O’Neil, S., Ben-Dov, E.,Jones, A.F., Murphy, L., Quail, M.A.,Holden, M.T., Harris, D., Zaritsky, A., andParkhill, J. (2002). Complete sequenceand organization of pBtoxis, the toxin-coding plasmid of Bacillus thuringiensissubsp. israelensis. Appl. Environ.Microbiol. 68, 5082–5095.9. Tang, M., Bideshi, D.K., Park, H.W., andFederici, B.A. (2006). Minireplicon frompBtoxis of Bacillus thuringiensis subsp.israelensis. Appl. Environ. Microbiol. 72,6948–6954.10. Larsen, R.A., Cusumano, C., Fujioka, A.,Lim-Fong, G., Patterson, P., andPogliano, J. (2007). Treadmilling ofa prokaryotic tubulin-like protein, TubZ,required for plasmid stability in Bacillusthuringiensis. Genes Dev. 21, 1340–1352.11. Theriot, J.A., and Mitchison, T.J. (1991).Actin microfilament dynamics inlocomoting cells. Nature 352, 126–131.12. Kim, S.Y., Gitai, Z., Kinkhabwala, A.,Shapiro, L., and Moerner, W.E. (2006).Single molecules of the bacterial actinMreB undergo directed treadmillingmotion in Caulobacter crescentus. Proc.Natl. Acad. Sci. USA 103, 10929–10934.13. Waterman-Storer, C.M., and Salmon, E.D.(1997). Microtubule dynamics:treadmilling comes around again. Curr.Biol. 7, 369–372.14. Rodionov, V.I., and Borisy, G.G. (1997).Microtubule treadmilling in vivo. Science275, 215–218.15. Shaw, S.L., Kamyar, R., andEhrhardt, D.W. (2003). Sustainedmicrotubule treadmilling in Arabidopsiscortical arrays. Science 300, 1715–1718.16. Thanedar, S., and Margolin, W. (2004).FtsZ exhibits rapid movement andoscillation waves in helix-like patternsin Escherichia coli. Curr. Biol. 14,1167–1173.17. Redick, S.D., Stricker, J., Briscoe, G., andErickson, H.P. (2005). Mutants of FtsZtargeting the protofilament interface:effects on cell division and GTPaseactivity. J. Bacteriol. 187, 2727–2736.18. Hayes, F., and Barilla, D. (2006). Thebacterial segrosome: a dynamicnucleoprotein machine for DNA traffickingand segregation. Nat. Rev. Microbiol. 4,133–143.Department of Microbiologyand Molecular Genetics,University of Texas Medical School,6431 Fannin Street, Houston,Texas 77030, USA.E-mail: William.Margolin@uth.tmc.eduDOI: 10.1016/j.cub.2007.06.020Current Biology Vol 17 No 16R636Circadian Clock: Time for a PhaseShift of Ideas?A recent study shows that cycling of cryptochrome proteins isdispensable for circadian clock function in mammalian cells. Is it timefor a paradigm shift in how we think about the circadian clockmechanism?Martha Merrow1,*and Till Roenneberg2Clocks rule our lives. One of themis the circadian clock — anadaptive, endogenous temporalprogramme which ensures that thesame processes occur at the sametime, day after day. Theseprocesses are based on a numberof rhythmic functions ranging fromgene expression and metabolismto behaviour and they are sopronounced that we arebiochemically ‘different’ peoplefrom day to night. Daily rhythmsare self-sustained; they evenoscillate in the absence of a cyclingenvironment with their periodbeing close to 24 hours, hencecircadian. The implications of thiscircadian programme should be ofinterest to all biologists anddoctors because time of day canpotentially impact on all kinds ofexperiments and medicaldiagnoses.The genetic revolution forcomplex behaviour started withSeymour Benzer who identifiedthe first clock gene, period, inDrosophila [1]. Since then, clockgenes have been described in allcircadian model systems, fromcyanobacteria to mice. Bydiscovering how the products ofthese clock genes interact,a cohesive mechanism emergedthat is apparently common toall circadian systems: anauto-regulatory negativefeedback loop involvingtranscription and translation(Transcription TranslationFeedback Loop, TTFL). Changesin any of the loop’s componentshave dominant effects on thecircadian rhythmicity, and it hasbeen hypothesised that thisfeedback loop generatescircadian rhythms at the cellularlevel (Figure 1A).In a recent paper in CurrentBiology, Fan et al. [2] describeprovocative results that — ifrepeated, extended andelaborated — challenge the currentfeedback loop model. In mice,levels of the TTFL componentsmPERIOD (PER) 1 and 2,mCRYPTOCHROME (CRY) 1 and 2,and BMAL1 typically oscillate overthe course of a day. The oscillationis thought to depend largely onthe CRY proteins, which repressCLK/BMAL1 mediatedtranscriptional activation. PERproteins also show negativefeedback but are less potent thaneither of the CRY proteins in cellculture systems [3]. Fan et al. [2]have engineered the two keynegative feedback components,the mCRY proteins, such that theymove freely into cells. Theysurprisingly find that, despite theapparently constant level of bothCRYs and CRY-induced BMAL1expression, Per2 transcriptioncontinues to cycle rhythmically.This indicates that one of thefundamental assumptionsconcerning the mouse circadianclock seems incorrect, namely thatoscillations of CRY protein levelsDispatchR637are essential for generatingcircadian rhythmicity.There are numerous possiblescenarios which could account forthe observations of Fan et al. [2]from which we have chosen five.Scenario 1: The effects on themolecular machinery of thecircadian clock using the newcell-permeable CRY proteins couldsimply be an artefact of the methoditself. Many things happen toclock proteins on their daily journeythrough time. They are modified,gang up with other proteins, getshuffled between compartmentsand so on. Does all of this alsohappen in cells that are floodedwith the two CRY proteins? Futureexperiments will elaborate on thefates of the imported proteins andhow they compare with those ofendogenous CRY proteins inuntreated cells. However, thesurprising observation thatthe constitutively present,cell-permeant CRYs supportBMAL1 oscillations in normallyarrhythmic Cry1;Cry2 mutantfibroblasts argues against thisscenario.Scenario 2: The traditionalfeedback loop hypothesis stillholds. Experiments in manycircadian systems have shown thatseveral interlocking loops forma ‘network of time’ and the authorsmay have simply uncovered yetanother loop within the network,one that reflects PER2 rhythmsversus CRY1 and CRY2 rhythms(Figure 1B). There are already manyindications of auxiliary loopsinvolving, for example, nuclearorphan receptors, DECs orPAR-proteins. The findings ofFan et al. [2] may be linked to oneof these or may lead to a novelextra-TTFL loop.Scenario 3: The experimentmight reveal a similar phenomenonas in Drosophila, whereconstitutive expression of bothso-called negative elements of theTTFL–PER and TIM- still allowsrhythmicity in locomotor activity aswell as in the levels of the clockproteins themselves [4]. Theassumption here is that therhythmicity of the proteins — interms of level, state orlocalization — is essential forcircadian behaviour, rather thanthe rhythmicity of theirArep rep actBactCrepact actrepact rep act actrep repPDPEPP kinkinPCurrent BiologyFigure 1. Possible scenarios explaining the dispensability of CRY protein oscillationsin the circadian clock.Five scenarios as described in the text are illustrated. (A) The Transcription TranslationFeedback Loop (TTFL) model is built on the elegantly simple hypothesis that clockgene transcription, effected by transcriptional activators (act) is self-regulated by feed-back repression (rep). (B) The TTFL network is far more complex, and the actual rela-tionships may be revealed through experiments such as those described in [3]. (C) Therole of transcription (dashed lines) may not be critical for all circadian properties.Rather, post-translational modifications, such as phosphorylation (P) or sub-cellularlocalisation (block arrow) could play major roles. (D) The TTFL components may beserving input and output functions in the clock signal transduction pathway. In thiscase, the identity of the actual rhythm generator (depicted as a sine wave) may stillbe unknown. (E) The TTFL may be regulated by a ‘phosphorylation oscillator’, namelyan interaction between a kinase (kin) and a phosphatase (PP) whereby functionalactivity of clock-critical kinases is modulated via phosphatases, which are in turnregulated by components of the TTFL that are targets of the kinases.transcription (Figure 1C).Additional support for this ideacomes from experiments exposingthe clock to different dailyenvironmental cycles in the lab,whereby in the fungus Neurospora,it is the clock gene protein levelsthat correlate with the ‘behavioural’phase (in this case, midnight), whilethe RNA levels appear simplydriven (masked) by the changinglight levels [5]. In the experimentshere, at least PER1 protein levelscan oscillate, so perhaps this is theessential rhythmic component,with the rhythms of the othercomponents contributingcharacteristics of the system thatcannot be appreciated in cellculture systems.Scenario 4: The TTFL is not thecore, rhythm-generatingmechanism, but rather servesboth an input and an output withfeedback function. As its input, itprocesses signals from theenvironment to the rhythmgenerator, which in turn regulatesthe input pathway. This wouldrender the TTFL also an output ofthe clock and in this role it wouldtranscriptionally control diverseoutputs (Figure 1D). Such analternative hypothesis was initiallyproposed in 1998 ([6] andreferences therein) based onmathematical modelling, andsince then numerous experimentalresults in many model systemssupport this scenario [7]. Lightreception and components of theTTFL are tightly associated. Insome cases, they serve bothfunctions, in other cases, theyhave changed their role from onefunction to the other duringevolution [8–10]. Light often leadsto rapid changes in the TTFLcomponents, concurring withphase shifts of the circadianoscillation. Others have shown,however, that residual circadianproperties persist whencomponents of the TTFL arerendered non-functional.In Drosophila, combination of thearrhythmic mutant allele of perwith cryb results in the restorationof some circadian properties,rather than an additionaldecrement [11]. In Neurospora,sporadic free running rhythmsCurrent Biology Vol 17 No 16R638[12,13] and also systematicentrainment characteristics canbe found in clock null mutants [14],and subsequently, molecularrhythms have been shown topersist in these strains [15]. Inmice, many mutants that had beenconsidered ‘clock null mutants’because they were arrhythmic(e.g., the mPer2;mCry2 doublemutant) become rhythmic whenput into constant dim light [16].Furthermore, conceptual modelsshow that alterations of inputfeedback components can havehuge effects on circadianoscillations [6]. Finally, feedbacksof the rhythm generator back ontoits inputs have been shown toshield the rhythm from noise bycompensation and, thus, addrobustness to the endogenousdaily cycle [6].Scenario 5: Some of thescenarios above indicate thattranscription and/or translationmay not need to be rhythmic toensure circadian function. Butwhere, then, is rhythmicitygenerated? A recent finding incyanobacteria suggests thatphosphorylation plays a crucialrole. Phosphorylation not onlyconfers specific half-lives toproteins, it also regulates theirsub-cellular localization and theirswitching between activity orinactivity — not only intranscription factors, which are somuch at the heart of the traditionalhypothesis of rhythm generation.In cyanobacteria, three proteinsand ATP were shown to besufficient recapitulate most of thecircadian clock properties in vitro[17]! Given the added complexityin eukaryotes, we may not expectto find a similar, simple molecularclock mechanism. However, theremay be elements ofa phosphorylation oscillator [18]that are preserved and remain tobe described, but sucha mechanism could have alsoco-evolved by recruiting any of themany autocatalytic kinases andphosphatases present in differentspecies. An indication thata phosphorylation oscillator mightbe a primordial circadianmechanism comes from severalindependent observationsconcerning human kinases andphosphatases. Human CKIautoregulates according tophosphorylation state and isdephosphorylated by PP5 ([18]and references therein), thusthese two components could forma feedback loop if they display thecorrect kinetics and if they wereconnected to an input pathway.PP5 activity, in turn, depends onbinding to CRY2 ([18] andreferences therein), whichfluctuates in concentration inmany cells. Even withoutoscillations in the amount ofCRY2, rhythms in the state ofphosphorylation could play a partin a phosphorylation oscillatormechanism and might explain theresults shown with the cell-permeable CRY1 and CRY2 byFan et al. [2].Why is it important tounderstand how the circadiansystem is put together? Severalreports have demonstrated linksbetween health and behaviourwith respect to chronotype. Arecent publication showed that theerectile dysfunction drug sildenafilhas a profound impact onre-synchronization of thecircadian system in hamsters [19].If we are to start medicating thecircadian system itself or usea robust circadian system tomedicate other pathologies, wehave to know what we’re aimingfor and how the parts of thesystem are put together.References1. Konopka, R., and Benzer, S. (1971). Clockmutants of Drosophila melanogaster.Proc. Natl. Acad. Sci. USA 68,2112–2116.2. Fan, Y., Hida, A., Anderson, D.A.,Izumo, M., and Johnson, C.H. (2007).Cycling of CRYPTOCHROME proteins isnot necessary for circadian clock functionin mammalian fibroblasts. Curr. Biol. 17,1091–1100.3. Kume, K., Zylka, M.J., Sriram, S.,Shearman, L.P., Weaver, D.R., Jin, X.,Maywood, E.S., Hastings, M.H., andReppert, S.M. (1999). mCRY1 and mCRY2are essential components of the negativelimb of the circadian clock feedback loop.Cell 98, 193–205.4. Yang, Z., and Sehgal, A. (2001). Role ofmolecular oscillations in generatingbehavioral rhythms in Drosophila. Neuron29, 453–467.5. Tan, Y., Dragovic, Z., Roenneberg, T., andMerrow, M. (2004). Entrainment of thecircadian clock: translational and post-translational control as key elements.Curr. Biol. 14, 433–438.6. Roenneberg, T., and Merrow, M. (1999).Circadian clocks and metabolism. J. Biol.Rhythms 14, 449–459.7. Lakin-Thomas, P.L. (2006).Transcriptional feedback oscillators:maybe, maybe not. J. Biol. Rhythms 21,83–92.8. Froehlich, A.C., Liu, Y., Loros, J.J., andDunlap, J.C. (2002). White Collar-1,a circadian blue light photoreceptor,binding to the frequency promoter.Science 297, 815–819.9. Krishnan, B., Levine, J.D., Lynch, M.K.S.,Dowse, H.B., Funes, P., Hall, J.C.,Hardin, P.E., and Dryer, S.E. (2001). A newrole for cryptochrome in a Drosophilacircadian oscillator. Nature 411,313–317.10. Yuan, Q., Metterville, D., Briscoe, A.D.,and Reppert, S.M. (2007). Insectcryptochromes: gene duplication and lossdefine diverse ways to construct insectcircadian clocks. Mol. Biol. Evol. 24,948–955.11. Collins, B.H., Dissel, S., Gaten, E.,Rosato, E., and Kyriacou, C.P. (2005).Disruption of Cryptochrome partiallyrestores circadian rhythmicity to thearrhythmic period mutant of Drosophila.Proc. Natl. Acad. Sci. USA 102,19021–19026.12. Loros, J.J., and Feldman, J.F. (1986). Lossof temperature compensation of circadianperiod length in the frq-9 mutant ofNeurospora crassa. J. Biol. Rhythms 1,187–198.13. Dragovic, Z., Tan, Y., Görl, M.,Roenneberg, T., and Merrow, M. (2002).Light reception and circadian behavior in‘blind’ and ‘clock-less’ mutants ofNeurospora crassa. EMBO J. 21,3643–3651.14. Roenneberg, T., Dragovic, Z., andMerrow, M. (2005). Demasking biologicaloscillators: properties and principles ofentrainment exemplified by theNeurospora circadian clock. Proc.Natl. Acad. Sci. USA 102,7742–7747.15. Correa, A., Lewis, Z.A., Greene, A.V.,March, I.J., Gomer, R.H., and Bell-Pedersen, D. (2003). Multiple oscillatorsregulate circadian gene expression inNeurospora. Proc. Natl. Acad. Sci. USA100, 13597–13602.16. Steinlechner, S., Jacobmeier, B.,Scherbarth, F., Dernbach, H., Kruse, F.,and Albrecht, U. (2002). Robustcircadian rhythmicity of Per1 and Per2mutant mice in constant light anddynamics of Per1 and Per2 geneexpression under long and shortphotoperiods. J. Biol. Rhythms 17,202–209.17. Nakajima, M., Imai, K., Ito, H.,Nishiwaki, T., Murayama, Y., Iwasaki, H.,Oyama, T., and Kondo, T. (2005).Reconstitution of circadian oscillation ofcyanobacterial KaiC phosphorylationin vitro. Science 308, 414–415.18. Merrow, M., Mazzotta, G., Chen, Z., andRoenneberg, T. (2006). The right place atthe right time: regulation of daily timing byphosphorylation. Genes Dev. 20,2629–2633.19. Agostino, P.V., Plano, S.A., andGolombek, D.A. (2007). Sildenafilaccelerates reentrainment of circadianrhythms after advancing light schedules.Proc. Natl. Acad. Sci. USA 104,9834–9839.1Biological Center, University ofGroningen, Postbus 14, 9750AA, Haren,Netherlands. 2Institute of MedicalPsychology, University of Munich,Goethestrasse 31, 80336 München,Germany.*E-mail: m.merrow@rug.nlDOI: 10.1016/j.cub.2007.06.041

Circadian clock:Time for a phase shift of ideas?

Proceedings - University of Groningen

   University of GroningenCircadian clockMerrow, Martha; Roenneberg, TillPublished in:Current BiologyDOI:10.1016/j.cub.2007.06.041IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2007Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Merrow, M., & Roenneberg, T. (2007). Circadian clock: Time for a phase shift of ideas? Current Biology,17(16), R636-R638. https://doi.org/10.1016/j.cub.2007.06.041CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 03-06-2022treadmill and to be implicated ina mitotic-like process.References1. Michie, K.A., and Löwe, J. (2006).Dynamic filaments of the bacterialcytoskeleton. Annu. Rev. Biochem. 75,467–492.2. Gitai, Z. (2007). Diversification andspecialization of the bacterialcytoskeleton. Curr. Opin. Cell. Biol. 19,5–12.3. Margolin, W. (2005). FtsZ and the divisionof prokaryotic cells and organelles. Nat.Rev. Mol. Cell. Biol. 6, 862–871.4. Schlieper, D., Oliva, M.A., Andreu, J.M.,and Löwe, J. (2005). Structure of bacterialtubulin BtubA/B: evidence for horizontalgene transfer. Proc. Natl. Acad. Sci. USA102, 9170–9175.5. Vaughan, S., Wickstead, B., Gull, K., andAddinall, S.G. (2004). Molecular evolutionof FtsZ protein sequences encoded withinthe genomes of archaea, bacteria, andeukaryota. J. Mol. Evol. 58, 19–29.6. Koehler, T.M. (2002). Bacillus anthracisgenetics and virulence gene regulation.Curr. Top. Microbiol. Immunol. 271,143–164.7. Tinsley, E., and Khan, S.A. (2006). A novelFtsZ-like protein is involved in replicationof the anthrax toxin-encoding pXO1plasmid in Bacillus anthracis. J. Bacteriol.188, 2829–2835.8. Berry, C., O’Neil, S., Ben-Dov, E.,Jones, A.F., Murphy, L., Quail, M.A.,Holden, M.T., Harris, D., Zaritsky, A., andParkhill, J. (2002). Complete sequenceand organization of pBtoxis, the toxin-coding plasmid of Bacillus thuringiensissubsp. israelensis. Appl. Environ.Microbiol. 68, 5082–5095.9. Tang, M., Bideshi, D.K., Park, H.W., andFederici, B.A. (2006). Minireplicon frompBtoxis of Bacillus thuringiensis subsp.israelensis. Appl. Environ. Microbiol. 72,6948–6954.10. Larsen, R.A., Cusumano, C., Fujioka, A.,Lim-Fong, G., Patterson, P., andPogliano, J. (2007). Treadmilling ofa prokaryotic tubulin-like protein, TubZ,required for plasmid stability in Bacillusthuringiensis. Genes Dev. 21, 1340–1352.11. Theriot, J.A., and Mitchison, T.J. (1991).Actin microfilament dynamics inlocomoting cells. Nature 352, 126–131.12. Kim, S.Y., Gitai, Z., Kinkhabwala, A.,Shapiro, L., and Moerner, W.E. (2006).Single molecules of the bacterial actinMreB undergo directed treadmillingmotion in Caulobacter crescentus. Proc.Natl. Acad. Sci. USA 103, 10929–10934.13. Waterman-Storer, C.M., and Salmon, E.D.(1997). Microtubule dynamics:treadmilling comes around again. Curr.Biol. 7, 369–372.14. Rodionov, V.I., and Borisy, G.G. (1997).Microtubule treadmilling in vivo. Science275, 215–218.15. Shaw, S.L., Kamyar, R., andEhrhardt, D.W. (2003). Sustainedmicrotubule treadmilling in Arabidopsiscortical arrays. Science 300, 1715–1718.16. Thanedar, S., and Margolin, W. (2004).FtsZ exhibits rapid movement andoscillation waves in helix-like patternsin Escherichia coli. Curr. Biol. 14,1167–1173.17. Redick, S.D., Stricker, J., Briscoe, G., andErickson, H.P. (2005). Mutants of FtsZtargeting the protofilament interface:effects on cell division and GTPaseactivity. J. Bacteriol. 187, 2727–2736.18. Hayes, F., and Barilla, D. (2006). Thebacterial segrosome: a dynamicnucleoprotein machine for DNA traffickingand segregation. Nat. Rev. Microbiol. 4,133–143.Department of Microbiologyand Molecular Genetics,University of Texas Medical School,6431 Fannin Street, Houston,Texas 77030, USA.E-mail: William.Margolin@uth.tmc.eduDOI: 10.1016/j.cub.2007.06.020Current Biology Vol 17 No 16R636Circadian Clock: Time for a PhaseShift of Ideas?A recent study shows that cycling of cryptochrome proteins isdispensable for circadian clock function in mammalian cells. Is it timefor a paradigm shift in how we think about the circadian clockmechanism?Martha Merrow1,*and Till Roenneberg2Clocks rule our lives. One of themis the circadian clock — anadaptive, endogenous temporalprogramme which ensures that thesame processes occur at the sametime, day after day. Theseprocesses are based on a numberof rhythmic functions ranging fromgene expression and metabolismto behaviour and they are sopronounced that we arebiochemically ‘different’ peoplefrom day to night. Daily rhythmsare self-sustained; they evenoscillate in the absence of a cyclingenvironment with their periodbeing close to 24 hours, hencecircadian. The implications of thiscircadian programme should be ofinterest to all biologists anddoctors because time of day canpotentially impact on all kinds ofexperiments and medicaldiagnoses.The genetic revolution forcomplex behaviour started withSeymour Benzer who identifiedthe first clock gene, period, inDrosophila [1]. Since then, clockgenes have been described in allcircadian model systems, fromcyanobacteria to mice. Bydiscovering how the products ofthese clock genes interact,a cohesive mechanism emergedthat is apparently common toall circadian systems: anauto-regulatory negativefeedback loop involvingtranscription and translation(Transcription TranslationFeedback Loop, TTFL). Changesin any of the loop’s componentshave dominant effects on thecircadian rhythmicity, and it hasbeen hypothesised that thisfeedback loop generatescircadian rhythms at the cellularlevel (Figure 1A).In a recent paper in CurrentBiology, Fan et al. [2] describeprovocative results that — ifrepeated, extended andelaborated — challenge the currentfeedback loop model. In mice,levels of the TTFL componentsmPERIOD (PER) 1 and 2,mCRYPTOCHROME (CRY) 1 and 2,and BMAL1 typically oscillate overthe course of a day. The oscillationis thought to depend largely onthe CRY proteins, which repressCLK/BMAL1 mediatedtranscriptional activation. PERproteins also show negativefeedback but are less potent thaneither of the CRY proteins in cellculture systems [3]. Fan et al. [2]have engineered the two keynegative feedback components,the mCRY proteins, such that theymove freely into cells. Theysurprisingly find that, despite theapparently constant level of bothCRYs and CRY-induced BMAL1expression, Per2 transcriptioncontinues to cycle rhythmically.This indicates that one of thefundamental assumptionsconcerning the mouse circadianclock seems incorrect, namely thatoscillations of CRY protein levelsDispatchR637are essential for generatingcircadian rhythmicity.There are numerous possiblescenarios which could account forthe observations of Fan et al. [2]from which we have chosen five.Scenario 1: The effects on themolecular machinery of thecircadian clock using the newcell-permeable CRY proteins couldsimply be an artefact of the methoditself. Many things happen toclock proteins on their daily journeythrough time. They are modified,gang up with other proteins, getshuffled between compartmentsand so on. Does all of this alsohappen in cells that are floodedwith the two CRY proteins? Futureexperiments will elaborate on thefates of the imported proteins andhow they compare with those ofendogenous CRY proteins inuntreated cells. However, thesurprising observation thatthe constitutively present,cell-permeant CRYs supportBMAL1 oscillations in normallyarrhythmic Cry1;Cry2 mutantfibroblasts argues against thisscenario.Scenario 2: The traditionalfeedback loop hypothesis stillholds. Experiments in manycircadian systems have shown thatseveral interlocking loops forma ‘network of time’ and the authorsmay have simply uncovered yetanother loop within the network,one that reflects PER2 rhythmsversus CRY1 and CRY2 rhythms(Figure 1B). There are already manyindications of auxiliary loopsinvolving, for example, nuclearorphan receptors, DECs orPAR-proteins. The findings ofFan et al. [2] may be linked to oneof these or may lead to a novelextra-TTFL loop.Scenario 3: The experimentmight reveal a similar phenomenonas in Drosophila, whereconstitutive expression of bothso-called negative elements of theTTFL–PER and TIM- still allowsrhythmicity in locomotor activity aswell as in the levels of the clockproteins themselves [4]. Theassumption here is that therhythmicity of the proteins — interms of level, state orlocalization — is essential forcircadian behaviour, rather thanthe rhythmicity of theirArep rep actBactCrepact actrepact rep act actrep repPDPEPP kinkinPCurrent BiologyFigure 1. Possible scenarios explaining the dispensability of CRY protein oscillationsin the circadian clock.Five scenarios as described in the text are illustrated. (A) The Transcription TranslationFeedback Loop (TTFL) model is built on the elegantly simple hypothesis that clockgene transcription, effected by transcriptional activators (act) is self-regulated by feed-back repression (rep). (B) The TTFL network is far more complex, and the actual rela-tionships may be revealed through experiments such as those described in [3]. (C) Therole of transcription (dashed lines) may not be critical for all circadian properties.Rather, post-translational modifications, such as phosphorylation (P) or sub-cellularlocalisation (block arrow) could play major roles. (D) The TTFL components may beserving input and output functions in the clock signal transduction pathway. In thiscase, the identity of the actual rhythm generator (depicted as a sine wave) may stillbe unknown. (E) The TTFL may be regulated by a ‘phosphorylation oscillator’, namelyan interaction between a kinase (kin) and a phosphatase (PP) whereby functionalactivity of clock-critical kinases is modulated via phosphatases, which are in turnregulated by components of the TTFL that are targets of the kinases.transcription (Figure 1C).Additional support for this ideacomes from experiments exposingthe clock to different dailyenvironmental cycles in the lab,whereby in the fungus Neurospora,it is the clock gene protein levelsthat correlate with the ‘behavioural’phase (in this case, midnight), whilethe RNA levels appear simplydriven (masked) by the changinglight levels [5]. In the experimentshere, at least PER1 protein levelscan oscillate, so perhaps this is theessential rhythmic component,with the rhythms of the othercomponents contributingcharacteristics of the system thatcannot be appreciated in cellculture systems.Scenario 4: The TTFL is not thecore, rhythm-generatingmechanism, but rather servesboth an input and an output withfeedback function. As its input, itprocesses signals from theenvironment to the rhythmgenerator, which in turn regulatesthe input pathway. This wouldrender the TTFL also an output ofthe clock and in this role it wouldtranscriptionally control diverseoutputs (Figure 1D). Such analternative hypothesis was initiallyproposed in 1998 ([6] andreferences therein) based onmathematical modelling, andsince then numerous experimentalresults in many model systemssupport this scenario [7]. Lightreception and components of theTTFL are tightly associated. Insome cases, they serve bothfunctions, in other cases, theyhave changed their role from onefunction to the other duringevolution [8–10]. Light often leadsto rapid changes in the TTFLcomponents, concurring withphase shifts of the circadianoscillation. Others have shown,however, that residual circadianproperties persist whencomponents of the TTFL arerendered non-functional.In Drosophila, combination of thearrhythmic mutant allele of perwith cryb results in the restorationof some circadian properties,rather than an additionaldecrement [11]. In Neurospora,sporadic free running rhythmsCurrent Biology Vol 17 No 16R638[12,13] and also systematicentrainment characteristics canbe found in clock null mutants [14],and subsequently, molecularrhythms have been shown topersist in these strains [15]. Inmice, many mutants that had beenconsidered ‘clock null mutants’because they were arrhythmic(e.g., the mPer2;mCry2 doublemutant) become rhythmic whenput into constant dim light [16].Furthermore, conceptual modelsshow that alterations of inputfeedback components can havehuge effects on circadianoscillations [6]. Finally, feedbacksof the rhythm generator back ontoits inputs have been shown toshield the rhythm from noise bycompensation and, thus, addrobustness to the endogenousdaily cycle [6].Scenario 5: Some of thescenarios above indicate thattranscription and/or translationmay not need to be rhythmic toensure circadian function. Butwhere, then, is rhythmicitygenerated? A recent finding incyanobacteria suggests thatphosphorylation plays a crucialrole. Phosphorylation not onlyconfers specific half-lives toproteins, it also regulates theirsub-cellular localization and theirswitching between activity orinactivity — not only intranscription factors, which are somuch at the heart of the traditionalhypothesis of rhythm generation.In cyanobacteria, three proteinsand ATP were shown to besufficient recapitulate most of thecircadian clock properties in vitro[17]! Given the added complexityin eukaryotes, we may not expectto find a similar, simple molecularclock mechanism. However, theremay be elements ofa phosphorylation oscillator [18]that are preserved and remain tobe described, but sucha mechanism could have alsoco-evolved by recruiting any of themany autocatalytic kinases andphosphatases present in differentspecies. An indication thata phosphorylation oscillator mightbe a primordial circadianmechanism comes from severalindependent observationsconcerning human kinases andphosphatases. Human CKIautoregulates according tophosphorylation state and isdephosphorylated by PP5 ([18]and references therein), thusthese two components could forma feedback loop if they display thecorrect kinetics and if they wereconnected to an input pathway.PP5 activity, in turn, depends onbinding to CRY2 ([18] andreferences therein), whichfluctuates in concentration inmany cells. Even withoutoscillations in the amount ofCRY2, rhythms in the state ofphosphorylation could play a partin a phosphorylation oscillatormechanism and might explain theresults shown with the cell-permeable CRY1 and CRY2 byFan et al. [2].Why is it important tounderstand how the circadiansystem is put together? Severalreports have demonstrated linksbetween health and behaviourwith respect to chronotype. Arecent publication showed that theerectile dysfunction drug sildenafilhas a profound impact onre-synchronization of thecircadian system in hamsters [19].If we are to start medicating thecircadian system itself or usea robust circadian system tomedicate other pathologies, wehave to know what we’re aimingfor and how the parts of thesystem are put together.References1. Konopka, R., and Benzer, S. (1971). Clockmutants of Drosophila melanogaster.Proc. Natl. Acad. Sci. USA 68,2112–2116.2. Fan, Y., Hida, A., Anderson, D.A.,Izumo, M., and Johnson, C.H. (2007).Cycling of CRYPTOCHROME proteins isnot necessary for circadian clock functionin mammalian fibroblasts. Curr. Biol. 17,1091–1100.3. Kume, K., Zylka, M.J., Sriram, S.,Shearman, L.P., Weaver, D.R., Jin, X.,Maywood, E.S., Hastings, M.H., andReppert, S.M. (1999). mCRY1 and mCRY2are essential components of the negativelimb of the circadian clock feedback loop.Cell 98, 193–205.4. Yang, Z., and Sehgal, A. (2001). Role ofmolecular oscillations in generatingbehavioral rhythms in Drosophila. Neuron29, 453–467.5. Tan, Y., Dragovic, Z., Roenneberg, T., andMerrow, M. (2004). Entrainment of thecircadian clock: translational and post-translational control as key elements.Curr. Biol. 14, 433–438.6. Roenneberg, T., and Merrow, M. (1999).Circadian clocks and metabolism. J. Biol.Rhythms 14, 449–459.7. Lakin-Thomas, P.L. (2006).Transcriptional feedback oscillators:maybe, maybe not. J. Biol. Rhythms 21,83–92.8. Froehlich, A.C., Liu, Y., Loros, J.J., andDunlap, J.C. (2002). White Collar-1,a circadian blue light photoreceptor,binding to the frequency promoter.Science 297, 815–819.9. Krishnan, B., Levine, J.D., Lynch, M.K.S.,Dowse, H.B., Funes, P., Hall, J.C.,Hardin, P.E., and Dryer, S.E. (2001). A newrole for cryptochrome in a Drosophilacircadian oscillator. Nature 411,313–317.10. Yuan, Q., Metterville, D., Briscoe, A.D.,and Reppert, S.M. (2007). Insectcryptochromes: gene duplication and lossdefine diverse ways to construct insectcircadian clocks. Mol. Biol. Evol. 24,948–955.11. Collins, B.H., Dissel, S., Gaten, E.,Rosato, E., and Kyriacou, C.P. (2005).Disruption of Cryptochrome partiallyrestores circadian rhythmicity to thearrhythmic period mutant of Drosophila.Proc. Natl. Acad. Sci. USA 102,19021–19026.12. Loros, J.J., and Feldman, J.F. (1986). Lossof temperature compensation of circadianperiod length in the frq-9 mutant ofNeurospora crassa. J. Biol. Rhythms 1,187–198.13. Dragovic, Z., Tan, Y., Görl, M.,Roenneberg, T., and Merrow, M. (2002).Light reception and circadian behavior in‘blind’ and ‘clock-less’ mutants ofNeurospora crassa. EMBO J. 21,3643–3651.14. Roenneberg, T., Dragovic, Z., andMerrow, M. (2005). Demasking biologicaloscillators: properties and principles ofentrainment exemplified by theNeurospora circadian clock. Proc.Natl. Acad. Sci. USA 102,7742–7747.15. Correa, A., Lewis, Z.A., Greene, A.V.,March, I.J., Gomer, R.H., and Bell-Pedersen, D. (2003). Multiple oscillatorsregulate circadian gene expression inNeurospora. Proc. Natl. Acad. Sci. USA100, 13597–13602.16. Steinlechner, S., Jacobmeier, B.,Scherbarth, F., Dernbach, H., Kruse, F.,and Albrecht, U. (2002). Robustcircadian rhythmicity of Per1 and Per2mutant mice in constant light anddynamics of Per1 and Per2 geneexpression under long and shortphotoperiods. J. Biol. Rhythms 17,202–209.17. Nakajima, M., Imai, K., Ito, H.,Nishiwaki, T., Murayama, Y., Iwasaki, H.,Oyama, T., and Kondo, T. (2005).Reconstitution of circadian oscillation ofcyanobacterial KaiC phosphorylationin vitro. Science 308, 414–415.18. Merrow, M., Mazzotta, G., Chen, Z., andRoenneberg, T. (2006). The right place atthe right time: regulation of daily timing byphosphorylation. Genes Dev. 20,2629–2633.19. Agostino, P.V., Plano, S.A., andGolombek, D.A. (2007). Sildenafilaccelerates reentrainment of circadianrhythms after advancing light schedules.Proc. Natl. Acad. Sci. USA 104,9834–9839.1Biological Center, University ofGroningen, Postbus 14, 9750AA, Haren,Netherlands. 2Institute of MedicalPsychology, University of Munich,Goethestrasse 31, 80336 München,Germany.*E-mail: m.merrow@rug.nlDOI: 10.1016/j.cub.2007.06.041

University of Groningen

   University of GroningenCircadian clockMerrow, Martha; Roenneberg, TillPublished in:Current BiologyDOI:10.1016/j.cub.2007.06.041IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2007Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Merrow, M., & Roenneberg, T. (2007). Circadian clock: Time for a phase shift of ideas? 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For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 26-04-2023treadmill and to be implicated ina mitotic-like process.References1. Michie, K.A., and Löwe, J. (2006).Dynamic filaments of the bacterialcytoskeleton. Annu. Rev. Biochem. 75,467–492.2. Gitai, Z. (2007). Diversification andspecialization of the bacterialcytoskeleton. Curr. Opin. Cell. Biol. 19,5–12.3. Margolin, W. (2005). FtsZ and the divisionof prokaryotic cells and organelles. Nat.Rev. Mol. Cell. Biol. 6, 862–871.4. Schlieper, D., Oliva, M.A., Andreu, J.M.,and Löwe, J. (2005). Structure of bacterialtubulin BtubA/B: evidence for horizontalgene transfer. Proc. Natl. Acad. Sci. USA102, 9170–9175.5. Vaughan, S., Wickstead, B., Gull, K., andAddinall, S.G. (2004). Molecular evolutionof FtsZ protein sequences encoded withinthe genomes of archaea, bacteria, andeukaryota. J. Mol. Evol. 58, 19–29.6. Koehler, T.M. (2002). Bacillus anthracisgenetics and virulence gene regulation.Curr. Top. Microbiol. Immunol. 271,143–164.7. Tinsley, E., and Khan, S.A. (2006). A novelFtsZ-like protein is involved in replicationof the anthrax toxin-encoding pXO1plasmid in Bacillus anthracis. J. Bacteriol.188, 2829–2835.8. Berry, C., O’Neil, S., Ben-Dov, E.,Jones, A.F., Murphy, L., Quail, M.A.,Holden, M.T., Harris, D., Zaritsky, A., andParkhill, J. (2002). Complete sequenceand organization of pBtoxis, the toxin-coding plasmid of Bacillus thuringiensissubsp. israelensis. Appl. Environ.Microbiol. 68, 5082–5095.9. Tang, M., Bideshi, D.K., Park, H.W., andFederici, B.A. (2006). Minireplicon frompBtoxis of Bacillus thuringiensis subsp.israelensis. Appl. Environ. Microbiol. 72,6948–6954.10. Larsen, R.A., Cusumano, C., Fujioka, A.,Lim-Fong, G., Patterson, P., andPogliano, J. (2007). Treadmilling ofa prokaryotic tubulin-like protein, TubZ,required for plasmid stability in Bacillusthuringiensis. Genes Dev. 21, 1340–1352.11. Theriot, J.A., and Mitchison, T.J. (1991).Actin microfilament dynamics inlocomoting cells. Nature 352, 126–131.12. Kim, S.Y., Gitai, Z., Kinkhabwala, A.,Shapiro, L., and Moerner, W.E. (2006).Single molecules of the bacterial actinMreB undergo directed treadmillingmotion in Caulobacter crescentus. Proc.Natl. Acad. Sci. USA 103, 10929–10934.13. Waterman-Storer, C.M., and Salmon, E.D.(1997). Microtubule dynamics:treadmilling comes around again. Curr.Biol. 7, 369–372.14. Rodionov, V.I., and Borisy, G.G. (1997).Microtubule treadmilling in vivo. Science275, 215–218.15. Shaw, S.L., Kamyar, R., andEhrhardt, D.W. (2003). Sustainedmicrotubule treadmilling in Arabidopsiscortical arrays. Science 300, 1715–1718.16. Thanedar, S., and Margolin, W. (2004).FtsZ exhibits rapid movement andoscillation waves in helix-like patternsin Escherichia coli. Curr. Biol. 14,1167–1173.17. Redick, S.D., Stricker, J., Briscoe, G., andErickson, H.P. (2005). Mutants of FtsZtargeting the protofilament interface:effects on cell division and GTPaseactivity. J. Bacteriol. 187, 2727–2736.18. Hayes, F., and Barilla, D. (2006). Thebacterial segrosome: a dynamicnucleoprotein machine for DNA traffickingand segregation. Nat. Rev. Microbiol. 4,133–143.Department of Microbiologyand Molecular Genetics,University of Texas Medical School,6431 Fannin Street, Houston,Texas 77030, USA.E-mail: William.Margolin@uth.tmc.eduDOI: 10.1016/j.cub.2007.06.020Current Biology Vol 17 No 16R636Circadian Clock: Time for a PhaseShift of Ideas?A recent study shows that cycling of cryptochrome proteins isdispensable for circadian clock function in mammalian cells. Is it timefor a paradigm shift in how we think about the circadian clockmechanism?Martha Merrow1,*and Till Roenneberg2Clocks rule our lives. One of themis the circadian clock — anadaptive, endogenous temporalprogramme which ensures that thesame processes occur at the sametime, day after day. Theseprocesses are based on a numberof rhythmic functions ranging fromgene expression and metabolismto behaviour and they are sopronounced that we arebiochemically ‘different’ peoplefrom day to night. Daily rhythmsare self-sustained; they evenoscillate in the absence of a cyclingenvironment with their periodbeing close to 24 hours, hencecircadian. The implications of thiscircadian programme should be ofinterest to all biologists anddoctors because time of day canpotentially impact on all kinds ofexperiments and medicaldiagnoses.The genetic revolution forcomplex behaviour started withSeymour Benzer who identifiedthe first clock gene, period, inDrosophila [1]. Since then, clockgenes have been described in allcircadian model systems, fromcyanobacteria to mice. Bydiscovering how the products ofthese clock genes interact,a cohesive mechanism emergedthat is apparently common toall circadian systems: anauto-regulatory negativefeedback loop involvingtranscription and translation(Transcription TranslationFeedback Loop, TTFL). Changesin any of the loop’s componentshave dominant effects on thecircadian rhythmicity, and it hasbeen hypothesised that thisfeedback loop generatescircadian rhythms at the cellularlevel (Figure 1A).In a recent paper in CurrentBiology, Fan et al. [2] describeprovocative results that — ifrepeated, extended andelaborated — challenge the currentfeedback loop model. In mice,levels of the TTFL componentsmPERIOD (PER) 1 and 2,mCRYPTOCHROME (CRY) 1 and 2,and BMAL1 typically oscillate overthe course of a day. The oscillationis thought to depend largely onthe CRY proteins, which repressCLK/BMAL1 mediatedtranscriptional activation. PERproteins also show negativefeedback but are less potent thaneither of the CRY proteins in cellculture systems [3]. Fan et al. [2]have engineered the two keynegative feedback components,the mCRY proteins, such that theymove freely into cells. Theysurprisingly find that, despite theapparently constant level of bothCRYs and CRY-induced BMAL1expression, Per2 transcriptioncontinues to cycle rhythmically.This indicates that one of thefundamental assumptionsconcerning the mouse circadianclock seems incorrect, namely thatoscillations of CRY protein levelsDispatchR637are essential for generatingcircadian rhythmicity.There are numerous possiblescenarios which could account forthe observations of Fan et al. [2]from which we have chosen five.Scenario 1: The effects on themolecular machinery of thecircadian clock using the newcell-permeable CRY proteins couldsimply be an artefact of the methoditself. Many things happen toclock proteins on their daily journeythrough time. They are modified,gang up with other proteins, getshuffled between compartmentsand so on. Does all of this alsohappen in cells that are floodedwith the two CRY proteins? Futureexperiments will elaborate on thefates of the imported proteins andhow they compare with those ofendogenous CRY proteins inuntreated cells. However, thesurprising observation thatthe constitutively present,cell-permeant CRYs supportBMAL1 oscillations in normallyarrhythmic Cry1;Cry2 mutantfibroblasts argues against thisscenario.Scenario 2: The traditionalfeedback loop hypothesis stillholds. Experiments in manycircadian systems have shown thatseveral interlocking loops forma ‘network of time’ and the authorsmay have simply uncovered yetanother loop within the network,one that reflects PER2 rhythmsversus CRY1 and CRY2 rhythms(Figure 1B). There are already manyindications of auxiliary loopsinvolving, for example, nuclearorphan receptors, DECs orPAR-proteins. The findings ofFan et al. [2] may be linked to oneof these or may lead to a novelextra-TTFL loop.Scenario 3: The experimentmight reveal a similar phenomenonas in Drosophila, whereconstitutive expression of bothso-called negative elements of theTTFL–PER and TIM- still allowsrhythmicity in locomotor activity aswell as in the levels of the clockproteins themselves [4]. Theassumption here is that therhythmicity of the proteins — interms of level, state orlocalization — is essential forcircadian behaviour, rather thanthe rhythmicity of theirArep rep actBactCrepact actrepact rep act actrep repPDPEPP kinkinPCurrent BiologyFigure 1. Possible scenarios explaining the dispensability of CRY protein oscillationsin the circadian clock.Five scenarios as described in the text are illustrated. (A) The Transcription TranslationFeedback Loop (TTFL) model is built on the elegantly simple hypothesis that clockgene transcription, effected by transcriptional activators (act) is self-regulated by feed-back repression (rep). (B) The TTFL network is far more complex, and the actual rela-tionships may be revealed through experiments such as those described in [3]. (C) Therole of transcription (dashed lines) may not be critical for all circadian properties.Rather, post-translational modifications, such as phosphorylation (P) or sub-cellularlocalisation (block arrow) could play major roles. (D) The TTFL components may beserving input and output functions in the clock signal transduction pathway. In thiscase, the identity of the actual rhythm generator (depicted as a sine wave) may stillbe unknown. (E) The TTFL may be regulated by a ‘phosphorylation oscillator’, namelyan interaction between a kinase (kin) and a phosphatase (PP) whereby functionalactivity of clock-critical kinases is modulated via phosphatases, which are in turnregulated by components of the TTFL that are targets of the kinases.transcription (Figure 1C).Additional support for this ideacomes from experiments exposingthe clock to different dailyenvironmental cycles in the lab,whereby in the fungus Neurospora,it is the clock gene protein levelsthat correlate with the ‘behavioural’phase (in this case, midnight), whilethe RNA levels appear simplydriven (masked) by the changinglight levels [5]. In the experimentshere, at least PER1 protein levelscan oscillate, so perhaps this is theessential rhythmic component,with the rhythms of the othercomponents contributingcharacteristics of the system thatcannot be appreciated in cellculture systems.Scenario 4: The TTFL is not thecore, rhythm-generatingmechanism, but rather servesboth an input and an output withfeedback function. As its input, itprocesses signals from theenvironment to the rhythmgenerator, which in turn regulatesthe input pathway. This wouldrender the TTFL also an output ofthe clock and in this role it wouldtranscriptionally control diverseoutputs (Figure 1D). Such analternative hypothesis was initiallyproposed in 1998 ([6] andreferences therein) based onmathematical modelling, andsince then numerous experimentalresults in many model systemssupport this scenario [7]. Lightreception and components of theTTFL are tightly associated. Insome cases, they serve bothfunctions, in other cases, theyhave changed their role from onefunction to the other duringevolution [8–10]. Light often leadsto rapid changes in the TTFLcomponents, concurring withphase shifts of the circadianoscillation. Others have shown,however, that residual circadianproperties persist whencomponents of the TTFL arerendered non-functional.In Drosophila, combination of thearrhythmic mutant allele of perwith cryb results in the restorationof some circadian properties,rather than an additionaldecrement [11]. In Neurospora,sporadic free running rhythmsCurrent Biology Vol 17 No 16R638[12,13] and also systematicentrainment characteristics canbe found in clock null mutants [14],and subsequently, molecularrhythms have been shown topersist in these strains [15]. Inmice, many mutants that had beenconsidered ‘clock null mutants’because they were arrhythmic(e.g., the mPer2;mCry2 doublemutant) become rhythmic whenput into constant dim light [16].Furthermore, conceptual modelsshow that alterations of inputfeedback components can havehuge effects on circadianoscillations [6]. Finally, feedbacksof the rhythm generator back ontoits inputs have been shown toshield the rhythm from noise bycompensation and, thus, addrobustness to the endogenousdaily cycle [6].Scenario 5: Some of thescenarios above indicate thattranscription and/or translationmay not need to be rhythmic toensure circadian function. Butwhere, then, is rhythmicitygenerated? A recent finding incyanobacteria suggests thatphosphorylation plays a crucialrole. Phosphorylation not onlyconfers specific half-lives toproteins, it also regulates theirsub-cellular localization and theirswitching between activity orinactivity — not only intranscription factors, which are somuch at the heart of the traditionalhypothesis of rhythm generation.In cyanobacteria, three proteinsand ATP were shown to besufficient recapitulate most of thecircadian clock properties in vitro[17]! Given the added complexityin eukaryotes, we may not expectto find a similar, simple molecularclock mechanism. However, theremay be elements ofa phosphorylation oscillator [18]that are preserved and remain tobe described, but sucha mechanism could have alsoco-evolved by recruiting any of themany autocatalytic kinases andphosphatases present in differentspecies. An indication thata phosphorylation oscillator mightbe a primordial circadianmechanism comes from severalindependent observationsconcerning human kinases andphosphatases. Human CKIautoregulates according tophosphorylation state and isdephosphorylated by PP5 ([18]and references therein), thusthese two components could forma feedback loop if they display thecorrect kinetics and if they wereconnected to an input pathway.PP5 activity, in turn, depends onbinding to CRY2 ([18] andreferences therein), whichfluctuates in concentration inmany cells. Even withoutoscillations in the amount ofCRY2, rhythms in the state ofphosphorylation could play a partin a phosphorylation oscillatormechanism and might explain theresults shown with the cell-permeable CRY1 and CRY2 byFan et al. [2].Why is it important tounderstand how the circadiansystem is put together? Severalreports have demonstrated linksbetween health and behaviourwith respect to chronotype. Arecent publication showed that theerectile dysfunction drug sildenafilhas a profound impact onre-synchronization of thecircadian system in hamsters [19].If we are to start medicating thecircadian system itself or usea robust circadian system tomedicate other pathologies, wehave to know what we’re aimingfor and how the parts of thesystem are put together.References1. Konopka, R., and Benzer, S. (1971). Clockmutants of Drosophila melanogaster.Proc. Natl. Acad. Sci. USA 68,2112–2116.2. Fan, Y., Hida, A., Anderson, D.A.,Izumo, M., and Johnson, C.H. (2007).Cycling of CRYPTOCHROME proteins isnot necessary for circadian clock functionin mammalian fibroblasts. Curr. Biol. 17,1091–1100.3. Kume, K., Zylka, M.J., Sriram, S.,Shearman, L.P., Weaver, D.R., Jin, X.,Maywood, E.S., Hastings, M.H., andReppert, S.M. (1999). mCRY1 and mCRY2are essential components of the negativelimb of the circadian clock feedback loop.Cell 98, 193–205.4. Yang, Z., and Sehgal, A. (2001). Role ofmolecular oscillations in generatingbehavioral rhythms in Drosophila. Neuron29, 453–467.5. Tan, Y., Dragovic, Z., Roenneberg, T., andMerrow, M. (2004). Entrainment of thecircadian clock: translational and post-translational control as key elements.Curr. Biol. 14, 433–438.6. Roenneberg, T., and Merrow, M. (1999).Circadian clocks and metabolism. J. Biol.Rhythms 14, 449–459.7. Lakin-Thomas, P.L. (2006).Transcriptional feedback oscillators:maybe, maybe not. J. Biol. Rhythms 21,83–92.8. Froehlich, A.C., Liu, Y., Loros, J.J., andDunlap, J.C. (2002). White Collar-1,a circadian blue light photoreceptor,binding to the frequency promoter.Science 297, 815–819.9. Krishnan, B., Levine, J.D., Lynch, M.K.S.,Dowse, H.B., Funes, P., Hall, J.C.,Hardin, P.E., and Dryer, S.E. (2001). A newrole for cryptochrome in a Drosophilacircadian oscillator. Nature 411,313–317.10. Yuan, Q., Metterville, D., Briscoe, A.D.,and Reppert, S.M. (2007). Insectcryptochromes: gene duplication and lossdefine diverse ways to construct insectcircadian clocks. Mol. Biol. Evol. 24,948–955.11. Collins, B.H., Dissel, S., Gaten, E.,Rosato, E., and Kyriacou, C.P. (2005).Disruption of Cryptochrome partiallyrestores circadian rhythmicity to thearrhythmic period mutant of Drosophila.Proc. Natl. Acad. Sci. USA 102,19021–19026.12. Loros, J.J., and Feldman, J.F. (1986). Lossof temperature compensation of circadianperiod length in the frq-9 mutant ofNeurospora crassa. J. Biol. Rhythms 1,187–198.13. Dragovic, Z., Tan, Y., Görl, M.,Roenneberg, T., and Merrow, M. (2002).Light reception and circadian behavior in‘blind’ and ‘clock-less’ mutants ofNeurospora crassa. EMBO J. 21,3643–3651.14. Roenneberg, T., Dragovic, Z., andMerrow, M. (2005). Demasking biologicaloscillators: properties and principles ofentrainment exemplified by theNeurospora circadian clock. Proc.Natl. Acad. Sci. USA 102,7742–7747.15. Correa, A., Lewis, Z.A., Greene, A.V.,March, I.J., Gomer, R.H., and Bell-Pedersen, D. (2003). Multiple oscillatorsregulate circadian gene expression inNeurospora. Proc. Natl. Acad. Sci. USA100, 13597–13602.16. Steinlechner, S., Jacobmeier, B.,Scherbarth, F., Dernbach, H., Kruse, F.,and Albrecht, U. (2002). Robustcircadian rhythmicity of Per1 and Per2mutant mice in constant light anddynamics of Per1 and Per2 geneexpression under long and shortphotoperiods. J. Biol. Rhythms 17,202–209.17. Nakajima, M., Imai, K., Ito, H.,Nishiwaki, T., Murayama, Y., Iwasaki, H.,Oyama, T., and Kondo, T. (2005).Reconstitution of circadian oscillation ofcyanobacterial KaiC phosphorylationin vitro. Science 308, 414–415.18. Merrow, M., Mazzotta, G., Chen, Z., andRoenneberg, T. (2006). The right place atthe right time: regulation of daily timing byphosphorylation. Genes Dev. 20,2629–2633.19. Agostino, P.V., Plano, S.A., andGolombek, D.A. (2007). Sildenafilaccelerates reentrainment of circadianrhythms after advancing light schedules.Proc. Natl. Acad. Sci. USA 104,9834–9839.1Biological Center, University ofGroningen, Postbus 14, 9750AA, Haren,Netherlands. 2Institute of MedicalPsychology, University of Munich,Goethestrasse 31, 80336 München,Germany.*E-mail: m.merrow@rug.nlDOI: 10.1016/j.cub.2007.06.041

ARTS repository - University of Groningen

   University of GroningenCircadian clockMerrow, Martha; Roenneberg, TillPublished in:Current BiologyDOI:10.1016/j.cub.2007.06.041IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2007Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Merrow, M., & Roenneberg, T. (2007). Circadian clock: Time for a phase shift of ideas? Current Biology,17(16), R636-R638. https://doi.org/10.1016/j.cub.2007.06.041CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 13-02-2023treadmill and to be implicated ina mitotic-like process.References1. Michie, K.A., and Löwe, J. (2006).Dynamic filaments of the bacterialcytoskeleton. Annu. Rev. Biochem. 75,467–492.2. Gitai, Z. (2007). Diversification andspecialization of the bacterialcytoskeleton. Curr. Opin. Cell. Biol. 19,5–12.3. Margolin, W. (2005). FtsZ and the divisionof prokaryotic cells and organelles. Nat.Rev. Mol. Cell. Biol. 6, 862–871.4. Schlieper, D., Oliva, M.A., Andreu, J.M.,and Löwe, J. (2005). Structure of bacterialtubulin BtubA/B: evidence for horizontalgene transfer. Proc. Natl. Acad. Sci. USA102, 9170–9175.5. Vaughan, S., Wickstead, B., Gull, K., andAddinall, S.G. (2004). Molecular evolutionof FtsZ protein sequences encoded withinthe genomes of archaea, bacteria, andeukaryota. J. Mol. Evol. 58, 19–29.6. Koehler, T.M. (2002). Bacillus anthracisgenetics and virulence gene regulation.Curr. Top. Microbiol. Immunol. 271,143–164.7. Tinsley, E., and Khan, S.A. (2006). A novelFtsZ-like protein is involved in replicationof the anthrax toxin-encoding pXO1plasmid in Bacillus anthracis. J. Bacteriol.188, 2829–2835.8. Berry, C., O’Neil, S., Ben-Dov, E.,Jones, A.F., Murphy, L., Quail, M.A.,Holden, M.T., Harris, D., Zaritsky, A., andParkhill, J. (2002). Complete sequenceand organization of pBtoxis, the toxin-coding plasmid of Bacillus thuringiensissubsp. israelensis. Appl. Environ.Microbiol. 68, 5082–5095.9. Tang, M., Bideshi, D.K., Park, H.W., andFederici, B.A. (2006). Minireplicon frompBtoxis of Bacillus thuringiensis subsp.israelensis. Appl. Environ. Microbiol. 72,6948–6954.10. Larsen, R.A., Cusumano, C., Fujioka, A.,Lim-Fong, G., Patterson, P., andPogliano, J. (2007). Treadmilling ofa prokaryotic tubulin-like protein, TubZ,required for plasmid stability in Bacillusthuringiensis. Genes Dev. 21, 1340–1352.11. Theriot, J.A., and Mitchison, T.J. (1991).Actin microfilament dynamics inlocomoting cells. Nature 352, 126–131.12. Kim, S.Y., Gitai, Z., Kinkhabwala, A.,Shapiro, L., and Moerner, W.E. (2006).Single molecules of the bacterial actinMreB undergo directed treadmillingmotion in Caulobacter crescentus. Proc.Natl. Acad. Sci. USA 103, 10929–10934.13. Waterman-Storer, C.M., and Salmon, E.D.(1997). Microtubule dynamics:treadmilling comes around again. Curr.Biol. 7, 369–372.14. Rodionov, V.I., and Borisy, G.G. (1997).Microtubule treadmilling in vivo. Science275, 215–218.15. Shaw, S.L., Kamyar, R., andEhrhardt, D.W. (2003). Sustainedmicrotubule treadmilling in Arabidopsiscortical arrays. Science 300, 1715–1718.16. Thanedar, S., and Margolin, W. (2004).FtsZ exhibits rapid movement andoscillation waves in helix-like patternsin Escherichia coli. Curr. Biol. 14,1167–1173.17. Redick, S.D., Stricker, J., Briscoe, G., andErickson, H.P. (2005). Mutants of FtsZtargeting the protofilament interface:effects on cell division and GTPaseactivity. J. Bacteriol. 187, 2727–2736.18. Hayes, F., and Barilla, D. (2006). Thebacterial segrosome: a dynamicnucleoprotein machine for DNA traffickingand segregation. Nat. Rev. Microbiol. 4,133–143.Department of Microbiologyand Molecular Genetics,University of Texas Medical School,6431 Fannin Street, Houston,Texas 77030, USA.E-mail: William.Margolin@uth.tmc.eduDOI: 10.1016/j.cub.2007.06.020Current Biology Vol 17 No 16R636Circadian Clock: Time for a PhaseShift of Ideas?A recent study shows that cycling of cryptochrome proteins isdispensable for circadian clock function in mammalian cells. Is it timefor a paradigm shift in how we think about the circadian clockmechanism?Martha Merrow1,*and Till Roenneberg2Clocks rule our lives. One of themis the circadian clock — anadaptive, endogenous temporalprogramme which ensures that thesame processes occur at the sametime, day after day. Theseprocesses are based on a numberof rhythmic functions ranging fromgene expression and metabolismto behaviour and they are sopronounced that we arebiochemically ‘different’ peoplefrom day to night. Daily rhythmsare self-sustained; they evenoscillate in the absence of a cyclingenvironment with their periodbeing close to 24 hours, hencecircadian. The implications of thiscircadian programme should be ofinterest to all biologists anddoctors because time of day canpotentially impact on all kinds ofexperiments and medicaldiagnoses.The genetic revolution forcomplex behaviour started withSeymour Benzer who identifiedthe first clock gene, period, inDrosophila [1]. Since then, clockgenes have been described in allcircadian model systems, fromcyanobacteria to mice. Bydiscovering how the products ofthese clock genes interact,a cohesive mechanism emergedthat is apparently common toall circadian systems: anauto-regulatory negativefeedback loop involvingtranscription and translation(Transcription TranslationFeedback Loop, TTFL). Changesin any of the loop’s componentshave dominant effects on thecircadian rhythmicity, and it hasbeen hypothesised that thisfeedback loop generatescircadian rhythms at the cellularlevel (Figure 1A).In a recent paper in CurrentBiology, Fan et al. [2] describeprovocative results that — ifrepeated, extended andelaborated — challenge the currentfeedback loop model. In mice,levels of the TTFL componentsmPERIOD (PER) 1 and 2,mCRYPTOCHROME (CRY) 1 and 2,and BMAL1 typically oscillate overthe course of a day. The oscillationis thought to depend largely onthe CRY proteins, which repressCLK/BMAL1 mediatedtranscriptional activation. PERproteins also show negativefeedback but are less potent thaneither of the CRY proteins in cellculture systems [3]. Fan et al. [2]have engineered the two keynegative feedback components,the mCRY proteins, such that theymove freely into cells. Theysurprisingly find that, despite theapparently constant level of bothCRYs and CRY-induced BMAL1expression, Per2 transcriptioncontinues to cycle rhythmically.This indicates that one of thefundamental assumptionsconcerning the mouse circadianclock seems incorrect, namely thatoscillations of CRY protein levelsDispatchR637are essential for generatingcircadian rhythmicity.There are numerous possiblescenarios which could account forthe observations of Fan et al. [2]from which we have chosen five.Scenario 1: The effects on themolecular machinery of thecircadian clock using the newcell-permeable CRY proteins couldsimply be an artefact of the methoditself. Many things happen toclock proteins on their daily journeythrough time. They are modified,gang up with other proteins, getshuffled between compartmentsand so on. Does all of this alsohappen in cells that are floodedwith the two CRY proteins? Futureexperiments will elaborate on thefates of the imported proteins andhow they compare with those ofendogenous CRY proteins inuntreated cells. However, thesurprising observation thatthe constitutively present,cell-permeant CRYs supportBMAL1 oscillations in normallyarrhythmic Cry1;Cry2 mutantfibroblasts argues against thisscenario.Scenario 2: The traditionalfeedback loop hypothesis stillholds. Experiments in manycircadian systems have shown thatseveral interlocking loops forma ‘network of time’ and the authorsmay have simply uncovered yetanother loop within the network,one that reflects PER2 rhythmsversus CRY1 and CRY2 rhythms(Figure 1B). There are already manyindications of auxiliary loopsinvolving, for example, nuclearorphan receptors, DECs orPAR-proteins. The findings ofFan et al. [2] may be linked to oneof these or may lead to a novelextra-TTFL loop.Scenario 3: The experimentmight reveal a similar phenomenonas in Drosophila, whereconstitutive expression of bothso-called negative elements of theTTFL–PER and TIM- still allowsrhythmicity in locomotor activity aswell as in the levels of the clockproteins themselves [4]. Theassumption here is that therhythmicity of the proteins — interms of level, state orlocalization — is essential forcircadian behaviour, rather thanthe rhythmicity of theirArep rep actBactCrepact actrepact rep act actrep repPDPEPP kinkinPCurrent BiologyFigure 1. Possible scenarios explaining the dispensability of CRY protein oscillationsin the circadian clock.Five scenarios as described in the text are illustrated. (A) The Transcription TranslationFeedback Loop (TTFL) model is built on the elegantly simple hypothesis that clockgene transcription, effected by transcriptional activators (act) is self-regulated by feed-back repression (rep). (B) The TTFL network is far more complex, and the actual rela-tionships may be revealed through experiments such as those described in [3]. (C) Therole of transcription (dashed lines) may not be critical for all circadian properties.Rather, post-translational modifications, such as phosphorylation (P) or sub-cellularlocalisation (block arrow) could play major roles. (D) The TTFL components may beserving input and output functions in the clock signal transduction pathway. In thiscase, the identity of the actual rhythm generator (depicted as a sine wave) may stillbe unknown. (E) The TTFL may be regulated by a ‘phosphorylation oscillator’, namelyan interaction between a kinase (kin) and a phosphatase (PP) whereby functionalactivity of clock-critical kinases is modulated via phosphatases, which are in turnregulated by components of the TTFL that are targets of the kinases.transcription (Figure 1C).Additional support for this ideacomes from experiments exposingthe clock to different dailyenvironmental cycles in the lab,whereby in the fungus Neurospora,it is the clock gene protein levelsthat correlate with the ‘behavioural’phase (in this case, midnight), whilethe RNA levels appear simplydriven (masked) by the changinglight levels [5]. In the experimentshere, at least PER1 protein levelscan oscillate, so perhaps this is theessential rhythmic component,with the rhythms of the othercomponents contributingcharacteristics of the system thatcannot be appreciated in cellculture systems.Scenario 4: The TTFL is not thecore, rhythm-generatingmechanism, but rather servesboth an input and an output withfeedback function. As its input, itprocesses signals from theenvironment to the rhythmgenerator, which in turn regulatesthe input pathway. This wouldrender the TTFL also an output ofthe clock and in this role it wouldtranscriptionally control diverseoutputs (Figure 1D). Such analternative hypothesis was initiallyproposed in 1998 ([6] andreferences therein) based onmathematical modelling, andsince then numerous experimentalresults in many model systemssupport this scenario [7]. Lightreception and components of theTTFL are tightly associated. Insome cases, they serve bothfunctions, in other cases, theyhave changed their role from onefunction to the other duringevolution [8–10]. Light often leadsto rapid changes in the TTFLcomponents, concurring withphase shifts of the circadianoscillation. Others have shown,however, that residual circadianproperties persist whencomponents of the TTFL arerendered non-functional.In Drosophila, combination of thearrhythmic mutant allele of perwith cryb results in the restorationof some circadian properties,rather than an additionaldecrement [11]. In Neurospora,sporadic free running rhythmsCurrent Biology Vol 17 No 16R638[12,13] and also systematicentrainment characteristics canbe found in clock null mutants [14],and subsequently, molecularrhythms have been shown topersist in these strains [15]. Inmice, many mutants that had beenconsidered ‘clock null mutants’because they were arrhythmic(e.g., the mPer2;mCry2 doublemutant) become rhythmic whenput into constant dim light [16].Furthermore, conceptual modelsshow that alterations of inputfeedback components can havehuge effects on circadianoscillations [6]. Finally, feedbacksof the rhythm generator back ontoits inputs have been shown toshield the rhythm from noise bycompensation and, thus, addrobustness to the endogenousdaily cycle [6].Scenario 5: Some of thescenarios above indicate thattranscription and/or translationmay not need to be rhythmic toensure circadian function. Butwhere, then, is rhythmicitygenerated? A recent finding incyanobacteria suggests thatphosphorylation plays a crucialrole. Phosphorylation not onlyconfers specific half-lives toproteins, it also regulates theirsub-cellular localization and theirswitching between activity orinactivity — not only intranscription factors, which are somuch at the heart of the traditionalhypothesis of rhythm generation.In cyanobacteria, three proteinsand ATP were shown to besufficient recapitulate most of thecircadian clock properties in vitro[17]! Given the added complexityin eukaryotes, we may not expectto find a similar, simple molecularclock mechanism. However, theremay be elements ofa phosphorylation oscillator [18]that are preserved and remain tobe described, but sucha mechanism could have alsoco-evolved by recruiting any of themany autocatalytic kinases andphosphatases present in differentspecies. An indication thata phosphorylation oscillator mightbe a primordial circadianmechanism comes from severalindependent observationsconcerning human kinases andphosphatases. Human CKIautoregulates according tophosphorylation state and isdephosphorylated by PP5 ([18]and references therein), thusthese two components could forma feedback loop if they display thecorrect kinetics and if they wereconnected to an input pathway.PP5 activity, in turn, depends onbinding to CRY2 ([18] andreferences therein), whichfluctuates in concentration inmany cells. Even withoutoscillations in the amount ofCRY2, rhythms in the state ofphosphorylation could play a partin a phosphorylation oscillatormechanism and might explain theresults shown with the cell-permeable CRY1 and CRY2 byFan et al. [2].Why is it important tounderstand how the circadiansystem is put together? Severalreports have demonstrated linksbetween health and behaviourwith respect to chronotype. Arecent publication showed that theerectile dysfunction drug sildenafilhas a profound impact onre-synchronization of thecircadian system in hamsters [19].If we are to start medicating thecircadian system itself or usea robust circadian system tomedicate other pathologies, wehave to know what we’re aimingfor and how the parts of thesystem are put together.References1. Konopka, R., and Benzer, S. (1971). Clockmutants of Drosophila melanogaster.Proc. Natl. Acad. Sci. USA 68,2112–2116.2. Fan, Y., Hida, A., Anderson, D.A.,Izumo, M., and Johnson, C.H. (2007).Cycling of CRYPTOCHROME proteins isnot necessary for circadian clock functionin mammalian fibroblasts. Curr. Biol. 17,1091–1100.3. Kume, K., Zylka, M.J., Sriram, S.,Shearman, L.P., Weaver, D.R., Jin, X.,Maywood, E.S., Hastings, M.H., andReppert, S.M. (1999). mCRY1 and mCRY2are essential components of the negativelimb of the circadian clock feedback loop.Cell 98, 193–205.4. Yang, Z., and Sehgal, A. (2001). Role ofmolecular oscillations in generatingbehavioral rhythms in Drosophila. Neuron29, 453–467.5. Tan, Y., Dragovic, Z., Roenneberg, T., andMerrow, M. (2004). Entrainment of thecircadian clock: translational and post-translational control as key elements.Curr. Biol. 14, 433–438.6. Roenneberg, T., and Merrow, M. (1999).Circadian clocks and metabolism. J. Biol.Rhythms 14, 449–459.7. Lakin-Thomas, P.L. (2006).Transcriptional feedback oscillators:maybe, maybe not. J. Biol. Rhythms 21,83–92.8. Froehlich, A.C., Liu, Y., Loros, J.J., andDunlap, J.C. (2002). White Collar-1,a circadian blue light photoreceptor,binding to the frequency promoter.Science 297, 815–819.9. Krishnan, B., Levine, J.D., Lynch, M.K.S.,Dowse, H.B., Funes, P., Hall, J.C.,Hardin, P.E., and Dryer, S.E. (2001). A newrole for cryptochrome in a Drosophilacircadian oscillator. Nature 411,313–317.10. Yuan, Q., Metterville, D., Briscoe, A.D.,and Reppert, S.M. (2007). Insectcryptochromes: gene duplication and lossdefine diverse ways to construct insectcircadian clocks. Mol. Biol. Evol. 24,948–955.11. Collins, B.H., Dissel, S., Gaten, E.,Rosato, E., and Kyriacou, C.P. (2005).Disruption of Cryptochrome partiallyrestores circadian rhythmicity to thearrhythmic period mutant of Drosophila.Proc. Natl. Acad. Sci. USA 102,19021–19026.12. Loros, J.J., and Feldman, J.F. (1986). Lossof temperature compensation of circadianperiod length in the frq-9 mutant ofNeurospora crassa. J. Biol. Rhythms 1,187–198.13. Dragovic, Z., Tan, Y., Görl, M.,Roenneberg, T., and Merrow, M. (2002).Light reception and circadian behavior in‘blind’ and ‘clock-less’ mutants ofNeurospora crassa. EMBO J. 21,3643–3651.14. Roenneberg, T., Dragovic, Z., andMerrow, M. (2005). Demasking biologicaloscillators: properties and principles ofentrainment exemplified by theNeurospora circadian clock. Proc.Natl. Acad. Sci. USA 102,7742–7747.15. Correa, A., Lewis, Z.A., Greene, A.V.,March, I.J., Gomer, R.H., and Bell-Pedersen, D. (2003). Multiple oscillatorsregulate circadian gene expression inNeurospora. Proc. Natl. Acad. Sci. USA100, 13597–13602.16. Steinlechner, S., Jacobmeier, B.,Scherbarth, F., Dernbach, H., Kruse, F.,and Albrecht, U. (2002). Robustcircadian rhythmicity of Per1 and Per2mutant mice in constant light anddynamics of Per1 and Per2 geneexpression under long and shortphotoperiods. J. Biol. Rhythms 17,202–209.17. Nakajima, M., Imai, K., Ito, H.,Nishiwaki, T., Murayama, Y., Iwasaki, H.,Oyama, T., and Kondo, T. (2005).Reconstitution of circadian oscillation ofcyanobacterial KaiC phosphorylationin vitro. Science 308, 414–415.18. Merrow, M., Mazzotta, G., Chen, Z., andRoenneberg, T. (2006). The right place atthe right time: regulation of daily timing byphosphorylation. Genes Dev. 20,2629–2633.19. Agostino, P.V., Plano, S.A., andGolombek, D.A. (2007). Sildenafilaccelerates reentrainment of circadianrhythms after advancing light schedules.Proc. Natl. Acad. Sci. USA 104,9834–9839.1Biological Center, University ofGroningen, Postbus 14, 9750AA, Haren,Netherlands. 2Institute of MedicalPsychology, University of Munich,Goethestrasse 31, 80336 München,Germany.*E-mail: m.merrow@rug.nlDOI: 10.1016/j.cub.2007.06.041

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Circadian clock: Time for a phase shift of ideas?

   University of GroningenCircadian clockMerrow, Martha; Roenneberg, TillPublished in:Current BiologyDOI:10.1016/j.cub.2007.06.041IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2007Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Merrow, M., & Roenneberg, T. (2007). Circadian clock: Time for a phase shift of ideas? Current Biology,17(16), R636-R638. https://doi.org/10.1016/j.cub.2007.06.041CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 16-07-2023treadmill and to be implicated ina mitotic-like process.References1. Michie, K.A., and Löwe, J. (2006).Dynamic filaments of the bacterialcytoskeleton. Annu. Rev. Biochem. 75,467–492.2. Gitai, Z. (2007). Diversification andspecialization of the bacterialcytoskeleton. Curr. Opin. Cell. Biol. 19,5–12.3. Margolin, W. (2005). FtsZ and the divisionof prokaryotic cells and organelles. Nat.Rev. Mol. Cell. Biol. 6, 862–871.4. Schlieper, D., Oliva, M.A., Andreu, J.M.,and Löwe, J. (2005). Structure of bacterialtubulin BtubA/B: evidence for horizontalgene transfer. Proc. Natl. Acad. Sci. USA102, 9170–9175.5. Vaughan, S., Wickstead, B., Gull, K., andAddinall, S.G. (2004). Molecular evolutionof FtsZ protein sequences encoded withinthe genomes of archaea, bacteria, andeukaryota. J. Mol. Evol. 58, 19–29.6. Koehler, T.M. (2002). Bacillus anthracisgenetics and virulence gene regulation.Curr. Top. Microbiol. Immunol. 271,143–164.7. Tinsley, E., and Khan, S.A. (2006). A novelFtsZ-like protein is involved in replicationof the anthrax toxin-encoding pXO1plasmid in Bacillus anthracis. J. Bacteriol.188, 2829–2835.8. Berry, C., O’Neil, S., Ben-Dov, E.,Jones, A.F., Murphy, L., Quail, M.A.,Holden, M.T., Harris, D., Zaritsky, A., andParkhill, J. (2002). Complete sequenceand organization of pBtoxis, the toxin-coding plasmid of Bacillus thuringiensissubsp. israelensis. Appl. Environ.Microbiol. 68, 5082–5095.9. Tang, M., Bideshi, D.K., Park, H.W., andFederici, B.A. (2006). Minireplicon frompBtoxis of Bacillus thuringiensis subsp.israelensis. Appl. Environ. Microbiol. 72,6948–6954.10. Larsen, R.A., Cusumano, C., Fujioka, A.,Lim-Fong, G., Patterson, P., andPogliano, J. (2007). Treadmilling ofa prokaryotic tubulin-like protein, TubZ,required for plasmid stability in Bacillusthuringiensis. Genes Dev. 21, 1340–1352.11. Theriot, J.A., and Mitchison, T.J. (1991).Actin microfilament dynamics inlocomoting cells. Nature 352, 126–131.12. Kim, S.Y., Gitai, Z., Kinkhabwala, A.,Shapiro, L., and Moerner, W.E. (2006).Single molecules of the bacterial actinMreB undergo directed treadmillingmotion in Caulobacter crescentus. Proc.Natl. Acad. Sci. USA 103, 10929–10934.13. Waterman-Storer, C.M., and Salmon, E.D.(1997). Microtubule dynamics:treadmilling comes around again. Curr.Biol. 7, 369–372.14. Rodionov, V.I., and Borisy, G.G. (1997).Microtubule treadmilling in vivo. Science275, 215–218.15. Shaw, S.L., Kamyar, R., andEhrhardt, D.W. (2003). Sustainedmicrotubule treadmilling in Arabidopsiscortical arrays. Science 300, 1715–1718.16. Thanedar, S., and Margolin, W. (2004).FtsZ exhibits rapid movement andoscillation waves in helix-like patternsin Escherichia coli. Curr. Biol. 14,1167–1173.17. Redick, S.D., Stricker, J., Briscoe, G., andErickson, H.P. (2005). Mutants of FtsZtargeting the protofilament interface:effects on cell division and GTPaseactivity. J. Bacteriol. 187, 2727–2736.18. Hayes, F., and Barilla, D. (2006). Thebacterial segrosome: a dynamicnucleoprotein machine for DNA traffickingand segregation. Nat. Rev. Microbiol. 4,133–143.Department of Microbiologyand Molecular Genetics,University of Texas Medical School,6431 Fannin Street, Houston,Texas 77030, USA.E-mail: William.Margolin@uth.tmc.eduDOI: 10.1016/j.cub.2007.06.020Current Biology Vol 17 No 16R636Circadian Clock: Time for a PhaseShift of Ideas?A recent study shows that cycling of cryptochrome proteins isdispensable for circadian clock function in mammalian cells. Is it timefor a paradigm shift in how we think about the circadian clockmechanism?Martha Merrow1,*and Till Roenneberg2Clocks rule our lives. One of themis the circadian clock — anadaptive, endogenous temporalprogramme which ensures that thesame processes occur at the sametime, day after day. Theseprocesses are based on a numberof rhythmic functions ranging fromgene expression and metabolismto behaviour and they are sopronounced that we arebiochemically ‘different’ peoplefrom day to night. Daily rhythmsare self-sustained; they evenoscillate in the absence of a cyclingenvironment with their periodbeing close to 24 hours, hencecircadian. The implications of thiscircadian programme should be ofinterest to all biologists anddoctors because time of day canpotentially impact on all kinds ofexperiments and medicaldiagnoses.The genetic revolution forcomplex behaviour started withSeymour Benzer who identifiedthe first clock gene, period, inDrosophila [1]. Since then, clockgenes have been described in allcircadian model systems, fromcyanobacteria to mice. Bydiscovering how the products ofthese clock genes interact,a cohesive mechanism emergedthat is apparently common toall circadian systems: anauto-regulatory negativefeedback loop involvingtranscription and translation(Transcription TranslationFeedback Loop, TTFL). Changesin any of the loop’s componentshave dominant effects on thecircadian rhythmicity, and it hasbeen hypothesised that thisfeedback loop generatescircadian rhythms at the cellularlevel (Figure 1A).In a recent paper in CurrentBiology, Fan et al. [2] describeprovocative results that — ifrepeated, extended andelaborated — challenge the currentfeedback loop model. In mice,levels of the TTFL componentsmPERIOD (PER) 1 and 2,mCRYPTOCHROME (CRY) 1 and 2,and BMAL1 typically oscillate overthe course of a day. The oscillationis thought to depend largely onthe CRY proteins, which repressCLK/BMAL1 mediatedtranscriptional activation. PERproteins also show negativefeedback but are less potent thaneither of the CRY proteins in cellculture systems [3]. Fan et al. [2]have engineered the two keynegative feedback components,the mCRY proteins, such that theymove freely into cells. Theysurprisingly find that, despite theapparently constant level of bothCRYs and CRY-induced BMAL1expression, Per2 transcriptioncontinues to cycle rhythmically.This indicates that one of thefundamental assumptionsconcerning the mouse circadianclock seems incorrect, namely thatoscillations of CRY protein levelsDispatchR637are essential for generatingcircadian rhythmicity.There are numerous possiblescenarios which could account forthe observations of Fan et al. [2]from which we have chosen five.Scenario 1: The effects on themolecular machinery of thecircadian clock using the newcell-permeable CRY proteins couldsimply be an artefact of the methoditself. Many things happen toclock proteins on their daily journeythrough time. They are modified,gang up with other proteins, getshuffled between compartmentsand so on. Does all of this alsohappen in cells that are floodedwith the two CRY proteins? Futureexperiments will elaborate on thefates of the imported proteins andhow they compare with those ofendogenous CRY proteins inuntreated cells. However, thesurprising observation thatthe constitutively present,cell-permeant CRYs supportBMAL1 oscillations in normallyarrhythmic Cry1;Cry2 mutantfibroblasts argues against thisscenario.Scenario 2: The traditionalfeedback loop hypothesis stillholds. Experiments in manycircadian systems have shown thatseveral interlocking loops forma ‘network of time’ and the authorsmay have simply uncovered yetanother loop within the network,one that reflects PER2 rhythmsversus CRY1 and CRY2 rhythms(Figure 1B). There are already manyindications of auxiliary loopsinvolving, for example, nuclearorphan receptors, DECs orPAR-proteins. The findings ofFan et al. [2] may be linked to oneof these or may lead to a novelextra-TTFL loop.Scenario 3: The experimentmight reveal a similar phenomenonas in Drosophila, whereconstitutive expression of bothso-called negative elements of theTTFL–PER and TIM- still allowsrhythmicity in locomotor activity aswell as in the levels of the clockproteins themselves [4]. Theassumption here is that therhythmicity of the proteins — interms of level, state orlocalization — is essential forcircadian behaviour, rather thanthe rhythmicity of theirArep rep actBactCrepact actrepact rep act actrep repPDPEPP kinkinPCurrent BiologyFigure 1. Possible scenarios explaining the dispensability of CRY protein oscillationsin the circadian clock.Five scenarios as described in the text are illustrated. (A) The Transcription TranslationFeedback Loop (TTFL) model is built on the elegantly simple hypothesis that clockgene transcription, effected by transcriptional activators (act) is self-regulated by feed-back repression (rep). (B) The TTFL network is far more complex, and the actual rela-tionships may be revealed through experiments such as those described in [3]. (C) Therole of transcription (dashed lines) may not be critical for all circadian properties.Rather, post-translational modifications, such as phosphorylation (P) or sub-cellularlocalisation (block arrow) could play major roles. (D) The TTFL components may beserving input and output functions in the clock signal transduction pathway. In thiscase, the identity of the actual rhythm generator (depicted as a sine wave) may stillbe unknown. (E) The TTFL may be regulated by a ‘phosphorylation oscillator’, namelyan interaction between a kinase (kin) and a phosphatase (PP) whereby functionalactivity of clock-critical kinases is modulated via phosphatases, which are in turnregulated by components of the TTFL that are targets of the kinases.transcription (Figure 1C).Additional support for this ideacomes from experiments exposingthe clock to different dailyenvironmental cycles in the lab,whereby in the fungus Neurospora,it is the clock gene protein levelsthat correlate with the ‘behavioural’phase (in this case, midnight), whilethe RNA levels appear simplydriven (masked) by the changinglight levels [5]. In the experimentshere, at least PER1 protein levelscan oscillate, so perhaps this is theessential rhythmic component,with the rhythms of the othercomponents contributingcharacteristics of the system thatcannot be appreciated in cellculture systems.Scenario 4: The TTFL is not thecore, rhythm-generatingmechanism, but rather servesboth an input and an output withfeedback function. As its input, itprocesses signals from theenvironment to the rhythmgenerator, which in turn regulatesthe input pathway. This wouldrender the TTFL also an output ofthe clock and in this role it wouldtranscriptionally control diverseoutputs (Figure 1D). Such analternative hypothesis was initiallyproposed in 1998 ([6] andreferences therein) based onmathematical modelling, andsince then numerous experimentalresults in many model systemssupport this scenario [7]. Lightreception and components of theTTFL are tightly associated. Insome cases, they serve bothfunctions, in other cases, theyhave changed their role from onefunction to the other duringevolution [8–10]. Light often leadsto rapid changes in the TTFLcomponents, concurring withphase shifts of the circadianoscillation. Others have shown,however, that residual circadianproperties persist whencomponents of the TTFL arerendered non-functional.In Drosophila, combination of thearrhythmic mutant allele of perwith cryb results in the restorationof some circadian properties,rather than an additionaldecrement [11]. In Neurospora,sporadic free running rhythmsCurrent Biology Vol 17 No 16R638[12,13] and also systematicentrainment characteristics canbe found in clock null mutants [14],and subsequently, molecularrhythms have been shown topersist in these strains [15]. Inmice, many mutants that had beenconsidered ‘clock null mutants’because they were arrhythmic(e.g., the mPer2;mCry2 doublemutant) become rhythmic whenput into constant dim light [16].Furthermore, conceptual modelsshow that alterations of inputfeedback components can havehuge effects on circadianoscillations [6]. Finally, feedbacksof the rhythm generator back ontoits inputs have been shown toshield the rhythm from noise bycompensation and, thus, addrobustness to the endogenousdaily cycle [6].Scenario 5: Some of thescenarios above indicate thattranscription and/or translationmay not need to be rhythmic toensure circadian function. Butwhere, then, is rhythmicitygenerated? A recent finding incyanobacteria suggests thatphosphorylation plays a crucialrole. Phosphorylation not onlyconfers specific half-lives toproteins, it also regulates theirsub-cellular localization and theirswitching between activity orinactivity — not only intranscription factors, which are somuch at the heart of the traditionalhypothesis of rhythm generation.In cyanobacteria, three proteinsand ATP were shown to besufficient recapitulate most of thecircadian clock properties in vitro[17]! Given the added complexityin eukaryotes, we may not expectto find a similar, simple molecularclock mechanism. However, theremay be elements ofa phosphorylation oscillator [18]that are preserved and remain tobe described, but sucha mechanism could have alsoco-evolved by recruiting any of themany autocatalytic kinases andphosphatases present in differentspecies. An indication thata phosphorylation oscillator mightbe a primordial circadianmechanism comes from severalindependent observationsconcerning human kinases andphosphatases. Human CKIautoregulates according tophosphorylation state and isdephosphorylated by PP5 ([18]and references therein), thusthese two components could forma feedback loop if they display thecorrect kinetics and if they wereconnected to an input pathway.PP5 activity, in turn, depends onbinding to CRY2 ([18] andreferences therein), whichfluctuates in concentration inmany cells. Even withoutoscillations in the amount ofCRY2, rhythms in the state ofphosphorylation could play a partin a phosphorylation oscillatormechanism and might explain theresults shown with the cell-permeable CRY1 and CRY2 byFan et al. [2].Why is it important tounderstand how the circadiansystem is put together? Severalreports have demonstrated linksbetween health and behaviourwith respect to chronotype. Arecent publication showed that theerectile dysfunction drug sildenafilhas a profound impact onre-synchronization of thecircadian system in hamsters [19].If we are to start medicating thecircadian system itself or usea robust circadian system tomedicate other pathologies, wehave to know what we’re aimingfor and how the parts of thesystem are put together.References1. Konopka, R., and Benzer, S. (1971). Clockmutants of Drosophila melanogaster.Proc. Natl. Acad. Sci. USA 68,2112–2116.2. Fan, Y., Hida, A., Anderson, D.A.,Izumo, M., and Johnson, C.H. (2007).Cycling of CRYPTOCHROME proteins isnot necessary for circadian clock functionin mammalian fibroblasts. Curr. Biol. 17,1091–1100.3. Kume, K., Zylka, M.J., Sriram, S.,Shearman, L.P., Weaver, D.R., Jin, X.,Maywood, E.S., Hastings, M.H., andReppert, S.M. (1999). mCRY1 and mCRY2are essential components of the negativelimb of the circadian clock feedback loop.Cell 98, 193–205.4. Yang, Z., and Sehgal, A. (2001). Role ofmolecular oscillations in generatingbehavioral rhythms in Drosophila. Neuron29, 453–467.5. Tan, Y., Dragovic, Z., Roenneberg, T., andMerrow, M. (2004). Entrainment of thecircadian clock: translational and post-translational control as key elements.Curr. Biol. 14, 433–438.6. Roenneberg, T., and Merrow, M. (1999).Circadian clocks and metabolism. J. Biol.Rhythms 14, 449–459.7. Lakin-Thomas, P.L. (2006).Transcriptional feedback oscillators:maybe, maybe not. J. Biol. Rhythms 21,83–92.8. Froehlich, A.C., Liu, Y., Loros, J.J., andDunlap, J.C. (2002). White Collar-1,a circadian blue light photoreceptor,binding to the frequency promoter.Science 297, 815–819.9. Krishnan, B., Levine, J.D., Lynch, M.K.S.,Dowse, H.B., Funes, P., Hall, J.C.,Hardin, P.E., and Dryer, S.E. (2001). A newrole for cryptochrome in a Drosophilacircadian oscillator. Nature 411,313–317.10. Yuan, Q., Metterville, D., Briscoe, A.D.,and Reppert, S.M. (2007). Insectcryptochromes: gene duplication and lossdefine diverse ways to construct insectcircadian clocks. Mol. Biol. Evol. 24,948–955.11. Collins, B.H., Dissel, S., Gaten, E.,Rosato, E., and Kyriacou, C.P. (2005).Disruption of Cryptochrome partiallyrestores circadian rhythmicity to thearrhythmic period mutant of Drosophila.Proc. Natl. Acad. Sci. USA 102,19021–19026.12. Loros, J.J., and Feldman, J.F. (1986). Lossof temperature compensation of circadianperiod length in the frq-9 mutant ofNeurospora crassa. J. Biol. Rhythms 1,187–198.13. Dragovic, Z., Tan, Y., Görl, M.,Roenneberg, T., and Merrow, M. (2002).Light reception and circadian behavior in‘blind’ and ‘clock-less’ mutants ofNeurospora crassa. EMBO J. 21,3643–3651.14. Roenneberg, T., Dragovic, Z., andMerrow, M. (2005). Demasking biologicaloscillators: properties and principles ofentrainment exemplified by theNeurospora circadian clock. Proc.Natl. Acad. Sci. USA 102,7742–7747.15. Correa, A., Lewis, Z.A., Greene, A.V.,March, I.J., Gomer, R.H., and Bell-Pedersen, D. (2003). Multiple oscillatorsregulate circadian gene expression inNeurospora. Proc. Natl. Acad. Sci. USA100, 13597–13602.16. Steinlechner, S., Jacobmeier, B.,Scherbarth, F., Dernbach, H., Kruse, F.,and Albrecht, U. (2002). Robustcircadian rhythmicity of Per1 and Per2mutant mice in constant light anddynamics of Per1 and Per2 geneexpression under long and shortphotoperiods. J. Biol. Rhythms 17,202–209.17. Nakajima, M., Imai, K., Ito, H.,Nishiwaki, T., Murayama, Y., Iwasaki, H.,Oyama, T., and Kondo, T. (2005).Reconstitution of circadian oscillation ofcyanobacterial KaiC phosphorylationin vitro. Science 308, 414–415.18. Merrow, M., Mazzotta, G., Chen, Z., andRoenneberg, T. (2006). The right place atthe right time: regulation of daily timing byphosphorylation. Genes Dev. 20,2629–2633.19. Agostino, P.V., Plano, S.A., andGolombek, D.A. (2007). Sildenafilaccelerates reentrainment of circadianrhythms after advancing light schedules.Proc. Natl. Acad. Sci. USA 104,9834–9839.1Biological Center, University ofGroningen, Postbus 14, 9750AA, Haren,Netherlands. 2Institute of MedicalPsychology, University of Munich,Goethestrasse 31, 80336 München,Germany.*E-mail: m.merrow@rug.nlDOI: 10.1016/j.cub.2007.06.041

Merrow, Martha,

Roenneberg, Till,

University of Groningen Digital Archive

Circadian Clock: Time for a Phase Shift of Ideas?

treadmill and to be implicated in
a mitotic-like process.
References
1. Michie, K.A., and Lo¨we, J. (2006).
Dynamic filaments of the bacterial
cytoskeleton. Annu. Rev. Biochem. 75,
467–492.
2. Gitai, Z. (2007). Diversification and
specialization of the bacterial
cytoskeleton. Curr. Opin. Cell. Biol. 19,
5–12.
3. Margolin, W. (2005). FtsZ and the division
of prokaryotic cells and organelles. Nat.
Rev. Mol. Cell. Biol. 6, 862–871.
4. Schlieper, D., Oliva, M.A., Andreu, J.M.,
and Lo¨we, J. (2005). Structure of bacterial
tubulin BtubA/B: evidence for horizontal
gene transfer. Proc. Natl. Acad. Sci. USA
102, 9170–9175.
5. Vaughan, S., Wickstead, B., Gull, K., and
Addinall, S.G. (2004). Molecular evolution
of FtsZ protein sequences encoded within
the genomes of archaea, bacteria, and
eukaryota. J. Mol. Evol. 58, 19–29.
6. Koehler, T.M. (2002). Bacillus anthracis
genetics and virulence gene regulation.
Curr. Top. Microbiol. Immunol. 271,
143–164.
7. Tinsley, E., and Khan, S.A. (2006). A novel
FtsZ-like protein is involved in replication
of the anthrax toxin-encoding pXO1
plasmid in Bacillus anthracis. J. Bacteriol.
188, 2829–2835.
8. Berry, C., O’Neil, S., Ben-Dov, E.,
Jones, A.F., Murphy, L., Quail, M.A.,
Holden, M.T., Harris, D., Zaritsky, A., and
Parkhill, J. (2002). Complete sequence
and organization of pBtoxis, the toxin-
coding plasmid of Bacillus thuringiensis
subsp. israelensis. Appl. Environ.
Microbiol. 68, 5082–5095.
9. Tang, M., Bideshi, D.K., Park, H.W., and
Federici, B.A. (2006). Minireplicon from
pBtoxis of Bacillus thuringiensis subsp.
israelensis. Appl. Environ. Microbiol. 72,
6948–6954.
10. Larsen, R.A., Cusumano, C., Fujioka, A.,
Lim-Fong, G., Patterson, P., and
Pogliano, J. (2007). Treadmilling of
a prokaryotic tubulin-like protein, TubZ,
required for plasmid stability in Bacillus
thuringiensis. Genes Dev. 21, 1340–1352.
11. Theriot, J.A., and Mitchison, T.J. (1991).
Actin microfilament dynamics in
locomoting cells. Nature 352, 126–131.
12. Kim, S.Y., Gitai, Z., Kinkhabwala, A.,
Shapiro, L., and Moerner, W.E. (2006).
Single molecules of the bacterial actin
MreB undergo directed treadmilling
motion in Caulobacter crescentus. Proc.
Natl. Acad. Sci. USA 103, 10929–10934.
13. Waterman-Storer, C.M., and Salmon, E.D.
(1997). Microtubule dynamics:
treadmilling comes around again. Curr.
Biol. 7, 369–372.
14. Rodionov, V.I., and Borisy, G.G. (1997).
Microtubule treadmilling in vivo. Science
275, 215–218.
15. Shaw, S.L., Kamyar, R., and
Ehrhardt, D.W. (2003). Sustained
microtubule treadmilling in Arabidopsis
cortical arrays. Science 300, 1715–1718.
16. Thanedar, S., and Margolin, W. (2004).
FtsZ exhibits rapid movement and
oscillation waves in helix-like patterns
in Escherichia coli. Curr. Biol. 14,
1167–1173.
17. Redick, S.D., Stricker, J., Briscoe, G., and
Erickson, H.P. (2005). Mutants of FtsZ
targeting the protofilament interface:
effects on cell division and GTPase
activity. J. Bacteriol. 187, 2727–2736.
18. Hayes, F., and Barilla, D. (2006). The
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133–143.
Department of Microbiology
and Molecular Genetics,
University of Texas Medical School,
6431 Fannin Street, Houston,
Texas 77030, USA.
E-mail: William.Margolin@uth.tmc.edu
DOI: 10.1016/j.cub.2007.06.020
Current Biology Vol 17 No 16
R636Circadian Clock: Time for a Phase
Shift of Ideas?
A recent study shows that cycling of cryptochrome proteins is
dispensable for circadian clock function in mammalian cells. Is it time
for a paradigm shift in how we think about the circadian clock
mechanism?Martha Merrow1,*
and Till Roenneberg2
Clocks rule our lives. One of them
is the circadian clock — an
adaptive, endogenous temporal
programme which ensures that the
same processes occur at the same
time, day after day. These
processes are based on a number
of rhythmic functions ranging from
gene expression and metabolism
to behaviour and they are so
pronounced that we are
biochemically ‘different’ people
from day to night. Daily rhythms
are self-sustained; they even
oscillate in the absence of a cycling
environment with their period
being close to 24 hours, hence
circadian. The implications of this
circadian programme should be of
interest to all biologists and
doctors because time of day can
potentially impact on all kinds ofexperiments and medical
diagnoses.
The genetic revolution for
complex behaviour started with
Seymour Benzer who identified
the first clock gene, period, in
Drosophila [1]. Since then, clock
genes have been described in all
circadian model systems, from
cyanobacteria to mice. By
discovering how the products of
these clock genes interact,
a cohesive mechanism emerged
that is apparently common to
all circadian systems: an
auto-regulatory negative
feedback loop involving
transcription and translation
(Transcription Translation
Feedback Loop, TTFL). Changes
in any of the loop’s components
have dominant effects on the
circadian rhythmicity, and it has
been hypothesised that this
feedback loop generatescircadian rhythms at the cellular
level (Figure 1A).
In a recent paper in Current
Biology, Fan et al. [2] describe
provocative results that — if
repeated, extended and
elaborated — challenge the current
feedback loop model. In mice,
levels of the TTFL components
mPERIOD (PER) 1 and 2,
mCRYPTOCHROME (CRY) 1 and 2,
and BMAL1 typically oscillate over
the course of a day. The oscillation
is thought to depend largely on
the CRY proteins, which repress
CLK/BMAL1 mediated
transcriptional activation. PER
proteins also show negative
feedback but are less potent than
either of the CRY proteins in cell
culture systems [3]. Fan et al. [2]
have engineered the two key
negative feedback components,
the mCRY proteins, such that they
move freely into cells. They
surprisingly find that, despite the
apparently constant level of both
CRYs and CRY-induced BMAL1
expression, Per2 transcription
continues to cycle rhythmically.
This indicates that one of the
fundamental assumptions
concerning the mouse circadian
clock seems incorrect, namely that
oscillations of CRY protein levels
Dispatch
R637are essential for generating
circadian rhythmicity.
There are numerous possible
scenarios which could account for
the observations of Fan et al. [2]
from which we have chosen five.
Scenario 1: The effects on the
molecular machinery of the
circadian clock using the new
cell-permeable CRY proteins could
simply be an artefact of the method
itself. Many things happen to
clock proteins on their daily journey
through time. They are modified,
gang up with other proteins, get
shuffled between compartments
and so on. Does all of this also
happen in cells that are flooded
with the two CRY proteins? Future
experiments will elaborate on the
fates of the imported proteins and
how they compare with those of
endogenous CRY proteins in
untreated cells. However, the
surprising observation that
the constitutively present,
cell-permeant CRYs support
BMAL1 oscillations in normally
arrhythmic Cry1;Cry2 mutant
fibroblasts argues against this
scenario.
Scenario 2: The traditional
feedback loop hypothesis still
holds. Experiments in many
circadian systems have shown that
several interlocking loops form
a ‘network of time’ and the authors
may have simply uncovered yet
another loop within the network,
one that reflects PER2 rhythms
versus CRY1 and CRY2 rhythms
(Figure 1B). There are already many
indications of auxiliary loops
involving, for example, nuclear
orphan receptors, DECs or
PAR-proteins. The findings of
Fan et al. [2] may be linked to one
of these or may lead to a novel
extra-TTFL loop.
Scenario 3: The experiment
might reveal a similar phenomenon
as in Drosophila, where
constitutive expression of both
so-called negative elements of the
TTFL–PER and TIM- still allows
rhythmicity in locomotor activity as
well as in the levels of the clock
proteins themselves [4]. The
assumption here is that the
rhythmicity of the proteins — in
terms of level, state or
localization — is essential for
circadian behaviour, rather than
the rhythmicity of theirA
rep rep act
B
act
C
repact actrep
act rep act actrep rep
P
D
P
E
PP kinkin
P
Current Biology
Figure 1. Possible scenarios explaining the dispensability of CRY protein oscillations
in the circadian clock.
Five scenarios as described in the text are illustrated. (A) The Transcription Translation
Feedback Loop (TTFL) model is built on the elegantly simple hypothesis that clock
gene transcription, effected by transcriptional activators (act) is self-regulated by feed-
back repression (rep). (B) The TTFL network is far more complex, and the actual rela-
tionships may be revealed through experiments such as those described in [3]. (C) The
role of transcription (dashed lines) may not be critical for all circadian properties.
Rather, post-translational modifications, such as phosphorylation (P) or sub-cellular
localisation (block arrow) could play major roles. (D) The TTFL components may be
serving input and output functions in the clock signal transduction pathway. In this
case, the identity of the actual rhythm generator (depicted as a sine wave) may still
be unknown. (E) The TTFL may be regulated by a ‘phosphorylation oscillator’, namely
an interaction between a kinase (kin) and a phosphatase (PP) whereby functional
activity of clock-critical kinases is modulated via phosphatases, which are in turn
regulated by components of the TTFL that are targets of the kinases.transcription (Figure 1C).
Additional support for this idea
comes from experiments exposing
the clock to different daily
environmental cycles in the lab,
whereby in the fungus Neurospora,
it is the clock gene protein levels
that correlate with the ‘behavioural’
phase (in this case, midnight), while
the RNA levels appear simply
driven (masked) by the changing
light levels [5]. In the experiments
here, at least PER1 protein levels
can oscillate, so perhaps this is the
essential rhythmic component,
with the rhythms of the other
components contributing
characteristics of the system that
cannot be appreciated in cell
culture systems.
Scenario 4: The TTFL is not the
core, rhythm-generating
mechanism, but rather serves
both an input and an output with
feedback function. As its input, it
processes signals from the
environment to the rhythm
generator, which in turn regulates
the input pathway. This would
render the TTFL also an output of
the clock and in this role it wouldtranscriptionally control diverse
outputs (Figure 1D). Such an
alternative hypothesis was initially
proposed in 1998 ([6] and
references therein) based on
mathematical modelling, and
since then numerous experimental
results in many model systems
support this scenario [7]. Light
reception and components of the
TTFL are tightly associated. In
some cases, they serve both
functions, in other cases, they
have changed their role from one
function to the other during
evolution [8–10]. Light often leads
to rapid changes in the TTFL
components, concurring with
phase shifts of the circadian
oscillation. Others have shown,
however, that residual circadian
properties persist when
components of the TTFL are
rendered non-functional.
In Drosophila, combination of the
arrhythmic mutant allele of per
with cryb results in the restoration
of some circadian properties,
rather than an additional
decrement [11]. In Neurospora,
sporadic free running rhythms
Current Biology Vol 17 No 16
R638[12,13] and also systematic
entrainment characteristics can
be found in clock null mutants [14],
and subsequently, molecular
rhythms have been shown to
persist in these strains [15]. In
mice, many mutants that had been
considered ‘clock null mutants’
because they were arrhythmic
(e.g., the mPer2;mCry2 double
mutant) become rhythmic when
put into constant dim light [16].
Furthermore, conceptual models
show that alterations of input
feedback components can have
huge effects on circadian
oscillations [6]. Finally, feedbacks
of the rhythm generator back onto
its inputs have been shown to
shield the rhythm from noise by
compensation and, thus, add
robustness to the endogenous
daily cycle [6].
Scenario 5: Some of the
scenarios above indicate that
transcription and/or translation
may not need to be rhythmic to
ensure circadian function. But
where, then, is rhythmicity
generated? A recent finding in
cyanobacteria suggests that
phosphorylation plays a crucial
role. Phosphorylation not only
confers specific half-lives to
proteins, it also regulates their
sub-cellular localization and their
switching between activity or
inactivity — not only in
transcription factors, which are so
much at the heart of the traditional
hypothesis of rhythm generation.
In cyanobacteria, three proteins
and ATP were shown to be
sufficient recapitulate most of the
circadian clock properties in vitro
[17]! Given the added complexity
in eukaryotes, we may not expect
to find a similar, simple molecular
clock mechanism. However, there
may be elements of
a phosphorylation oscillator [18]
that are preserved and remain to
be described, but such
a mechanism could have also
co-evolved by recruiting any of the
many autocatalytic kinases and
phosphatases present in different
species. An indication that
a phosphorylation oscillator might
be a primordial circadian
mechanism comes from several
independent observations
concerning human kinases and
phosphatases. Human CKIautoregulates according to
phosphorylation state and is
dephosphorylated by PP5 ([18]
and references therein), thus
these two components could form
a feedback loop if they display the
correct kinetics and if they were
connected to an input pathway.
PP5 activity, in turn, depends on
binding to CRY2 ([18] and
references therein), which
fluctuates in concentration in
many cells. Even without
oscillations in the amount of
CRY2, rhythms in the state of
phosphorylation could play a part
in a phosphorylation oscillator
mechanism and might explain the
results shown with the cell-
permeable CRY1 and CRY2 by
Fan et al. [2].
Why is it important to
understand how the circadian
system is put together? Several
reports have demonstrated links
between health and behaviour
with respect to chronotype. A
recent publication showed that the
erectile dysfunction drug sildenafil
has a profound impact on
re-synchronization of the
circadian system in hamsters [19].
If we are to start medicating the
circadian system itself or use
a robust circadian system to
medicate other pathologies, we
have to know what we’re aiming
for and how the parts of the
system are put together.
References
1. Konopka, R., and Benzer, S. (1971). Clock
mutants of Drosophila melanogaster.
Proc. Natl. Acad. Sci. USA 68,
2112–2116.
2. Fan, Y., Hida, A., Anderson, D.A.,
Izumo, M., and Johnson, C.H. (2007).
Cycling of CRYPTOCHROME proteins is
not necessary for circadian clock function
in mammalian fibroblasts. Curr. Biol. 17,
1091–1100.
3. Kume, K., Zylka, M.J., Sriram, S.,
Shearman, L.P., Weaver, D.R., Jin, X.,
Maywood, E.S., Hastings, M.H., and
Reppert, S.M. (1999). mCRY1 and mCRY2
are essential components of the negative
limb of the circadian clock feedback loop.
Cell 98, 193–205.
4. Yang, Z., and Sehgal, A. (2001). Role of
molecular oscillations in generating
behavioral rhythms in Drosophila. Neuron
29, 453–467.
5. Tan, Y., Dragovic, Z., Roenneberg, T., and
Merrow, M. (2004). Entrainment of the
circadian clock: translational and post-
translational control as key elements.
Curr. Biol. 14, 433–438.
6. Roenneberg, T., and Merrow, M. (1999).
Circadian clocks and metabolism. J. Biol.
Rhythms 14, 449–459.
7. Lakin-Thomas, P.L. (2006).
Transcriptional feedback oscillators:maybe, maybe not. J. Biol. Rhythms 21,
83–92.
8. Froehlich, A.C., Liu, Y., Loros, J.J., and
Dunlap, J.C. (2002). White Collar-1,
a circadian blue light photoreceptor,
binding to the frequency promoter.
Science 297, 815–819.
9. Krishnan, B., Levine, J.D., Lynch, M.K.S.,
Dowse, H.B., Funes, P., Hall, J.C.,
Hardin, P.E., and Dryer, S.E. (2001). A new
role for cryptochrome in a Drosophila
circadian oscillator. Nature 411,
313–317.
10. Yuan, Q., Metterville, D., Briscoe, A.D.,
and Reppert, S.M. (2007). Insect
cryptochromes: gene duplication and loss
define diverse ways to construct insect
circadian clocks. Mol. Biol. Evol. 24,
948–955.
11. Collins, B.H., Dissel, S., Gaten, E.,
Rosato, E., and Kyriacou, C.P. (2005).
Disruption of Cryptochrome partially
restores circadian rhythmicity to the
arrhythmic period mutant of Drosophila.
Proc. Natl. Acad. Sci. USA 102,
19021–19026.
12. Loros, J.J., and Feldman, J.F. (1986). Loss
of temperature compensation of circadian
period length in the frq-9 mutant of
Neurospora crassa. J. Biol. Rhythms 1,
187–198.
13. Dragovic, Z., Tan, Y., Go¨rl, M.,
Roenneberg, T., and Merrow, M. (2002).
Light reception and circadian behavior in
‘blind’ and ‘clock-less’ mutants of
Neurospora crassa. EMBO J. 21,
3643–3651.
14. Roenneberg, T., Dragovic, Z., and
Merrow, M. (2005). Demasking biological
oscillators: properties and principles of
entrainment exemplified by the
Neurospora circadian clock. Proc.
Natl. Acad. Sci. USA 102,
7742–7747.
15. Correa, A., Lewis, Z.A., Greene, A.V.,
March, I.J., Gomer, R.H., and Bell-
Pedersen, D. (2003). Multiple oscillators
regulate circadian gene expression in
Neurospora. Proc. Natl. Acad. Sci. USA
100, 13597–13602.
16. Steinlechner, S., Jacobmeier, B.,
Scherbarth, F., Dernbach, H., Kruse, F.,
and Albrecht, U. (2002). Robust
circadian rhythmicity of Per1 and Per2
mutant mice in constant light and
dynamics of Per1 and Per2 gene
expression under long and short
photoperiods. J. Biol. Rhythms 17,
202–209.
17. Nakajima, M., Imai, K., Ito, H.,
Nishiwaki, T., Murayama, Y., Iwasaki, H.,
Oyama, T., and Kondo, T. (2005).
Reconstitution of circadian oscillation of
cyanobacterial KaiC phosphorylation
in vitro. Science 308, 414–415.
18. Merrow, M., Mazzotta, G., Chen, Z., and
Roenneberg, T. (2006). The right place at
the right time: regulation of daily timing by
phosphorylation. Genes Dev. 20,
2629–2633.
19. Agostino, P.V., Plano, S.A., and
Golombek, D.A. (2007). Sildenafil
accelerates reentrainment of circadian
rhythms after advancing light schedules.
Proc. Natl. Acad. Sci. USA 104,
9834–9839.
1Biological Center, University of
Groningen, Postbus 14, 9750AA, Haren,
Netherlands. 2Institute of Medical
Psychology, University of Munich,
Goethestrasse 31, 80336 Mu¨nchen,
Germany.
*E-mail: m.merrow@rug.nl
DOI: 10.1016/j.cub.2007.06.041


   University of GroningenCircadian clockMerrow, Martha; Roenneberg, TillPublished in:Current BiologyDOI:10.1016/j.cub.2007.06.041IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2007Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Merrow, M., & Roenneberg, T. (2007). Circadian clock: Time for a phase shift of ideas? Current Biology,17(16), R636-R638. https://doi.org/10.1016/j.cub.2007.06.041CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019treadmill and to be implicated ina mitotic-like process.References1. Michie, K.A., and Lo¨we, J. (2006).Dynamic filaments of the bacterialcytoskeleton. Annu. Rev. Biochem. 75,467–492.2. Gitai, Z. (2007). Diversification andspecialization of the bacterialcytoskeleton. Curr. Opin. Cell. Biol. 19,5–12.3. Margolin, W. (2005). FtsZ and the divisionof prokaryotic cells and organelles. Nat.Rev. Mol. Cell. Biol. 6, 862–871.4. Schlieper, D., Oliva, M.A., Andreu, J.M.,and Lo¨we, J. (2005). Structure of bacterialtubulin BtubA/B: evidence for horizontalgene transfer. Proc. Natl. Acad. Sci. USA102, 9170–9175.5. Vaughan, S., Wickstead, B., Gull, K., andAddinall, S.G. (2004). Molecular evolutionof FtsZ protein sequences encoded withinthe genomes of archaea, bacteria, andeukaryota. J. Mol. Evol. 58, 19–29.6. Koehler, T.M. (2002). Bacillus anthracisgenetics and virulence gene regulation.Curr. Top. Microbiol. Immunol. 271,143–164.7. Tinsley, E., and Khan, S.A. (2006). A novelFtsZ-like protein is involved in replicationof the anthrax toxin-encoding pXO1plasmid in Bacillus anthracis. J. Bacteriol.188, 2829–2835.8. Berry, C., O’Neil, S., Ben-Dov, E.,Jones, A.F., Murphy, L., Quail, M.A.,Holden, M.T., Harris, D., Zaritsky, A., andParkhill, J. (2002). Complete sequenceand organization of pBtoxis, the toxin-coding plasmid of Bacillus thuringiensissubsp. israelensis. Appl. Environ.Microbiol. 68, 5082–5095.9. Tang, M., Bideshi, D.K., Park, H.W., andFederici, B.A. (2006). Minireplicon frompBtoxis of Bacillus thuringiensis subsp.israelensis. Appl. Environ. Microbiol. 72,6948–6954.10. Larsen, R.A., Cusumano, C., Fujioka, A.,Lim-Fong, G., Patterson, P., andPogliano, J. (2007). Treadmilling ofa prokaryotic tubulin-like protein, TubZ,required for plasmid stability in Bacillusthuringiensis. Genes Dev. 21, 1340–1352.11. Theriot, J.A., and Mitchison, T.J. (1991).Actin microfilament dynamics inlocomoting cells. Nature 352, 126–131.12. Kim, S.Y., Gitai, Z., Kinkhabwala, A.,Shapiro, L., and Moerner, W.E. (2006).Single molecules of the bacterial actinMreB undergo directed treadmillingmotion in Caulobacter crescentus. Proc.Natl. Acad. Sci. USA 103, 10929–10934.13. Waterman-Storer, C.M., and Salmon, E.D.(1997). Microtubule dynamics:treadmilling comes around again. Curr.Biol. 7, 369–372.14. Rodionov, V.I., and Borisy, G.G. (1997).Microtubule treadmilling in vivo. Science275, 215–218.15. Shaw, S.L., Kamyar, R., andEhrhardt, D.W. (2003). Sustainedmicrotubule treadmilling in Arabidopsiscortical arrays. Science 300, 1715–1718.16. Thanedar, S., and Margolin, W. (2004).FtsZ exhibits rapid movement andoscillation waves in helix-like patternsin Escherichia coli. Curr. Biol. 14,1167–1173.17. Redick, S.D., Stricker, J., Briscoe, G., andErickson, H.P. (2005). Mutants of FtsZtargeting the protofilament interface:effects on cell division and GTPaseactivity. J. Bacteriol. 187, 2727–2736.18. Hayes, F., and Barilla, D. (2006). Thebacterial segrosome: a dynamicnucleoprotein machine for DNA traffickingand segregation. Nat. Rev. Microbiol. 4,133–143.Department of Microbiologyand Molecular Genetics,University of Texas Medical School,6431 Fannin Street, Houston,Texas 77030, USA.E-mail: William.Margolin@uth.tmc.eduDOI: 10.1016/j.cub.2007.06.020Current Biology Vol 17 No 16R636Circadian Clock: Time for a PhaseShift of Ideas?A recent study shows that cycling of cryptochrome proteins isdispensable for circadian clock function in mammalian cells. Is it timefor a paradigm shift in how we think about the circadian clockmechanism?Martha Merrow1,*and Till Roenneberg2Clocks rule our lives. One of themis the circadian clock — anadaptive, endogenous temporalprogramme which ensures that thesame processes occur at the sametime, day after day. Theseprocesses are based on a numberof rhythmic functions ranging fromgene expression and metabolismto behaviour and they are sopronounced that we arebiochemically ‘different’ peoplefrom day to night. Daily rhythmsare self-sustained; they evenoscillate in the absence of a cyclingenvironment with their periodbeing close to 24 hours, hencecircadian. The implications of thiscircadian programme should be ofinterest to all biologists anddoctors because time of day canpotentially impact on all kinds ofexperiments and medicaldiagnoses.The genetic revolution forcomplex behaviour started withSeymour Benzer who identifiedthe first clock gene, period, inDrosophila [1]. Since then, clockgenes have been described in allcircadian model systems, fromcyanobacteria to mice. Bydiscovering how the products ofthese clock genes interact,a cohesive mechanism emergedthat is apparently common toall circadian systems: anauto-regulatory negativefeedback loop involvingtranscription and translation(Transcription TranslationFeedback Loop, TTFL). Changesin any of the loop’s componentshave dominant effects on thecircadian rhythmicity, and it hasbeen hypothesised that thisfeedback loop generatescircadian rhythms at the cellularlevel (Figure 1A).In a recent paper in CurrentBiology, Fan et al. [2] describeprovocative results that — ifrepeated, extended andelaborated — challenge the currentfeedback loop model. In mice,levels of the TTFL componentsmPERIOD (PER) 1 and 2,mCRYPTOCHROME (CRY) 1 and 2,and BMAL1 typically oscillate overthe course of a day. The oscillationis thought to depend largely onthe CRY proteins, which repressCLK/BMAL1 mediatedtranscriptional activation. PERproteins also show negativefeedback but are less potent thaneither of the CRY proteins in cellculture systems [3]. Fan et al. [2]have engineered the two keynegative feedback components,the mCRY proteins, such that theymove freely into cells. Theysurprisingly find that, despite theapparently constant level of bothCRYs and CRY-induced BMAL1expression, Per2 transcriptioncontinues to cycle rhythmically.This indicates that one of thefundamental assumptionsconcerning the mouse circadianclock seems incorrect, namely thatoscillations of CRY protein levelsDispatchR637are essential for generatingcircadian rhythmicity.There are numerous possiblescenarios which could account forthe observations of Fan et al. [2]from which we have chosen five.Scenario 1: The effects on themolecular machinery of thecircadian clock using the newcell-permeable CRY proteins couldsimply be an artefact of the methoditself. Many things happen toclock proteins on their daily journeythrough time. They are modified,gang up with other proteins, getshuffled between compartmentsand so on. Does all of this alsohappen in cells that are floodedwith the two CRY proteins? Futureexperiments will elaborate on thefates of the imported proteins andhow they compare with those ofendogenous CRY proteins inuntreated cells. However, thesurprising observation thatthe constitutively present,cell-permeant CRYs supportBMAL1 oscillations in normallyarrhythmic Cry1;Cry2 mutantfibroblasts argues against thisscenario.Scenario 2: The traditionalfeedback loop hypothesis stillholds. Experiments in manycircadian systems have shown thatseveral interlocking loops forma ‘network of time’ and the authorsmay have simply uncovered yetanother loop within the network,one that reflects PER2 rhythmsversus CRY1 and CRY2 rhythms(Figure 1B). There are already manyindications of auxiliary loopsinvolving, for example, nuclearorphan receptors, DECs orPAR-proteins. The findings ofFan et al. [2] may be linked to oneof these or may lead to a novelextra-TTFL loop.Scenario 3: The experimentmight reveal a similar phenomenonas in Drosophila, whereconstitutive expression of bothso-called negative elements of theTTFL–PER and TIM- still allowsrhythmicity in locomotor activity aswell as in the levels of the clockproteins themselves [4]. Theassumption here is that therhythmicity of the proteins — interms of level, state orlocalization — is essential forcircadian behaviour, rather thanthe rhythmicity of theirArep rep actBactCrepact actrepact rep act actrep repPDPEPP kinkinPCurrent BiologyFigure 1. Possible scenarios explaining the dispensability of CRY protein oscillationsin the circadian clock.Five scenarios as described in the text are illustrated. (A) The Transcription TranslationFeedback Loop (TTFL) model is built on the elegantly simple hypothesis that clockgene transcription, effected by transcriptional activators (act) is self-regulated by feed-back repression (rep). (B) The TTFL network is far more complex, and the actual rela-tionships may be revealed through experiments such as those described in [3]. (C) Therole of transcription (dashed lines) may not be critical for all circadian properties.Rather, post-translational modifications, such as phosphorylation (P) or sub-cellularlocalisation (block arrow) could play major roles. (D) The TTFL components may beserving input and output functions in the clock signal transduction pathway. In thiscase, the identity of the actual rhythm generator (depicted as a sine wave) may stillbe unknown. (E) The TTFL may be regulated by a ‘phosphorylation oscillator’, namelyan interaction between a kinase (kin) and a phosphatase (PP) whereby functionalactivity of clock-critical kinases is modulated via phosphatases, which are in turnregulated by components of the TTFL that are targets of the kinases.transcription (Figure 1C).Additional support for this ideacomes from experiments exposingthe clock to different dailyenvironmental cycles in the lab,whereby in the fungus Neurospora,it is the clock gene protein levelsthat correlate with the ‘behavioural’phase (in this case, midnight), whilethe RNA levels appear simplydriven (masked) by the changinglight levels [5]. In the experimentshere, at least PER1 protein levelscan oscillate, so perhaps this is theessential rhythmic component,with the rhythms of the othercomponents contributingcharacteristics of the system thatcannot be appreciated in cellculture systems.Scenario 4: The TTFL is not thecore, rhythm-generatingmechanism, but rather servesboth an input and an output withfeedback function. As its input, itprocesses signals from theenvironment to the rhythmgenerator, which in turn regulatesthe input pathway. This wouldrender the TTFL also an output ofthe clock and in this role it wouldtranscriptionally control diverseoutputs (Figure 1D). Such analternative hypothesis was initiallyproposed in 1998 ([6] andreferences therein) based onmathematical modelling, andsince then numerous experimentalresults in many model systemssupport this scenario [7]. Lightreception and components of theTTFL are tightly associated. Insome cases, they serve bothfunctions, in other cases, theyhave changed their role from onefunction to the other duringevolution [8–10]. Light often leadsto rapid changes in the TTFLcomponents, concurring withphase shifts of the circadianoscillation. Others have shown,however, that residual circadianproperties persist whencomponents of the TTFL arerendered non-functional.In Drosophila, combination of thearrhythmic mutant allele of perwith cryb results in the restorationof some circadian properties,rather than an additionaldecrement [11]. In Neurospora,sporadic free running rhythmsCurrent Biology Vol 17 No 16R638[12,13] and also systematicentrainment characteristics canbe found in clock null mutants [14],and subsequently, molecularrhythms have been shown topersist in these strains [15]. Inmice, many mutants that had beenconsidered ‘clock null mutants’because they were arrhythmic(e.g., the mPer2;mCry2 doublemutant) become rhythmic whenput into constant dim light [16].Furthermore, conceptual modelsshow that alterations of inputfeedback components can havehuge effects on circadianoscillations [6]. Finally, feedbacksof the rhythm generator back ontoits inputs have been shown toshield the rhythm from noise bycompensation and, thus, addrobustness to the endogenousdaily cycle [6].Scenario 5: Some of thescenarios above indicate thattranscription and/or translationmay not need to be rhythmic toensure circadian function. Butwhere, then, is rhythmicitygenerated? A recent finding incyanobacteria suggests thatphosphorylation plays a crucialrole. Phosphorylation not onlyconfers specific half-lives toproteins, it also regulates theirsub-cellular localization and theirswitching between activity orinactivity — not only intranscription factors, which are somuch at the heart of the traditionalhypothesis of rhythm generation.In cyanobacteria, three proteinsand ATP were shown to besufficient recapitulate most of thecircadian clock properties in vitro[17]! Given the added complexityin eukaryotes, we may not expectto find a similar, simple molecularclock mechanism. However, theremay be elements ofa phosphorylation oscillator [18]that are preserved and remain tobe described, but sucha mechanism could have alsoco-evolved by recruiting any of themany autocatalytic kinases andphosphatases present in differentspecies. An indication thata phosphorylation oscillator mightbe a primordial circadianmechanism comes from severalindependent observationsconcerning human kinases andphosphatases. Human CKIautoregulates according tophosphorylation state and isdephosphorylated by PP5 ([18]and references therein), thusthese two components could forma feedback loop if they display thecorrect kinetics and if they wereconnected to an input pathway.PP5 activity, in turn, depends onbinding to CRY2 ([18] andreferences therein), whichfluctuates in concentration inmany cells. Even withoutoscillations in the amount ofCRY2, rhythms in the state ofphosphorylation could play a partin a phosphorylation oscillatormechanism and might explain theresults shown with the cell-permeable CRY1 and CRY2 byFan et al. [2].Why is it important tounderstand how the circadiansystem is put together? Severalreports have demonstrated linksbetween health and behaviourwith respect to chronotype. Arecent publication showed that theerectile dysfunction drug sildenafilhas a profound impact onre-synchronization of thecircadian system in hamsters [19].If we are to start medicating thecircadian system itself or usea robust circadian system tomedicate other pathologies, wehave to know what we’re aimingfor and how the parts of thesystem are put together.References1. Konopka, R., and Benzer, S. (1971). Clockmutants of Drosophila melanogaster.Proc. Natl. Acad. Sci. USA 68,2112–2116.2. Fan, Y., Hida, A., Anderson, D.A.,Izumo, M., and Johnson, C.H. (2007).Cycling of CRYPTOCHROME proteins isnot necessary for circadian clock functionin mammalian fibroblasts. Curr. Biol. 17,1091–1100.3. Kume, K., Zylka, M.J., Sriram, S.,Shearman, L.P., Weaver, D.R., Jin, X.,Maywood, E.S., Hastings, M.H., andReppert, S.M. (1999). mCRY1 and mCRY2are essential components of the negativelimb of the circadian clock feedback loop.Cell 98, 193–205.4. Yang, Z., and Sehgal, A. (2001). Role ofmolecular oscillations in generatingbehavioral rhythms in Drosophila. Neuron29, 453–467.5. Tan, Y., Dragovic, Z., Roenneberg, T., andMerrow, M. (2004). Entrainment of thecircadian clock: translational and post-translational control as key elements.Curr. Biol. 14, 433–438.6. Roenneberg, T., and Merrow, M. (1999).Circadian clocks and metabolism. J. Biol.Rhythms 14, 449–459.7. Lakin-Thomas, P.L. (2006).Transcriptional feedback oscillators:maybe, maybe not. J. Biol. Rhythms 21,83–92.8. Froehlich, A.C., Liu, Y., Loros, J.J., andDunlap, J.C. (2002). White Collar-1,a circadian blue light photoreceptor,binding to the frequency promoter.Science 297, 815–819.9. Krishnan, B., Levine, J.D., Lynch, M.K.S.,Dowse, H.B., Funes, P., Hall, J.C.,Hardin, P.E., and Dryer, S.E. (2001). A newrole for cryptochrome in a Drosophilacircadian oscillator. Nature 411,313–317.10. Yuan, Q., Metterville, D., Briscoe, A.D.,and Reppert, S.M. (2007). Insectcryptochromes: gene duplication and lossdefine diverse ways to construct insectcircadian clocks. Mol. Biol. Evol. 24,948–955.11. Collins, B.H., Dissel, S., Gaten, E.,Rosato, E., and Kyriacou, C.P. (2005).Disruption of Cryptochrome partiallyrestores circadian rhythmicity to thearrhythmic period mutant of Drosophila.Proc. Natl. Acad. Sci. USA 102,19021–19026.12. Loros, J.J., and Feldman, J.F. (1986). Lossof temperature compensation of circadianperiod length in the frq-9 mutant ofNeurospora crassa. J. Biol. Rhythms 1,187–198.13. Dragovic, Z., Tan, Y., Go¨rl, M.,Roenneberg, T., and Merrow, M. (2002).Light reception and circadian behavior in‘blind’ and ‘clock-less’ mutants ofNeurospora crassa. EMBO J. 21,3643–3651.14. Roenneberg, T., Dragovic, Z., andMerrow, M. (2005). Demasking biologicaloscillators: properties and principles ofentrainment exemplified by theNeurospora circadian clock. Proc.Natl. Acad. Sci. USA 102,7742–7747.15. Correa, A., Lewis, Z.A., Greene, A.V.,March, I.J., Gomer, R.H., and Bell-Pedersen, D. (2003). Multiple oscillatorsregulate circadian gene expression inNeurospora. Proc. Natl. Acad. Sci. USA100, 13597–13602.16. Steinlechner, S., Jacobmeier, B.,Scherbarth, F., Dernbach, H., Kruse, F.,and Albrecht, U. (2002). Robustcircadian rhythmicity of Per1 and Per2mutant mice in constant light anddynamics of Per1 and Per2 geneexpression under long and shortphotoperiods. J. Biol. Rhythms 17,202–209.17. Nakajima, M., Imai, K., Ito, H.,Nishiwaki, T., Murayama, Y., Iwasaki, H.,Oyama, T., and Kondo, T. (2005).Reconstitution of circadian oscillation ofcyanobacterial KaiC phosphorylationin vitro. Science 308, 414–415.18. Merrow, M., Mazzotta, G., Chen, Z., andRoenneberg, T. (2006). The right place atthe right time: regulation of daily timing byphosphorylation. Genes Dev. 20,2629–2633.19. Agostino, P.V., Plano, S.A., andGolombek, D.A. (2007). Sildenafilaccelerates reentrainment of circadianrhythms after advancing light schedules.Proc. Natl. Acad. Sci. USA 104,9834–9839.1Biological Center, University ofGroningen, Postbus 14, 9750AA, Haren,Netherlands. 2Institute of MedicalPsychology, University of Munich,Goethestrasse 31, 80336 Mu¨nchen,Germany.*E-mail: m.merrow@rug.nlDOI: 10.1016/j.cub.2007.06.041

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Circadian clock:Time for a phase shift of ideas?

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