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CELL BIOLOGY: ON CRYPTOCHROMES

The following points are made by E. van der Schalie and C.B. Green (Current Biology 2005 15:R785):

1) The protein cryptochrome is most often mentioned by circadian biologists. In organisms from cyanobacteria to humans, circadian rhythms are responsible for controlling temporal relationships of cellular, physiological, and behavioral processes and synchronizing these processes with important environmental cues, such as light and temperature. These rhythms are approximately 24 hours in duration and endogenous to the organism, persisting even when placed in constant conditions. The ~24 hour period of circadian oscillation is driven by a molecular clock found in individual cells and consists of interdependent transcriptional/translational feedback loops.

2) The term "cryptochrome" began as a generic label for photoreceptors in plants that were responsible for plant responsiveness to blue light. After the first blue-light receptor was cloned in Arabidposis thaliana, the term cryptochrome came to mean a photolyase-like photoreceptor, due to its sequence similarity to photolyase, a DNA repair enzyme activated by blue light. Cryptochromes (CRYs) are thought to have evolved from photolyases several times independently; for example, Drosophila CRYs and mammalian CRYs are more related to photolyases than to each other. CRY/photolyase family proteins have highly conserved amino termini and bind two chromophores -- flavin and either pterin or deazaflavin.

3) The functional diversity between CRYs in different species is surprising, considering that the amino terminus is highly conserved and only the carboxy-terminal tails vary widely in size and amino acid composition. CRY's role in the molecular circadian clock differs among plants, invertebrates, non-mammalian vertebrates, and mammals. For example, in plants, CRY is a blue-light photoreceptor that controls many rhythmic behaviors. Similarly, in the fruit fly, CRY acts predominantly as a photoreceptor, but in some tissues is also part of the core clock mechanism. In lower vertebrates and mammals, CRYs are part of the molecular clock.

4) Unlike in plants and Drosophila, in vertebrates no light-dependent actions of cryptochromes have been identified, although chromophore-binding residues are conserved. The debate as to whether or not CRYs act as photoreceptors in vertebrates, especially mammals, continues. Additionally, mammalian CRYs exist in two forms, CRY1 and CRY2, both of which can potently repress the CLOCK-BMAL1 heterodimeric transcription factor, but have opposing effects on the period of the clock.[1-4]

References (abridged):

1. Cashmore, A.R., Jarillo, J.A., Wu, Y., and Liu, D. (1999). Cryptochromes: Blue light receptors for plants and animals. Science 284, 760-765

2. Green, C.B. (2004). Cryptochromes: Tail-ored for distinct functions. Curr. Biol. 14, R847-R849

3. Lowrey, P.L. and Takahashi, J.S. (2004). Mammalian circadian biology: elucidating genome-wide levels of temporal organization. Annu. Rev. Genom. Hum. Genet. 5, 407-441

4. Young, M.W. and Kay, S.A. (2001). Time zones: a comparative genetics of circadian clocks. Nat. Rev. Genet. 2, 702-715

Current Biology http://www.current-biology.com

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COORDINATION OF CIRCADIAN TIMING IN MAMMALS

The following points are made by S.M. Reppert and D.R. Weaver (Nature 2002 418:935):

1) Circadian rhythms, as exemplified by the sleep/wake cycle, are the outward manifestation of an internal timing system. The full force of genetic, molecular and biochemical approaches, complemented by precise behavioral observations, has rapidly advanced our knowledge of circadian timing in mammals. The focal point of this system is a master clock, located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus, which orchestrates the circadian program(1). Principal advances in understanding the molecular and biochemical basis of circadian timing have provided a rapidly evolving model of the underlying "clockwork". Recent developments have also revolutionized our view of SCN input and output mechanisms. These include the discovery of a new visual pathway from retina to the SCN that entrains (synchronizes) circadian rhythms to the solar day, and the elucidation of ways in which the SCN clock ultimately generates output rhythms in physiology and behavior.

2) Defining the molecular basis of circadian timing in mammals has profound implications. In terms of fundamental brain mechanisms, the circadian system is among the most tractable models for providing a complete understanding of the cellular and molecular events connecting genes to behavior. Thorough dissection of the genetic basis of circadian behavior may help to decipher this connection for more complex behaviors. Understanding the molecular clock could increase our knowledge of how gene mutations of the molecular clock contribute to psychopathology (for example, major depression and seasonal affective disorder)(2). Similarly, such understanding should lead to new strategies for pharmacological manipulation of the human clock to improve the treatment of jet lag and ailments affecting shift workers, and of clock-related sleep and psychiatric disorders.

3) Circadian timing in mammals is organized in a hierarchy of multiple circadian oscillators. The oscillatory machinery of the master clock is contained within single neurons(3), and it is possible that most of the approximately 20,000 neurons that comprise the bilateral SCN are "clock cells". Molecular evidence is beginning to emerge for functionally distinct populations of clock cells within the SCN(4-5).

4) In summary: Time in the biological sense is measured by cycles that range from milliseconds to years. Circadian rhythms, which measure time on a scale of 24 h, are generated by one of the most ubiquitous and well-studied timing systems. At the core of this timing mechanism is an intricate molecular mechanism that ticks away in many different tissues throughout the body. However, these independent rhythms are tamed by a master clock in the brain, which coordinates tissue-specific rhythms according to light input it receives from the outside world.

References (abridged):

1. Reppert, S. M. & Weaver, D. R. Molecular analysis of mammalian circadian rhythms. Ann. Rev. Physiol. 63, 647-676 (2001)

2. Bunney, W. E. & Bunney, B. G. Molecular clock genes in man and lower animals: possible implications for circadian abnormalities in depression. Neuropsychopharmacology 22, 335-345 (2000)

3. Welsh, D. K., Logothetis, D. E., Meister, M. & Reppert, S. M. Individual neurons dissociated from rat suprachiasmatic nucleus express independently phase circadian firing rhythms. Neuron 14, 697-706 (1995)

4. Jagota, A., de la Iglesia, H. O. & Schwartz, W. J. Morning and evening circadian oscillations in the suprachiasmatic nucleus in vitro. Nature Neurosci. 3, 372-376 (2000)

5. Low-Zeddies, S. S. & Takahashi, J. S. Chimera analysis of the Clock mutation in mice shows that complex cellular integration determines circadian behavior. Cell 105, 25-42 (2001)

Nature http://www.nature.com/nature

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NEUROSCIENCE: ON CIRCADIAN MECHANISMS IN DROSOPHILA

The following points are made by William J. Schwartz (Nature 2004 431:751):

1) Animals have an internal timekeeping mechanism that precisely regulates 24-hour (circadian) rhythms of body function and behavior, and synchronizes them to the day-night cycle. A constellation of "clock" genes lies at the core of this timepiece, and these genes interact in complex intracellular feedback loops to produce oscillations in their own expression(1). But how are such molecular cycles translated into the adaptable temporal programs that are characteristic of whole organisms? In fruitflies (Drosophila melanogaster), for example, how does a daily intracellular molecular oscillation drive a rhythm of rest and activity that is overtly bimodal, with pronounced bouts of activity around morning and evening that can anticipate the times of lights-on and lights-off? Recent work (2,3) has demonstrated that such behavior arises at an intercellular (tissue) level of organization, with discrete sets of clock-gene-expressing brain cells differentially involved in the response to dawn and dusk.

2) One well-known clock gene in fruitflies is period (per), which, in the brains of adult flies, is expressed in photoreceptor cells, glial (non-neuronal) cells and in a few clusters of neurons(4). These neuron clusters lie in specific areas of the brain: there are three groups of dorsal neurons (DN1, DN2 and DN3), and two groups of lateral neurons, on each side of the brain. One group of five to eight lateral neurons lies towards the top of the brain (that is, dorsolaterally; these are called LNd neurons). The other group, the LNv neurons, lies towards the bottom of the brain (ventrolaterally); it includes four to six large cells and five small cells. The LNv neurons (except for one of the five small cells) express the neurotransmitter molecule known as pigment-dispersing factor (PDF), whereas none of the LNd or dorsal neurons does.

3) Attention has focused on the LNv neurons as the essential circadian "pacemaker" cells that set fly activity rhythms, especially so because removing them leads to defective behavioral rhythmicity in constant darkness(5). But data from work on flies lacking PDF and on other mutants, as well as studies in which flies were engineered to express neuronal genes that block electrical activity or synaptic transmission, suggest that a multi-neuronal network is also somehow involved.

4) Stoleru et al(2) and Grima et al(3) sought to dissect this network, using mutant flies and genetic crosses to target specific genes to specific cells. Stoleru et al(2) succeeded in delivering a cell-death gene to the LNv or LNd neurons in flies, killing these cells. Tests of the flies' cycles of rest and activity showed that the insects' behavior was still rhythmic in a light-dark cycle -- but the rhythms were different. The LNv-lacking flies anticipated only lights-off in the evening, not lights-on in the morning. Meanwhile, the flies nominally lacking LNd anticipated lights-on but not lights-off. In constant darkness, the strains showed unimodal evening- or morning-phased rhythms, respectively (although rhythmicity could not be sustained in the LNv-less flies).

5) Grima et al(3) used a different, non-ablative strategy. They started with flies that were deficient in the gene per and therefore arrhythmic, and then forced the re-expression of per only in the LNv neurons, or in both the LNv and LNd neurons. LNv-restricted per expression rescued behavioral rhythmicity, but only lights-on was anticipated. Expression in the small LNv cells seemed to be sufficient for this, even in constant darkness. Lights-off was also anticipated when per was also expressed in about half of the LNd cells. Thus, two independent strategies have led to the same conclusion: morning and evening bouts of activity are differentially controlled by the LNv and LNd cells, respectively.

References (abridged):

1. Hall, J. C. Adv. Genet. 48, 1-280 (2003)

2. Stoleru, D., Peng, Y., Agosto, J. & Rosbash, M. Nature 431, 862-868 (2004)

3. Grima, B., Chélot, E., Xia, R. & Rouyer, F. Nature 431, 869-873 (2004)

4. Helfrich-Foerster, C. Microsc. Res. Tech. 62, 94-102 (2003)

5. Renn, S. C., Park, J. H., Rosbash, M., Hall, J. C. & Taghert, P. H. Cell 99, 791-802 (1999)

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