Stoichiometric relationship among clock proteins determines robustness of circadian rhythms

The mammalian circadian oscillator is primarily driven by an essential negative feedback loop comprising a positive component, the CLOCK-BMAL1 complex, and a negative component, the PER-CRY complex. Numerous studies suggest that feedback inhibition of CLOCK-BMAL1 is mediated by time-dependent physical interaction with its direct target gene products PER and CRY, suggesting that the ratio between the negative and positive complexes must be important for the molecular oscillator and rhythm generation. We explored this idea by altering expression of clock components in fibroblasts derived from Per2(Luc) and Per mutant mice, a cell system extensively used to study in vivo clock mechanisms. Our data demonstrate that the stoichiometric relationship between clock components is critical for the robustness of circadian rhythms and provide insights into the mechanistic organization of the negative feedback loop. Our findings may explain why certain mutant mice or cells are arrhythmic, whereas others are rhythmic, and suggest that robustness of circadian rhythms can be increased even in wild-type cells by modulating the stoichiometry.     

Cryptochromes: sensory reception, transduction, and clock functions subserving circadian systems

Cryptochromes (CRYs) are blue-light-absorbing proteins involved in a variety of biological phenomena. In animals, CRYs exhibit a certain versatility with regard to these organisms' circadian rhythms, as has been revealed by the effects of mutations and molecular manipulations. The rhythm system of Drosophila uses one gene's worth of CRY protein to transmit light into a circadian clock within the brain, which controls the fly's sleep-wake cycles. In fact, the relevant pacemaking neurons are themselves circadian photoreceptive structures. In peripheral tissues and others located posterior to the brain, Drosophila CRY may be a photoreceptive molecule and also part of the pacemaker mechanism. Mice have two CRY-encoding genes. They are expressed in many tissues, including the retina and a clock structure within the brain. In the former location, mouse CRY may play a circadian-photoreceptive role, along with that mediated by rhodopsins found elsewhere in the retina. In the latter tissue, the hypothalamic suprachiasmatic nucleus, mouse CRYs are closely connected to the multimolecule murine clock mechanism.     

Animal type 1 cryptochromes. Analysis of the redox state of the flavin cofactor by site-directed mutagenesis

It has recently been realized that animal cryptochromes (CRYs) fall into two broad groups. Type 1 CRYs, the prototype of which is the Drosophila CRY, that is known to be a circadian photoreceptor. Type 2 CRYs, the prototypes of which are human CRY 1 and CRY 2, are known to function as core clock proteins. The mechanism of photosignaling by the Type 1 CRYs is not well understood. We recently reported that the flavin cofactor of the Type 1 CRY of the monarch butterfly may be in the form of flavin anion radical, FAD(*-), in vivo. Here we describe the purification and characterization of wild-type and mutant forms of Type 1 CRYs from fruit fly, butterfly, mosquito, and silk moth. Cryptochromes from all four sources contain FAD(ox) when purified, and the flavin is readily reduced to FAD(*-) by light. Interestingly, mutations that block photoreduction in vitro do not affect the photoreceptor activities of these CRYs, but mutations that reduce the stability of FAD(*-) in vitro abolish the photoreceptor function of Type 1 CRYs in vivo. Collectively, our data provide strong evidence for functional similarities of Type 1 CRYs across insect species and further support the proposal that FAD(*-) represents the ground state and not the excited state of the flavin cofactor in Type 1 CRYs.     

Flies, clocks and evolution.

The negative feedback model for gene regulation of the circadian mechanism is described for the fruitfly, Drosophila melanogaster. The conservation of function of clock molecules is illustrated by comparison with the mammalian circadian system, and the apparent swapping of roles between various canonical clock gene components is highlighted. The role of clock gene duplications and divergence of function is introduced via the timeless gene. The impressive similarities in clock gene regulation between flies and mammals could suggest that variation between more closely related species within insects might be minimal. However, this is not borne out because the expression of clock molecules in the brain of the giant silk moth, Antheraea pernyi, is not easy to reconcile with the negative feedback roles of the period and timeless genes. Variation in clock gene sequences between and within fly species is examined and the role of co-evolution between and within clock molecules is described, particularly with reference to adaptive functions of the circadian phenotype.     

松果体昼夜节律生物钟分子机制的研究进展

在各种非哺乳类脊椎动物中 ,松果体起着中枢昼夜节律振荡器的作用。近来 ,在鸟类松果体中相继发现了几种钟基因 ,如Per、Cry、Clock和Bmal等 ,其表达的时间变化规律与哺乳类视交叉上核 (SCN)的非常相似。钟的振荡由其自身调控反馈环路的转录和翻译组成 ,鸟类松果体和哺乳类SCN似乎具有共同的钟振荡基本分子构架 ;若干钟基因产物作为正向或负向调节子影响钟的振荡 ;昼夜性的控时机制同时也需要翻译后事件的参与。这些过程对钟振荡器的稳定性和 /或钟导引的光输入通路有着重要的调控作用