4.5.1 CRY plays different roles in the central and peripheral circadian oscillators in Drosophila
In cryb flies luciferase reported per rhythm was found to be lost, which mainly originated from the compound eyes (Stanewsky et al., 1998). The olfactory response sensitivity rhythm and rhythmic clock gene expression were disrupted in cryb flies in DD (Krishnan et al., 2001), suggesting that CRY is more than a photoreceptor at least in this tissue, and that it occupies a place in the clock work mechanism. cry is required for maintaining circadian oscillations in the peripheral tissues.
In LD, TIM expression was constitutively high in the photoreceptor cells of cryb flies (Stanewsky et al., 1998) but reinstating CRY expression in the photoreceptor cells restored the synchronized TIM oscillations (Emery et al., 2000b). The TIM oscillations in Malpighian tubules did not respond to light pulses in cryb flies (Ivanchenko et al., 2001). This findings suggest that CRY is also a photoreceptor in the peripheral clocks. On contrary in LD in adult cryb flies the TIM oscillations continued in the s--LNvs and in DN1s (Helfrich--Förster et al., 2001). This means that CRY plays different roles in the central and peripheral circadian oscillators. In the central oscillators, CRY is not essential for light entrainment of TIM because its role may be substituted by other yet unknown circadian photoreceptor(s) (CRY--independent mechanism). Instead, in the peripheral oscillators, CRY is required for light--mediated degradation of TIM (CRY--dependent mechanism). A direct interaction of CRY with TIM in light, in vitro (Ceriani et al., 1999) and in vivo (Busza et al., 2004) supports this belief. CRY may also interact with PER in a light--dependent manner (Rosato et al., 2001).
The phosphorylated forms of both PER and TIM form a stable heterodimer.
However, interaction of CRY with TIM (may be with PER, too) results in TIM
degradation in a light--dependent manner (Fig. 4; Naidoo et al., 1999). Possibly, the CRY interaction activates a tyrosine kinase mediated phosphorylation, followed by ubiquitination of TIM resulting in its decomposition in the proteasome (Lin et al., 2001; Grima et al., 2002). CRY is also degraded by the proteasome in a light--dependent manner, but light induced ubiquitination of TIM precedes the CRY degradation (Lin et al., 2001). CRY degradation requires electron transfer, fulfilled by a CRY cofactor, FAD.
In conclusion, the light mediated CRY interaction with TIM, and the subsequent degradation of the latter may be responsible for high amplitude molecular cycling of the clock gene products in LD. This may be true mostly for the peripheral oscillators because the central oscillators can maintain clock gene cycling independent of CRY.
4.5.2 CRY is a core clock component in the mammalian clock
There are two CRY encoding genes in mammals, i.e. mCry1 and mCry2. Mice which are double knockouts for mCry1 and mCry2 show complete loss of circadian rhythmicity (van der Horst et al., 1999), suggesting that mCrys are part of the central circadian clock mechanism. These genes are rhythmically expressed in the suprachiasmatic nuclei (SCN--the central circadian pacemaker in mammals), with a similar phase like the mammalian period genes (mPers) but in the opposite phase to Bmal 1--the cyc equivalent in mammals (Miyamoto and Sancar, 1998;
Kume et al., 1999). Inhibition of BMAL 1:CLK mediated transcription results in high levels of mCRY1 and mCRY2 proteins in the SCN. Consistent with a negative role of mCRYs in the feed--back loop, mCry RNA levels are severely reduced and non--cycling in the SCN of Clk mutant mice (Kume et al., 1999). This indicates that mCRYs play a negative feed--back role like TIM in Drosophila (Stanewsky, 2003).
The mCRYs are able to block the BMAL 1:CLK mediated transcription in vitro, independent of mPER, thus indicating a direct interplay between mCRYs and the BMAL 1:CLK (Kume et al., 1999; Shearman et al., 2000). Therefore, CRY is a crucial clock component in the mammalian circadian clock. It is suggested that CRY has substituted for Drosophila TIM in the mammalian circadian clock (Stanewsky, 2003).
In the peripheral clock (skeletal muscles) of Clk mutant mice the mCry1 and mCry2 RNA expression rhythms were dampened and phase advanced, and totally abolished, respectively (Kume et al., 1999). Moreover, the presence of the canonical E--box in the mCry1 promoter suggested that CLK directly mediate the mCry1 transcription by binding to the E--box (Kume et al., 1999). In mCry double knockout mice the mPER1 and mPER2 were predominantly seen in the cytoplasmic compartment, suggesting mCRYs are required for the nuclear transport of mPERs (Lee et al., 2001). Taken together, these data indicate that mCry1 and mCry2 are part of the clock work mechanism in the peripheral clocks, too.
4.5.3 Is CRY a photoreceptor for the mammalian circadian clock?
In mammals, eyes are required for circadian photoreception because enucleation eliminates circadian responses to light, but the rods and cones are not necessary (Forster and Helfrich--Förster, 2001). The pupillary constriction following light exposure to eyes does not require rods and cones. But the maximum sensitivity for this response was around 479 nm implicating an opsin--type photopigment is involved (Lucas et al., 2001a). Melanopsin was suggested as the responsible photopigment because it is expressed in the inner retina (Provencio et al., 2000). It is also found in a subset of retinal ganglion cells, which are part of the retinal hypothalamic tract connecting the eye with the SCN (Hannibal et al., 2002; Hatter et al., 2002). These subsets of ganglion cells are intrinsically light sensitive and their photic--response correlates with the photic entrainment (Berson et al., 2002;
Hatter et al., 2002). Recently, it has been demonstrated that human melanopsin can function as a photopigment, but in a way typical for invertebrate opsins (Melyan et al., 2005; Panda et al., 2005; Qiu et al., 2005). In fact melanopsin shows highest sequence similarity to invertebrate opsins (Provencio et al., 2000).
The mCRYs are also potential candidates for the photic entrainment of the circadian clock because they are expressed in the ganglion cell layer and inner nuclear layer of retina (Miyamoto and Sancar, 1998). Another line of evidence comes from the observation that mCry double knockout mice are able to entrain to the LD, while in DD they displayed arrhythmic behavior (van der Horst et al., 1999). This LD behavior was argued as masking response to light (van der Horst
et al., 1999; Mrosovsky, 2001). However, this masking response was eliminated in the mCry double knockout mice in a rodless background, while rodless mice alone were able to entrain to the LD (Selby et al., 2000). The above finding indicated that both rods and mCRYs are involved in the masking behavior. Therefore, rods and mCRYs could contribute to the photic signaling. However, the spectral evidence suggested that the photic entrainment in rodless and coneless mice may not be mediated by CRY (Berson et al., 2002) despite the fact that determining the precise action spectrum of CRY chromophore remains difficult (Cashmore, 2003) because flavin exists in three different redox states (Lin et al., 1995). More recently, it was shown that vitamin A--depleted, retinol binding protein (required for transporting the retinol from liver to extraheptic tissues like eye) knockout (rbp-/-) mice exhibit either normal photic entrainment or become diurnal. In contrast, (vitamin A--repleted) rbp-/- cry1-/- cry2-/- mice have reduced light sensitivity, while (vitamin A--depleted) rbp-/- cry1-/- cry2-/- mice, that are additionally on a vitamin A--depleted diet, presumably lack functional opsins and cryptochromes were affected in most of their behavioral and molecular responses to light (Thompson et al., 2004). Therefore, the authors have suggested that both cryptochromes and opsins regulate non--visual photic responses. In conclusion, mCRYs may be part of the multiple photopigments responsible for the light detection for photic entrainment of the SCN clock in mammals (Stanewsky, 2003; Forster and Helfrich--Förster, 2001).