to oxidized flavin and can be reduced by light to the anionic radical flavin form [10,11], which correlates with biological activity [9]. This leads to the suggestion that light activation of animal Type I cryptochromes may occur by a mechanism similar to plant cryptochromes, in that the receptor cycles between inactive (oxidized flavin) and active (radical flavin) states that differentially interact with signaling partners [12,13]. Further evidence for such a mechanism is obtained from fluorescence and EPR spectroscopic studies of whole cell cultures overexpressing Dmcry, which indicate oxidized to radical flavin interconversion subsequent to illumination [9]. The biological role of DmCRY is therefore assigned primarily as a light sensor to the circadian clock. Nevertheless, some isolated reports of possible dark function of DmCRY, (e.g. during functioning of the antenna clock or of roles in temperature compensation), raise the question of whether there can be additional roles of Type I cryptochrome that do not require illumination [14,15] and may act by unrelated molecular mechanisms [15,16]. In marked contrast, Type II cryptochromes are central components of the mammalian circadian oscillator where their role as transcriptional repressor occurs entirely independently of light. This is shown by studies with cryptochrome knockout mice, which demonstrate that in whole organisms, cryptochromes are required for rhythmicity of the circadian clock in constant darkness [17–20]. The same is true of rhythmicity in mammalian cell cultures [21], where cryptochrome is required for clock function resulting from non-photic entrainment stimuli such as serum shock [22,23]. Type II cryptochromes from a variety of organisms, including insects, function as transcriptional repressors in transfection assays, suggesting a marked degree of conservation in function across species lines [24]. However, unlike the function of type I cryptochromes in such assays, transcriptional activity by Type II cryptochromes occurs entirely independently of light. Type II cryptochromes are also not subject to proteolysis subsequent to illumination in S2 insect cell expression assays as are Type I cryptochromes [8,24]. raising the question whether this class of ‘blue light receptor’ is indeed capable of responding to light at all. Recently however, a number of studies have shown the possibility of light responsivity also in Type II cryptochromes. Firstly, studies of whole cell cultures overexpressing human (HsCRY1) cryptochrome have shown by both fluorescence and EPR spectroscopies that Type II cryptochromes can indeed undergo photoreduction from oxidized to radical flavin states in vivo. Furthermore, illumination of Type II cryptochrome in recombinant flies leads to rapid degradation, indicating a light- induced lability similar to that of Type I cryptochrome (DmCRY) in this same system [9]. Finally, recent reports have shown that Type II cryptochrome from the Monarch Butterfly and human (HsCRY1) can rescue light-dependent magnetoreception in transgenic Drosophila, supporting the notion that light sensing can occur in Type II cryptochromes [25,26]. In this work, the question of the distinction between Type I and Type II cryptochromes is re-examined using a two-fold approach. Firstly, the function of Homo sapiens cryptochrome (HsCRY1) was analyzed in Drosophila, a small organism that is transparent to light and therefore more amenable to photobiological approaches than cryptochrome-deficient mice, which are for the most part impermeable to light (they are dense animals and covered in hair) and behaviourally and developmentally far more complex. Since cryptochromes are highly conserved across species lines (as is their function as transcriptional repressor of certain promoters [1], it was reasoned that homologs of appropriate signaling interme- diates are likely to be recognized and targeted by HsCRY1 even in Human Cryptochrome Light Responsivity a heterologous system such as Drosophila. The goal was to determine the light dependence of biological phenotypes conferred by this transgene, thus determining whether HsCRY1 could function primarily as a light sensor in an appropriate context. As a corollary to these studies of biological activity in transgenic flies, HsCRY1 protein was purified and evaluated in vitro for ability to undergo photochemical modifications in response to light. The goal was to evaluate whether existing models of cry activation through formation of a flavin radical signaling state could be reconciled with HsCRY1 photochemistry. As a result of these two complementary and distinct approaches, the molecular basis for the different light sensitivities of Type I and Type II crypto- chromes was correlated with the stability of flavin radical in the purified proteins, consistent with a fundamentally similar mech- anism of light activation. As an unexpected additional finding, novel functions for both TypeI and TypeII cryptochromes have been discovered which are unrelated to previously known roles in the circadian clock. Results Expression of HsCRY1 in transgenic flies To determine possible biological roles of HsCRY1 in a heterologous expressing system, two distinct constructs comprising the Hscry1 gene were cloned into a Drosophila expression vector. One construct comprises full-length Hscry1 [9] and in a second construct (Hsdm), the C-terminal domain of Hscry1 is replaced with that of DmCRY (Fig. 1a) to allow for possible improvement in interactions with Drosophila signaling partners. Both constructs were fused downstream of the UAS promoter element and introduced into flies by established methods [27]. Constructs were introduced into both wild type (wt) and cryb mutant [28] flies and expression induced from the UAS element by crossing to tim-Gal4 expressing flies [29]. In F1 progeny, Hscry1 expression is therefore targeted to all of the clock cell types where timeless is expressed. Expression of HsCRY1 was verified by Western blot analysis in dark adapted flies using anti-HsCRY1 antibody in F1 progeny lines (Fig. 1b). Upon transfer to light, HsCRY1 levels dropped rapidly, and continued to decline, such that within 40 minutes less than 20% of initial protein levels remained, consistent with previous reports [9]. This response is similar to that for DmCRY [8] and Arabidopsis CRY2, whose light activated form is recognized and targeted via the E3 ubiquitin ligase COP1 to the proteasome upon light activation [30]. Such light activated degradation was not observed for HsDM protein, whose levels actually increased somewhat on transfer to light (Fig. 1b), suggesting that the hybrid protein may not adopt a conformation that leads to protein instability/degradation following light activation. HsCRY1 does not complement the circadian phenotype of DmCRY We next performed phenotypic analysis of transgenic flies to evaluate possible biological roles of HsCRY1 in the functioning of the fly circadian clock. Endogenous DmCRY acts as a light input to the circadian clock [28,31]. In contrast to wild type (wt) flies, Dmcry mutant flies remain rhythmic in continuous high light due to loss of the light-dependent damping and of the free-running circadian rhythm that would normally occur in wild type [31,32]. However, flies expressing either Hsdm or Hscry1 in a cryb genetic background maintained robust rhythmicity in LL over a period of several days (Table 1) with a period length slightly longer to that of wt flies. Therefore, HsCRY1 does not appear to complement DmCRY in the regular functioning of the Drosophila circadian PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e31867