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Circadian regulation of the light input pathway in Neurospora crassa

2001; Springer Nature; Volume: 20; Issue: 3 Linguagem: Inglês

10.1093/emboj/20.3.307

1460-2075

Martha Merrow,

Spaceflight effects on biology

Article1 February 2001free access Circadian regulation of the light input pathway in Neurospora crassa Martha Merrow Martha Merrow Institute for Medical Psychology, University of Munich, D-80336 Munich, Germany Search for more papers by this author Lisa Franchi Lisa Franchi Department of Biology, New York University, New York, NY, USA Dipartimento di Biotecnologie Cellulari ed Ematologia, Università di Roma, 00185 Rome, Italy Search for more papers by this author Zdravko Dragovic Zdravko Dragovic Institute for Medical Psychology, University of Munich, D-80336 Munich, Germany Search for more papers by this author Margit Görl Margit Görl Institute for Physiolgical Chemistry, University of Munich, D-80336 Munich, Germany Search for more papers by this author Judy Johnson Judy Johnson Institute for Immunology, University of Munich, D-80336 Munich, Germany Search for more papers by this author Michael Brunner Michael Brunner Institute for Physiolgical Chemistry, University of Munich, D-80336 Munich, Germany Search for more papers by this author Giuseppe Macino Corresponding Author Giuseppe Macino Dipartimento di Biotecnologie Cellulari ed Ematologia, Università di Roma, 00185 Rome, Italy Search for more papers by this author Till Roenneberg Corresponding Author Till Roenneberg Institute for Medical Psychology, University of Munich, D-80336 Munich, Germany Search for more papers by this author Martha Merrow Martha Merrow Institute for Medical Psychology, University of Munich, D-80336 Munich, Germany Search for more papers by this author Lisa Franchi Lisa Franchi Department of Biology, New York University, New York, NY, USA Dipartimento di Biotecnologie Cellulari ed Ematologia, Università di Roma, 00185 Rome, Italy Search for more papers by this author Zdravko Dragovic Zdravko Dragovic Institute for Medical Psychology, University of Munich, D-80336 Munich, Germany Search for more papers by this author Margit Görl Margit Görl Institute for Physiolgical Chemistry, University of Munich, D-80336 Munich, Germany Search for more papers by this author Judy Johnson Judy Johnson Institute for Immunology, University of Munich, D-80336 Munich, Germany Search for more papers by this author Michael Brunner Michael Brunner Institute for Physiolgical Chemistry, University of Munich, D-80336 Munich, Germany Search for more papers by this author Giuseppe Macino Corresponding Author Giuseppe Macino Dipartimento di Biotecnologie Cellulari ed Ematologia, Università di Roma, 00185 Rome, Italy Search for more papers by this author Till Roenneberg Corresponding Author Till Roenneberg Institute for Medical Psychology, University of Munich, D-80336 Munich, Germany Search for more papers by this author Author Information Martha Merrow1, Lisa Franchi2,3, Zdravko Dragovic1, Margit Görl4, Judy Johnson5, Michael Brunner4, Giuseppe Macino 3 and Till Roenneberg 1 1Institute for Medical Psychology, University of Munich, D-80336 Munich, Germany 2Department of Biology, New York University, New York, NY, USA 3Dipartimento di Biotecnologie Cellulari ed Ematologia, Università di Roma, 00185 Rome, Italy 4Institute for Physiolgical Chemistry, University of Munich, D-80336 Munich, Germany 5Institute for Immunology, University of Munich, D-80336 Munich, Germany *Corresponding authors. E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2001)20:307-315https://doi.org/10.1093/emboj/20.3.307 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info FREQUENCY (FRQ) is a critical element of the circadian system of Neurospora. The white collar genes are important both for light reception and circadian function. We show that the responsiveness of the light input pathway is circadianly regulated. This circadian modulation extends to light-inducible components and functions that are not rhythmic themselves in constant conditions. FRQ interacts genetically and physically with WHITE COLLAR-1, and physically with WHITE COLLAR-2. These findings begin to address how components of the circadian system interact with basic cellular functions, in this case with sensory transduction. Introduction Circadian systems coordinate the temporal program of organisms in all phyla to accommodate and anticipate the daily changes of the environment (Pittendrigh, 1993). Experimentally, circadian rhythms are characterized by their self-sustained, ∼24 h oscillation in constant conditions. Rhythms are entrained (in nature to 24 h) by appropriate environmental signals (zeitgeber), of which light is the most studied (Roenneberg and Foster, 1997). An intact circadian system includes input pathways, a mechanism that generates rhythmicity (rhythm generator) and outputs. Physiological experiments show that light input pathways (LIPs) to the rhythm generator may themselves be under circadian control. For example, in the marine unicell Gonyaulax, one of the circadian LIPs is only active during the subjective night (for definition of subjective day and night, see legend to Figure 1; Roenneberg and Taylor, 1994; Roenneberg and Deng, 1997). There are also indications that light receptors of circadian systems are rhythmic, e.g. cryptochrome in Drosophila (Emery et al., 1998) or phytochrome B in plants (Bognar et al., 1999). Figure 1.Circadian time course of frq and wc-1 RNA and protein levels. frq+ was grown at 25°C in constant darkness. Samples were harvested at 2 h intervals and frq RNA (A), FRQ protein (B), wc-1 RNA (C) and WC-1 protein (D) levels were measured. Specific RNA signals were normalized to rRNA (RNA/ribo. rel. units) and specific protein signals were normalized to amido black-stained blots (rel. protein concentration). In rhythmic time series (A, B and C), the highest value was set equal to 1; in (C), the average signal was set equal to 0.5. See Materials and methods for curve fits. The initial light to dark transfer of the cells corresponds to dusk and is designated circadian time (CT) 12. See Materials and methods for determination of CT. Gray areas indicate subjective night (i.e. the half of the full circadian cycle that corresponds to darkness in a light:dark cycle); open areas are subjective day. Download figure Download PowerPoint Thus, circadian light input is probably not a straightforward transduction of signals to the rhythm generator, but rather an active, circadianly regulated mechanism. In principle, input pathways can influence circadian rhythmicity by responding both to external zeitgebers and to the endogenous circadian system. Modeling shows that mutations in components of rhythmic input pathways change period as determined in constant conditions and contribute to self-sustainment. These theoretical results (Roenneberg and Merrow, 1998, 1999) are in accordance with reports showing that mutations in genes encoding input elements can change period or even result in arhythmicity in constant conditions (Millar et al., 1995; Somers et al., 1998; Iwasaki et al., 2000). Either of these observations would also be consistent with mutations in components that are central to the rhythm generator. In all molecular/genetic model systems, a negative feedback loop is essential for self-sustained circadian rhythmicity in constant conditions and is generally considered to be central to the rhythm generator. These loops involve expression of genes to proteins (transcription, translation and modification, e.g. phosphorylation) which, in turn, inhibit their own expression. Such a transcription/translation feedback loop is also necessary for self-sustainment of circadian rhythms in Neurospora, involving the genes frequency (frq), white collar-1 (wc-1) and white collar-2 (wc-2) as central players (Dunlap, 1999). Initially, wc-1 and wc-2 were identified in screens for lack of light reception (Harding and Turner, 1981; Degli-Innocenti and Russo, 1984). They are both light-inducible, DNA-binding, putative transcription factors (Ballario et al., 1996; Linden and Macino, 1997), and WC-1 shares similarity with a class of proteins from diverse species that actively process electrons or photons (Huala et al., 1997). Also, WC-1 and WC-2 regulate basal levels of frq (Crosthwaite et al., 1997), which was identified in screens for circadian period mutants (Feldman and Hoyle, 1973). FRQ negatively regulates its own transcription in constant conditions (Aronson et al., 1994b), providing a mechanistic basis for continuous oscillations (self-sustained rhythmicity). However, circadian properties remain in the absence of the frq/FRQ transcription/translation feedback loop. FRQ-deficient strains are capable of circadian entrainment (a special, circadian form of synchronization) in temperature cycles, whereas light entrainment fails, indicating a functional role for FRQ in the LIP (Merrow et al., 1999) and opening up the possibility of additional circadian machinery that functions in the absence of FRQ (see also Lakin-Thomas and Brody, 2000). Here, we characterize how FRQ is involved in light signal transduction. Two distinct physiological responses to light (conidial banding and carotenogenesis) represent two separate destinations of this light transduction pathway. The former has an absolute requirement for FRQ to respond to light, the latter only requires FRQ for circadian regulation and overall magnitude of light induction. We further show genetic and physical interaction of FRQ and the WC proteins, demonstrating that circadian regulation and light signal transduction are hard wired together. Results Regulation of LIP components The components that have been characterized most thoroughly for their early involvement in light responses in Neurospora are WC-1 and WC-2. Both proteins are critical for normal circadian rhythmicity; however, wc-1 mRNA is regulated more robustly by light than wc-2 (Linden and Macino, 1997). Thus, for questions regarding the relationship between light and circadian regulation, we focused our studies on interactions of wc-1 and frq. One of the important circadian features of frq RNA and protein is their rhythmicity in constant darkness (DD; Figure 1A and B; Aronson et al., 1994b; Garceau et al., 1997). We analyzed frq and wc-1 RNA and protein levels under these conditions. The RNA levels of wc-1 were variable, but not circadian (Figure 1C; Lee et al., 2000). WC-1 protein levels, however, changed with circadian time (Figure 1D; Lee et al., 2000). The period of the WC-1 oscillation is specific for different circadian period mutants (Lee et al., 2000). The differences for frq and wc-1 RNA and protein profiles in constant conditions show that their regulation is distinct. This is also indicated by the fact that maximum WC-1 protein levels coincide with the FRQ minimum (Figure 1B and D; Lee et al., 2000). Regulation of FRQ and WC-1 is interdependent Basal frq/FRQ levels are low in wc-1 and wc-2 mutants (Crosthwaite et al., 1997). Here, these levels were determined in a Δwc-1 strain (RIPed to a functional knockout; see Materials and methods and Talora et al., 1999), confirming that frq RNA and FRQ protein (Figure 2A and B) levels are substantially lower in the Δwc-1 than in a wc-1+ strain. These observations suggest that regulation of frq lies downstream of WC-1. Figure 2.Regulation of frq and wc-1. Mycelial pads from frq+, Δwc-1, frq10 and frq9 were incubated in darkness for 28 h and then RNA and protein extracts were prepared. (A) frq RNA is reduced in Δwc-1. frq RNA was detected by northern blotting (upper panel), quantified and normalized to rRNA levels (lower panel). The columns in the graphs represent averages of four experiments (standard deviations are given at the top of each bar). The controls frq10 and frq9 show no frq or elevated RNA, respectively, as expected. (B) FRQ protein is reduced in Δwc-1. FRQ protein was detected by western blotting. To control for equal loading of the gel, a portion of the blot was stained with amido black (lower panel). The controls frq10 and frq+LL (constant light) show no FRQ protein or elevated levels, respectively. (C) wc-1 RNA is reduced in frq10 and frq9. RNA was analyzed as described above. (D) WC-1 protein levels are reduced in frq10 as shown by western blotting. The amido black-stained membrane indicated equal loading (lower panel). Download figure Download PowerPoint Recently, the activators of genes in the Drosophila circadian transcription/translation feedback loop were shown to depend on 'downstream' gene products for their expression (Bae et al., 1998; Glossop et al., 1999). A comparative analysis would suggest that this might be a common regulatory mechanism in circadian molecular networks. The rhythmicity of WC-1 (Figure 1D; Lee et al., 2000) suggests a similar interactive network between FRQ and the white collar gene products. We therefore determined wc-1 RNA levels in frq10 (a frq null strain, see Materials and methods for description). Basal RNA levels were lower than those observed in frq+ grown in DD (Figure 2C; in contrast to Lee et al., 2000), and WC-1 protein levels were reduced similarly (Figure 2D; and as in Lee et al., 2000). Thus, at least under some conditions (see Discussion), FRQ apparently is required to maintain basal levels of wc-1 RNA and protein in DD. FRQ deficiency and light responsiveness We probed the functional consequences of frq deficiency, and the resultant depressed WC-1 levels, on light-induced physiology by investigating several light-inducible outputs: conidial band formation, carotenogenesis and expression of specific RNAs. Figure 3A shows light regulation of conidial banding in a wild-type strain (tubes 1 and 2). As previously reported, frq null strains do not respond to light:dark (LD) cycles with succinct formation of conidial bands or their synchronization (Figure 3A, tube 3; Chang and Nakashima, 1997; Merrow et al., 1999; Lakin-Thomas and Brody, 2000; Roenneberg and Merrow, 2001). In contrast, temperature cycles entrain conidial band formation in frq9 (Merrow et al., 1999). Figure 3.A bifurcated light input pathway: FRQ is required for conidiation but not for carotenogenesis. (A) FRQ rescues light-regulated conidiation in frq10. Race tubes were inoculated with frq+ (bd) or frq10 qa-2p-frq (bd frq10 his3::his3qa-2p-frqhis), which expresses a His-tagged version of FRQ under the control of the inducible qa-2 promoter. The race tubes were incubated in 12 h light:12 h dark (LD 12:12) cycles with or without quinic acid (QA). Approximately 4.5 cycles are shown. (B) QA-induced expression of FRQ is unaffected by light. frq10 qa-2p-frq was harvested in either the light or dark portion of the third day of an LD 12:12 cycle. At time 0, FRQ expression was induced by QA. Mycelia were harvested after 6 and 12 h and FRQ was analyzed by western blotting (left panel). Signals were quantified by densitometry. Relative expression levels (rel. FRQ conc., right panel) are the average of triplicate samples, normalized for loading based on amido black-stained membranes. (C) Induction of carotenoids by light does not require FRQ. Carotenoids were induced in frq+ (thick line), frq10 (thin line) and Δwc-1 with 4 μE/m2/s of light for 5 h. Samples were extracted with hexane and absorption spectra were determined. The frq+ extract was diluted 2-fold relative to the other samples. (D) The fluence threshold of carotenogenesis is independent of FRQ. Carotenoids were induced in frq+ (filled circles) and frq10 (open circles) over the indicated range of fluences. Absorption of carotenoids was determined at 445 nm. Values >1.0 were measured as dilutions for accuracy. The black arrow indicates the fluence rate at which half-maximal light induction of carotenoids occurs for both strains. For comparison, the gray arrow indicates the fluence threshold for light-dependent conidial band formation in frq+ (Merrow et al., 1999; Roenneberg and Merrow, 2001). Download figure Download PowerPoint To clarify whether FRQ, as such, is required for light-induced conidial band formation or whether this function depends on the intact negative feedback regulation of the frq/FRQ loop, we constitutively expressed FRQ in frq10 (using a qa-2p-frq fusion construct similar to that of Aronson et al., 1994b). Although under these conditions this response is not entirely normal, a light-regulated conidiation response was rescued (Figure 3A, tube 4); this strain remains arhythmic in DD (data not shown; and Aronson et al., 1994b). The timing of conidiation following the light signal (its phase angle) is similar to that for the frq+ strain. The reconstitution of light responsiveness could be mediated by light-induced rhythmicity of FRQ levels, even in the absence of transcriptional regulation. However, when FRQ was induced in the frq10 strain, there was no difference in phosphorylation state (judged by mobility in SDS–PAGE; Figure 3B, left panel) or protein accumulation (right panel) in light versus darkness (Figure 3B). Thus, expression of FRQ is required for light regulation of conidiation, even in the absence of negative feedback of FRQ on frq. Given that there is not an obvious qualitative or quantitative difference in FRQ protein induced in light or dark (Figure 3B), it is possible that rhythmicity of the protein is not essential for this response. Because it was reported previously that the frq null strains do show light-induced gene expression (Arpaia et al., 1993, 1995), we investigated light-induced mycelial carotenogenesis (De Fabo et al., 1976). Figure 3C shows that this light response remained qualitatively intact in frq10: the absorption spectrum of hexane-extracted, light-induced tissue was the same in frq10 and frq+. However, final carotenoid concentrations were approximately half in frq10 compared with frq+ (the two superimposed spectra represent extracts of different dilutions; see Figure 3C legend). Note that light-dependent carotenoid synthesis is entirely absent in wc-1 mutants (Harding and Turner, 1981; Linden et al., 1999). When fluence response curves for light induction of carotenogenesis in frq+ and frq10 were compared, the amplitude of the saturation response in frq10 was about half that of frq+ (Figure 3D). The sensitivity of both strains (fluence rate at half-maximal response, black arrow) was, however, identical. Comparison of the fluence threshold for carotenogenesis with that for light-driven synchronization of conidiation (Figure 3D, gray arrow; Merrow et al., 1999; Roenneberg and Merrow, 2001) suggests two, distinct, light-regulated pathways. Without FRQ, light-regulated conidial band formation is absent even in high light intensity LD cycles, but carotenoids are induced normally based on fluence threshold, although overall accumulation is about half. So, while both branches of this pathway are light blind without WC-1, they are each modulated differently by FRQ (see Figure 7A). Carotenoid production is the result of a complex, multi-step process. In contrast, one of the earliest detectable events after light exposure of Neurospora is induction of wc-1 RNA, which occurs in <2 min at high light intensities (P.Ballario, unpublished data). Induction of wc-1 RNA was, therefore, used to monitor early events in light signal reception and transduction, and contrasts the endpoint that carotenogenesis represents. The amplitudes (maximum response relative to baseline) of light-induced RNA in frq+ and frq10 appeared to be similar (Figure 4); however, the peak light-induced wc-1 levels were low in frq10, reaching, at most, the basal DD levels of wc-1 in frq+ (compare with Figure 2C). al-1 RNA was also induced weakly by light in frq10 (data not shown). al-1 encodes a downstream enzyme on the carotenogenesis pathway (phytoene dehydrogenase) (Schmidhauser et al., 1990). Given that RNA induction is a relatively rapid and discrete response compared with carotenogenesis, the disparity in strength of carotenoid and RNA induction is not directly comparable. Figure 4.Robust light induction of wc-1 RNA requires FRQ. frq+ and frq10 were incubated for 28 h in the dark and then exposed to 4 μE/m2/s of light. Samples were harvested over 2 h. (A) wc-1 and rRNAs were detected by northern blot analysis. (B) wc-1 RNA was quantified and normalized to the amount of rRNA. The maximal signal was set equal to 1. Data are the average of duplicate samples and representative of three experiments. Download figure Download PowerPoint Circadian regulation of light responses Light responses depend on FRQ and WC-1 (Figures 3A, C and D, and 4), and both proteins show a circadian rhythm in abundance (Figure 1B and D). We therefore investigated the physiological light responses described above at different times of the circadian cycle. Time courses measured over 2 h in the middle of the subjective day and night showed large differences in light-induced gene expression. While basal levels of wc-1 were indistinguishable (see also Figure 1C), the amplitude of the response in the subjective day was less than half when compared with the subjective night (Figure 5A). Figure 5.Light responses are regulated by the circadian system. Time of day-specific light induction of wc-1 RNA (A), frq RNA (B) and al-1 RNA (C). Mycelial pads were incubated for 16 h (∼CT05, unfilled circles) and 27 h (∼CT17, filled circles) in the dark and then exposed to light (0.4 μE/m2/s). After the indicated time periods, RNA was prepared, analyzed by northern blotting and quantified based on rRNA values. The maximal signal was set equal to 1. (D) Light-induced carotenogenesis over the course of a circadian cycle. frq+ (filled squares) and frq10 (open squares) mycelia were transferred from light to dark. After the indicated time in darkness, samples were exposed to light for 5 h to induce carotenogenesis. Carotenoids were extracted with hexane and quantified by absorption at 445 nm. Circadian time of frq+ is shown on top; the gray background indicates subjective night. Download figure Download PowerPoint frq RNA levels depend on WC-1 and WC-2, for both basal expression and rapid and robust light induction (Crosthwaite et al., 1997). In contrast to wc-1, frq RNA is induced to approximately the same maximal levels at the opposite circadian times (Figure 5B; Crosthwaite et al., 1995). Because frq levels are circadian (Figure 1A; Aronson et al., 1994b; Garceau et al., 1997), frq is already at different levels at the beginning of light incubation. Finally, al-1 light induction resembles the pattern of wc-1 RNA induction, but the subjective night/day ratio is ∼10-fold (Figure 5C). Interestingly, wc-1 and al-1 RNA profiles, and to a lesser extent frq, show a transiency in prolonged light exposure (Schmidhauser et al., 1990; Arpaia et al., 1995; Linden et al., 1999), resembling classical adaptation responses. RNA levels are down-regulated within 1–2 h and, at least for wc-1, remain constant for 10 h of illumination (data not shown). The adaptation profile is apparent at both circadian times that were evaluated. Lastly, we determined light-induced mycelial carotenogenesis at different circadian times. Light-dependent accumulation of carotenoids was also circadianly regulated, peaking before subjective dawn (notably, the circadian modulation of light-induced of carotenogenesis is absent in frq10, Figure 5D). Thus, circadian regulation of light responsiveness is evident at numerous levels, from some of the earliest known events of light detection to enzymatically regulated, downstream outputs. FRQ is physically associated with the WC proteins WC-1 and WC-2 are found in the same complex in vivo (Talora et al., 1999). We wondered if the involvement of FRQ in light responses could stem from a physical association with either of these proteins. We probed anti-WC-2 immunoprecipitates of cell extracts and found that FRQ binds to WC-2 (Figure 6A). The amount of FRQ in the complex correlated with its circadian accumulation in crude cell extracts (Figure 1B). For reference, FRQ in total cell extracts from frq+ is shown at two circadian times: one harvested at subjective dawn with low levels of highly phosphorylated FRQ, the other at late subjective day with large amounts of less phosphorylated FRQ (Figure 6A, left panel, right lanes). In this experiment, highly phosphorylated FRQ apparently does not participate in complex formation, although in some others it did. Using anti- WC-1, we were also able to immunoprecipitate FRQ from cell extracts of mycelia grown in constant light (LL; Figure 6B). Thus, FRQ is in complexes containing WC-2 and/or WC-1 in vivo and WC-2 is found in association with WC-1 (Talora et al., 1999). Figure 6.Interaction of FRQ with WC-2 and WC-1. (A) Co-precipitation of FRQ with WC-2 (right panel) and control cell extracts (left panel). For immunoprecipitations, cell extracts were prepared from age-matched mycelial pads grown in DD for the indicated amounts of time. Samples were immunoprecipitated with anti-WC-2 serum (right panel), separated by SDS–PAGE and probed with antibodies against FRQ on western blots. Control cell extracts include those from wc-2-234w, frq10 and a strain wild-type at frq and wc-2 loci (frq+) at DD12 (∼CT01) and DD20 (∼CT10). These were probed for FRQ and indicate the relative abundance and phosphorylation state of FRQ protein at these times. (B) Co-precipitation of FRQ with WC-1. frq+, frq10, Δwc-1 and wc-2-234w were grown in constant light (LL). Extracts were prepared and subjected to immunoprecipitation with antibodies against WC-1. The precipitates were analyzed by SDS–PAGE and blots were probed with antibodies against FRQ. The cell extract of frq+ cultures represents 4% of the input in the co-precipitation experiment and is shown in the right lane as an estimate of the efficiency of the co-precipitation. Download figure Download PowerPoint Figure 7.The molecular components of the Neurospora circadian system and the light input pathway (LIP) are inseparable. (A) Our results are compatible with a bifurcation within the LIP, with one branch leading to light-dependent (or, in the absence of light, circadian) conidiation and the other to light-induced carotenogenesis. In terms of regulation by light, WC-1 is required for both branches, while FRQ is only essential for regulated conidiation. Both are important for circadian rhythmicity. In addition, the circadian system modulates the branch regulating carotenoid synthesis, possibly via FRQ. Within the LIP, the WCs and FRQ form a functional unit responsible for both light reception and circadian modulation. (B) Genetic interactions within the Neurospora LIP. Although not specified, a basal activation of wc-1 and frq is presumed, in addition to the inferred positive effect on wc-1 RNA levels by FRQ or WC-1 (see Figure 2). Also, WC-2 is involved in frq regulation (Crosthwaite et al., 1997), but this aspect was not addressed in this work. Download figure Download PowerPoint Discussion We previously observed circadian entrainment by temperature cycles in Neurospora strains deficient in FRQ (Merrow et al., 1999). The same strains, however, fail to synchronize to light cycles (Figure 3A; Chang and Nakashima, 1997; Merrow et al., 1999; Lakin-Thomas and Brody, 2000), suggesting that FRQ is required in processing light signals, in addition to controlling essential circadian properties, such as self-sustained rhythmicity in DD. Here, we address several questions about the role of frq/FRQ in the LIP and the circadian system of Neurospora crassa. What is the nature of the interaction between the WC proteins and their complex (WCC) and FRQ? What is the role of FRQ in light transduction and how does it contribute to entrainment? Figure 7 is a diagrammatic view of the Neurospora LIP, specifically with respect to WC-1, WC-2 and FRQ at the protein level in Figure 7A and at the level of gene regulation of wc-1 and frq in Figure 7B. Interdependent regulation of frq and wc-1 by their proteins The circadian and light input pathways in Neurospora interact genetically. Robust frq RNA and protein expression depends on WC-1 and WC-2 proteins, which are essential for light responses in Neurospora (Figure 2A and B; Crosthwaite et al., 1997). However, frq expression is not simply downstream of the WC gene products: we find that basal, DD levels of wc-1 (Figure 2C) and WC-1 depend on FRQ (Figure 2D; Lee et al., 2000). Since FRQ levels are robustly circadian (Figure 1B; Garceau et al., 1997), one would expect rhythmic wc-1 RNA in DD, but this is not the case (Figure 1C). A recent report (Lee et al., 2000) also finds that wc-1 RNA is constitutive but further describes that basal wc-1 levels are similar in frq+ and frq10. This is in contrast to what we observe. The most obvious difference in the experimental protocols is the time at which the samples are harvested: after 28 h in darkness in the protocol described here, versus only 6 h in that presented by Lee et al. (2000). There could be residual light-induced wc-1 RNA in frq10 (Figure 4) that persists for at least 6 h in DD; sustainment of those RNA levels may depend on FRQ. WC-1 protein oscillates with a circadian period, out of phase with FRQ (Figure 1D; and as shown in Lee et al., 2000), and its regulation includes a post-transcriptional control that is initiated by FRQ expression (Lee et al., 2000). Thus, WC-1 regulates FRQ, and FRQ regulates WC-1, indicating the backbone of the circadian transcription/translation feedback loop in Neurospora (see Figure 7B). A bifurcated light input pathway The effect of FRQ on light transduction was investigated by comparing various