Handbook of Photosensory Receptors (BRIGGS:PHOTORECEPTORS O-BK) || Neurospora Photoreceptors

18Neurospora Photoreceptors

Jay C. Dunlap and Jennifer J. Loros

18.1Introduction and Overview

Filamentous fungi are well known to display a variety of responses to light, chief ly toblue light. Among these organisms, Neurospora crassa has emerged as a tractable andinformative model system. The photobiology of Neurospora has been well reviewed,with more recent surveys tending to focus on the genetic and molecular studiesaimed at identifying the photoreceptor and describing the spectrum of genes regu-lated by light (Ballario and Macino, 1997; Degli Innocenti and Russo, 1984a; Lindenet al., 1997). The principal blue light photoreceptor was identified recently as WC-1(Froehlich et al., 2002; He et al., 2002), and its identity highlighted the LOV proteindomain already associated with blue light photoreception in plants (Christie et al.,1998; Huala et al., 1997). In addition, the recent availability of the Neurospora ge-nomic sequence, followed by the genomes of other fungi, has yielded additional pu-tative photoreceptors (Borkovich et al., 2004; Galagan et al., 2003). This chapter willbegin with a brief overview of blue light photobiology in fungi and then go on to de-scribe the nature of the known and suspected photoreceptors.

18.2The Photobiology of Fungi in General and Neurospora in Particular

18.2.1Photoresponses are Widespread

Although most research on photobiology in the fungi has been driven by Neurospo-ra, a great deal of excellent research has also used Phycomyces blakesleeanus which al-so elaborates carotenoids in response to blue light as well as displaying well-describeddevelopmental and phototropic responses (Cerda-Olmedo, 2001). Research in this or-ganism has been hampered by the lack of a reliable transformation system(Obraztsova et al., 2004). In addition to the many light responses in Neurospora and

Handbook of Photosensory Receptors. Edited by W. R. Briggs, J. L. SpudichCopyright © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN 3-527-31019-3

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Phycomyces, asexual spore production is light-induced in P. blakesleeanus, Trichoder-ma harzianum, and Aspergillus nidulans as well as in N. crassa, and both cell-wallbranching and fruiting-body production are induced by light in Schizophyllum com-mune as well as in N. crassa (reviewed in Lauter, 1996). Interestingly, while blue-lightresponses are known in all of these organisms, Aspergillus also exhibits a red (far-redreversible) light response in its requirement for light in the formation of asexualspores (Mooney and Yager, 1990); the photoreceptor for this response is not known.

18.2.2Photobiology of Neurospora

Light signals inf luence many aspects of both the sexual and asexual (vegetative)stages of life (Figure 18.1), and a brief description of the life and times of Neurospo-ra (Davis, 2000) will serve to set the stage. Neurospora comes in two mating types, Aand a. Sexual spores are activated by fires in the wild, the heat allowing germination;Neurospora is classified as a Pyrenomycete. The organism spends most of its life

18 Neurospora Photoreceptors

Figure 18.1 The life cycle of Neurospora.Light inf luences many aspects of both the sex-

ual and asexual cycles. See text for detail.Adapted from Davis (2000) with permission.

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growing vegetatively on the charred substrate as a syncytium with incomplete cellwalls separating cellular compartments. As a vegetative culture, Neurospora can ex-ist as surface mycelia or can elaborate aerial hyphae. The tips of aerial hyphae formmorphological distinct structures called conidia that act as asexual spores and are eas-ily dispersed by wind. When nutrients get scarce, vegetative Neurospora of eithermating type induce sexuality by forming a fruiting body (a protoperithecium) whichcan be fertilized by a nucleus from a piece of mycelium or conidium of the oppositemating type. The mature reproductive fruiting body is the perithecium. Pairs of nu-clei representing each parent replicate in tandem and eventually fuse to make a tran-sient diploid that immediately undergoes meiosis to produce an 8-spored ascus, eachascus containing the products of meiosis from a single diploid nucleus.

The many effects of light on Neurospora are shown in Figure 18.2. During the asex-ual phase of the life cycle, light acts acutely to induce conidiation. More conidia areproduced in light and they are produced faster (Klemm and Ninneman, 1978; Lauter,1996). Carotenogenesis in mycelia is light-induced (Harding and Shropshire, 1980),and this response is quite rapid, being observable within the first 30 minutes after ex-posure to light. Pigmentation of the conidia is constitutive though, and in a strain de-fective for light perception or transduction of the light signal this results in the pro-duction of white mycelia underlying yellow/orange conidia, a phenotype that has

18.2 The Photobiology of Fungi in General and Neurospora in Particular

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BlueLight

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Figure 18.2 Summary of light responses in Neurospora.(Figure courtesy of C. Schwerdtfeger; all rights reserved.)

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been of central importance in the genetic identification of the photoreceptors as de-scribed below. Although aerial hyphae are reported to display a phototropism (e.g.Siegel et al., 1968) in that they preferentially form on the lighted side of a dish, it isnot clear to what extent this response is distinct from the overall light induction ofconidiation. Reports also exist of changes in membrane conductivity (hyperpolariza-tion and an increase in input resistance) in response to light (e.g. Potapova et al.,1984).

The most global effect of light during the asexual cycle (in the center of Figure 18.1)is to set the phase of the endogenous biological clock which acts to regulate a varietyof aspects of the life cycle of the organism (reviewed in Loros and Dunlap, 2001; Sar-gent and Briggs, 1967). Light is used to set the phase of the clock that controls the dai-ly timing of a developmental switch that leads to conidiation; a light-to-dark transferis interpreted as dusk, and a dark to light transfer as dawn. Continued light also actsto suppress the expression of the clock. Conidiation involves a major morphologicalchange that requires many novel gene products. Although the production of asexualspores is the best characterized light-phased circadian rhythm in Neurospora, otherpersisting rhythms at the physiological level have been described, which include theproduction of CO2, lipid and diacylglycerol metabolism [e.g. (Lakin-Thomas andBrody, 2000; Ramsdale and Lakin-Thomas, 2000; Roeder et al., 1982)], a number ofenzymatic activities [e.g. (Hochberg and Sargent, 1974; Martens and Sargent, 1974)],heat shock proteins (Rensing et al., 1987), and even growth rate (Sargent et al., 1966).These can be considered as secondary light responses.

The overall initiation of the sexual phase of the life cycle is enhanced by light (DegliInnocenti and Russo, 1983). During this time, carotenogenesis of perithecial walls islight induced (Perkins, 1988) and once mature perithecia are formed, ejection ofspores from them is induced by light as is the direction in which the spores are shot:that is, the tips (‘beaks’) of the perithecia display a distinct phototropism (Hardingand Melles, 1983). The number of spores shot from perithecia is also regulated by thelight-phased circadian clock.

The involvement of light in the above-mentioned processes is acute and obvious,but Neurospora is also capable of more subtle responses to light characteristic ofadded regulatory sophistication: Neurospora responds to changes in the level of am-bient light, from dark to dim or dim to bright in a process known as photoadaptationthat is manifested in two ways. First, when the organism initially sees light, a re-sponse leading to transcription of light-induced genes is triggered that peaks within15 to 30 minutes, but this response generally decays away within two hours or so, andif the organism is exposed to light within this two-hour period, no additional re-sponse is seen. Also, if the lights remain on, the response decays away, but after thetwo-hour latency period, the organism can respond again if the ambient level of lightis increased.

Figure 18.2 not only summarizes these light responses but also indicates some ofthe genes associated with them. This overview also serves to emphasize the point thatmost of the well-characterized responses are tied directly to light-induced transcrip-tion.


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18.3Light Perception – the Nature of the Primary Blue Light Photoreceptor

18.3.1Flavins as Chromophores

All known photoresponses in Neurospora are specific to light in the blue-green re-gion of the spectrum. Action spectra developed in early efforts to identify the pho-toreceptor (Sargent, 1985) were consistent with the involvement of either carotenoidsor f lavins; however, since loss of the carotenogenesis genes al-1, al-2, and al-3 elimi-nated nearly all of these pigments from cells but had no effect on the light responses(Russo, 1986), interest soon focused on f lavins as chromophores. Consistent withthis possibility, f lavin biosynthesis mutants rib-1 and rib-2 showed reduced andf lavin-dependent light responses including induction of carotenogenesis and phaseshifting and photosuppression of circadian rhythmicity (Paietta and Sargent, 1981).Also, supplementation of the mutants with 1-deazaribof lavin and roseof lavin yield-ed light responses having appropriately altered action spectra (Paietta and Sargent,1983a). A number of f lavoproteins have been suggested as possible blue light pho-toreceptors including nitrate reductase (Klemm and Ninneman, 1978) and cryp-tochromes. In the end, however, it was neither of these but was instead an entirelynovel type of molecule, and genetic analysis of the phenomenon pointed the way toits identification.

18.3.2Genetic Dissection of the Light Response

Screens for genes encoding the photoreceptor were based on the observation thatblind strains are completely normal except for the fact that mycelia elaborate nocarotenoids. Thus, colonies on agar are white if examined early (before conidia form)and slants kept in the light on the top of a lab bench will have white mycelia on thesurface of the agar beneath a ring of yellow/orange conidia. Because of this appear-ance, when examined from the side, the collar of agar and mycelia at the top of theslant of such mutants will be white, hence the origin of the white collar genes. Suchscreens executed in the Russo and Macino laboratories identified a number of blindwhite collar strains all mapping to two loci, white collar-1 and white collar-2 (wc-1 andwc-2). True loss-of-function wc mutants appear to be blind to all known light re-sponses, including the light induction of carotenogenesis (Harding and Shropshire,1980), light-induction of conidiation (Ninneman, 1991), induction of protoperithecia,and pointing of perithecial beaks (Harding and Melles, 1983; Degli Innocenti andRusso, 1984b). Further, photoadaptation is also lost because there is no primary lightresponse to be adapted. A recent report of photoresponses in wc null strains (Dragov-ic et al., 2002) was traced to poor genetic craftsmanship in that the strains used werenot true nulls; see (Lee et al., 2003) and (Cheng et al., 2003b) for more discussion.Genes encoding proteins that by sequence look like additional photoreceptors have

18.3 Light Perception – the Nature of the Primary Blue Light Photoreceptor

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been detected in the Neurospora genome, so novel assays for light responses mayidentify additional photoreceptor genes as described below.

Although wc-1 and wc-2 null mutants appear to be blind, a number of genes affect-ing photoresponses have been identified. The poky gene encodes a mitochondrial 19SRNA and its mutation appears to cause a b-type cytochrome deficiency that in someway reduces sensitivity for photosuppression of clock-regulated conidiation (Brain etal., 1977). Using a clever colony-based assay for the circadian rhythm, Paietta and Sar-gent identified several light-insensitive (lis) mutants that showed reduced photosup-pression of the circadian rhythm (Paietta and Sargent, 1983b). Lastly, the vivid gene,now known to encode the photoreceptor required for photoadaptation (Schwerdt-feger and Linden, 2001), was described based upon a spontaneous mutation that re-sulted in a much more intense orange color (Hall et al., 1993).

18.3.3New Insights into Photoreceptors from Genomics

Ongoing genomics efforts in Neurospora have turned up several additional putativephotoreceptors (Figure 18.3) that cannot as yet be assigned to any photobiology. Thefirst of these was NOP-1, a Neurospora opsin that appeared during an extensive ESTsequencing project aimed at identifying genes regulated by the circadian clock (Zhuet al., 2001). Analysis of the protein following heterologous expression in Pischiashowed that it bound a retinal cofactor and could undergo a photocycle, but the workfailed to identify a solid light-regulatory function in Neurospora (Bieszke et al., 1999).The complete genomic sequence of Neurospora includes several bacteriophy-tochromes and a cryptochrome (Galagan et al., 2003) (Borkovich et al., 2004), genesfor each of which are expressed. The CRY protein (Froehlich et al., 2004a) is found inboth the cytoplasm and nucleus, is strongly light induced in a WC-1-mediated path-way, is circadianly regulated, and has been shown to bind FAD in vitro. Since CRYsare often characterized as proteins having sequences similar to DNA photolyases butlacking in photolyase function, it is of significance that complete deletion of CRY hasno impact on photoreactivation repair (A. Froehlich, J. Loros, J. Dunlap, in prepara-tion).

Also expressed are two genes encoding proteins having extensive similarities tobacteriophytochromes (Figure 18.3), members of the Cph1 family of phytochromes,with two PAS domains and a PHY domain followed by an ATPase and a response-reg-ulator domain (Froehlich et al., 2004b). An N-terminal fragment of 515 amino acidsof PHY-2 expressed in E. coli has been shown to bind either biliverdin or PCB and toundergo spectral shifts when exposed to red or far red light, and after red exposurethere is a gradual decay back to the red absorbing form (Froehlich, A., Vierstra, R.,Loros, J., and Dunlap, J. C., unpublished). Unfortunately, there are as yet no clock orlight-related phenotypes that can be associated with loss of either PHY or CRY, andmicroarray analyses have so far failed to detect transcripts whose regulation is alteredeither by red or far-red light, or by loss of either PHY or CRY.

Sequence homologs to WC-1 have also appeared in other fungi. Although Saccha-romyces has no apparent photobiology, the recent expansion of genomics efforts


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from yeasts first to Neurospora and more recently to other complex filamentous fun-gi has identified a number of WC-1 homologs elsewhere. The common feature ofthese molecules is the LOV domains used as the photosensory module (see below).Based on strong sequence similarities, functional homologs of WC-1 probably existin a number of fungi including Podospora, Sordaria, Aspergillus, Cochliobolus, Magna-porthe, Coprinus and Fusarium, although functional studies are still rare.

18.4How do the Known Photoreceptors Work?

18.4.1WC-1 and WC-2 contain PAS Domains and Act as a Complex

Molecular dissection of photoresponses in Neurospora began with the cloning of thewc genes by Macino and colleagues (Ballario et al., 1996; Linden and Macino, 1997).WC-1 is a 117 kD protein (Ballario et al., 1996) that contains a Zn-finger DNA-bind-ing domain, a polyglutamine stretch in addition to two acidic domains of the typestypical for transcriptional activators, and three PAS domains (Lee et al., 2000). In gen-eral, PAS domains are believed to mediate protein–protein interactions as has beenshown to be the case with the WC proteins. The second and third PAS domains ofWC-1 are of this type, but the first is a LOV domain, a subclass of PAS domains as-sociated with proteins that sense light, oxygen, and voltage (Christie et al., 1999; Tay-lor and Zhulin, 1999). In a BLAST search, WC-1 shows strong similarity to a numberof photoreceptors via the LOV domain and also to animal circadian clock proteins;subsequent work has shown that, consistent with its dark function in the circadian

18.4 How do the Known Photoreceptors Work?

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Figure 18.3 Real and putative photoreceptorsin Neurospora. The identity and approximatelocation of various functional domains in Neu-rospora proteins are shown. In the list, onlyWC-1, VVD and CRY are known to bind chro-mophores and only the first two of these to be

true photoresponse mediators, although WC-2is required for the function of WC-1. The re-maining genes have been identified as a resultof various genomics-based efforts. See text fordetails.

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clock, WC-1 is a sequence and functional homolog of the mammalian clock proteinBMAL1 with which it is 48% similar or identical over the entire length of BMAL1(Leeet al., 2000). WC-1 is a nuclear protein whose intracellular location is not regulated bylight, whose expression is regulated post-transcriptionally (Lee et al., 2000), andwhose activity and stability are controlled both through phosphorylation (Arpaia etal., 1999; Lee et al., 2000) and protein–protein interactions (Cheng et al., 2002; De-nault et al., 2001; Talora et al., 1999). In contrast to WC-1, WC-2 is constitutively ex-pressed, giving rise to a 57 kDa (530 amino acid) nuclear protein having a single ac-tivation domain, PAS domain, and Zn-finger that does not appear to be highly regu-lated (Denault et al., 2001; Schwerdtfeger and Linden, 2000). WC-1 and WC-2 inter-act in the nucleus via their PAS domains to form the White Collar Complex (WCC)(Talora et al., 1999; Schwerdtfeger and Linden, 2000; Ballario et al., 1998; Denault etal., 2001) and with FRQ (Denault et al., 2001; Cheng et al., 2001) in the feedback loopcomprising the circadian oscillator (see below). WC-1 is the limiting factor in thecomplex, since WC-2 is always present in excess (Denault et al., 2001; Cheng et al.,2001), but WC-2 mediates the interactions between the regulated components of theWCC.

18.4.2WC-1 is the Blue Light Photoreceptor

Based on the genetics, Harding and Shropshire and later Macino and colleagues pro-posed that the white collar genes encoded a photoreceptor (Ballario et al., 1998; Bal-lario et al., 1996; Harding and Shropshire, 1980; Linden and Macino, 1997), a soundguess that was later verified (Froehlich et al., 2002; He et al., 2002). The genetics wereequally consistent with a model that posited either WC-1 and WC-2 as the photore-ceptor or as essential elements in the signal transduction cascade leading from a pho-toreceptor to downstream responses. Interestingly, the identification in 1997 of an ex-clusively dark function for the WC proteins in the circadian clock (Crosthwaite et al.,1997) appeared to support the second model. In any case, to prove that a protein is aphotoreceptor, of course, it is necessary not only to prove that mutants lacking it haveno photoresponses, and to show that it binds a chromophore, but also that the pro-tein itself undergoes photochemistry with a f luence and wavelength response con-sistent with the known biology. These were the tests used to establish WC-1 as theblue-light photoreceptor in Neurospora (Froehlich et al., 2002).

18.4.2.1 WC-1 utilizes FAD as a ChromophoreThe sequence of the LOV domain of WC-1 contains the diagnostic NCRFLQ regionthat is conserved among all of the f lavin-binding phototropins, and was expected tobind a f lavin. It was thus satisfying when the WCC was epitope tagged, purified fromNeurospora, and shown to contain a noncovalently bound f lavin adenine dinu-cleotide (FAD) (He et al., 2002) (Figure 18.4). This result was consistent with the ob-servation that FAD, but not FMN, is essential for function of the in vitro transcribedprotein (Froehlich et al., 2002), and with the earlier biochemical genetic studies.


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18.4.2.2 Proof of Photoreceptor FunctionAn in vitro assay of photoreceptor function was used to establish unequivocally therole of WC-1 as a photoreceptor. Froehlich et al. (2002) first dissected the light-regu-latory regions of a known highly light-inducible gene, the circadian clock gene frq,which turned out to have two separate light-regulatory elements (LREs). Light induc-tion of frq was known to require both WC-1 and WC-2. Using nuclear protein extractsfrom both wild-type and definitive null strains, antisera that recognized both WC-1and WC-2, and the LREs as targets in electrophoretic gel mobility shift assays (EM-SAs), they showed that the WCC bound to each LRE. Mutants lacking either WC hadno binding capacity, and FRQ was not found in the complex, although it is known tobind to the WCC in solution both in vivo and in vitro (Froehlich et al., 2002). The sig-nal biochemical observation, however, was that the apparent size (mobility) of thecomplex in the shifted band was different when nuclear extracts were made in thelight versus in the dark, and moreover that this difference could be recapitulatedwhen extracts of dark grown cultures were extracted in the dark or under red light andlater exposed to light – that is, the extracts themselves retained their ability to be pho-toresponsive (Figure 18.5). Based on these results, the biochemical nature of the pho-toreceptor could be determined, after controls were completed verifying that the invitro reaction corresponded to the in vivo photobiology.

The important controls (Figures 18.6 and 18.7) established that the action spec-trum for the conversion of dark complex to light complex had the same f luence andwavelength response (action spectrum) as had previously been shown for photosup-pression of circadian rhythmicity (Sargent and Briggs, 1967) and also for blue light-induced phase shifting of the circadian clock (Crosthwaite et al., 1995). These data es-


Figure 18.4 The affinity–purified white collarcomplex from Neurospora is associated withFAD based on f luorescence excitation peaks

at 370 and 450 nm (left), and an emissionpeak at 520 (middle) that is pH sensitive(right) (from He at al., 2002).

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tablished the photoresponse in the test tube as indistinguishable from the biologicalresponses. From this point it was logically straightforward to express the proteins invitro in a coupled transcription/translation system, so that no other Neurospora pro-teins would be present, and to show that the light response (the change in mobilityon the gel shift) could be achieved. In the absence of any added chromophore, bothlight and dark mobility complexes were seen, but the addition of FAD (but not FMN)promoted the formation of the dark complex in the dark and the light complex in thelight.

Taken together, the data suggest the model shown in Figure 18.8 for the initial pri-mary photoreception response. The complex of WC-1 and WC-2 binds to DNA at aspecific consensus sequence, the light responsive element or LRE. Because the ap-parent mobility of the complex increases on exposure to light even when the complexis composed of proteins made in vitro in a test tube, the simplest interpretation is thatlight results in a multimerization of the components WC-1 and WC-2. This interpre-tation is consistent with the observation that self association of WC-1 in complexes isseen but self association of WC-2 is not seen (Cheng et al., 2003b). The proteins bothcontain GATA-type Zn fingers and the WCC binds to LREs having imperfect repeatsof GATN (not GATA) (Froehlich et al., 2002), a sequence similar to one previouslyidentified for the Neurospora al-3 gene (Carattoli et al., 1994). Since the photoactivepart of WC-1 is the LOV domain, everything supposed about the photochemistry ofthe events is based on better-studied LOV domains in the phototropins (Chapter 13,Christie and Briggs; Chapter 15, Crosson). Thus, it is expected that when the FADchromophore absorbs blue light, the C(4a) position of the FAD undergoes a transient


Figure 18.5 Electrophoretic mobility gel shiftassays (EMSAs) using Neurospora nuclear ex-tracts reveal an in vitro light-sensitive complexformation. Nuclear protein extracts were madefrom cultures grown the dark (D) or light (L)and used in EMSAs run either in D or L. Ex-

tracts from light cultures yield a slower mobili-ty band (open arrow). Lane 3 shows that evenafter extraction, extracts retain the ability to re-spond to light and generate the lower mobilitycomplex.


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Figure 18.6 The in vitro light-induced WCC/LRE mobility change occurs at biologically rel-evant light intensities. The amount of materialin the EMSA faster-mobility dark band (closed

symbol) or slower mobility light band (opensymbol) is plotted versus the amount of light(Crosthwaite et al, 1995). Figure adapted fromFroehlich et al.(2002).

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Figure 18.7 The in vitro light-induced WCC/LRE mobility change occurs at biologically rel-evant wavelengths. An action spectrum wasderived by using the amount of light seen togive a half maximal response in Figure 18.6,but of different wavelengths. The in vitro reac-

tion shows a peak in the blue that matches theaction spectrum previously observed for pho-tosuppression of circadian banding (Sargentand Briggs, 1967). Figure adapted fromFroehlich et al. (2002).

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covalent interaction with the cysteine in the NCRFLQ sequence of WC-1, thereby in-ducing a conformational change in the protein. Consistent with this expectation,point mutations changing this Cys to Ser or to Met yield a blind WC-1 (Cheng et al.,2003a). Since Froehlich et al. showed that the response occurs in a test tube contain-ing only in vitro translated proteins and FAD, the WCC by itself must be sufficientwith FAD to function as the photoreceptor. This conformational change in turn ap-pears to bring about the quaternary interaction between WCCs, apparently resultingin formation of a multimer and in enhanced transcriptional activation of WCC-bound light responsive promoters.

18.4.3Post-activation Regulation of WC-1

Following exposure to light WC-1 undergoes quantitative and qualitative changes thatfurther modulate its activity. Macino and colleagues have advanced a model, based oninhibitor studies, in which WC-1 is phosphorylated and degraded soon after lights-on, followed by synthesis of non-phosphorylated WC-1 (Talora et al., 1999). The ki-netics of light induction of most rapidly induced genes closely parallel this pattern,and the decrease in phosphorylated WC -1 is accompanied by a refractory period tolight stimulation. In vvdnull strains (see below) total WC-1 decreases after light expo-sure but phosphorylated WC -1 levels remain high instead of decreasing, and the re-fractory period is missing; this is consistent with a model in which phosphorylatedWC-1 is the active form of the protein (Heintzen et al., 2001; Schwerdtfeger and Lin-den, 2001; Talora et al., 1999).


+1

proximalLRE

distalLREfrq promoter

FAD FAD

FAD FAD +1

proximalLRE

distalLREfrq promoter

FAD FAD

in the dark after lights-on

Figure 18.8 A model for blue light photore-ceptor activation of transcription in Neurospo-ra. Two light-regulatory sequences within thefrq promoter in Neurospora bind to complexes

of WC-1 and WC-2. The large oval representsWC-1 binding to FAD as the chromophore,and the small oval represents WC-2. See textfor details. (Figure courtesy of A. Froehlich.)

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18.4.4A Non-photobiological Role for WC-1 and the WCC

As an aside, it should be noted that, exclusive of their role as the primary and initialblue-light photoreceptor complex in Neurospora, WC-1 and WC-2 serve as positive el-ements in the circadian feedback loop. Circadian rhythmicity is inextricably linkedwith light responses in most organisms: even though the clock will by design con-tinue to run in the absence of light cues, it is light and dark transitions that most com-monly provide the cues required to phase the internal clock oscillation correctly sothat internal subjective day as experienced in the cell corresponds to external day asdefined by the earth’s rotation.

The WCC is the positive factor that drives expression of the negative element in thenegative feedback loop comprising the core of the circadian oscillator (Crosthwaite etal., 1997). Brief ly, in the transcriptional/translational feedback loops comprising thecore of circadian oscillators of eukaryotes in the fungal/animal lineage, negative ele-ments (like FRQ in Neurospora, PER/TIM in Drosophila, and PER1, PER2, CRY1, andCRY2 in mammals) block activation by heterodimeric PAS domain-containing posi-tive elements (WC-1/WC-2 in Neurospora, dCLK/CYC in Drosophila, and CLOCK/BMAL1 in mammals). In turn, the positive elements activate expression of the nega-tive elements, thereby giving rise to the negative feedback loop that lies at the core ofthe clock (Dunlap, 1998). Predictions that follow from this role of the WCC in theclock have been confirmed: (1) proteins such as WC-2 should play a role in tempera-ture compensation (Collett et al., 2001); (2) defects in WC-2 should lengthen the cir-cadian period length in the dark; (3) WC-2, FRQ, and WC-1 should physically inter-act in solution. Thus, in the Neurospora circadian clock, FRQ acts to depress the levelof its own transcript at least in part by interfering with the formation of the WCC,thereby blocking activation of the frq gene by the WCC (Froehlich et al., 2003). The es-sential role of the WCC in the operation of the circadian clock, and of heterodimersof PAS domain proteins (such as CLOCK/BMAL in mammals or CLK/CYC inDrosophila) in circadian feedback loops was not known until the work of Crosthwaitein 1997 (Crosthwaite et al., 1997). That report provided the first precedent for the roleof PAS:PAS heterodimers as activators in a circadian feedback loop, although it wasfollowed just two weeks later by the cloning and description of the mammalianCLOCK gene (King et al., 1997) in which similar conclusions in the mammalian cir-cadian system were independently reached.

In having an important and clearly distinct dark-only function, the WCC is proba-bly unique among eukaryotic photoreceptors. To begin to gauge the importance ofthis regulation, Lewis et al (Lewis et al., 2002) expressed WC-1 under a regulatablepromoter and examined gene regulation using microarrays. Although many geneswere found to be regulated directly or indirectly in the dark by WC-1, most were notsubsequently found to be light-induced. Conversely they found 22 light-inducedgenes that included 4 (like frq) that were also activated in the dark by WC-1. These da-ta would be consistent with a role for WCC as a master regulator that acts to turn ondownstream regulators that, in turn, more directly regulate target genes.


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18.5VIVID, a Second Photoreceptor that Modulates Light Responses

18.5.1Types of Photoresponse Modulation

Although WC-1 is required for all known light responses, its actions are not sufficientto mediate all Neurospora photobiology. For there to be any light-dependent modula-tion of a light response, it follows that there must be additional photoreceptors. TheVIVID protein (VVD) fulfills this role by modulating the organism’s response to lightsignals subsequent to an initial exposure, a process known as photoadaptation(Schwerdtfeger and Linden, 2001; Schwerdtfeger and Linden, 2003; Shrode et al.,2001). VVD is, like WC-1 and WC-2, a member of the PAS protein superfamily(Heintzen et al., 2001) and its action describes an autoregulatory negative feedbackloop that closes outside of the core circadian oscillator to affect both the clock andphotoresponses. VVD as a photoreceptor binds a f lavin (either FMN or FAD) as achromophore (Schwerdtfeger and Linden, 2003), and as might be inferred from itsplace in the regulatory scheme, when vvd is lost all photoresponses in the organismare elevated.

When Neurospora sees light it exhibits an immediate early light response, for in-stance the rapid induction of vvd or al-1 gene expression that peaks within 15–30 min-utes and then decays away even when the light stays on (Figure 18.9). Interestingly,the photoresponse system remains refractory such that a second light exposure of thesame f luence any time within two hours has no additional inductive effect, althoughexposure to brighter light can yield induction (Figure 18.10); this process requires denovo light-induced synthesis of VVD as well as phosphorylation of unspecified pro-


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minutes in lightFigure 18.9 vvd is rapidly light induced, andloss of vvd slows the rate of decay of light-in-duced transcripts. On the right, P and S standfor two alleles, P which makes no transcriptand S that makes transcript but no protein;

W denotes wild type. Below the Northerns areshown the results of densitometric analysis re-lating the relative abundance of the vvd tran-script seen above (From Heintzen et al.,2001).

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teins by protein kinase C. When VVD is lost, the rate of decay of light–induced geneexpression is slowed, and more importantly perhaps, second exposures to light with-in two hours of a first exposure have additive effects (Schwerdtfeger and Linden,2001; Schwerdtfeger and Linden, 2003; Shrode et al., 2001). As an aside it is worthnoting that not all genes are subject to photoadaptation; for instance, the clock genefrq is simply induced by light (Crosthwaite et al., 1995) and its level does not decayover time to the same extent as other more typical genes.

Because VVD is strongly induced by light and in turn acts as a repressor of light re-sponses, VVD affects circadian entrainment, the process by which the internal clockis set by the daily light/dark cycle. vvd is expressed at a significant level only duringthe first day in constant darkness, and VVD thus probably accounts for the findingthat light signals have little if any clock-resetting effects when delivered on the firstday after a light-to-dark transfer. Furthermore, vvd expression is controlled in part bythe clock, and because of this control, the effect of a brief exposure to light on geneexpression or on the clock will depend on the subjective time of day when the light isseen. This is an aspect of regulation known as circadian gating. VVD contributes togating but loss of VVD does not result in complete loss of gating, suggesting thatthere are other factors involved (Heintzen et al., 2001). Clock regulation of the im-mediate and transient repressor VVD contributes to circadian entrainment by mak-ing dark-to-light transitions more discrete (Heintzen et al., 2001).

18.5 VIVID, a Second Photoreceptor that Modulates Light Responses

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Figure 18.10 The ability to respond to increas-es in ambient light is lost in vvdnull strains.15 µE m–2 s–1 of light was delivered at time 0,followed after 4 hrs by an increase to55 µE m–2 s–1. RNA was isolated from culturesand analyzed by Northern with vvd or al-1 geneprobes as shown. In WT the increased light

elicited an increased transcriptional responsewhich was not seen in a vvd-null strain. Thevvd-null strain used is allele SS692 whichmakes transcript but no VVD protein. Adaptedfrom Schwerdtfeger and Linden (2001 and2003).

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18.5.2Proof of VVD Photoreceptor Function

VVD is a small protein, basically just a short N-terminal extension preceding a LOVdomain (Figure 18.3). Its sequence thus suggested a photoreceptor function even be-fore Schwerdtfeger and colleagues reported chromophore binding and a photocycleas seen in Figure 18.11 (Schwerdtfeger and Linden, 2003). VVD undergoes a classicphotocycle; based on precedents set with the LOV domain of PHOT1, the expectationis that the C(4a) carbon of the f lavin bound by VVD undergoes a transient covalentaddition to the Cys in the NCRFLQ f lavin binding site, resulting in structuralchanges in the protein that affect its activity and ability to interact with partners(Briggs and Christie, 2002; Harper et al., 2003). In support of this model, mutationof the Cys to Ala abrogates the ability of VVD to undergo the photocycle (Schwerdt-feger and Linden, 2003). A photoresponsive role for this LOV domain has also beensupported by domain-swap experiments in which LOV domains from various pro-teins were swapped into WC-1 and the chimeras assayed in vivo for rescue of frq lightinduction and other related responses (Cheng et al., 2003a). A current model is thatVVD will interact with the transcriptional activator WCC, down-regulating its activi-ty and thereby inf luencing the expression of WCC-controlled genes, although thishas not yet been verified biochemically.


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Figure 18.11 VVD binds a f lavin and under-goes a photocycle. Repeated absorption spec-tra of VVD in solution taken (1) 10 s; (2)10 min; (3) 2 h and (4) 5 h after initial expo-

sure to 100 µmol photons m–2 s–1 for 30 s.Three isosbestic points are shown by arrows.From Schwerdtfeger and Linden (2003).

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18.6Complexities in Light Regulatory Pathways

The straightforward expectation initially arising from experiments was that the WCCwould bind to LREs in the promoters of light-regulated genes and activate them inconcert, in most cases being modified by the action of VVD. However, the story doesnot appear to be this simple. Diverse induction kinetics are seen with light inducedgenes, and some genes are much more sensitive to the dose of WCC than are others.For instance, point mutations in the Zn-finger DNA-binding domain of WC-2 abol-ished light induction of some target genes but allowed induction of frq to proceed un-affected (Collett et al., 2002). Similarly, mutations in WC-1 having little effect on frqinduction can abrogate light responses of other genes (Lee et al., 2003; Cheng et al.,2003a), and when expression of the WC proteins is placed under the control of in-ducible promoters, normal light-induced expression of frq is seen at basal levels of in-duction (Lee et al., 2003; Cheng et al., 2002).

An example from the literature illustrates errors that have arisen from this unex-pected dosage dependence. In the MK1 allele of WC-1, a frame shift mutation placesa STOP codon in the N-terminal region before the LOV domain; however, a very lowlevel of WC-1 (less than 1% of the normal amount) is made due to reinitiation oftranslation and is sufficient to rescue low dosage functions such as light-induction offrq (Lee et al., 2003; Cheng et al., 2002). A similar wc-1 allele, having point mutationsin the N-terminal region of the gene that introduced STOP codons, was assumed tobe a null and used to assess the importance of WC-1 to light-induction of frq and op-eration of the clock. Not appreciating the differential requirements for WCC levels inlight-induction, or the importance of using true knockout strains, the authors mis-takenly reported that “blind” mutants of Neurospora still showed robust light re-sponses (Dragovic et al., 2002); actually WC-1 was still being made, as in MK1. Moregenerally, it now appears that light-induced genes can be sorted into three groupsbased on their sensitivity to WC levels. The group most sensitive to WCC dose in-cludes the genes encoding components of the carotenoid biosynthesis pathways (al-1, al-2,and al-3) and photoconidiation (e.g. con-6 and con-10) pathways. Less sensitiveis vvd. In a class by itself, frq requires log orders less WCC than the albino genes. Themolecular basis of this sensitivity difference remains obscure.

18.7Summary and Conclusion

Neurospora has for a long time been useful for dissecting the role of light in biologi-cal systems. Most photoresponses in Neurospora can be traced to light induction oftranscription, and to date, responses only to blue light have been identified. Initial re-sponses are due to the blue-light photoreceptor WC-1 that uses an FAD cofactorbound in a LOV domain; WC-1 partners with WC-2 to make a heterodimer which actsas a light-inducible transcription factor. Unique perhaps among photoreceptors, theWCC also displays a prominent function in the dark by playing the central role of key

18.6 Complexities in Light Regulatory Pathways

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transcriptional activator in the negative feedback loop at the core of the circadian sys-tem. The other verified photoreceptor, VVD, uses a f lavin bound in a LOV domainand acts independently as a photoreceptor to modulate the WCC response. Withinthe past few years, genomic studies in Neurospora have identified an opsin, a cryp-tochrome, and two phytochromes that may act in light responses, but to date no sig-nificant photobiological phenotypes have been associated with loss of these genes.

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Handbook of Photosensory Receptors (BRIGGS:PHOTORECEPTORS O-BK) || Neurospora Photoreceptors