Báo cáo khoa học: A cryptochrome-based photosensory system in the siliceous sponge Suberites domuncula (Demospongiae) docx

A cryptochrome-based photosensory system in the siliceous sponge Suberites domuncula (Demospongiae) Werner E. G. Mu ¨ ller 1 , Xiaohong Wang 2 , Heinz C. Schro ¨ der 1 , Michael Korzhev 1 , Vladislav A. Grebenjuk 1 , Julia S. Markl 1 , Klaus P. Jochum 3 , Dario Pisignano 4 and Matthias Wiens 1 1 Institute for Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg University, Mainz, Germany 2 National Research Center for Geoanalysis, Beijing, China 3 Max-Planck-Institute for Chemistry, Mainz, Germany 4 Scuola Superiore ISUFI, Universita ` del Salento and National Nanotechnology Laboratory, Istituto Nazionale di Fisica della Materia-Consiglio Nazionale Delle Ricerche, Lecce, Italy Keywords optical waveguide; photosensor; Porifera; sponges; Suberites domuncula Correspondence W. E. G. Mu ¨ ller, Institute for Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg University, Medical School, Duesbergweg 6, D-55099 Mainz, Germany Fax: +49 6131 39 25243 Tel: +49 6131 39 25910 E-mail: wmueller@uni-mainz.de Website: http://www.biotecmarin.de/ Database Sequences CRYPTO_SUBDO (Suberites domuncula) and CRYPTO_CRAME (Cratero- morpha meyeri) have been submitted to the EMBL ⁄ GenBank database under the acces- sion numbers FN421335 (CRYPTO_SUBDO) and FN421336 (CRYPTO_CRAME). Sequence HPRT_SUBDO (hypoxanthine phosphoribosyl-transferase 1) has been submitted to the EMBL ⁄ GenBank database under the accession number FN564031 Note This contribution is dedicated to Professor M. Pavans de Ceccatty (Universite ´ Claude Bernard, Lyon/Montpellier) in memory of his groundbreaking studies on the ‘coordination in sponges’ (Received 19 September 2009, revised 8 November 2009, accepted 17 December 2009) doi:10.1111/j.1742-4658.2009.07552.x Based on the light-reactive behavior of siliceous sponges, their intriguing quartz glass-based spicular system and the existence of a light-generating luciferase [Mu ¨ ller WEG et al. (2009) Cell Mol Life Sci 66, 537–552], a pro- tein potentially involved in light reception has been identified, cloned and recombinantly expressed from the demosponge Suberites domuncula. Its sequence displays two domains characteristic of cryptochrome, the N-ter- minal photolyase-related region and the C-terminal FAD-binding domain. The expression level of S. domuncula cryptochrome depends on animal’s exposure to light and is highest in tissue regions rich in siliceous spicules; in the dark, no cryptochrome transcripts ⁄ translational products are seen. From the experimental data, it is proposed that sponges might employ a luciferase-like protein, the spicular system and a cryptochrome as the light source, optical waveguide and photosensor, respectively. Abbreviations CPD, cyclobutane pyrimidine dimer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GBS, giant basal spicule; HPRT, hypoxanthine phosphoribosyl-transferase 1. 1182 FEBS Journal 277 (2010) 1182–1201 ª 2010 The Authors Journal compilation ª 2010 FEBS Introduction During the evolutionary transition from unicellular to multicellular organisms, the common metazoan ances- tor acquired most of the structural ⁄ functional regula- tory systems and molecular pathways present in ‘modern’ Metazoa [1]. Sponges (phylum Porifera), con- sidered to belong to the most basal Metazoa, have a surprisingly complex genetic repertoire with an intri- cate network of highly differentiated interacting cells [2]. Even though some characteristics of diploblasts and triploblasts, for example, the neuronal basis for contraction or light perception [3–6], are missing in sponges, coordinated reactions to light and mechanical stimuli can be observed [7]. Increasing experimental evidence indicates that at least some molecular basal components of a neuron-like system exist in Porifera, such as metabotropic glutamate ⁄ 4-aminobutyrate-like receptors [8], protosynaptic protein homologs or post- synaptic scaffold proteins [9]. These findings suggest that the phylum Porifera possesses a sophisticated intercellular communication and signaling system which nevertheless differs from the integrated neuronal network of other Metazoa [8,9]. In particular, reactions to light observed in sponges have led to studies on potential sensor systems for light [5] and mechanical stimuli [10]; it is proposed that such signal transmissions are based on electric signal propa- gation [11]. To elucidate the phototactic behavior of sponge larvae [11], it has been shown that larvae of the demosponge Reniera sp. respond to blue light (440 nm), and to a lesser extent to orange–red light (600 nm), with coordinated reactions. Interestingly, the study suggests the involvement of photoreceptive pigments and several candidate photoreactive pigments, including carote- noids, have been identified in demosponges [12]. In 1921, endogenous light formation after tactile stimulation was observed in the demosponge Grantia sp. [13]. It was proposed that light in sponges might be generated either by symbiotic bacteria [14] or by a sponge-specific endogenous photoprotein [15]. In this line, a sponge luciferase was very recently cloned and expressed. In the presence of the substrate luciferin, the poriferan enzyme generates light with emission peaks at 548 and 590 nm [16]. The existence of a corre- sponding poriferan light-guiding system, which is based upon siliceous skeletal elements (spicules), is well established. This spicular framework of the classes Demospongiae and Hexactinellida [17,18] is composed of biosilica [19]. Its inorganic polymerous component, poly(silicate), is formed enzymatically via the enzyme silicatein in demosponges and in hexactinellids [20–22]. Poriferan biosilica reaches a purity similar to that of quartz glass [23] and allows for the transmission of light as an optical fiber [24]. More specifically, spicules can act as single-mode, few-mode or multimode fibers [14]. They efficiently transmit light between wave- lengths of 615 and 1310 nm [15]. To date, no poriferan genes or gene products related to those that usually control the morphogenesis of visual systems in triploblasts (e.g. Pax 6) [25] have been discovered. Recently, the molecular basis of an alternative photoreceptor system was identified in trip- loblastic Metazoa in general [26], and corals in particu- lar, as a representative taxon of early-branching, diploblastic Metazoa [27]. This photoreceptor system is based upon cryptochrome(s) and has been described as a flavoprotein-signaling receptor [28]. Cryptochromes control the circadian rhythm in plants and animals [28]. They belong to the protein family of photolyases, which is divided into three groups, according to their functions in repairing light-induced DNA damage [27,29,30]. First, cyclobutane pyrimidine dimers (CPD) are repaired by the CPD photolyases; second, 6,4- pyrimidine-pyrimidones (6,4 photoproducts, induced by UV irradiation) are mended by (6-4) photolyases, only known to be present in eukaryotes [31]; and third, CPDs in single-stranded DNA are excised by photoly- ases present in bacteria, plants and animals. Structurally, photolyase proteins are composed of a ⁄ b domains and the helical domain [32] that bind cofactor(s), the chromophore(s) [32]. Usually, the cata- lytic chromophore is FADH 2 , which is tethered to the helical domain. A second chromophore, working as a light-harvesting antenna in plants, for example 8-hydroxy-5-deazaflavin, 5,10-methenyltetrahydro-folic acid or again flavin mononucleotide ⁄ FAD [33], is bound to the cryptochromes. Cryptochromes can be divided into three classes according to sequence similarities: (a) metazoan cryp- tochromes, (b) plant cryptochromes and (c) crypto- chrome-DASH proteins of bacteria, fungi, plants and animals. Cryptochrome-DASH proteins display DNA- specific photolyase activity [34]. By contrast, members of the first cryptochrome subfamily are not part of any DNA repair mechanism even though they are closely related to (6-4) photolyases [30]. Major progress in our understanding of the role(s) of metazoan crypto- chromes derived from the studies of Levy et al. [27] and Hoang et al. [26]. By analyzing (potential) blue-light-sensing photoreceptors in the coral Acro- pora millepora, the authors showed that expression levels of two cryptochrome genes, cry1 and cry2, were significantly upregulated during exposure to light [27]. W. E. G. Mu ¨ ller et al. Cryptochrome-based photosensory system of sponges FEBS Journal 277 (2010) 1182–1201 ª 2010 The Authors Journal compilation ª 2010 FEBS 1183 Based on this finding, a dominant role for cry1 and cry2 in controlling the circadian rhythm in Cnidaria has been assumed. This assumption is supported by the observation that insect cells, transfected with human or Drosophila cryptochrome genes, respond to blue light [26]. In addition, it has been shown that light causes a change in the redox state of flavin bound to cryptochrome receptors [26]. In view of these data, it is proposed that the vertebrate cryptochrome system might represent a hitherto unknown light-activated nonvisual perception system [35]. In this study, we report cryptochromes of siliceous sponges (consisting of the two classes Demospongiae and Hexactinellida). Because the demosponge Sube- rites domuncula can be cultivated under controlled lab- oratory conditions [36], and a cell culture system (primmorphs) has been established [37], functional studies were performed with this species. Primmorphs are 3D cell aggregates, comprising both rapidly prolif- erating and differentiating cells. Furthermore, light transmission of spicules can be studied exemplarily with the macroscopic spicules of hexactinellids [14,15]. In particular, the giant basal spicules (GBS) of Mono- rhaphis chuni can reach 3 m in length, with a diameter of 12 mm [38]. Comparative analyses of sequence data of the poriferan cryptochrome genes isolated from S. domuncula (demosponge) and Crateromorpha meyeri (hexactinellid) revealed a considerable phylogenetic relationship to the coral cry1 and cry2 genes. In addi- tion, the gene products display characteristic structural features, the N-terminal photolyase-related region, pro- posed to bear two chromophore-binding domains and the C-terminal FAD-binding domain. Having prepared recombinant S. domuncula cryptochrome and antibod- ies against this protein, it was possible to prove that S. domuncula cryptochrome expression is increased in tissue regions that had been exposed to light, in partic- ular in spicule-rich layers. Therefore, we propose that poriferan siliceous spicules represent a network of light waveguides with the luciferase molecule as the light producing element and cryptochrome as the photo- receptor. Results Spicules as optical glass fibers S. domuncula (Demospongiae) specimens are usually associated with a hermit crab, living in a mollusk shell (Fig. 1A), that provides free motility. However,  10% of the animals used in this study had lost the crab, which forced them into sessile behavior (Fig. 1A). The specimens were  5–6 cm in size. A B C E F G JK H I D Fig. 1. Spicules as optical glass fibers. (A) Specimens of the demo- sponge Suberites domuncula. Although most specimens are associ- ated with hermit crabs, allowing the sponge to live on a ‘mobile’ substrate, some have lost the crab, consequently forcing them into a sessile way of life. (B) Giant basal spicule (GBS) of Monorha- phis chuni. (C,D) Localization of tylostyles at the surface of S. do- muncula. Colloidal gold particles were used to highlight spicules that protrude with their knobs from the surface of the animals (<; ><). (C) Transversal section of S. domuncula tissue; the packed zones of spicules are marked (><; sz). (D) Sponge surface. (E) S. domuncula tylostyle illuminated by a white light source (wl) that was coupled to its knob. (F) GBS illuminated using a green laser light source (gl). Epibiontic corals (co), surrounding the spicule, remain opaque. (G–I) The majority of the tylostyles from S. domun- cula spicules have perfect terminal knobs (k) (G), whereas some tylostyles (H) display a more complex morphology with a collar (c) between the knob (k) and the monaxonal spicule rod (sp). (I) After etching with HF the different building blocks, knob (k), collar (c) and spicule (sp), become more prominent. (J,K) Net of fused choano- somal spicules (Euplectella aspergillum), highlighting that light guided within spicules is split at fusion sites (fs). Cryptochrome-based photosensory system of sponges W. E. G. Mu ¨ ller et al. 1184 FEBS Journal 277 (2010) 1182–1201 ª 2010 The Authors Journal compilation ª 2010 FEBS S. domuncula comprises relatively small spicules (< 400 lm). By contrast, some hexactinellid spicules are gigantic, reaching a length of up to 3 m and a diameter of 12 mm, for example the GBS of M. chuni, around which the sponge tissue grows (Fig. 1B). In S. domuncula, the tylostyles (spicules with a globu- lar swelling at one end and a sharp tip at the other; 150–320 lm in length) are regularly arranged in pali- sade-like arrays at the periphery of the poriferan body (Fig. 1C,D). There, zones of packed spicules reach a thickness of up to 5 mm. By contrast, tylostyles in the central part of the body, the medulla, are oriented in a slanted direction along the aquiferous canal system [39]. All tylostyles display a globular knob, which is located almost exclusively at the end of the monaxonial spicules (Fig. 1G). In rare cases, it is fixed to a narrow collar (Fig. 1H). By using a nanopositioning and nanomeasur- ing machine, analyses of such globular knobs were pos- sible at the nanometer scale. The majority of terminal knobs, with a spherical ⁄ elliptical geometry, have a sur- prisingly regular shape, reminiscent of a collecting lens. Their diameters vary slightly between 6.53 and 7.28 lm (in the longitudinal direction of the spicule) and 8.54 and 9.21 lm (in the perpendicular direction) (n = 12). These globular knobs are fused to monaxonial rods with a diameter of 6.14–6.57 lm. The outer circumfer- ences of the subterminal collars range between 6.9 and 7.2 lm. Limited dissolution of the silica mantel indi- cates that terminal knobs and subterminal collars are formed as independent units (Fig. 1I). Siliceous spicules of hexactinellids have the potential to guide light [15]. For example, GBS of M. chuni (the syntypus deposited by Schulze [40]) showed that coher- ent light is guided through the spicule associated with the siliceous rod, but not through epibiontic corals (Fig. 1F). In some hexactinellids, for example Euplec- tella aspergillum, secondary fusion of spicules is obser- ved. By illuminating this choanosomal spicular network, it can be seen that the light beam is split at the fusion sites of the choanosomal skeletal spicules (Fig. 1J,K). Similarly, illumination of the tylostyles of the demo- sponge S. domuncula with a white light source demon- strates that the light beam is transmitted and directed along their longitudinal axis (Fig. 1E). Spicules in sponge tissue In general, demosponge tissues contain small microscl- eres (siliceous skeletal elements of sizes < 10 lm) and larger macroscleres (between > 10 and < 300 lm). All spicules are initially formed intracellularly and, after having reached sizes of > 8 lm, are completed extracellularly [41,42]. S. domuncula primmorphs repre- sent a highly suitable model to study the organization of spicules within sponge tissue, because this species generates exclusively tylostyles. In this study, prim- morphs were used 5 days after re-aggregation of disso- ciated, single cells to investigate the establishment of contact between spicules and cells. The cells involved in spiculogenesis, termed sclerocytes, release both the silica precursors ⁄ enzyme substrate and the enzyme sili- catein [43]. Silicatein and silica are required for the appositional layering of biosilica during spicule growth, in order to reach the final spicular morphol- ogy. TEM showed that the cells are scattered along the spicule surface (Fig. 2), but are mainly present at t t k 1 µm 5 µm 1 µm 5 µm -ac sp sp col 1 µm sp ac sp sp sp m m m A B C D E F -ac 1 µm Fig. 2. Localization of spicules within Suberites domuncula primmorphs. Primmorphs were formed over a 5-day period and then used for sectioning and SEM analysis (A–C) Sections through the knobs (k) and the spiny tips (t) of tylostyles (sp). The cells, sclerocytes, are scat- tered along the surface of tylostyles. (m) Mesohyl (intercellular matrix). (D) Immature spicule, comprising a large oval axial canal (ac) contain- ing the axial filament. The spicule is embedded in the bulky mesohyl, which is traversed by collagen fibers (co). (E) At a later stage the axial filament is contracted and adopts a triangular profile. (F) At the final stage of spiculogenesis, the 3.5 lm spicule contains a small (0.5 lm diameter) axial canal. W. E. G. Mu ¨ ller et al. Cryptochrome-based photosensory system of sponges FEBS Journal 277 (2010) 1182–1201 ª 2010 The Authors Journal compilation ª 2010 FEBS 1185 both ends of spicules, the knob (Fig. 2A) and the pointed tip (Fig. 2B,C). Cross-sections through imma- ture spicules revealed a large oval axial canal (1 lm diameter), homogeneously filled with the proteinaceous axial filament (Fig. 2D). During maturation, this axial canal develops a triangular form, whereas the axial fil- ament concurrently contracts to < 0.2 lm in diameter (Fig. 2E). In adult spicules, the diameter of the axial canal reduces and, in most cases, it becomes round again (Fig. 2F). Notably, sclerocytes are not intimately associated with spicules. Instead, there is a gap of 50–100 nm between them (Fig. 2F). Cells and spicules are embed- ded in a bulky extracellular matrix, the mesohyl, which is composed of structural proteins, for example colla- gen and soluble proteins such as galectin [1]. Cloning and analysis of sponge cDNA encoding cryptochromes Complete cDNAs coding for putative cryptochrome homologues were isolated from the demosponge S. domuncula and the hexactinellid C. meyeri. The S. domuncula cDNA (SDCRYPTO; 1565 nucleotides) comprises an ORF (CRYPTO_SUBDO) from nucleo- tides 1-3(Met) to 1552-1554 (Fig. 3A). Northern blot- ting confirmed that the cDNA was completely isolated, with a size of 1.9 kb (see below). The deduced polypep- tide (518 amino acids) had a predicted molecular mass of 59 070 Da (isoelectric point 6.47). Domain search analysis (http://myhits.isb-sib.ch/cgi-bin/motif_scan) revealed two main features, the N-terminal photolyase- related region (photolyase) (amino acids 20–200) and the FAD-binding domain (amino acids 237–507). Both domains showed a high similarity score (Expect value [E]) [44] of E = 3.1e )25 and 1e )42 , respectively. CRYPTO_SUBDO had highest sequence similarity to the cryptochrome 3 sequence of Danio rerio (BAA96850.1; E =1e )95 ) and cryptochrome CRY1 of Acropora millepora (ABP97098.1; E =4e )87 ). The C. meyeri cDNA (CMCRYPTO; 1675 nucleo- tides) comprised an ORF from nucleotides 22-24(Met) to 1584-1586, encoding the putative polypeptide CRAME_CRYPTO (521 amino acids). The calculated size of CRAME_CRYPTO is 59 070 Da (isoelectric point 6.47). Again, transcript size (1.9 kb) was con- firmed on northern blots (not shown). The two afore- mentioned domains were found between amino acids 3 and 134 (photolyase-related region; E =2e )06 ) and from amino acids 205 to 475 (FAD-binding domain; E = 3.2e )35 ). In general, the hexactinellid sequence had a lower similarity to other cryptochromes than CRYPTO_SUBDO, for example D. rerio Cry4 (AAI64413.1; E =5e )53 )orA. millepora CRY2 (ABP97099.1; E =3e )49 ). For phylogenetic analysis, we used an extended data set that had originally been applied to the study of coral cryptochromes [27]. The resulting phylogenetic tree was rooted with the blue light photoreceptor cryp- tochrome 1 of Arabidopsis thaliana. The tree revealed a distinct branch near the root that contained all mem- bers of the class II photolyases, including distantly related bacterial enzymes. By contrast, the molecules of C. meyeri, A. vastus and S. domuncula were grouped at the base of those branches that include metazoan cryptochromes (Fig. 3B). The close relationship between CRYPTO_SUBDO and the coral crypto- chrome CRY2 was remarkable. Fig. 3. Poriferan cryptochromes. (A) The deduced poriferan cryptochrome protein sequences CRYPTO_SUBDO (Suberites domuncula) and CRYPTO_CRAME (Crateromorpha meyeri), and the photolyase-related protein from Aphrocallistes vastus (PHL64_APHVA; NCBI accession no. 28625001), were aligned with the two coral (Acropora millepora) cryptochromes, CRY1 (CRY1_ACRO; 145881069) and CRY2 (CRY2_ ACRO; 145881071). Residues conserved (identical or similar with respect to their physicochemical properties) in all sequences are shown in white on black; those which share similarity in four sequences are shown in black on gray. The characteristic domains, the N-terminal photol- yase-related region (photolyase) and the FAD-binding domain, are marked. (B) For phylogenetic analyses, the aforementioned sequences were used in combination with other representative members of the metazoan cryptochrome family, Danio rerio cryptochrome 4 (CRY4_ DARE; 8698594), cryptochrome 3 (CRY3_DARE; 8698592), cryptochrome 2a (CRY2a_DARE; 8698588), cryptochrome 1a (CRY1a_DARE; 8698584); Gallus gallus cryptochrome 1 (CRY1_ CHICKEN; 19550963), cryptochrome 2 (CRY2_CHICKEN; 19550965); Homo sapiens crypto- chrome 2 (CRY2_HUMAN; 27469701); Mus musculus cryptochrome 2 (CRY2_MOUSE; 5670009); Anopheles gambiae cryptochrome 1 (CRY1_ANOGA; 78191295); Drosophila melanogaster blue light photoreceptor (CRY_DROME; 3986298) and Bactrocera tryoni cryptochrome (CRY_BACTR; 51944883). In addition, the following photolyase sequences were integrated, the 6 : 4-type photolyases of D. rerio (PHL64_DARE; 8698596) and Xenopus laevis (PHL64_XENLA; 8809676) and the D. melanogaster photolyase (PHL_DROME;1304062). Finally, members of the class II photolyases were included, the DNA photolyase from Rhodopirellula baltica (PHL_RHOBA; 32447829), Methanobacterium thermoautotrophicum (2507184; PHR_METTH), Arabidopsis thaliana (PHR-CPD_ARATH; 1617219), the D. rerio crypto- chrome-DASH (CRYda_DARE; 41688004), and the cryptochrome 1 blue-light photoreceptor of A. thaliana (CRY1_ARATH; 2499553). The latter sequence was used as outgroup to root the resulting phylogenetic tree. The degree of support of internal branches was assessed by bootstrapping (1000 replicates) and the evolutionary distance calculated (0.1 amino acid substitutions per position in the sequence). Cryptochrome-based photosensory system of sponges W. E. G. Mu ¨ ller et al. 1186 FEBS Journal 277 (2010) 1182–1201 ª 2010 The Authors Journal compilation ª 2010 FEBS A B W. E. G. Mu ¨ ller et al. Cryptochrome-based photosensory system of sponges FEBS Journal 277 (2010) 1182–1201 ª 2010 The Authors Journal compilation ª 2010 FEBS 1187 Recombinant S. domuncula cryptochrome and cryptochrome antibodies To facilitate functional analyses, recombinant S. domuncula cryptochrome and cryptochrome anti- bodies had to be generated. For this purpose, a partial SDCRYPTO cDNA was expressed in Escherichia coli, using l-arabinose as the transcription-inducing agent. The bacterial crude extract was prepared and analyzed by SDS ⁄ PAGE (Fig. 4A). In l-arabinose-induced sam- ples (Fig. 4A, lane a), as well as in noninduced samples (because of leaky expression; Fig. 4A, lane b), a prom- inent band was detected at  19 kDa. This band was also detected after purification of protein extracts through affinity chromatography (Fig. 4A, lane c). The size of this protein corresponded to the calculated size of the recombinant protein, including His-tag and vec- tor-specific sequences (18 970 Da). Subsequently, poly- clonal antibodies (termed PoAb-aCRYPTO_SUBDO) were raised against the purified recombinant protein and used to detect wild-type cryptochrome in poriferan protein extracts. Thus, on western blots PoAb-aCRYP- TO_SUBDO recognized a 60-kDa protein (Fig. 4B, lanes a,b), which matched the calculated size of CRYPTO_SUBDO (59 070 Da). Controls demon- strated that the preimmune serum did not cross-react with the 60-kDa protein (Fig. 4B, lane c). Light-induced expression of cryptochrome To investigate the light-induced expression of crypto- chrome (transcription and translation) in S. domuncula, specimens that had lost their hermit crabs and hence turned to a sessile living form (Fig. 1A), were adapted to complete darkness over a 5-day period. In addition, the poriferan 3D cell-culture system (primmorphs) was used. For this purpose, dissociated single cells (Fig. 5A) were first transferred to a Ca 2+ -containing medium, in which primmorphs subsequently formed (Fig. 5B,C, after 3 and 5 days, respectively). In order to stimulate spiculogenesis, primmorphs were transferred to a silicate cushion for an additional 6 days (Fig. 5D). Ultimately, all samples were exposed to light for 1–8 h, using a short-pass filter (spectral range, 330–900 nm) or long-pass filter (spectral range, 700–1100 nm). Afterwards, RNA was extracted. Subsequent north- ern blot analyses revealed that after dark adaptation (5 days) of primmorphs and tissues, no expression of cryptochrome was detectable (Fig. 6). However, after 2 h of light exposure (330–900 nm), an increased SDCRYPTO expression level could be seen which increased further after 4 or 8 h of light exposure, both in tissue (Fig. 6A) and in primmorphs (Fig. 6B). Interestingly, a change in light quality (700–1100 nm) did not affect the expression pattern AB Fig. 4. Protein detection of the Suberites domuncula crypto- chrome. (A) Preparation of recombinant cryptochrome. Escherichia coli was transformed with SDCRYPTO cDNA, as described in Materials and methods. Protein expression was analyzed in the presence (+) or absence (–) of L-arabinose, using 15% polyacryl- amide gel containing SDS (lanes a and b); equal amounts of protein were loaded onto the gel. The His-tagged recombinant protein (19 kDa) was purified by affinity chromatography on Ni-IDA col- umns and then applied to SDS ⁄ PAGE (lane c). M, size marker. (B) Immunodetection of cryptochrome in crude protein extracts from S. domuncula via western blots. Proteins of crude extracts were size-separated by SDS ⁄ PAGE and stained with Coomassie Brilliant Blue (lane a). In parallel, proteins were transferred to membranes. There, PoAb-aCRYPTO_SUBDO detected the 60-kDa cryptochrome protein (lane b). As a control, preimmune serum was applied to the blots (lane c). M, size marker. AB CD Fig. 5. Suberites domuncula primmorphs. (A) Suspension of single cells obtained after dissociation of sponge tissue in Ca 2+ ⁄ Mg 2+ -free artificial seawater. Formation of primmorphs after 3 days (B) or 5 days (C), respectively, in Ca 2+ ⁄ Mg 2+ -supplemented medium. The 3D cell aggregates were transferred to a silicate cushion (D) for fur- ther experiments. Size bars are given. Cryptochrome-based photosensory system of sponges W. E. G. Mu ¨ ller et al. 1188 FEBS Journal 277 (2010) 1182–1201 ª 2010 The Authors Journal compilation ª 2010 FEBS of SDCRYPTO (Fig. 6C). The expression of tubulin, which was used as an internal control, remained unchanged, irrespective of the duration of light expo- sure (Fig. 6E). Alternatively, an animal was exposed to light for 4 h (330–900 nm) and then immediately transferred to darkness. After 2 h of dark adapta- tion, significantly reduced transcription was seen, whereas after 8 h of darkness no transcripts could be identified using this method (Fig. 6D). In a final set of RNA experiments, qPCR was applied to determine cryptochrome transcription over 24 h, including light–dark transition (Fig. 7). Subse- quently, cryptochrome expression was correlated to the expression of the housekeeping gene tubulin. Thus, during 12 h light exposure, cryptochrome expression increased to 0.53 (± 0.02; n = 5) and then decreased, until after 12 h of darkness a ratio of 0.15 (± 0.025) was calculated. Accordingly, cryptochrome expression during light exposure was up to 3.5-fold higher than in darkness. This ratio remained invariant when the housekeeping genes glyceraldehyde-3-phosphate dehy- drogenase (GAPDH) or hypoxanthine phosphoribosyl- transferase 1 (HPRT) were used as a reference. In a parallel approach, cryptochrome protein expres- sion was analyzed by immunodetection on western blots. In these studies, expression of CRYPTO_SUB- DO could not be detected in dark-adapted prim- morphs (Fig. 8B, lane a). However, extracts from primmorphs that had been exposed to light for 2, 4 or 8 h showed the characteristic 60 kDa band of crypto- chrome (Fig. 8B, lanes b to days, respectively). To demonstrate the specificity of PoAb-aCRYPTO_SUB- DO, preimmune serum was used in parallel with pro- tein extracts of primmorphs that had been exposed to light for 8 h. Whereas preimmune serum did not immunodetect any proteins (Fig. 8A, lane a) PoAb- aCRYPTO_SUBDO elicited a positive signal at 60 kDa. In situ localization of cryptochrome Immobile S. domuncula specimens were exposed to light (330–900 nm) for 24 h. For immunohistological analyses, tissue sections were reacted with anti-crypto- chrome IgG. The resulting immunostaining displayed a distinct zonation. The brightest reactions were seen  50 lm below the surface of the animals in a thick (500 lm) zone that was characterized by the ordered accumulation of a spicule (tylostyle) phalanx A B C D E Fig. 6. Gene expression analyses of Suberites domuncula tissue and primmorphs. Dark-adapted specimens were exposed to light (330–900 or 700–1100 nm). RNA was extracted, size-separated, blotted and probed for SDCRYPTO, using a digoxigenin-labeled probe. RNA was analyzed from tissue (A) or primmorphs (B) that had remained in the dark (0 h) or been exposed to light (330– 900 nm) for 2, 4 or 8 h (0 h, +2 h, +4 h, +8 h). (C) RNA was used from the tissues of animals challenged with light of longer wave- lengths (700–1100 nm) for the same times. (D) Animals were exposed to light (330–900 nm) for 4 h [+4 h), followed by a period of darkness for 2 or 8 h ()2h,)8 h). (E) Internal control. To ensure that the same amount of RNA was loaded onto the gels, size-sepa- rated RNA was probed for transcripts of the housekeeping gene b-tubulin (SDTUB). Transcript sizes are indicated. Fig. 7. Cryptochrome expression analyses of Suberites domuncula tissue. Dark-adapted samples were exposed to light (330–900 nm) for 8 h (08:00 to 16:00) and then kept in darkness for 12 h (16:00 to 04:00). Following RNA isolation, expression levels of crypto- chrome and tubulin (housekeeping gene) were determined through quantitative real-time PCR and subsequently correlated to deter- mine relative expression levels. W. E. G. Mu ¨ ller et al. Cryptochrome-based photosensory system of sponges FEBS Journal 277 (2010) 1182–1201 ª 2010 The Authors Journal compilation ª 2010 FEBS 1189 (Fig. 9A,B). Close inspection showed that in addition to the cells surrounding the spicules, the extracellular matrix was also stained. This observation suggests that cryptochrome not only exists intracellularly, but is also present in the extracellular matrix, in which cells and adjacent spicules are embedded. The staining of cellu- lar and spicular structures was specific, because appli- cation of preimmune serum did not elicit any immunostaining (Fig. 9C,D). In a further series of experiments, sections of near- surface tissue that had been exposed to light were reacted with anti-cryptochrome IgG. Micrographs show the strongest accumulation of immunocomplexes adja- cent to the spicules (Fig. 10A,E,G). In parallel, the cell nuclei were visualized by 4¢,6-diamidino-2-phenylindole AB Fig. 8. Cryptochrome protein expression in primmorphs. (A) Speci- ficity of PoAb-aCRYPTO_SUBDO. Protein extracts of primmorphs that had been exposed to light (330–900 nm) for 8 h were size separated and blotted onto membranes. Lane a, application of preimmune serum (pi) to the membranes; lane b, PoAb-aCRYPTO_ SUBDO (i) binding the Suberites domuncula cryptochrome 60 kDa protein (resulting immunocomplexes were detected with labeled secondary antibodies). (B) Protein extracts of dark-adapted prim- morphs (lane a) or primmorphs exposed to light (330–900 nm) for 2 (lane b), 4 (lane c) or 8 h (lane d) were analyzed on western blots, using PoAb-aCRYPTO_SUBDO. Size markers are indicated. AB CD Fig. 9. Immunohistological detection of cryptochrome in Suberites domuncula tissue. After adaptation to light (330–900 nm), animals were irradiated for 24 h; the direction of the light emission is indi- cated by an arrow. (A) Immunostaining of a tissue section (sp) with anti-cryptochrome IgG PoAb-aCRYPTO_SUBDO. (B) Corresponding Nomarsky interference image. (C) Application of preimmune serum to an adjacent section (control). (D) Corresponding Nomarsky inter- ference image. The surface of the sponge is marked (s). Size bars are given. AB CD EF GH Fig. 10. Localization of cryptochrome in Suberites domuncula tissue. Slices of S. domuncula tissue (following adaptation to light at 330–900 nm for 24 h) were prepared and (A,E,G) reacted with antibodies (PoAb-aCRYPTO_SUBDO); (B,F,H) corresponding views are shown in which the cell nuclei had been visualized by 4¢,6- diamidino-2-phenylindole. Control sections were incubated with preimmune serum and Cy3-conjugated IgG and inspected (C) or illuminated with fluorescence light to identify 4¢,6-diamidino- 2-phenylindole -stained nuclei (D). Size bars are given. Cryptochrome-based photosensory system of sponges W. E. G. Mu ¨ ller et al. 1190 FEBS Journal 277 (2010) 1182–1201 ª 2010 The Authors Journal compilation ª 2010 FEBS staining (Fig. 10B,F,H). Higher magnification reveals staining of both the cells and the extracellular matrix (Fig. 10E,G). In controls, preimmune serum and Cy3- conjugated IgG were used, resulting in a very weak background staining because of unspecific binding (Fig. 10C). Concurrent staining with 4¢,6-diamidino-2- phenylindole highlights the localization of nuclei and, consequently, of cells in the vicinity of spicules (Fig. 10D). For in situ hybridization, labeled single-stranded sense or antisense probes were applied to mounted S. domuncula tissue samples. Animals that had been dark adapted for 3 days revealed very weak staining after application of the antisense probe (Fig. 11A). Following exposure of animals to light (330–900 nm) for 2 (Fig. 11B) and 8 h, binding of the antisense probe elicited an increasingly strong staining pattern, first observed in the region directly exposed to the light source (Fig. 11D). By contrast, no staining was observed through the sense probe (control; Fig. 11C). Discussion In bacteria, the sequence of the intermediate reaction state of bacteriorhodopsin generated during the photo- cycle has been elucidated to a large extent [45–47]. There is evidence that a single protein conformational change in the cytoplasmic region occurs within a few milliseconds after illumination, and is paralleled by deprotonation of the Schiff base. In the protonated state, this base covalently links a single molecule of retinal to the protein. An analogous photocycle system was studied in plants with the light-responsive protein. This comprises the mononucleotide light-binding fla- vin, oxygen and voltage domain proteins, which have been implicated in phototropic movement [48,49], chlo- roplast relocation [50] and stomatal opening of guard cells [51]. Biochemical evidence that luciferase is involved in circadian rhythms was found in the marine dinoflagellate, Gonyaulax polyedra [52,53]. Subse- quently, the molecular basis of these processes was elu- cidated by Krieger et al. [54], and then completed by molecular sequencing [55]. In mammals, the protein cryptochrome is one regulator in the complex molecu- lar system of the circadian clock [56]. Focusing on the phylum Porifera, the closest relative of the common metazoan ancestor, seminal studies on luminescence were performed by Harvey [57,58]. He described a case of ‘doubtful’ luminous sponge with the hexactinellid Crateromorpha meyeri [59] and the demosponge Grantia sp. [13,57]. Whereas light produc- tion in C. meyeri was attributed to an annelid and, hence considered as a secondary luminescence, Grantia sp. was classified as self-luminous. However, in view of the recently gathered biochemical and molecular bio- logical data, it seems likely that the sponges, with S. domuncula as a potential reference species, are inherently bioluminescent. Siliceous sponges represent the only animal taxon that comprises a complex array of fiber-optic like structures. The biosiliceous material of these skeletal elements not only confers unique physical and mechan- ical properties, but also reaches quartz glass quality [23], which is one reason for the exceptional potential of spicules to operate as optical fibers ex vivo [14,15,60]. Further, recent studies indicate fluorescence properties of spicules in the long-wavelength region [61]. These observations led to the assumption that the poriferan spicular network might be the light-transmit- ting part of an alternative photosensory system [15]. This was supported by the recent finding that sponges themselves, and not their symbiotic bacteria [14], pro- duce light which can be coupled into spicules [16]. This study aims to identify and characterize putative poriferan photoreceptors. Recent studies of corals [27], but also of human and insect cell models [26], suggest the involvement of cryptochromes in a light-sensing response via photoreduction of chromophores. The existence of a poriferan protein homologue was reported in 2003 for the hexactinellid Aphrocallistes vastus [62], in which it was shown that the (6 : 4) pho- tolyases-based system is expressed most highly at the A B C D Fig. 11. In situ hybridization analyses of Suberites domuncula tis- sue. The animals were exposed to light (330–900 nm) for 0 (A), 2 (B,C) or 8 h (D). They were then subjected to whole-mount hybrid- ization, as described in Materials and methods. For hybridization of samples A, B and D the digoxigenin-labeled antisense probe was used, whereas specimen C was treated with the sense probe (con- trol). The direction of light emission is indicated by an arrow. Size bars, 5 mm. W. E. G. Mu ¨ ller et al. Cryptochrome-based photosensory system of sponges FEBS Journal 277 (2010) 1182–1201 ª 2010 The Authors Journal compilation ª 2010 FEBS 1191 [...]... 1266–1245) and the TubB-Probe FAM-5¢-TGTTGGCAACAGCACTGCC ATCCAAGAG-3¢-TAMRA (nucleotides 1177–1204) were used The product size was 141 bp For GAPDH, FAM5¢-CAAGAAGGCTTCAGAAGACCAGACATTGAAGA AC-3¢-TAMRA (nucleotides 854–887), SdGAPDH-F 5¢-TC CAAACCAGCCAAGTACGATG-3¢ (forward primer; nucleotides 816–837) and SdGAPDH-R 5¢-AGTGAGTGT CTCCCCTGAAGTC-3¢ (reverse; nucleotides 945–924) were employed, resulting in a product... AAAGCAGGCTTAGAGTTTGCACTCTATACG-3¢ and attB2_ASP ⁄ Crypto_dom 5¢-GGGGACCACTTTGTACAA GAAAGCTGGGTACTATTGCCTGATTTGACGTAT-3¢ at an initial denaturation at 95 °C for 3 min, followed by 35 amplification cycles at 95 °C for 30 s, 56 °C for 35 s, 72 °C for 45 s, with a decreasing temperature of 0.1 °C in every cycle, and a final extension at 72 °C for 7 min After recombination (in frame with Metstart and 6· His-tag of the expression... Muller WEG (2002) ¨ ¨ Synthesis of the neurotoxin quinolinic acid in apoptotic tissue from Suberites domuncula: cell biological, molecular biological and chemical analyses Mar Biotechnol 4, 546–558 Schmidt I, Hausotte T, Gerhardt U, Manske E & Jager ¨ G (2007) Investigations and calculations into decreasing the uncertainty of a nanopositioning and nanomeasuring machine (NPM-Machine) Meas Sci Technol 18,... photons that are, subsequently, transported within the rod-like part of spicules S domuncula spicules are localized in a highly ordered pattern, immediately below the surface cell layers, in palisade-like arrays Interestingly, the orientation of tylostyles is such that the pointed rods are directed towards the center of the animal, the medulla, whereas the knobs are directed towards the surface This... were cleaned by soaking in nitric acid ⁄ sulfuric acid (1 : 4 v ⁄ v) for 2 days, followed by washing in distilled water until the pH value was 6 The hexactinellid samples were obtained from J Li (Institute of Oceanology, Academia Sinica, Qingdao, China) and from the Museum fur Naturkunde (Berlin, Germany) In one ¨ experiment, tylostyles were exposed to hydrofluoric acid vapor to display the inner architecture... from the Cryptochrome-based photosensory system of sponges phylip package [74] Distance matrices were calculated using the Dayhoff PAM matrix model as described previously [75] The degree of support for internal branches was further assessed by bootstrapping [73] The graphic presentations were prepared with genedoc [76] Preparation of recombinant S domuncula cryptochrome The partial ORF (amino acids... SDCRYPTO was recombinantly expressed in E coli, using GatewayTechnology in combination with the pDEST17 vector, as described previously [44,77] For this purpose, SDCRYPTO cDNA was first inserted into the donor vector pDONR221 (Invitrogen) by BP-recombination after amplification via two primers (SDCRYPTO-specific sequences underlined): attB1_SP ⁄ Crypto_dom 5¢-GGGGACAAGTTTGTACAAA AAAGCAGGCTTAGAGTTTGCACTCTATACG-3¢... luciferase represents only a fraction of the total wavelength range that can be transferred through spicules At present, we assume that this observation has a specific biological relevance First, it is most efficient that in seawater the coupling of a light-generating and a light-receiving system occurs within a range far from maximally absorbed wavelengths (i.e the red region of the electromagnetic spectrum)... pDEST17) the clone SDCRYPTO was expressed in the host strain BL21-AI (Invitrogen), growing in Luria–Bertani medium with 50 lgÆmL)1 carbenicillin, in the presence or absence of 0.2% (w ⁄ v) l-arabinose (overnight at 37 °C) The bacterial pellet was then lysed in ‘BugBuster (primary amine-free) Protein Extraction Reagent’ (Novagen) with 1 lLÆmL)1 benzonase (Novagen ⁄ Merck KGaA, Darmstadt, Germany), supplemented... DNAse was inactivated by DNAse inactivation reagent (‘DNA-free’ kit; Ambion Inc, Austin, TX, USA) Firststrand cDNA was synthesized by M-MLV reverse transcriptase (Promega, Madison, WI, USA) Each reaction (40 lL) contained  10 lg of total RNA, 0.5 mm dNTPs, 100 pmol of oligo(dT)18 and 400 U reverse transcriptase in 50 mm Tris ⁄ HCl buffer Reactions were incubated at 42 °C for 1 h, followed by inactivation . 5¢-GGGGACAAGTTTGTACAAA AAAGCAGGCTTA GAGTTTGCACTCTATACG-3¢ and attB2_ASP ⁄ Crypto_dom 5¢-GGGGACCACTTTGTACAA GAAAGCTGGGTACTA TTGCCTGATTTGACGTAT-3¢ at an initial denaturation at 95 °C for 3 min, followed by 35 amplification. FAM-5¢-TGTTGGCAACAGCACTGCC ATCCAAGAG-3¢-TAMRA (nucleotides 1177–1204) were used. The product size was 141 bp. For GAPDH, FAM- 5¢-CAAGAAGGCTTCA GAAGACCAGAC ATTGAAG A AC-3¢-TAMRA (nucleotides 854–887), SdGAPDH-F 5¢-TC CAAACCAGCCAAGTACGATG-3¢. comprising a large oval axial canal (ac) contain- ing the axial filament. The spicule is embedded in the bulky mesohyl, which is traversed by collagen fibers (co). (E) At a later stage the axial filament