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

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

siliceous sponge Suberites domuncula (Demospongiae) Werner E G Mu¨ller1, Xiaohong Wang2, Heinz C Schro¨der1, Michael Korzhev1, Vladislav A

Grebenjuk1, Julia S Markl1, Klaus P Jochum3, Dario Pisignano4and Matthias Wiens1

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-terN-ter-minal 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.

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 FADH2, 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 milleAcro-pora, the authors showed that expression levels of two cryptochrome genes, cry1 and cry2, were significantly upregulated during exposure to light [27]

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 domunculacan 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

C

E

F

G

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).

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] Silisili-catein 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

sp ac

sp

m

-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.

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) or A 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 cryptocrypto-chrome 2 (CRY2_MOUSE; 5670009); Anopheles gambiae cryptocrypto-chrome 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).

B

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 samsam-ples

(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 Ca2+-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

Fig 4 Protein detection of the Suberites domuncula

crypto-chrome (A) Preparation of recombinant cryptocrypto-chrome 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.

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 Ca2+⁄ Mg 2+

-supplemented medium The 3D cell aggregates were transferred to a silicate cushion (D) for fur-ther experiments Size bars are given.

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 ( )2 h, )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.

(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 spicucellu-lar 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

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 primprim-morphs 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.

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.

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.

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

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.