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 ª2010 The Authors Journal compilation ª2010 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⁄bdomains 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 ª2010 The Authors Journal compilation ª2010 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.
AB
C
E
F
G
JK
HI
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 ª2010 The Authors Journal compilation ª2010 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
mm
m
ABC
DEF
-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 ª2010 The Authors Journal compilation ª2010 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 ª2010 The Authors Journal compilation ª2010 FEBS
