SANS (USH1G) expression in developing and mature mammalian retina.

Abstract

The human Usher syndrome (USH) is the most common form of combined deaf-blindness. Usher type I (USH1), the most severe form, is characterized by profound congenital deafness, constant vestibular dysfunction and prepubertal-onset of retinitis pigmentosa. Five corresponding genes of the six USH1 genes have been cloned so far. The USH1G gene encodes the SANS (scaffold protein containing ankyrin repeats and SAM domain) protein which consists of protein motifs known to mediate protein-protein interactions. Recent studies indicated SANS function as a scaffold protein in the protein interactome related to USH. Here, we generated specific antibodies for SANS protein expression analyses. Our study revealed SANS protein expression in NIH3T3 fibroblasts, murine tissues containing ciliated cells and in mature and developing mammalian retinas. In mature retinas, SANS was localized in inner and outer plexiform retinal layers, and in the photoreceptor cell layer. Subcellular fractionations, tangential cryosections and immunocytochemistry revealed SANS in synaptic terminals, cell-cell adhesions of the outer limiting membrane and ciliary apparati of photoreceptor cells. Analyses of postnatal developmental stages of murine retinas demonstrated SANS localization in differentiating ciliary apparati and in fully developed cilia, synapses, and cell-cell adhesions of photoreceptor cells. Present data provide evidence that SANS functions as a scaffold protein in USH protein networks during ciliogenesis, at the mature ciliary apparatus, the ribbon synapse and the cell-cell adhesion of mammalian photoreceptor cells. Defects of SANS may cause dysfunction of the entire network leading to retinal degeneration, the ocular symptom characteristic for USH patients.

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SANS (USH1G) expression in developing and mature
mammalian retina
Nora Overlack1, Tina Maerker1, Martin Latz, Kerstin Nagel-Wolfrum, Uwe Wolfrum*
Department of Cell and Matrix Biology, Institute of Zoology, Johannes Gutenberg-University of Mainz, Germany
Received 17 July 2007; received in revised form 16 August 2007
Abstract
The human Usher syndrome (USH) is the most common form of combined deaf-blindness. Usher type I (USH1), the most severe
form, is characterized by profound congenital deafness, constant vestibular dysfunction and prepubertal-onset of retinitis pigmentosa.
Five corresponding genes of the six USH1 genes have been cloned so far. The USH1G gene encodes the SANS (scaffold protein con-
taining ankyrin repeats and SAM domain) protein which consists of protein motifs known to mediate protein–protein interactions.
Recent studies indicated SANS function as a scaffold protein in the protein interactome related to USH.
Here, we generated specific antibodies for SANS protein expression analyses. Our study revealed SANS protein expression in
NIH3T3 fibroblasts, murine tissues containing ciliated cells and in mature and developing mammalian retinas. In mature retinas, SANS
was localized in inner and outer plexiform retinal layers, and in the photoreceptor cell layer. Subcellular fractionations, tangential cryo-
sections and immunocytochemistry revealed SANS in synaptic terminals, cell–cell adhesions of the outer limiting membrane and ciliary
apparati of photoreceptor cells. Analyses of postnatal developmental stages of murine retinas demonstrated SANS localization in
differentiating ciliary apparati and in fully developed cilia, synapses, and cell–cell adhesions of photoreceptor cells.
Present data provide evidence that SANS functions as a scaffold protein in USH protein networks during ciliogenesis, at the mature
ciliary apparatus, the ribbon synapse and the cell–cell adhesion of mammalian photoreceptor cells. Defects of SANS may cause dysfunc-
tion of the entire network leading to retinal degeneration, the ocular symptom characteristic for USH patients.
? 2007 Elsevier Ltd. All rights reserved.
Keywords: Usher syndrome; Photoreceptor cells; Connecting cilium; Synapse; Ciliogenesis; Retinal development
1. Introduction
The human Usher syndrome (USH) is the most frequent
cause of combined hereditary deaf-blindness. USH is
genetically heterogeneous with at least 12 chromosomal
loci involved (Ebermann et al., 2007; Reiners, Nagel-Wolf-
rum, Jurgens, Marker, & Wolfrum, 2006). Depending on
the degree of clinical symptoms, USH can be divided into
three types USH1, USH2, and USH3 (Ahmed, Riazuddin,
Riazuddin, & Wilcox, 2003; Davenport & Omenn, 1977;
Petit, 2001). USH1 represents the most severe form, char-
acterized by profound congenital deafness, constant vestib-
ular dysfunction andprepubertal-onset
pigmentosa (RP) (Kremer, van Wijk, Ma ¨rker, Wolfrum,
& Roepman, 2006; Reiners et al., 2006).
The gene products of the nine identified USH genes are
assigned to various protein classes and families as recently
reviewed in Reiners et al. (2006) and Kremer et al. (2006):
USH1B encodes for the molecular motor myosin VIIa.
Harmonin (USH1C), SANS (scaffold protein containing
ankyrin repeats and SAM domain, USH1G) and whirlin,
more recently identified as USH2D (Ebermann et al.,
2007) belong to the group of scaffold proteins. Cadherin
23 (USH1D) and protocadherin 15 (USH1F) represent
cell–cell adhesion proteins, whereas USH2A and USH2C
ofretinitis
0042-6989/$ - see front matter ? 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.visres.2007.08.021
*Corresponding author. Address: Johannes Gutenberg-Universita ¨t,
Institut fu ¨r Zoologie, D-55099 Mainz, Germany. Fax: +49 6131 39 23815.
E-mail address: wolfrum@uni-mainz.de (U. Wolfrum).
1These authors contributed equally to this work.
www.elsevier.com/locate/visres
Available online at www.sciencedirect.com
Vision Research 48 (2008) 400–412

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encode for the large transmembrane proteins, the USH2A
isoform b and the very large G protein coupled receptor 1b
(VLGR1b). The four-transmembrane-domain protein cla-
rin-1 (USH3A) is so far the only identified member of
USH3.
Previous analyses elucidated the assembly of all USH1
and USH2 proteins into an USH protein network mediated
by the USH1C gene product—the scaffold protein harmo-
nin (Adato et al., 2005; Boeda et al., 2002; Reiners, Marker,
Jurgens, Reidel, & Wolfrum, 2005; Reiners et al., 2003,
Reiners, van Wijk, et al., 2005; Siemens et al., 2002; Weil
et al., 2003). Since all proteins of the network were found
at the synapse of photoreceptor cells, a role of this network
in maintaining synaptic integrity was proposed (Reiners,
van Wijk, et al., 2005). In inner ear hair cells the USH pro-
tein network is involved in the development of stereocilia
and in signal transduction (Adato et al., 2005; Boeda
et al., 2002; Kremer et al., 2006). Lately, the scaffold protein
SANS came into focus of interest to mediate further protein
complexes (Adato et al., 2005). Most recent work identified
SANS as an organizer of a harmonin independent USH
protein network at the ciliary apparatus of vertebrate pho-
toreceptor cells (Maerker et al., 2007).
The USH1G gene product SANS is composed of differ-
ent domains (Fig. 1a) capable of mediating protein-protein
interactions (Nourry, Grant, & Borg, 2003; Sedgwick &
Smerdon, 1999; Stapleton, Balan, Pawson, & Sicheri,
1999; Weil et al., 2003). Three N-terminal ankyrin repeats
are followed by a central domain, a SAM (sterile alpha
motif) domain and a class I PBM (PDZ-binding motif) at
the C-terminus. The central domain is interacting with
both MyTH4 (myosin tail homology 4) and FERM (4.1,
ezrin, radixin, moesin) domains of myosin VIIa and medi-
ates SANS homodimerization. The SAM domain interacts
with harmonin PDZ1 and PDZ3 (Adato et al., 2005),
whereas the C-terminus of SANS containing the SAM
domain and the PBM class I interact with whirlin PDZ1
and PDZ2 (van Wijk et al., 2006) (Fig. 1b).
So far, the SANS protein expression and its function
was predominantly studied in inner ear hair cells. Analyses
in SANS deficient jackson shaker mice revealed disorga-
nized stereocilia bundles in the hair cells of the cochlea
(Kikkawa et al., 2003). Further studies elucidated SANS
localization in the apical part and the synapses of outer
and inner hair cells as well as in the basal body of outer
hair cells (Adato et al., 2005). Due to the latter localization
pattern SANS was proposed as an important component
for proper development of hair cells. Since the SANS pro-
tein expression was still elusive, the present study was per-
formed to analyze the localization of SANS during retinal
development and its subcellular distribution in mature
retina.
Here, we show that SANS is not only expressed in
the cochlea, but also in other tissues containing ciliated
cells, as the retina, the brain, the lung, the testis, and
the olfactory epithelium. Immunohistochemistry of adult
murine retina indicated SANS localization in the inner
plexiform layer and outer plexiform layer, as well as
in the photoreceptor cell layer. The subcellular expres-
sion of SANS was further analyzed by different indepen-
dent methods revealing SANS expression at the ciliary
complex and at the synapse of photoreceptor cells.
During postnatal development of the retina SANS was
localized inbasalbodies
photoreceptor cells. Present data provide evidence that
the SANS protein functions as an integral component
of USH protein networks in diverse compartments of
photoreceptor cells.
andmaturatingciliaof
2. Methods
2.1. Animals and tissue preparation
All experiments described herein conform to the statement by the
Association for Research in Vision and Ophthalmology (ARVO) as to
care and use of animals in research. Adult C57BL/6J mice, Wistar Kyoto
albino rats and Xenopus laevis were maintained under a 12 h light–dark
cycle, with food and water ad libitum. After sacrifice of the animals in
CO2(rodents) or chloroform (Xenopus) and decapitation, subsequently
entire eyeballs were dissected or retinas were removed through a slit in
the cornea prior to further analysis. For Western blot analyses, appropri-
ate tissues were homogenized in modified RIPA buffer (10 mM Tris, 1 mM
CaCl2, 0.5% NP-40, 0.5% desoxycholinacid, 0.1% SDS, 150 mM NaCl,
Fig. 1. Domain structure of the USH1G protein SANS and simplified
scheme of the protein interactome related to human Usher syndrome. (a)
SANS (Scaffold protein containing ankyrin repeats and SAM domain)
consists of three N-terminal ankyrin repeats (ANK1-3), a central domain
(CEN) and a C-terminal SAM (sterile alpha motif) domain. The last three
amino acids comprise a PBM class I (PDZ-binding motif I) indicated by
an asterisk. The region chosen for antibody generation is accentuated. (b)
Scheme of a simplified version of the Usher protein interactome in relation
to SANS. Interaction partners of the USH scaffold proteins harmonin,
SANS and whirlin were described in detail in Kremer et al. (2006).
Recently, whirlin was identified as USH2D (Ebermann et al., 2007) and its
binding to MPP1 was more recently shown (Gosens et al., 2007). On the
basis of simplification several protein-protein interactions are not shown.
Confirmed interaction partners are indicated by solid lines, putative
associations by dotted lines.
N. Overlack et al. / Vision Research 48 (2008) 400–412
401

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10 mM NaF, 20 mM b-glycerolphosphate, pH 7.4) containing a protease
inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Pig and
bovine eyeballs were obtained from the local slaughterhouse.
2.2. Antibodies
Polyclonal antibodies to SANS were generated in rabbits against a
recombinant expressed murine SANS fragment (amino acids 1–46).
Expression of the fusion proteins and the purification of antibodies were
performed as described elsewhere (Reiners et al., 2003). Antibodies against
acetylated a-tubulin, c-tubulin, actin, and PSD-95 (clone 7E3-1B8) were
acquired from Sigma–Aldrich (Deisenhofen, Germany). Anti-cytochrome
c antibodies (COX IV) were purchased from Invitrogen (Karlsruhe, Ger-
many), anti-synaptophysin (SVP38), and anti-b-catenin antibodies were
obtained from Santa Cruz Biotechnology (Santa Cruz, USA). Monoclonal
antibodies against centrins (clone 20H5, detecting all four centrin iso-
forms) and opsin (clone K16-155) were previously described (Adamus,
Arendt, Zam, McDowell, & Hargrave, 1988; Adamus, Zans, Arendt, Pal-
czewski, McDowell, & Hargrave, 1991; Wolfrum & Salisbury, 1998; Wolf-
rum & Schmitt, 2000). Secondary antibodies were purchased from
Invitrogen, Sigma–Aldrich or Rockland (Gilbertsville, USA). Preadsorp-
tion of anti-SANS antibodies was performed by incubation of antibodies
for 1–2 h at room temperature with 1 mg/ml of the specific antigen used
for immunization. After short centrifugation preadsorbed antibodies were
applied to the retina cryosection or Western blot membrane in the appro-
priate dilution, and were treated like others.
2.3. Isolation of ROS
Rod outer segments (ROS) were purified from bovine retinas using the
discontinuous sucrose gradient method as previously described (Paper-
master, 1982; Pulvermu ¨ller et al., 2002). Since immunofluorescence
microscopy showed no differences between dark and light adaptation,
the procedure was performed under light. The purity of ROS was con-
firmed by Western blot analyses. Briefly, retinas were vortexed in 1.4 ml
homogenizing medium (1 M sucrose, 65 mM NaCl, 0.2 mM MgCl2,
5 mM Tris–acetate, pH 7.4) and centrifuged at 2000g for 4 min at 4 ?C.
The supernatant was diluted with twice of its volume in 0.01 M Tris–ace-
tate, pH 7.4, and gently mixed. ROS were pelleted at 2000g for 4 min at
4 ?C and resuspended in the first density gradient solution (1.10 g/ml
sucrose, 0.1 mM MgCl2, 1 mM Tris–acetate, pH 7.4). Crude ROS were
carefully overlayed on top of a three-step gradient (1.11, 1.13, and
1.15 g/ml sucrose with 0.1 mM MgCl2, 1 mM Tris–acetate, pH 7.4) and
centrifuged at 85,000g for 30 min. The interface containing ROS was
recovered, diluted with 0.01 mM Tris–acetate, pH 7.4, and ROS were pel-
leted again at 50,000g for 20 min. The pellet containing ROS was resus-
pended and stored at ?80 ?C prior to use in Western blot analyses.
2.4. Isolation of the ciliary apparatus by differential density gradient
centrifugation
Isolation of ciliary apparati were performed as previously described
(Fleischman, 1981; Horst, Forestner, & Besharse, 1987; Schmitt & Wolf-
rum, 2001; Wolfrum & Schmitt, 2000). Briefly, bovine retinas were isolated
and kept at ?80 ?C. Frozen retinas were thawed in HBS buffer (115 mM
NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM Hepes, pH 7), neuronal cells were
cracked by gentle shaking for 1 min, filtered (400 lm, Millipore, Schwal-
bach, Germany) and enriched by a Sorvall RC-5B centrifuge, fixed angle
rotor SS34, for 20 min at 4 ?C and 48,000g. The pellet was resuspended in
50% sucrose (w/v), overlaid by HBS buffer and ultracentrifuged (Beck-
man-Coulter Optima max, rotor MLS 50) at 4 ?C, 31,000g for 1 h. Cell
fragments on the top of sucrose cushion were collected, added to a contin-
uous sucrose gradient 25–50% and overlaid with buffer. After centrifuga-
tion for 2 h at 4 ?C, 31,000g, two bands were collected. Probes were
sedimented by decreasing the sucrose concentration. Pellets were resus-
pended in cytoskeleton extraction buffer (100 mM Hepes, 10 mM MgSO4,
10 mM EGTA, 100 mM KCl, 5% DMSO, 20 mM DTT, 2% Triton X-100
adjusted to pH 7.5) and extracted on ice for 1 h. Ciliary apparati were sep-
arated by centrifugation for 3 h at 4 ?C and 31,000g on a discontinuous
sucrose gradient composed of 40, 50, and 60% (w/v) sucrose in a modified
cytoskeleton extraction buffer (100 mM Hepes, 10 mM MgSO4, 10 mM
EGTA, 100 mM KCl).
2.5. Isolation of crude synaptosomes
Crude synaptosomes were prepared as described elsewhere (Hirao
et al., 1998; Reiners et al., 2003). In brief, the brain of an adult mouse
was homogenized in 800 ll extraction buffer (0.32 M sucrose in 4 mM
Hepes, pH 7.4) and centrifuged at 800g for 10 min at 4 ?C. The superna-
tant was centrifuged at 9200g for 15 min at 4 ?C. The pellet was resus-
pended in 800 ll extraction buffer (0.32 M sucrose in 4 mM Hepes, pH
7.4) and centrifuged at 10,200g for 15 min at 4 ?C. The crude synaptosome
fraction was recovered in the pellet and resuspended in 800 ll buffer con-
taining 20 mM Hepes/NaOH (pH 8.0), 100 mM NaCl, 5 mM EDTA, and
1% Triton X-100 and centrifuged at 100,000g for 30 min at 4 ?C. The
supernatant was used in Western blot analyses.
2.6. Serial tangential sectioning
Western blot analyses of serial tangential sections were performed as
described by Reiners et al. (2003). Briefly, after removal of the vitreous,
dissected rat eye cups were cut at four opposite sites and flat-mounted
between two glass slides separated by 0.5 mm spacers. The bottom slide
facing the basal membrane of the retina was roughened with sandpaper
to improve adhesion. The top slide facing the outer side of the eye cup
was covered with teflon spray to improve later release. The glass slide
and eye cup sandwich was hold together by two small binder clips and fro-
zen immediately on dry ice. The bottom slide with the eye cup was
mounted into a cryomicrotome and sequential 10 lm tangential sections
of the eye cups were collected in 100 ll SDS–PAGE sample buffer. Ali-
quots of 10 ll per lane were subjected to Western blot analyses.
2.7. Western blot analyses
For denaturing gel electrophoresis, the samples were mixed with SDS-
PAGE sample buffer (62.5 mM Tris–HCl, pH 6.8; 10% glycerol; 2% SDS;
5% b-mercaptoethanol; 1 mM EDTA; 0.025% bromphenol blue). Twenty-
five micrograms of protein extract per lane were separated on 12% poly-
acrylamide gels and transferred onto PVDF membranes (Millipore).
Immunoreactivities were detected with the appropriate primary and corre-
sponding secondary antibodies (IRDye 680 or 800, Rockland) employing
the Odyssey infra red imaging system (LI-COR Biosciences, Lincoln, NE,
USA). In case of the use of the ECL detection system (Amersham Biotech/
GE Healthcare, Freiburg, Germany) donkey anti-rabbit secondary anti-
bodies coupled to horseradish peroxidase were applied to Western blot
membranes. Band sizes were calculated using Total Lab software (Phoret-
ics, UK). As a molecular marker a prestained ladder (Sigma–Aldrich),
ranging from 11 to 170 kDa was used.
2.8. Immunofluorescence microscopy
Eyes from developing and adult mice were cryofixed directly in melting
isopentane and cryosectioned as previously described (Wolfrum, 1991).
Cryosections were placed on poly-L-lysine-precoated coverslips and incu-
bated with 0.01% Tween 20 in PBS. PBS washed sections were blocked
with blocking solution (0.5% cold water fish gelatin, 0.1% ovalbumin in
PBS) for 30 min, and then incubated with primary antibodies in blocking
solution overnight at 4 ?C. Washed sections were subsequently incubated
with secondary antibodies conjugated to Alexa 488 or Alexa 568 (Molec-
ular Probes, Leiden, Netherlands) and DAPI (Sigma–Aldrich) for visual-
ization of the nuclei, in blocking solution for 1–2 h at room temperature in
the dark. After washing with PBS, sections were mounted in Mowiol 4.88
(Hoechst, Frankfurt, Germany). In none of the appropriate controls a
reaction was observed. Mounted retinal sections were examined with a
402
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Leica DMRB microscope (Leica microsystems, Bensheim, Germany).
Images were obtained with a Hamamatsu ORCA ER CCD camera (Ham-
amatsu, Herrsching, Germany) and processed with Adobe Photoshop CS
(Adobe Systems, San Jose, USA).
2.9. Cell culture
NIH3T3 cells were cultured in Dulbecco’s modified Eagle’s medium
supplemented with 10% heat-inactivated fetal calf serum (FCS) and
2 mM glutamine. Immunofluorescence analyses were carried out on cells
seeded on coverslips, followed by fixation with methanol and proceeded
as previously described (Nagel-Wolfrum et al., 2004).
3. Results
3.1. Generation and validation of specific anti-SANS
antibodies
Knowledge of expression profiles of proteins and their
subcellular localization provide important insights in their
specific function. So far, little was known about the expres-
sion and subcellular distribution of SANS. We generated a
polyclonal antiserum against recombinant expressed mur-
ine SANS protein as a tool to study the expression and sub-
cellular localization of SANS. Western blot analyses of
mouse retina extracts revealed that affinity purified anti-
SANS antibodies decorated a band at approximately
55 kDa (Fig. 2A), corresponding to the predicted size of
SANS (52 kDa). To validate the specificity of anti-SANS
antibodies, Western blot analyses of extracts of mouse ret-
ina were performed with anti-SANS antibodies pread-
sorbed with the antigen used for immunization. The
appropriate band for SANS was abolished (Fig. 2A), indi-
cating the specificity of the anti-SANS antibodies. We fur-
ther validated the specificity of the affinity purified
antibodies on retinal cryosections (Fig. 2C–E). The specific
SANS labeling described below was abolished when pread-
sorbed antibodies were applied to sections (Fig. 2E).
3.2. SANS protein is expressed in ciliated tissues
With these specific anti-SANS antibodies in hand, we
analyzed the expression of SANS for the first time on pro-
tein level in various murine tissues (Fig. 3). Our Western
blot analyses revealed SANS-specific bands of 55 kDa in
protein extracts of the retina, the cochlea, the brain, the
lung, the testis, the olfactory epithelium and of NIH3T3
cells. In addition to this 55 kDa band, a fade band at
Fig. 2. Validation of anti-SANS antibodies by Western blot analyses and indirect immunofluorescence analyses of mouse retinas. (A) Western blot
analysis of protein extract of mature mouse retina with affinity purified antibodies against SANS. A specific band with the molecular weight of
approximately 55 kDa was obtained (lane 1). This band was completely abolished after preadsorption of anti-SANS antibodies with the corresponding
antigen (lane 2). Coincubation with anti-actin was used as a loading control. (B) Schematic representation of a vertebrate rod photoreceptor cell.
Vertebrate photoreceptors are composed of distinct morphological and functional compartments. The photosensitive outer segment (OS) is connected by
the connecting cilium (arrowheads) with the biosynthetic active inner segment (IS). The cell body is localized in the outer nuclear layer (ONL) and contains
the nucleus (N) and the synaptic terminal in the outer plexiform layer (OPL) of the retina. (C) Differential interference contrast (DIC) image visualizing the
different retinal layers. (D) Indirect immunofluorescence analysis of anti-SANS on a longitudinal cryosection through a mature mouse retina. Anti-SANS
immunofluorescence was localized in the photoreceptor cell layer and in the plexiform layers. In photoreceptor cells, SANS was detected at the ciliary
complex, in the inner segment, the outer limiting membrane and at synapses in the OPL. No labeling was observed in the retinal pigmented epithelium
(RPE) and the ganglion cell layer (asterisk in C). (E) Parallel section to (C and D), incubated with preadsorbed anti-SANS antibodies. After
preadsorption, anti-SANS staining was abolished. Scale bar: 20 lm.
N. Overlack et al. / Vision Research 48 (2008) 400–412
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?72 kDa was reproducible found in cochlea protein
extracts. In contrast, no bands were obtained in kidney
and liver tissue samples. In conclusion, our data indicate
that the SANS protein is preferentially expressed in tissues
containing ciliated cells.
3.3. SANS is localized in the photoreceptor cell layer, the
inner plexiform layer and the outer plexiform layer of the
mammalian retina
To determine the subcellular distribution of SANS in
the retina, cryosections through mouse eyes were analyzed
by immunofluorescence microscopy using affinity-purified
anti-SANS antibodies. SANS expression was present in
the photoreceptor cell layer, the outer limiting membrane,
the inner plexiform layer and the outer plexiform layer of
the retina (Fig. 2D). In contrast, no staining was observed
in cells of the retinal pigmented epithelium (Fig. 2D). The
same staining pattern by indirect immunofluorescence with
anti-SANS antibodies were obtained in cryosections
through the mature retina of other mammals, namely rats
and pigs (data not shown). Furthermore, SANS expression
was found in the photoreceptor cell layer, the outer limiting
membrane and the plexiform layers of retinas of the
amphibian X. laevis (data not shown).
3.4. SANS protein localization at the ciliary apparatus, the
outer limiting membrane and the synapse of photoreceptor
cells
To analyze the expression of SANS in photoreceptor
cells immunohistochemical and biochemical analyses were
performed. In a first set of experiments, the subcellular
distribution of SANS was elucidated by immunohisto-
chemistry of mature murine photoreceptors (Fig. 4).
Immunofluorescence double labeling experiments with
antibodies against SANS and marker proteins of subcellu-
lar compartments of photoreceptor cells were performed.
Labeling with anti-pan-centrin antibodies (marker for the
connecting cilium and basal body complex; Gießl et al.,
2004)andanti-SANSantibodies revealedpartial
colocalization (Fig. 4A–C). This indicated SANS as a
component of the connecting cilium and of the basal body
complex. Furthermore, SANS was colocalized with b-cate-
nin, a marker for the cell–cell adhesion in the inner seg-
ment at the outer limiting membrane (Golenhofen &
Drenckhahn, 2000; Mehalow et al., 2003; Montonen,
Aho, Uitto, & Aho, 2001) (Fig. 4D–F). In addition, anti-
bodies against PSD-95 were applied. Although, PSD-95
is ubiquitously found in the post-synaptic dense differenti-
ations (Kornau, Seeburg, & Kennedy, 1997), PSD-95 is
known to be abundantly expressed in pre-synaptic termi-
nals of photoreceptor cells (Koulen, Garner, & Wassle,
1998). Double labeling with anti-PSD-95 and anti-SANS
antibodies revealed an overlapping staining pattern at syn-
aptic terminals of photoreceptor cells (Fig. 4G–I). The
obtained labeling for SANS was more distinct at the pre-
synapse (Fig. 4G–I), indicating its localization in the syn-
aptic terminal of rod and cone photoreceptor cells. A
diminished localization for SANS was observed at post-
synapses in second order neurons, in bipolar and horizon-
tal cells.
3.5. SANS presence in subcellular fractionations of
synaptosomes and photoreceptor cilia
To occlude the antigen masking occasionally observed
in tissue sections we determined the subcellular localiza-
tion of SANS in photoreceptor cells by Western blot
analyses. Therefore, we carried out subcellular fractiona-
tions of photoreceptor cells and tangential cryosections
through the rat retina. Western blot analyses revealed
the presence of SANS in protein extracts of mouse and
bovine retinas, in brain synaptosome fraction of mouse
and in the ciliary fraction of bovine photoreceptor cells
(Fig. 5a). However, in the fraction of isolated rod outer
segments (ROS) a weak band was obtained which
occurred most likely due to contamination of the ROS
fraction with ciliary components. Differential density gra-
dient centrifugation assays revealed that SANS was
cosedimented with the ciliary marker acetylated a-tubulin
(Fig. 5b).
Fig. 3. SANS expression in murine ciliated tissues. (a and b) Western blot analyses of protein extracts of adult murine retina, cochlea, brain, kidney, lung,
testis, liver, olfactory epithelium and NIH3T3 cells with anti-SANS antibodies. A specific band with a molecular weight of approximately 55 kDa was
detected in NIH3T3 cells and in all analyzed tissues, apart from kidney and liver. The SANS-positive tissues contain ciliated cells. In the Western blots
shown in (a), actin was used as loading control.
404
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3.6. Analysis of serial tangential cryosections of the retina
confirmed SANS localization in the ciliary apparatus and
synapse of photoreceptor cells
To validate the localization of SANS in photoreceptor
cells we analyzed the protein distribution in serial tangen-
tial cryosections through the rat retina by Western blot
analyses. For this purpose, we used antibodies against
opsin, cytochrome c, and synaptophysin as markers to dis-
tinguish between the subcellular photoreceptor compart-
ments (Reiners et al., 2003). Our analyses revealed that
SANS was localized in the synaptic region and the inner
segment, whereas SANS was not detected in the anti-opsin
positive outer segment sections (Fig. 5c). Since SANS
bands were obtained in sections at the transition of the
compartment markers for the inner segment and outer seg-
ment, we concluded the presence of SANS in the ciliary
apparatus of photoreceptor cells. This conclusion was fur-
ther supported by the present semi-quantification of SANS
protein in serial tangential sections. For this purpose, the
optical density of Western blot bands was ascertained.
These analyses revealed that SANS was enriched in the cil-
iary region more than two fold in comparison to other sub-
compartments of photoreceptor cells (Fig. 5d). The applied
Fig. 4. Double labeling revealed SANS localization in the photoreceptor cilium, the outer limiting membrane and outer plexiform synapses of murine
retinas. (A–I) Indirect double immunofluorescence analyses of photoreceptor compartments in retinal cryosections. (A–C) Double labeling with anti-
SANS (A) and anti-pan-centrin antibodies (B), a marker for the ciliary apparatus (connecting cilium and the basal body). (C) Merge image of SANS and
centrin labeling superposed with the nuclear DNA staining by DAPI. SANS and centrin were partially colocalized in the ciliary apparatus of
photoreceptor cells. (D–F) Double labeling of anti-SANS (D) and anti-b-catenin antibodies (E), a molecular marker for the outer limiting membrane. (F)
Merge image of SANS and b-catenin labeling superposed with DAPI staining. SANS and b-catenin were colocalized at the outer limiting membrane
(arrowhead). (G–I) Double labeling with anti-SANS (G) and anti-PSD-95 antibodies (H), a marker for post-synapses, also staining pre-synaptic terminals
in retinas. (I) Merge image of SANS and PSD-95 labeling superposed with DAPI staining. SANS and PSD-95 staining was colocalized in the synaptic
terminals of photoreceptor cells (asterisk) and partially overlaps in the post-synaptic region of 2nd order neurons (bipolar cells and horizontal cells)
(arrow). Scale bar: 10 lm.
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independent methods confirmed our previous immunohis-
tochemical localization of SANS in the ciliary apparatus
and at the synaptic region of mammalian photoreceptor
cells.
3.7. SANS protein expression during postnatal maturation of
the murine retina
We analyzed the expression and localization of SANS
during various developmental stages of postnatal murine
retinas. The expression of SANS protein was detected by
Western blot analyses in all selected postnatal stages,
namely PN0, PN7, PN14, and PN20 (Fig. 6). In addition,
we investigated the localization of SANS during retinal
development by indirect immunofluorescence and DAPI
counterstaining of the nuclear DNA (Fig. 7). Longitudinal
sections of PN0 mouse eyes revealed SANS expression at
the most apical tip of the neuroblast layer proximal to
the retinal pigmented epithelium (Fig. 7A and E). Double
labeling with anti-pan-centrin antibodies identified the
punctuated anti-SANS stained structures as centrioles
and basal bodies in photoreceptor precursors (Fig. 7I
and M). The labeling of the differentiating ciliary appara-
tus of photoreceptor cells persists during all following
developmental stages investigated (Fig. 7A–P). In the
outer plexiform layer anti-SANS immunofluorescence
was observed from PN14 on (Fig. 7C/G and D/H). At this
retinal developmental stage synapses are formed in the
outer plexiform layer in the mouse retina. SANS was local-
ized at the cell–cell adhesion of the outer limiting mem-
brane not before PN20 (Fig. 7D/H and L/P), when the
mouse retina is fully maturated (von Kriegstein & Schmitz,
2003).
3.8. SANS localization at centrosomes and spindle poles of
NIH3T3 cells
Our Western blot analyses revealed SANS expression in
NIH3T3 fibroblasts. This prompted us to analyze the sub-
Fig. 5. Biochemical fractionations and analysis of serial tangential sections of retinas confirm SANS subcellular localization in photoreceptor cells. (a)
Western blot analyses of subcellular fractions of mouse and bovine retinas. Prominent staining for SANS (?55 kDa) was present in total retina extract and
the ciliary fraction of bovine photoreceptor cells (fraction 6 in b), whereas a weak band for SANS was obtained in rod outer segments (ROS). SANS was
also detected in retina extract and in the synaptosome fraction of mice brain. (b) Western blot analyses of detergent-lysed bovine ciliary fractions harvested
after differential gradient centrifugation. SANS cosediments with the ciliary marker acetylated a-tubulin. (c) Western blot analyses of tangential
cryosections through a rat retina. Each lane corresponds to a 10 lm thick slice of the photoreceptor layer. Western blots with antibodies against opsin,
cytochrome c and synaptophysin were used to determine the photoreceptor compartments assigning the outer segment, the inner segment and the synaptic
region. SANS was detected in all tangential sections apart from anti-opsin positive slices of the outer segment. (d) SANS quantification by TotalLab
software. The highest SANS protein concentration was present in sections at the transition of the compartment markers for the inner segment (marker:
cytochrome c) and outer segment (marker: opsin)—the ciliary region of photoreceptor cells.
Fig. 6. SANS expression during postnatal differentiation of mouse retina.
Western blot analyses with anti-SANS and anti-actin antibodies (loading
control) of murine retina at different postnatal developmental stages,
namely PN0, PN7, PN14, and PN20. SANS was detected at 55 kDa in all
analyzed postnatal developmental stages.
406
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cellular localization of SANS in cultured NIH3T3 fibro-
blasts to gain insight whether SANS has in addition an
impact in non specialized cell types. Indirect immunofluo-
rescence microscopy revealed partial colocalization of
SANS with the centrosomal marker c-tubulin at the cen-
trosome of interphase cells (Fig. 8B and D). Further indi-
rect immunocytochemical
indicated that SANS was located at spindle pole bodies,
essential for proper cell division (Fig. 8F and G). The
obtained data point towards a general function of SANS
at centrosome related structures, like basal bodies of cilia
or centrioles.
analyses of mitoticcells
4. Discussion
Recent studies indicated that SANS functions as a scaf-
fold in the USH protein interactome (Adato et al., 2005;
Weil et al., 2003; summarized in: Reiners et al., 2006; Kre-
mer et al., 2006). So far, besides SANS homomer forma-
tion, interactions with harmonin isoforms a and b
(USH1C), myosin VIIa (USH1B), and whirlin (USH2D)
have been demonstrated (Adato et al., 2005; van Wijk
et al., 2006; Weil et al., 2003; Maerker et al., 2007).
In the present study, we demonstrate that SANS protein
expression is not restricted to the tissues mainly affected by
Fig. 7. SANS expression in photoreceptor cells of the maturating retina. (A–H) Indirect immunofluorescence with anti-SANS antibodies in cryosections
through mouse retinas at different developmental stages. (E–H) Different nuclear layers in the developing retina were identified by counterstaining with
DAPI. (A and E) In the PN0 retina, only the retinal pigmented epithelium (RPE), neuroblast layer (NBL), inner plexiform layer (IPL) and ganglion cell
layer (GCL) are distinguishable. SANS labeling showed a punctuated staining pattern in the apical part of the developing retina, beneath the RPE cells. (B
and F) In PN7, the neuroblast layer (NBL) is already divided in outer nuclear layer (ONL), the outer plexiform layer (OPL) and the inner nuclear layer
(INL). Punctuated SANS staining was visible proximal to the RPE. (C and G) In PN14 eyes, SANS was localized in the ciliary region of photoreceptor
cells (asterisk) and in the OPL, where synapses are differentiated. (D and H) In PN20 eyes, SANS labeling was localized at the ciliary region of
photoreceptor cells (asterisk), in the OPL and in a thin line underneath the inner segment (IS), representing the outer limiting membrane (arrowhead).
Outer segment = OS. (I–P) High magnification analyses of double immunofluorescence of ciliary regions (asterisks) in diverse developmental stages of
murine photoreceptor cells shown in (A–H). (I–L) Indirect immunofluorescence of anti-SANS antibodies (green). (M–P) Merged images of anti-SANS
(green) and anti-pan-centrin antibodies (red; a frequently used marker for centrioles, basal bodies and cilia of vertebrate photoreceptor cells). (I and M) In
PN0 retinas, SANS was partially colocalized with centrins in basal bodies as soon as these are formed, beneath the apical membrane of photoreceptor
precursor cells. (J and N) In PN7, SANS labeling revealed partial overlap with centrins in basal bodies and ciliary sprouts present in differentiating
photoreceptor cells at this developmental stage. (K and O) In PN14, SANS was partially colocalized with centrins in basal bodies and the developing
connecting cilium. (L and P) In PN20, SANS was partially colocalized with centrins in basal bodies and the connecting cilium. In addition, SANS was
stained at the outer limiting membrane (arrowhead), which was not stained with anti-pan-centrin antibodies. Scale bars: 10 lm. (For interpretation of the
references to the color in this figure legend the reader is referred to the web version of this article.)
N. Overlack et al. / Vision Research 48 (2008) 400–412
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USH, the retina and inner ear. Western blot analyses
revealed SANS specific bands at 55 kDa in further tissues,
namely the brain, the lung, the testis and in the olfactory
epithelium. Interestingly, we also obtained a band at
?72 kDa in protein extracts of cochleae. So far, we do
not know the nature of this protein band. It may result
from unspecific cross reactivity of the affinity purified
anti-SANS antibody restricted to the cochlea protein
extracts. Or, the band may represent the labeling of a
higher molecular splice variant of SANS. Such alterna-
tively spliced isoforms are well known from other USH
proteins (Kremer et al., 2006; Petit, 2001; Reiners et al.,
2006). Nevertheless, the obtained results for SANS protein
expression correlate to previous mRNA expression analy-
ses by RT-PCR (Johnston et al., 2004; Weil et al., 2003).
All SANS positive tissues have the presence of ciliated cells
in common. In SANS negative tissues, kidney and liver
which also contain cells with primary cilia the expression
of the SANS homologous Harp (harmonin-interacting,
ankyrin repeat-containing protein) was shown (Johnston
et al., 2004). Thus, Harp may resume the functional role
of SANS in these tissues.
The tissue expression of the SANS protein is in line with
the rather wide expression profile of other USH1 and 2
proteins (reviewed in: Reiners et al., 2006). This is in agree-
ment with several studies on USH patients which indicate
that USH can also affect other tissues, namely brain areas,
olfactory and tracheal epithelia as well as sperm cells (see
overview in: (Reiners et al., 2006). Such studies gathered
histopathological data from USH patients displaying cili-
ary abnormalities not only in photoreceptor cells but also
in olfactory epithelium and sperms. Based on these obser-
vations it has been suggested that USH is related to ciliary
dysfunction (Arden & Fox, 1979), which is supported by
the present study. SANS protein expression in tissues con-
taining ciliated cells indicates that the pathophysiology of
USH1G may also encroach cilia in cells of these tissues.
Our data show that SANS is expressed in the photore-
ceptor cell layer, the inner plexiform layer and the outer
plexiform layer of the mammalian retina. Applied subcellu-
larbiochemical fractionations
approaches determined the subcellular localization of
SANS in photoreceptor cells at the ciliary apparatus and
the synapse as well as at adhesion complexes of the outer
and immunological
Fig. 8. SANS localization at centrosomes and spindle poles of NIH3T3 cells. (A–D) Double labeling of SANS (B) and c-tubulin (C) in NIH3T3 cells by
indirect immunofluorescence. (A) Differential interference contrast (DIC) image. (B) SANS staining was present throughout the cytoplasm and
concentrated in a perinuclear spot. (C) Anti-c-tubulin antibodies stained centrosomes. (D) Merged immunofluorescence images demonstrated partial
colocalization of SANS and c-tubulin at centrosomes. Nuclear DNA was stained with DAPI. (E–G) Localization of SANS in dividing NIH3T3 cells. (E)
Differential interference contrast image. (F) SANS immunofluorescence analysis revealed SANS localization at spindle poles of dividing NIH3T3 cells. (G)
Double labeling with anti-SANS antibodies and DAPI. Scale bar: 10 lm.
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limiting membrane. Immunofluorescence analyses revealed
that SANS was colocalized with markers for these subcel-
lular photoreceptor compartments. Latter data were con-
firmed by results achieved by Western blot analyses of
tangential cryosections. Furthermore, the localization of
SANS in the ciliary apparatus and at the synapse of photo-
receptor cells was corroborated by the enrichment of
SANS in fractions of these compartments obtained by
sucrose density gradient centrifugation. In all analyzed
subcellular compartments of photoreceptor cells, networks
of USH proteins were previously described (reviewed in:
(Reiners et al., 2006). SANS may function as an integral
component of these USH protein networks in diverse pho-
toreceptor compartments. The integration of SANS in the
USH protein interactome is shown in a schematic represen-
tation in Fig. 1b. The functional relevance of compartment
specific interactions of SANS with its partner proteins will
be discussed in the following paragraphs.
The ciliary apparatus of photoreceptor cells consists of a
basal body complex from which the non-motile connecting
cilium originates (reviewed in: (Besharse & Horst, 1990;
Roepman & Wolfrum, 2007). The connecting cilium is
placed in a strategic position at the joint between the inner
segment and the outer segment of the photoreceptor cell.
All exchanges between the inner and outer segment occur
through the narrow and slender connecting cilium (Wolf-
rum, 1995). Currently, two in principle different alternative
mechanisms of active transport through the connecting cil-
ium towards the photoreceptor outer segment are discussed
(Williams, 2002; Roepman & Wolfrum, 2007): (i) A micro-
tubule-based translocation mediated by kinesin II associ-
ated withintraflagellar transport
(Marszalek et al., 2000; Rosenbaum & Witman, 2002).
(ii) Previous studies indicated that the USH network pro-
tein myosin VIIa (USH1B) is capable to transport cargo
along actin filaments within the ciliary membrane (Liu,
Udovichenko, Brown, Steel, & Williams, 1999; Wolfrum
& Schmitt, 2000; Maerker et al., 2007; present study). In
both alternative transport mechanisms SANS may partici-
pate. We have previously shown that SANS directly inter-
acts with myosin VIIa (Adato et al., 2005). Similar
localization of both proteins in the connecting cilium
(Liu, Vansant, Udovichenko, Wolfrum, & Williams,
1997; Wolfrum & Schmitt, 2000) indicates that these inter-
actions may take place within the ciliary compartment of
photoreceptor cells. In this protein complex, SANS may
provide the linkage to the prominent microtubule cytoskel-
eton of the cilium. An association of SANS with microtu-
bules has been previously discussed (Adato et al., 2005;
Roepman & Wolfrum, 2007). This is further supported
by present results on SANS localization at microtubule
organization centers, namely centrosomes and spindle
poles in NIH3T3 cells and by a recent study by Maerker
et al. (2007).
However, the integration of SANS into a ciliary USH
protein network linked by myosin VIIa to actin filaments
does not exclude an involvement of SANS in processes
(IFT) complexes
related to IFT complexes predominantly associated with
microtubules. In photoreceptor cells, SANS and IFT pro-
teins were both detected in the connecting cilium and in
the basal body region (present study; Pazour et al., 2002;
Maerker et al., 2007). In the basal body region, IFT pro-
teins are thought to recruit cargo from inner segment trans-
port carriers for the transport route through the connecting
cilium (Pazour et al., 2002). SANS may also participate in
processes of cargo handover between inner segment trans-
port and the delivery through the connecting cilium (Maer-
ker et al., 2007). A role for SANS in the transport of
vesicles in inner ear hair cells has been previously sug-
gested, where SANS was found in the periphery of the
cuticular plate and in the basal body complex of the kino-
cilium of outer hair cells (Adato et al., 2005).
Our present study revealed that SANS is not only asso-
ciated with the cilium of adult photoreceptor cells, but also
during maturation of photoreceptor cells. During develop-
mental stages PN0 and PN7, SANS was exclusively present
at the apical tip of the neuroblast layer. During this time
period ciliogenesis proceeds, basal bodies (centrioles) are
placed in the apical inner segments and ciliary sprouts
are formed at the photoreceptor apices (Uga & Smelser,
1973; Woodford & Blanks, 1989). Present double immuno-
fluorescence analyses of SANS and centrins indicate that
SANS is localized in basal bodies and growing cilia of mat-
urating photoreceptor cells. This is in agreement with an
association of SANS with the basal body complex of the
kinocilium in differentiating outer hair cells of the inner
ear (Adato et al., 2005). The presence of SANS in basal
bodies of ciliated cells in tissues is further strengthened
by the localization of SANS at the centrosomes and spindle
poles of NIH3T3 cells. In general, basal bodies from which
cilia arise are homologous to the mother centriole of cen-
trosomes (Dawe, Farr, & Gull, 2007).
The present study revealed the localization of SANS at
the outer limiting membrane also known as the membrana
limitans externa (Spitznas, 1970). The outer limiting mem-
brane is a layer of cell–cell adhesion contacts, mechanically
strengthening the adhesion between photoreceptor cells
and Mueller glia cells. Previous studies indicated that
besides SANS other USH proteins are present in the outer
limiting membrane (Ahmed et al., 2003; Reiners, Marker,
et al., 2005; van Wijk et al., 2006). Homo- or heterophilic
interaction of the ectodomains of protocadherin 15
(USH1F), cadherin 23 (USH1D), USH2A isoform b, and
VLGR1b (USH2C) are thought to design connectors
between the adjacent membranes of photoreceptor cells
and Mueller glia cells. These contacts may be facilitated
through intracellular tether of their cytoplasmic domains
by parallel direct binding to the PDZ domains 1 and 2 of
the scaffold protein whirlin (USH2D) (van Wijk et al.,
2006). The direct interaction of SANS with whirlin may
provide a conjunction of this protein network at the outer
limiting membrane to microtubules.
Recently, we described the direct interaction of whirlin
with MPP1, a membrane associated guanylate kinase
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(MAGUK) protein of the large Crumbs protein complex at
the outer limiting membrane (Gosens et al., 2007). There-
fore, the USH protein network at the outer limiting mem-
brane may be part of this multiprotein complex mainly
organized by Crumbs1 and MPP5 (Gosens et al., 2007;
Kantardzhieva et al., 2005). This complex is thought to
serve in cell polarity and cell adhesion processes that are
intimately connected and governs the formation and main-
tenance of the layered structure of vertebrate retina
(Gosens et al., 2007; Kantardzhieva et al., 2005). The iden-
tified USH proteins may contribute to the function of the
specialized Crumbs protein cell–cell adhesion complex at
the outer limiting membrane. In conclusion, SANS may
fulfill a role in bridging this cell–cell adhesion complex to
the microtubule cytoskeleton and may participate in trans-
port processes, necessary for the development and mainte-
nance of the outer limiting membrane.
Our previous studies demonstrated localization of all
identified USH1 and 2 proteins at photoreceptor and hair
cell synapses (Reiners, van Wijk, et al., 2005, Reiners
et al., 2006; van Wijk et al., 2006). Here, we confirm SANS
as a further molecular component of the ribbon synapse of
rod and cone photoreceptor cells. Present studies of devel-
opmental stages of the murine retina revealed that SANS
protein expression was not found until PN14 when the syn-
aptogenesis of ribbon synapses is terminated (von Krieg-
stein & Schmitz, 2003). Since our present data did not
cover all stages during the critical period of synaptogenesis,
we can not state whether or not SANS participates in the
latter process. However, our data strengthen that SANS
is part of the USH protein network present at the synapse
in mature photoreceptor cells (reviewed in (Reiners et al.,
2006). The current data set indicates that the PDZ domain
containing USH scaffold proteins, harmonin and whirlin
target the network components to the specialized ribbon
synapses and tether their physiologic function there (Rein-
ers et al., 2003, Reiners, van Wijk, et al., 2005; van Wijk
et al., 2006). In the pre- and post-synaptic membrane of
specialized photoreceptor synapses USH cadherins, cad-
herin 23, and protocadherin 15, as well as the transmem-
brane proteins USH2A and VLGR1b are thought to
interact via their extracellular domains and keep the synap-
tic membranes in spatial proximity (Reiners et al., 2006).
As in synapses in general, cytoplasmic scaffold proteins
may anchor these adhesion molecules, but also transmem-
brane proteins as well as channels and receptors into the
synaptic membrane (Garner, Nash, & Huganir, 2000).
Our present results suggest that SANS is also part of this
scaffolding machinery in ribbon synapses. By direct bind-
ing to the USH proteins harmonin, whirlin and myosin
VIIa (see Fig. 1b) (Adato et al., 2005; van Wijk et al.,
2006), SANS may connect the synaptic USH protein net-
work with the microtubule cytoskeleton. However, in this
association with microtubules, SANS may also participate
in synaptic molecule trafficking and serve in the molecular
handover from the long range microtubule-based neuronal
transport system to the actin filament associated short
range transport system of the synaptic terminal, probably
governed by the actin-based molecular motor myosin VIIa.
In conclusion, the scaffold protein SANS is a crucial
component of photoreceptor cells involved in various
structural and developmental processes associated with
the microtubule cytoskeleton. Our data indicate that SANS
participates in ciliogenesis during outer segment differenti-
ation, in formation and maintenance of retina and photo-
receptor cell polarity and in functions in the ribbon
synapse of photoreceptor cells. For its essential specific
functions SANS is integrated in protein networks in the cil-
iary apparatus, the outer limiting membrane and the rib-
bon synapse of photoreceptor cells. Defects of SANS
may cause dysfunctions of entire USH protein networks
and may lead overall to degeneration of the sensory sys-
tems in the inner ear and retina, symptoms characteristic
for USH patients.
Acknowledgements
This work was supported by the DFG GRK 1044 (to
U.W.), Forschung contra Blindheit—Initative Usher Syn-
drom (to T.M. and U.W.), the FAUN-Stiftung, Nu ¨rnberg
(to U.W.). Authors thank Gabi Stern-Schneider and Ulrike
Maas for skillful technical assistance, Philipp Trojan for
critical reading of the manuscript and J. Salisbury and P.
Hargrave for providing us with anti-pan-centrin and anti-
opsin antibodies.
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