(Investigative Ophthalmology and Visual Science. 2001;42:850-852.)
© 2001
by The Association for Research in Vision and Ophthalmology, Inc.
Localization of Caveolin-1 in Photoreceptor Synaptic Ribbons
Shu Kachi1,
Akio Yamazaki2 and
Jiro Usukura3,4
1 From the Departments of Ophthalmology and
2 Anatomy, Nagoya University, School of Medicine, Japan; and the
3 Departments of Ophthalmology and
4 Pharmacology, Kresge Eye Institute, Wayne State University, School of Medicine, Detroit, Michigan.
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Abstract
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PURPOSE. The purpose of this study is to determine whether caveolin-1 is a
constituent of photoreceptor synaptic ribbons.
METHODS. Immunoblot assay and electron microscopic immunocytochemistry were used
to localize caveolin-1 in synaptic ribbons.
RESULTS. Synaptic ribbons were localized close to the active site of presynaptic
membranes and surrounded by a halo of synaptic vesicles. Immunosignals
of caveolin-1 were clearly detected on the synaptic ribbons in rod and
cone photoreceptors. However, the signal was seen neither on synaptic
vesicles nor on presynaptic plasma membranes.
CONCLUSIONS. Caveolin-1 is a component protein of synaptic ribbons and may be
involved in the regulation of transmitter
release.
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Introduction
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The photoreceptor synaptic-terminal is characterized by
synaptic ribbons, which are located at fusion sites of the
photoreceptor synapse and surrounded by halos of synaptic vesicles. The
ribbon is a platelike structure that consists of a bilayer of highly
ordered elementary particles and tethers vesicles with fine
filaments.1
The ribbon seems to be changed based on
illumination conditions.2
3
These morphologic observations
suggest that synaptic ribbons may be involved in transmitter release.
However, mechanisms of transmitter release, ribbon morphologic changes,
and formation are unknown. One reason is that information about the
composition of synaptic ribbons has been very limited.
Caveolin is an intrinsic membrane protein with a molecular weight of
approximately 25 kDa.4
Three types of the protein,
caveolin-1, caveolin-2 and caveolin-3, have been identified, and
caveolin-1 and -2 are abundantly expressed in many
tissues.5
Caveolin is involved in many signal transduction
mechanisms, including protein phosphorylation by protein kinases and
G-protein–mediated signal transduction.6
Caveolin also
controls cholesterol transport.7
In addition, caveolin
seems to be related to caveolae formation,8
indicating
that caveolin plays important roles in the specific localization of
proteins and other components involved in signal transduction. In this
report, we show that caveolin-1 is localized in synaptic ribbons. We
suggest that caveolin may be a protein crucial for regulating
transmitter release and formation of synaptic ribbons.
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Materials and Methods
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Animals
Bovine and mouse retinas were used. Bovine eyes were obtained in
a local slaughterhouse, and retinas were used for detection of
caveolin-1 by immunoblot assay and immunocytochemistry. Mouse (strain
BALB/c) retinas were isolated and used for immunocytochemistry. All
animal procedures were performed in accordance with the ARVO Statement
for the Use of Animals in Ophthalmic and Vision Research.
Antibodies
An affinity-purified rabbit polyclonal antibody raised against
an N-terminal recombinant fragment of caveolin-1 was used for
immunocytochemistry and immunoblot analysis. This antibody is specific
to caveolin-1 and reacts with the protein in mouse, rat, bovine, and
human tissues. In some experiments, an antibody against caveolin-2 was
also used. The antibody was prepared against a C-terminal fragment of
caveolin-2. These antibodies were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA) and Transduction Laboratories
(Lexington, KY), respectively.
Preparation of OPL and ROS Fractions
The outer plexiform layer (OPL) fraction was obtained according
to the methods described previously.9
Briefly, bovine
retinas suspended in 35 ml of buffer A (15 mM
Na2HPO4, 1 mM EGTA, 1 mM
MgCl2, 1 mM phenylmethylsulfonyl fluoride
[PMSF], pH 7.4) were homogenized by a polytron homogenizer
(Iuchiseieido, Co., Osaka, Japan) on ice. The homogenate (20 ml) was
overlaid on 10 ml of buffer A containing 50% sucrose and centrifuged
(50 minutes, 4°C, 15,000 rpm) using a rotor (RPR-20; Hitachi Co.,
Ltd., Mito, Japan). The layer between the sucrose solution and
the overlaid solution was collected and suspended in the same volume of
buffer A. This mixture was overlaid again onto a linear sucrose
gradient (35%–50%) in buffer A and centrifuged (75 minutes, 4°C,
13,000 rpm) using a second rotor (SW28). The OPL fraction was
collected at approximately 40% sucrose. The rod outer segment
(ROS) fraction was obtained by a floatation method, as described
previously.10
Immunoblot Assay
Proteins of OPL and ROS fractions (protein 10 µg) were
separated by sodium dodecyl sulfate–polyacryamide gel electrophoresis
(SDS-PAGE) and transferred onto nitrocellulose membranes. The
nitrocellulose membranes were incubated with an anti-caveolin antibody
solution (1 hour, room temperature) and visualized with alkaline
phosphatase-conjugated anti-rabbit IgG.
Electron Microscopic Immunocytochemistry
Bovine and mouse retinas were fixed with 4% paraformaldehyde
and 1% glutaraldehyde in phosphate buffer adjusted to pH 7.4 for 2
hours and dehydrated with an ascending series of ethanols (up to 95%).
Dehydrated samples were embedded in Lowicryl K4M (Polysciences,
Warrington, PA) or LR White embedding medium (Electron Microscopy
Sciences, Fort Washington, PA). Ultrathin sections (65 nm) were
incubated with an anti-caveolin antibody solution (0.2 µg/ml) for 2
hours (room temperature). After they were washed several times, the
sections were incubated with 10 nm gold-conjugated goat anti-rabbit IgG
(1:20 dilution: Amersham, Amersham, UK). For control experiments, an
anti-caveolin antibody preabsorbed with a caveolin peptide was used as
the primary antibody.
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Results
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Immunoblot Assay
The anti-caveolin-1 antibody recognized only one protein in a
retinal homogenate and in the OPL fraction (Fig. 1)
. Its molecular weight was slightly higher than 25 kDa. The immunologic
signal in the OPL fraction was stronger than that of the retinal
homogenate and was not detected in the ROS fraction. These results
indicate that caveolin-1 is present in retinas, especially in the OPL
fraction. Caveolin-2, another isoform, was not detected in the retina
(data not shown). We note that only a monomeric form of caveolin-1 was
recognized in these retinal fractions, although caveolin-1 isolated
from other tissues and cells tends to aggregate or to form oligomers in
the process of SDS-PAGE.8
11
12
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Figure 1. Specificity of anti-caveolin-1 antibody used in this study and presence
of caveolin-1 in bovine retina. Anti-caveolin antibody recognized a
protein of approximately 25 kDa in a whole retinal homogenate (retina)
and OPL fraction. No band was found in the ROS fraction.
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Electron Microscopic Localization of Caveolin-1
Subcellular localization of caveolin-1 in photoreceptor synaptic
terminals of bovine and mice retinas was examined by electron
microscopic immunocytochemistry. As shown in Figure 2
, in synaptic terminals of rod and cone photoreceptors, synaptic ribbons
were localized close to the active sites of presynaptic membranes and
surrounded by halos of synaptic vesicles. The immunolabeling was mainly
found on the synaptic ribbons. The outer segments were not labeled.
Although some labeling was scattered in the synaptic cytoplasm, it was
barely above background level. No labeling was found in the controls
(Fig. 2C) . These observations indicate that caveolin-1 is a constituent
of synaptic ribbons in both rod and cone synaptic terminals.
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Figure 2. Immunocytochemical electron micrograph of
synaptic terminals in bovine (A, B, and
C) and mouse (D) photoreceptor cells.
Anti-caveolin-1 antibody binding sites were found exclusively on
synaptic ribbons (arrows) in both rod and cone synaptic
terminals. (A) Bovine rod, (B) bovine cone, and
(C) control (bovine rod) photoreceptor synaptic terminals.
(D) Mouse rod photoreceptor synaptic terminal. Scale bar,
500 nm.
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Discussion
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Generally, synapsin tethers synaptic vesicles,13
although it is not found in photoreceptor synapses.13
Instead, synaptic ribbons tether vesicles with fine filaments in
photoreceptor synaptic terminals.1
It is believed that
synaptic ribbons are involved in the transmitter release by tethering
synaptic vesicles. The present study indicates that caveolin-1 is
localized in synaptic ribbons. This finding is crucial for studying
functions of synaptic ribbons, because caveolin specifically binds to
various proteins involved in signal transduction such as G-proteins,
protein kinases,5
6
and a calcium pump.14
15
It is conceivable that caveolin-1 in synaptic ribbons is involved in
transmitter release by forming complexes with proteins related to
signal transduction.
Our results indicate that caveolin-1 was located in synaptic ribbons.
However, the caveolin-1 detected may be slightly different from the
protein reported previously9
because oligomerization of
the protein was not detected by SDS-PAGE, and its molecular weight was
slightly higher than 25 kDa. It is possible that caveolin-1 found in
synaptic ribbons has a slightly different amino acid sequence (a
caveolin-1 homologue), and/or the protein in synaptic ribbons is
modified.
It is not known how synaptic ribbons are formed. One reason is
that constituents of the synaptic ribbon have not been completely
identified. Schmitz et al.9
showed that a synaptic ribbon
fraction contains several proteins. Nguyen and Balkema16
demonstrated that antigenic epitope against the amino acid sequence
DTYQHPPKD colocalized with 
-actinin at the synaptic ribbon. Muresan
et al.17
have indicated that KIF3A, a member of the
heteromeric family of kinesins, is a filament that tethers vesicles,
suggesting that tubulin may be involved in synaptic ribbon formation.
In this study, we found that caveolin-1 is localized in synaptic
ribbons. A 30-kDa protein found by Schmitz et al.9
in
synaptic ribbons may be the caveolin-1 described in the present study.
Caveolin-1 was originally identified as an intrinsic membrane
protein,4
5
suggesting that the synaptic ribbon could form
from a membrane. However, immunocytochemical data (Fig. 2)
suggest that
vesicles and vacuoles do not contain caveolin-1. Information about
incorporation of caveolin-1 into synaptic ribbons may be crucial for
revealing the mechanism of synaptic ribbon formation.
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Acknowledgements
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The authors thank William H. Miller for critical
reading of the manuscript.
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Footnotes
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Supported by an unrestricted grant from Research to Prevent Blindness (AY).
Submitted for publication March 17, 2000; revised June 23, September 29, and November 7, 2000; accepted November 15, 2000.
Commercial relationships policy: N.
Corresponding author: Jiro Usukura, Department of Anatomy and Cell Biology, Nagoya University, School of Medicine, 65 Tsurumai, Nagoya 466-8550, Japan. usukuraj{at}tsuru.med.nagoya-u.ac.jp
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Abstract
Introduction
