* Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, c/o JCR Pharmaceuticals Co., Ltd., Nishi-ku,
Kobe 651-22, Japan; Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Japan; § Department of Virology II, National Institutes of Health, Tokyo 162, Japan;
Department of Anatomy and Neurobiology,
Graduate School, Kyoto University, Kyoto 606-01, Japan
We purified from rat brain a novel actin filament (F-actin)-binding protein of ~180 kD (p180), which was specifically expressed in neural tissue. We named p180 neurabin (neural tissue-specific F-actin- binding protein). We moreover cloned the cDNA of neurabin from a rat brain cDNA library and characterized native and recombinant proteins. Neurabin was a protein of 1,095 amino acids with a calculated molecular mass of 122,729. Neurabin had one F-actin-binding domain at the NH2-terminal region, one PSD-95, DlgA, ZO-1-like domain at the middle region, a domain known to interact with transmembrane proteins, and domains predicted to form coiled-coil structures at the COOH-terminal region. Neurabin bound along the sides of F-actin and showed F-actin-cross-linking activity. Immunofluorescence microscopic analysis revealed that neurabin was highly concentrated in the synapse of the developed neurons. Neurabin was also concentrated in the lamellipodia of the growth cone during the development of neurons. Moreover, a study on suppression of endogenous neurabin in primary cultured rat hippocampal neurons by treatment with an antisense oligonucleotide showed that neurabin was involved in the neurite formation. Neurabin is a candidate for key molecules in the synapse formation and function.
DURING the development of the nervous system, the
distal tip of the elongating axon In the developing nervous system, the actin cytoskeleton
is prominent in two structural domains of the growth cone,
filopodia and lamellipodia (Mitchison and Kirschner,
1988 When the growth cone contacts with the target cell, the
target cell regulates the development of the presynaptic
nerve terminal and the formation of the functional synapse
(Bowe and Fallon, 1995 To understand the regulation of the actin cytoskeleton
during and after the development of the nervous system, it
is of crucial importance to identify F-actin-binding proteins implicated in the synapse formation and function.
Therefore, we attempted here to isolate neural tissue-specific F-actin-binding proteins. We isolated a novel neural
tissue-specific F-actin-binding protein from rat brain,
which may be involved in neurite formation, and named it
neurabin (neural tissue-specific F-actin-binding protein).
125I-Labeled F-Actin Blot Overlay
125I-Labeled F-actin blot overlay was done as described (Chia et al., 1991 For competition experiments, 125I-labeled F-actin was prepared as described above except that the concentration of gelsolin was reduced to 7.2 µg/ml (molar ratio, 250:1). 20 µg/ml of 125I-labeled F-actin was incubated
for 30 min at room temperature with 0.42 µg/ml of myosin subfragment 1 (S1) (Sigma Chemical Co.) in a solution containing 20 mM Pipes, pH 7.0, 58 mM KCl, 2 mM MgCl2, 130 µM CaCl2, and 0.8 µM phalloidin. Where
indicated, 4 mM MgATP was added to the mixture. After the incubation,
the mixture was diluted with an equal volume of TBS containing 10% defatted powder milk, and it was added to the blot membrane, followed by incubation for 1 h at room temperature.
Purification of Neurabin
All the purification procedures were carried out at 0-4°C. The synaptic
soluble fraction was prepared from 240 adult rat brains as described previously (Mizoguchi et al., 1990
Peptide Mapping of Neurabin and Molecular Cloning
of Its cDNA
The purified Mono S sample (~30 µg of protein) was subjected to SDS-PAGE (8% polyacrylamide gel). A protein band corresponding to a protein
of ~180 kD was cut out from the gel and digested with a lysyl endopeptidase, and the digested peptides were separated by TSKgel ODS-80Ts (4.6 x 150 mm; Tosoh, Tokyo, Japan) reverse phase high pressure liquid column chromatography as described (Imazumi et al., 1994 For coiled-coil prediction, the MTK and MTIDK matrices of the
COILS version 2.1 algorithm (Lupas et al., 1991 Expression and Purification of Full-Length and
Truncated Forms of Neurabin
Prokaryote and eukaryote expression vectors were constructed in
pGexKG (Guan and Dixon, 1991 For the GST fusion proteins, the GST fusion constructs were transformed into Escherichia coli. The GST fusion proteins were purified by
use of glutathione-Sepharose beads. For the myc-tagged proteins, COS7
cells were transfected with the DEAE-dextran method (Hata and Südhof,
1995 F-Actin-Cross-linking Activity
Low shear viscometry was performed as described (Pollard and Cooper,
1982 Electron microscopy was performed as described (Endo and Masaki,
1982 Activity of Cosedimentation with F-Actin
G-Actin was polymerized by incubation for 30 min at room temperature
in a polymerization buffer (20 mM imidazol/Cl, pH 7.0, 2 mM MgCl2,
1 mM ATP, 0.5 mM DTT, and 90 mM KCl). GST-neurabin-1 (aa 1-144),
GST-neurabin-2 (aa 1-210), or His6-neurabin in an indicated amount was
incubated for 30 min at room temperature with 0.3 mg/ml of F-actin in a
solution containing 20 mM imidazol/Cl, pH 7.0, 2 mM MgCl2, 1 mM ATP,
0.4 mM DTT, 27 mM KCl, 100 mM NaCl, and 0.2 mM EGTA, and the
mixture (100 µl) was placed over a 50-µl cushion of 30% sucrose in the
polymerization buffer. After the sample was centrifuged at 130,000 g for
20 min, the supernatant was removed from the cushion and the pellet was
brought to the original volume in an SDS sample buffer. The comparable
amounts of the supernatant and pellet fractions were subjected to SDS-PAGE, followed by protein staining with Coomassie brilliant blue to quantitate the fusion protein cosedimented with F-actin using a densitometer.
Gel Filtration and Sucrose Density
Gradient Ultracentrifugation
In gel filtration analysis, His6-neurabin (2 µg of protein) was applied to a
Superdex 200 PC 3.2/30 column (Pharmacia Biotechnology, Inc.) equilibrated with a buffer containing 20 mM Tris/Cl, pH 7.5, 150 mM NaCl, 1 mM EGTA, and 1 mM DTT. Elution was performed with the same buffer
at a flow rate of 50 µl/min. Fractions of 50 µl each were collected. In sucrose density gradient ultracentrifugation analysis, His6-neurabin (0.2 ml,
5 µg of protein) was applied on a 4.8-ml, continuous sucrose density gradient (5-20% sucrose in 20 mM Tris/Cl, pH 7.5, 1 mM EGTA, and 1 mM
DTT) and centrifuged at 235,000 g for 6 h at 4°C with a swing rotor
(P55ST2; Hitachi Ltd.). After the ultracentrifugation, fractions of 150 µl
each were collected. Each fraction was subjected to protein staining with
silver and Western blot analysis using the anti-neurabin-1 antibody described below.
Primary Cultured Rat Hippocampal Neurons
Primary cultured rat hippocampal neurons were prepared as described
(Takeuchi et al., 1997 Antibodies and Immunofluorescence Staining
A rabbit polyclonal antibody against neurabin was raised against GST-
neurabin-7 (aa 609-868, anti-neurabin-1) or GST-neurabin-8 (aa 890-
1095, anti-neurabin-2). The antiserum was affinity purified with each GST
fusion protein covalently coupled to NHS-activated Sepharose (Pharmacia Biotechnology, Inc.). The specificities of these antibodies were confirmed by Western blot analyses on the pCMV-neurabin-transfected
COS7 cells versus mock cells. A monoclonal anti- For the immunofluorescence microscopy of cultured cells, the cells
were washed with PBS, fixed with 4% paraformaldehyde in PBS for 1 h,
washed with solution A (20 mM phosphate buffer, pH 7.2, and 0.45 M
NaCl), and then treated for 30 min with a solution containing 20 mM Tris/
Cl, pH 7.5, 140 mM NaCl, 0.1% Triton X-100, and 20% defatted powder
milk. The samples were incubated for 2 h with the rabbit anti-neurabin-1,
mouse anti- The immunofluorescence microscopy of adult rat neural tissue was
done as described (Mizoguchi et al., 1990 Antisense Phosphorothioate Oligonucleotide
Six antisense phosphorothioate oligonucleotides (PONs) complementary
to the neurabin sequence surrounding the initiation codon and the corresponding sense PONs were designed based on the neurabin sequence (see
Fig. 9 A). Their sequences did not have significant homology to any other
sequences in the database. They were synthesized on a synthesizer (Expedite; PerSeptive Biosystems, Cambridge, MA) and purified by two stepwise reverse phase high pressure liquid column chromatographies
(Beaton et al., 1991
For antisense experiments, the primary cultured rat hippocampal neurons were prepared as described above and attached on poly-L-lysine- coated glass coverslips in MEM with 10% horse serum for 1-2 h (Torre et
al., 1994 Other Procedures
G-Actin was purified from rabbit skeletal muscle as described (Pardee
and Spudich, 1982 Purification of Neurabin and Molecular Cloning of
Its cDNA
To identify novel F-actin-binding proteins from rat brain,
we attempted to detect F-actin-binding proteins using a
blot overlay method with 125I-labeled F-actin. The homogenates of various rat tissues were subjected to SDS-PAGE
followed by the blot overlay. Several radioactive bands with
various molecular masses were detected (Fig. 1 A). Of these
125I-labeled F-actin-binding proteins, two protein bands of
~180 (p180) and 140 kD were detected only in brain. p180
was highly purified by column chromatographies, including
Q-Sepharose, phenyl-Sepharose, hydroxyapatite, Mono Q,
and Mono S column chromatographies. On the final Mono
S column chromatography, the 125I-labeled F-actin-binding protein band well coincided with a protein of ~180 kD,
which was identified by protein staining (Fig. 1 B).
When the peptide mapping of the Mono S sample of
p180 was performed, over 30 peptide peaks were observed. Of the peptide peaks, the aa sequences of the nine
peptides were determined. Computer homology search revealed that they were not found in current protein database. On the basis of these aa sequences, we isolated a
clone from a rat brain cDNA library and determined its
nucleotide sequence. This clone contained a predicted initiation codon preceded by an in-frame stop codon, and a
3,285-bp coding region followed by a stop codon. The encoded protein consisted of 1,095 aa and showed a calculated molecular mass of 122,729 (these sequence data are
available from GenBank/EMBL/DDBJ under accession
number U72994) (Fig. 2). It included all the aa sequences
of the peptides. The molecular mass calculated from the
predicted aa sequence was far less than that estimated by
SDS-PAGE. To confirm whether this clone contained a
full-length cDNA of p180, we constructed the eukaryotic
expression vector with this cDNA and expressed the protein in COS7 cells. The expressed protein showed the mobility similar to that of native p180 on SDS-PAGE and the
125I-labeled F-actin-binding activity (Fig. 3 A). Although
the reason for the remarkable difference between the molecular mass value calculated from the predicted aa sequence and that estimated by SDS-PAGE is not known,
we concluded that this clone encoded the full-length
cDNA of p180.
We named p180 neurabin since p180 was specifically expressed in neural tissue and showed F-actin-binding activity as described below. Neurabin had one PSD-95, DlgA,
ZO-1-like (PDZ) domain (aa 505-595), a domain known
to interact with transmembrane proteins (Saras and Heldin, 1996 To determine the F-actin-binding domain of neurabin,
we prepared fusion proteins of several truncated forms of
neurabin with GST and examined the binding of 125I-
labeled F-actin to these fusion proteins. GST-neurabin-1
(aa 1-144) and GST-neurabin-2 (aa 1-210) showed the
125I-labeled F-actin-binding activity, whereas the fusion
proteins of other truncated forms of neurabin did not
show the activity (Fig. 3 C). To confirm that neurabin interacted with F-actin through the F-actin-binding domain
in intact cells, we compared the localization of truncated
forms of neurabin with that of F-actin. When full-length neurabin was expressed in COS7 cells as a myc-tagged
protein (myc-neurabin), it was colocalized with F-actin
(Fig. 3 D, a and d). A similar result was obtained when a
truncated form of neurabin (myc-neurabin-1, aa 1-144),
which possessed the F-actin-binding domain alone, was expressed (Fig. 3 D, b and e). In contrast, when another
truncated form of neurabin (myc-neurabin-2, aa 145-1095),
which lacked the F-actin-binding domain, was expressed,
the protein showed a diffuse distribution in the cytoplasm,
but was partly concentrated at the F-actin-rich region of
the cell periphery (Fig. 3 D, c and f). Taken together with
the findings that GST-neurabin-1 (aa 1-144) showed the
125I-labeled F-actin-binding activity and was cosedimented
with F-actin as described below, these results indicate that
neurabin has one F-actin-binding domain at the NH2-terminal region (aa 1-144) (Fig. 3 C), and that neurabin interacts with F-actin through this domain in intact cells. This
F-actin-binding domain showed no significant homology
to any protein in current protein database.
Biochemical Properties of Neurabin
In addition to 125I-labeled F-actin blot overlay, the binding
of neurabin to F-actin was examined by cosedimentation
of neurabin with F-actin. When GST-neurabin-2 (aa 1-210)
was incubated with F-actin followed by ultracentrifugation, the fusion protein was recovered with F-actin in the
pellet (Fig. 4 A). A similar result was obtained with GST-
neurabin-1 (aa 1-144) and His6-neurabin (data not shown).
The stoichiometry of the binding of His6-neurabin to actin
was calculated to be one His6-neurabin molecule per about eight actin molecules (Fig. 4 B). The Kd value was calculated to be ~8 x 10
The F-actin-cross-linking activity of the Mono S sample
of neurabin was examined by the falling ball method for
low shear viscometry. Neurabin increased the viscosity in
time- and dose-dependent manners, suggesting that neurabin
showed the cross-linking activity (Fig. 4 D). A similar result was obtained with His6-neurabin purified from Sf9
cells. Whereas the Mono S sample of native neurabin was contaminated with a protein of ~130 kD (Fig. 1 B), these
results indicate that neurabin itself indeed shows the
F-actin-cross-linking activity. This cross-linking activity of
neurabin was confirmed by transmission electron microscopy of negatively stained specimens. Neurabin caused
F-actin to associate into bundles (Fig. 4 E).
The molecular mass values of neurabin were estimated
by gel filtration and sucrose density gradient ultracentrifugation in the presence of a reducing agent. When His6-neurabin was subjected to Superdex 200 column chromatography, it appeared at a position corresponding to ~440
kD (Stokes' radius, about 66 Å) (Fig. 5 A). On sucrose
density gradient ultracentrifugation, His6-neurabin appeared at a position corresponding to ~380 kD (S value,
~15.6) (Fig. 5 B). The molecular mass value of neurabin
was calculated to be ~440 kD from both the Stokes' radius
and S value (Siegel and Monty, 1966
Tissue Distribution of Neurabin
Northern blot analysis showed that ~9.5 kb mRNA was
hybridized in rat brain (Fig. 6 A). In rat testis, mRNAs
with smaller sizes were weakly hybridized, but the significance of these weak signals is unknown. Other rat tissues
examined, including heart, spleen, lung, liver, skeletal
muscle, and kidney, did not show any detectable signal.
Western blot analysis showed that a polyclonal antibody
(anti-neurabin-1), which was raised against GST-neurabin-7
(aa 609-868), recognized a protein band of ~180 kD and
two proteins of ~140 kD in rat brain (Fig. 6 B). No reactive band was observed in other rat tissues examined. The
protein bands with the smaller molecular masses in rat
brain appear to be proteolytic products of neurabin (see
Discussion).
Localization of Neurabin in Cultured
Hippocampal Neurons
It was first examined by Western blot analysis whether
neurabin was expressed in primary cultured rat hippocampal neurons ranging in age 1-10 d. Neurabin was expressed
in the neurons and the expression amounts were almost
constant throughout the course of development in culture,
whereas those of the SV proteins, including synapsin I and
synaptophysin, increased (Fig. 7 A). Neurabin showed double immunoreactivity bands. This may be attributed to
the posttranslational modifications, such as phosphorylation. The cultured hippocampal neurons were then used to
assess the localization of neurabin by the immunofluorescence staining using the anti-neurabin-1 antibody. After
24 h in culture, almost all the neurons extend an axon and several minor processes that eventually become dendrites
(Dotti et al., 1988
Localization of Neurabin in Rat Neural Tissue
The in situ distribution and localization of neurabin were
examined by the immunofluorescence staining of cerebellum, hippocampus, retina, neuromuscular junction, and
adrenal gland of adult rat using the anti-neurabin-1 antibody. The staining patterns of neurabin were compared
with those of synaptophysin, known to be highly concentrated at the presynaptic nerve terminal (Navone et al., 1986
Involvement of Neurabin in Neurite Formation
To examine the physiological function of neurabin, we attempted to suppress the expression of endogenous neurabin
in primary cultured rat hippocampal neurons by treatment
with its antisense oligonucleotides. We designed six antisense PONs complementary to the neurabin sequence surrounding the initiation codon (Fig. 9 A). The corresponding sense PONs were also constructed. Each PON was
added to the serum-free medium at 50 µM after plating, and again at 25 µM every 12 h. After 48 h, we examined by
the immunofluorescence staining using the anti-neurabin-1
antibody that antisense PON was effective to suppress the
neurabin expression. Of these antisense PONs, antisense-1
and -2 were effective, whereas other antisense PONs, including antisense-3, -4, -5, and -6, as well as all the sense
PONs, were not (data not shown). Therefore, the effect of
antisense-1 on the hippocampal neurons was precisely studied. When the hippocampal neurons were treated with
neurabin antisense-1, the expression of neurabin was reduced as estimated by Western blot analysis using the
anti-neurabin-1 antibody (Fig. 9 B). The treatment of the
neurons with neurabin sense-1 did not affect the expression level of neurabin. In these neurons, the level of We have purified here from rat brain a 125I-labeled F-actin-
binding protein of ~180 kD and characterized it. Because
this protein is specifically expressed in neural tissue, including central and peripheral nerves, and adrenal chromaffin cells, we have named it neurabin. Structural analysis of neurabin has revealed that it consists of at least one
F-actin-binding domain at the NH2-terminal region, one
PDZ domain at the middle region, and domains for predicted coiled-coil structures at the COOH-terminal region.
Western blot analysis using the anti-neurabin-1 antibody in rat brain detected a protein band of ~180 kD and
two protein bands of ~140 kD as shown in Fig. 6 B. The
molecular mass value of neurabin calculated from the predicted aa was ~120 kD. Therefore, there is a possibility
that the 180- and 140-kD bands may be partially and fully
denatured forms on the gel, respectively. However, this possibility is unlikely and the 140-kD proteins appear to
be proteolytic products of neurabin because of the following observations. (a) Before the sample was subjected to
SDS-PAGE, the sample was always boiled for 10 min in an
SDS sample buffer containing 2-mercaptoethanol. Even
when the sample was treated with 8 M urea, neurabin
showed a molecular mass of ~180 kD on SDS-PAGE (data
not shown); (b) The 140-kD proteins were separated from
the 180-kD protein by hydroxyapatite column chromatography (data not shown); (c) When the major 140-kD protein was highly purified and the peptide mapping was performed, its peptide map was almost identical to that of neurabin, but the 140-kD protein lacked some peptide
peaks (data not shown). The aa sequences of the peptide
peaks of the 140-kD protein were identical to those of
neurabin (data not shown); (d) The amounts of the 140-kD proteins in rat brain varied from preparation to preparation; and (e) When the cell extract from the neurabin
cDNA-transfected COS7 cells was subjected to Western blot analysis, the antibody recognized the expressed neurabin and its proteolytic products. The molecular mass values of these proteolytic products in the COS7 cells were
~140 kD (data not shown).
We have concluded that neurabin functions as an F-actin-
binding protein in intact cells and that the F-actin-binding
domain is located at the NH2-terminal region (aa 1-144),
on the basis of the following observations: (a) Full-length
neurabin and its NH2-terminal region (aa 1-144) showed
the 125I-labeled F-actin-binding activity; (b) full-length
neurabin and its NH2-terminal region (aa 1-144) were
cosedimented with F-actin; (c) full-length neurabin showed
the F-actin-cross-linking activity; and (d) when full-length
neurabin and its NH2-terminal region (aa 1-144) were expressed in COS7 cells, both proteins were colocalized with
F-actin. There is no significant homology between the primary structures of F-actin-binding domains of neurabin
and any known F-actin-binding protein, but three-dimensional structure of the F-actin-binding domain of neurabin
may be similar to those of known F-actin-binding proteins.
We have moreover shown here that neurabin shows
F-actin-cross-linking activity. Many actin-binding proteins
have thus far been isolated, and of these proteins, the
The PDZ domain has originally been identified in PSD-95/SAP90, one of membrane-associated guanylate kinases
(Cho et al., 1992 We have found here that neurabin is expressed in both
the developing and developed neurons; in the developing
neurons it is highly concentrated in the lamellipodia of the
growth cone, whereas in the developed neurons it is highly
concentrated in the synapse. We have moreover shown
that suppression of the neurabin expression in the hippocampal neurons by treatment with the antisense oligonucleotide inhibits the neurite formation. These results, together with the biochemical properties of neurabin,
suggest two possible roles: (a) it is involved in the vesicle
trafficking in both the developing and developed neurons;
and (b) it is involved in the formation and the maintenance of the synaptic junction. To clarify the role of
neurabin, it is essential to identify its interacting molecule(s) other than F-actin. We have shown that myc-
neurabin-2 (aa 145-1095), which lacks the F-actin-binding
domain, is partly concentrated at the F-actin-rich region
of the cell periphery in the COS7 cells. This result suggests
that there is a molecule(s) other than F-actin that directly
interacts with neurabin through the PDZ domain and/or the coiled-coil domains, which is present at the F-actin-
rich region at the cell periphery. Identification of such an
interacting molecule(s) is now under investigation.
the growth
cone
actively migrates toward its target cell in
response to the combined actions of attractive and repulsive guidance molecules in the extracellular environment (Garrity and Zipursky, 1995
; Keynes and Cook, 1995
;
Chiba and Keshishian, 1996
; Culotti and Kolodkin, 1996
;
Friedman and O'Leary, 1996
; Tessier-Lavigne and Goodman, 1996
). When the growth cone contacts with the target cell, it is transformed into the functional presynaptic
terminal (Garrity and Zipursky, 1995
; Chiba and Kishishian, 1996). The actin cytoskeleton has been shown to play
crucial roles in these processes of the synapse formation
(Mitchison and Kirschner, 1988
; Smith, 1988
; Bentley and
O'Connor, 1994
; Lin et al., 1994
; Mackay et al., 1995
; Tanaka
and Sabry, 1995
).
; Smith, 1988
; Bentley and O'Connor, 1994
; Lin et al.,
1994
; Mackay et al., 1995
; Tanaka and Sabry, 1995
). In
these domains, actin filament (F-actin)1 assembled at the
leading edge are transported into the center of the growth
cone and disassembled there. It has been suggested that
this retrograde flow of F-actin is crucial for the growth cone motility. Drugs that disrupt F-actin have also been
shown to cause the lamellipodial and filopodial collapse
and block the ability of neurons to extend the growth cone
in the correct direction (Marsh and Letourneau, 1984
; Forscher and Smith, 1988
; Bentley and Toroian-Raymond,
1986
; Chien et al., 1993
). These results suggest that the actin cytoskeleton regulates not only the growth cone motility but also the growth cone directionality. Recently, a variety of guidance molecules and their receptors have been identified (Garrity and Zipursky, 1995
; Keynes and Cook,
1995
; Chiba and Keshishian, 1996
; Culotti and Kolodkin,
1996
; Friedman and O'Leary, 1996
; Tessier-Lavigne and
Goodman, 1996
). However, which molecules of the actin
cytoskeleton are essential for the growth cone motility and
directionality is not well understood.
; Chiba and Keshishian, 1996
). In
the established nervous system, the presynaptic and postsynaptic membranes get aligned in space and constitute the
synaptic junction (Burns and Augustine, 1995
; Garner and
Kindler, 1996
). Electron microscopic studies have revealed the ultrastructural features of the synaptic junction (Burns and Augustine, 1995
; Garner and Kindler, 1996
).
The presynaptic cytoplasm is characterized by synaptic
vesicles (SVs). SVs are not distributed uniformly; SVs
cluster together in the vicinity of the presynaptic plasma
membrane, where F-actin forms a network and is associated with the presynaptic plasma membrane (Hirokawa et
al., 1989
). Most SVs within the cluster are linked through thin strands to each other, to F-actin, or to both (Hirokawa et al., 1989
). A subset of SVs within the cluster are
attached by fine filamentous threads to neurotransmitter
release zone at the presynaptic plasma membrane (Hirokawa et al., 1989
). The presynaptic submembranous cytoskeleton is assumed to be involved in recruiting Ca2+
channels and the components of the SV fusion complex,
delivering SVs to the neurotransmitter release zone, and
keeping them in place (Burns and Augustine, 1995
; Garner and Kindler, 1996
). At the inner surface of the post-synaptic plasma membrane, there is an electron dense
thickening, called postsynaptic density. The postsynaptic
density is assumed to be involved in the selective targeting
and accumulation of ion channels and receptors (Burns and Augustine, 1995
; Garner and Kindler, 1996
). It is also
assumed that the presynaptic and postsynaptic submembranous cytoskeleton elements are linked to cell adhesion
molecules to regulate the synaptic stabilization and plasticity (Fields and Itoh, 1996
; Garner and Kindler, 1996
).
The presynaptic and postsynaptic submembranous cytoskeleton elements are thought to be composed of spectrin/fodrin, ankyrin,
-adducin, and protein 4.1 isoforms and to
be linked to F-actin through these cytoskeleton proteins
(Garner and Kindler, 1996
). However, little is known
about which molecules of the submembranous cytoskeleton are essential for the synaptic transmission and/or the
synaptic stabilization.
Materials and Methods
;
Pestonjamasp et al., 1995
). Briefly, purified actin monomer (G-actin) was
labeled with 125I-Bolton Hunter reagent. 125I-Labeled G-actin (1 mg/ml,
average specific activity, 63.3 µCi/mg) was polymerized with 18 µg/ml of
gelsolin (Sigma Chemical Co., St. Louis, MO) (molar ratio, 100:1) by incubation for 10 min at 4°C in a solution containing 20 mM Pipes, pH 7.0, 50 mM KCl, and 2 mM MgCl2. Phalloidin was then added to give a final concentration of 40 µM. The mixture was then incubated for another 15 min
at room temperature and stored at 4°C as 125I-labeled F-actin. The sample
to be tested was subjected to SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked in TBS containing 5% defatted powder milk. The membrane was then incubated for 1 h at room temperature with 10 µg/ml of 125I-labeled F-actin in TBS containing 5%
defatted powder milk, and 4 µM phalloidin. After the incubation, the
membrane was washed with TBS containing 0.5% Tween 20, followed by
autoradiography using an image analyzer (Fujix BAS-2000II; Fuji Photo
Film Co., Tokyo, Japan).
) and stored at
80°C until use. One sixth of
the fraction (420 ml, 360 mg of protein) was adjusted to 0.2 M NaCl with 4 M
NaCl, and applied to a Q-Sepharose FF column (2.6 x 10 cm; Pharmacia
Biotechnology Inc., Piscataway, NJ) equilibrated with buffer A (20 mM
Tris/Cl, pH 7.5, and 1 mM DTT) containing 0.2 M NaCl. After the column was washed with 250 ml of buffer A containing 0.2 M NaCl, elution was
performed with 350 ml of buffer A containing 0.5 M NaCl at a flow rate of
5 ml/min. Fractions of 10 ml each were collected. Neurabin appeared in
fractions 5-19. These fractions (150 ml, 152 mg of protein) were collected,
and NaCl was added to give a final concentration of 2 M. The sample was
applied to a phenyl-Sepharose column (2.6 x 10 cm; Pharmacia) equilibrated with buffer A containing 2 M NaCl. After the column was washed
with 250 ml of the same buffer, elution was performed with a 360-ml linear
gradient of NaCl (2.0-0 M) in buffer A, followed by 180 ml of buffer A, at a flow rate of 3 ml/min. Fractions of 6 ml each were collected. Neurabin
appeared in fractions 53-59. The active fractions (42 ml, 6.1 mg of protein)
were collected, and CHAPS was added to give a final concentration of
0.6%. The sample was then applied to a HPLC hydroxyapatite column
(0.78 x 10 cm; Koken Co. Ltd., Tokyo, Japan) equilibrated with buffer B
(20 mM potassium phosphate, pH 7.8, 1 mM DTT, 0.6% CHAPS, and
10% glycerol). After the column was washed with 120 ml of the same
buffer, elution was performed with a 75-ml linear gradient of potassium
phosphate (20-100 mM) in buffer B, and a subsequent 75-ml linear gradient (100-300 mM) in buffer B, followed by a 50-ml linear gradient (300-
500 mM) in buffer B, at a flow rate of 0.5 ml/min. Fractions of 2.5 ml each
were collected. Neurabin appeared in fractions 68-74. The active fractions
(17.5 ml, 0.22 mg of protein) were collected. The rest of the synaptic soluble fraction was also subjected to the successive column chromatographies
in the same manner as described above. The active fractions of the six hydroxyapatite column chromatographies were combined. One third of the
combined sample was diluted with an equal volume of buffer C (20 mM
Tris/Cl, pH 7.5, 0.5 mM EDTA, 1 mM DTT, and 0.6% CHAPS), and then
applied to a Mono Q HR 5/5 column (Pharmacia Biotechnology, Inc.)
equilibrated with buffer C. After the column was washed with 15 ml of
buffer C, elution was performed with 5 ml of buffer C containing 0.2 M
NaCl and a subsequent 15-ml linear gradient of NaCl (0.2-0.5 M) in buffer C, followed by 5 ml of buffer C containing 1.0 M NaCl, at a flow rate of 0.5 ml/min. Fractions of 0.5 ml each were collected. Neurabin appeared in
fractions 31-34. The active fractions (2 ml, 0.11 mg of protein) were collected. The rest of the combined sample of the hydroxyapatite column
chromatographies was also subjected to the Mono Q column chromatography in the same manner as described above. The active fractions of the
three Mono Q column chromatographies were combined and diluted with
an equal volume of buffer D (20 mM Hepes, pH 7.5., 0.5 mM EGTA, and
1 mM DTT). The sample was applied to a Mono S PC 1.6/5 column (Pharmacia Biotechnology, Inc.) equilibrated with buffer D. After the column
was washed with 2.0 ml of buffer D, elution was performed with a 1.5-ml
linear gradient of NaCl (0-1.0 M), followed by 0.5 ml of buffer D containing 1.0 M NaCl, at a flow rate of 50 µl/min. Fractions of 50 µl each were
collected. Neurabin appeared in fractions 16-17 (Fig. 1 B). The active
fractions (100 µl, 30 µg of protein) were collected and stored at
80°C until use.
Fig. 1.
125I-Labeled F-actin-binding activities of various rat tissues and Mono S column chromatography of p180 (neurabin).
(A) 125I-Labeled F-actin-binding activities of various rat tissues.
The homogenates of various tissues (30 µg of protein each) were
subjected to SDS-PAGE (8% polyacrylamide gel), followed by
125I-labeled F-actin blot overlay. (B) Mono S column chromatography. (a) Absorbance at 280 nm. (b) 125I-Labeled F-actin blot
overlay. An aliquot 1-µl of each fraction was subjected to SDS-PAGE (8% polyacrylamide gel), followed by 125I-labeled F-actin
blot overlay. (c) Protein staining with Coomassie brilliant blue. A
3-µl aliquot of each fraction was subjected to SDS-PAGE (8%
polyacrylamide gel), followed by protein staining with Coomassie
brilliant blue. Arrowheads indicate p180. Protein markers used
are myosin (206), -galactosidase (117), BSA (89), and ovalbumin (47).
[View Larger Version of this Image (33K GIF file)]
). The amino acid
(aa) sequences of the peptides were determined with a peptide sequencer.
A rat brain cDNA library in
ZAPII (Stratagene, La Jolla, CA) was
screened using the oligonucleotide probes designed from the partial aa sequences. DNA sequencing was performed by the dideoxy nucleotide termination method using a DNA sequencer (ABI 373; Applied Biosystems,
Inc., Foster City, CA).
; Lupas, 1996
) were used
with a 28-residue window. Weighting options were applied and a probability curve was generated.
), pCMV5, pCMV-myc (Takeuchi et al.,
1997
), and pAcYM1-His6 using standard molecular biology methods (Sambrook et al., 1989
). Various glutathione-S-transferase (GST) fusion
constructs of neurabin contained the following aa residues: pGex-
neurabin-1, aa 1-144; pGex-neurabin-2, aa 1-210; pGex-neurabin-3, aa
145-293; pGex-neurabin-4, aa 286-615; pGex-neurabin-5, aa 505-791; pGex-neurabin-6, aa 665-1095; pGex-neurabin-7, aa 609-868; and pGex-
neurabin-8, aa 890-1095. pCMV-neurabin contained full-length neurabin.
Various myc-tagged constructs of neurabin contained the following aa residues to express the proteins with the NH2-terminal myc-epitope (MEQKLISEEDL); pCMV-myc-neurabin, full length; pCMV-myc-neurabin-1, aa 1-144; pCMV-myc-neurabin-2, aa 145-1095. The His6-tagged construct (pAcYM1-His6-neurabin) contained full-length neurabin to express the protein with the COOH-terminal six histidine residues.
). For the His6-tagged protein, Spodoptera frugiperda (Sf9) cells were
transfected with baculovirus carrying the His6-tagged construct (Kikuchi
et al., 1995
). The Sf9 cells were homogenized with buffer E (20 mM Tris/
Cl, pH 7.5, 1 mM DTT, 20 µg/ml leupeptin, 1 µg/ml pepstatin, and 20 µg/
ml aprotinin), and centrifuged at 100,000 g for 1 h. The supernatant was
subjected to the phenyl-Sepharose column chromatography in the same
manner as used for the purification of native neurabin. Each fraction was
subjected to Western blot analysis using an antibody against neurabin
(anti-neurabin-1) described below. The active fractions were collected,
and 2 M imidazol/Cl, pH 7.2, was added to give a final concentration of 10 mM. The sample was subjected to Ni-agarose (Pharmacia Biotechnology,
Inc.) column chromatography according to the manufacturer's protocol.
The active fractions were collected and further subjected to the Mono S
PC 1.6/5 column chromatography in the same manner as used for the purification of native neurabin. The active fractions were collected and used
as His6-neurabin.
; Kato et al., 1996
). Briefly, the Mono S sample of native neurabin or
His6-neurabin in an indicated amount was mixed with 0.28 mg/ml of G-actin
in a solution containing 20 mM Tris/Cl, pH 7.2, 140 mM NaCl, 0.1 mM
ATP, 0.5 mM DTT, and 1 mM EGTA, and the solution was sucked into a
0.1-ml micropipette. After the incubation at 25°C for various periods of
time, the time for a stainless steel ball to fall a fixed distance in the pipette
was measured.
; Kato et al., 1996
). Briefly, 0.28 mg/ml of G-actin was incubated at
25°C for 45 min with the Mono S sample of native neurabin (20 µg/ml of
protein) in a solution containing 20 mM Tris/Cl, pH 7.2, 140 mM NaCl,
2 mM MgCl2, 0.1 mM ATP, 0.5 mM DTT, and 1 mM EGTA. The sample
was negatively stained with 2% uranyl acetate and viewed with an electron microscope (model H-7100; Hitachi Ltd., Tokyo, Japan).
). Briefly, hippocampi were isolated from rat embryo
(20-d gestation), dissociated, plated on poly-L-lysine-coated glass coverslips, and then cultured in MEM with 10% horse serum. After 4 d in culture, the medium was replaced with MEM supplemented with N2 supplement, 1 mg/ml of ovalbumin, 1 mM pyruvate, and 5 mM cytosine
arabinoside, cultured for the indicated period, and then subjected to
Western blot analysis and immunofluorescence microscopy.
-actinin antibody was
prepared as described (Kato et al., 1996
). Monoclonal anti-myc-epitope
(American Type Culture Collection, Rockville, MD), anti-synaptotagmin I
(Wako Pure Chemical Industries, Ltd., Osaka, Japan), anti-synaptophysin
(Boehringer Mannheim Corp., Indianapolis, IN), and anti-
-tubulin
(Amersham Corp., Arlington Heights, IL) antibodies and a polyclonal
anti-synapsin I (Chemicon International, Inc., Temecula, CA) were obtained from commercial sources.
-tubulin, mouse anti-synaptotagmin I, and/or mouse anti-myc antibodies. After the samples were washed with solution A, they
were incubated for 30 min with fluorescence-conjugated, anti-rabbit and/
or anti-mouse antibodies, including FITC-conjugated, rhodamine-conjugated, and/or Cy5-conjugated antibodies. For the double staining with
F-actin, these second antibodies were mixed with rhodamine-phalloidin. After the incubation, the sample was washed with solution A, embedded, and then viewed with a confocal imaging system (MRC-1024; Bio-Rad Laboratories, Hercules, CA).
). Briefly, rats were perfused
with 4% paraformaldehyde in PBS, and postfixed with 4% paraformaldehyde in PBS. Their tissues were sectioned in a cryostat, mounted on glass
slides, and then air dried. The samples were incubated for 12 h with the
rabbit anti-neurabin-1 or -2 antibody and the mouse anti-synaptophysin
antibody, followed by incubation for 12 h with Texas red-conjugated anti-
rabbit and FITC-conjugated anti-mouse antibodies.
).
Fig. 9.
Inhibition of the neurite formation by an antisense
phosphorothioate oligonucleotide complementary to the neurabin
sequence in primary cultured rat hippocampal neurons. (A) Antisense PONs complementary to the neurabin sequence. (B) Western
blot analysis. The samples (5 µg of protein each) were subjected
to Western blot analysis using the anti-neurabin-1 or anti--actinin
antibody. Protein markers used are myosin (206),
-galactosidase
(117), and BSA (89). (C) Immunofluorescence microscopy. The
neurons were treated with antisense-1 or sense-1, followed by the
double staining using rhodamine-phalloidin and the anti-
-tubulin
antibody. The anti-
-tubulin antibody was visualized with an
FITC-conjugated, anti-mouse antibody. (a and d) with antisense-1;
(b and e) with sense-1; and (c and f) control. (a-c) F-actin and (d-f)
-tubulin. These results are representative of three independent
experiments. The neurite formation in thirty neurons was statistically analyzed in each experiment. Bars, 20 µm.
[View Larger Versions of these Images (24 + 43K GIF file)]
). The coverslips with attached cells were then transferred to
dishes containing glia-conditioned MEM with N2 supplements, 1 mg/ml of
ovalbumin, and 1 mM pyruvate (Goslin and Banker, 1991
) in the presence
of 50 µM of each PON. Each PON was then added to the medium every
12 h, and half of the medium was renewed 24 h later. After 48 h, the neurons treated with each PON were subjected to Western blot analysis and
immunofluorescence microscopy.
). Protein concentrations were determined with BSA as
a reference protein (Bradford, 1976
).
Results
Fig. 2.
Deduced amino acid sequence of neurabin. The aa sequences of the nine peptide peaks derived from the purified sample of neurabin are indicated by underlines.
[View Larger Version of this Image (89K GIF file)]
Fig. 3.
Molecular characterization of neurabin. (A)
125I-Labeled F-actin-binding
activity of recombinant p180
(neurabin). The pCMV-neurabin was transfected to
COS7 cells, and the cell extract was subjected to SDS-PAGE (5-15% polyacrylamide gradient gel), followed
by 125I-labeled F-actin blot
overlay. The arrowhead indicates p180 (neurabin). The
radioactive bands of 125I-
labeled F-actin-binding activity shown by asterisks are
endogenous proteins of COS7
cells. Protein markers used are
myosin (206), -galactosidase
(117), BSA (89), and ovalbumin (47). (B) Schematic drawing of neurabin structure and its probabilities of
forming coiled-coil structures.
Probabilities were calculated
for each residue with the
weighted MTIDK matrix using a 28-residue window. (C)
125I-Labeled F-actin-binding
activity of various truncated
forms of neurabin. The purified proteins (0.2 µg of protein each) were subjected to
SDS-PAGE (5-15% polyacrylamide gradient gel), followed by 125I-labeled F-actin
blot overlay. GST-neurabin-1,
aa 1-144; GST-neurabin-2, aa 1-210; GST-neurabin-3,
aa 145-293; GST-neurabin-4, aa 286-615; GST-neurabin-5, aa 505-791; and GST-neurabin-6, aa 665-1095. Protein markers used are
BSA (89) and ovalbumin (47). (D) Localizations of neurabin and F-actin in COS7 cells. Full-length and truncated forms of neurabin
were expressed as myc-tagged proteins in COS7 cells. The cells were doubly stained using the anti-myc antibody and rhodamine-phalloidin. The anti-myc antibody was visualized with a Cy5-conjugated anti-mouse antibody. (a-c) myc-tagged protein; and (d-f) F-actin.
(a and d) myc-neurabin (full-length); (b and e) myc-neurabin-1 (aa 1-144); and (c and f) myc-neurabin-2 (aa 145-1095). Bars, 20 µm.
[View Larger Versions of these Images (27 + 78 + 65K GIF file)]
). In the COOH-terminal region following the
PDZ domain, several sequence stretches had high probabilities of forming coiled-coil structures (Lupas et al., 1991
;
Lupas, 1996
) (Fig. 3 B). Especially, the coiled-coil probabilities of four sequence stretches (first one [Fig. 3 B, a], aa
675-736; second one [Fig. 3 B, b], aa 739-779; third one
[Fig. 3 B, c], aa 787-834; and fourth one [Fig. 3 B, d], aa
1041-1080) were calculated to be 0.9-1.0. The probability
of the sequence stretch (aa 598-628) was calculated to be
0.6-0.7. This stretch is unlikely to form a coiled-coil structure, because there was a difference of >0.2-0.3 in the
probabilities between the MTK and MTIDK matrices
(Lupas et al., 1991
; Lupas, 1996
). The NH2-terminal region
of neurabin did not show any tendency to form a coiled-coil structure.
7 M. It was examined by competition
experiments whether neurabin bound along the sides of
F-actin or at the ends. The binding of the Mono S sample
of neurabin to 125I-labeled F-actin was completely inhibited by an excessive amount of myosin S1, a well-characterized protein that binds along the sides of F-actin (Rayment et al., 1993
; Schröder et al., 1993
) (Fig. 4 C). This
inhibition was reversed by the addition of MgATP because
MgATP dissociates the actin-myosin complex (Fraser et al., 1975
). These results indicate that neurabin binds along
the sides of F-actin.
Fig. 4.
Biochemical properties of neurabin. (A) Cosedimentation of GST-neurabin-2 with F-actin. GST-neurabin-2 (aa 1-210)
(10 µg of protein) was mixed with F-actin, followed by ultracentrifugation. S, supernatant; and P, pellet. Arrow and arrowhead
indicate actin and GST-neurabin-2, respectively. Asterisks indicate the proteolytic products of GST-neurabin-2, which were not
cosedimented with F-actin. Protein markers used are ovalbumin
(45) and carbonic anhydrase (31). (B) Binding of His6-neurabin
to F-actin. Various amounts of His6-neurabin were mixed with
F-actin, followed by ultracentrifugation. Amounts of the free and
bound His6-neurabin were calculated by determining the protein
amounts from the supernatant and pellet fractions with a densitometer. (C) Inhibition by myosin S1 of the binding of neurabin
to 125I-labeled F-actin. The Mono S sample of native neurabin
(0.1 µg of protein) was subjected to SDS-PAGE (8% polyacrylamide gel), followed by the blot overlay with 125I-labeled F-actin
pretreated with myosin S1 in the presence or absence of ATP.
Protein markers used are myosin (206), -galactosidase (117),
and BSA (89). (D) F-actin-cross-linking activity of neurabin estimated by low shear viscometry. G-actin was mixed with the Mono S sample of native neurabin or His6-neurabin in an indicated amount and incubated for 45 min, followed by low shear
viscometry (left).
, With native neurabin; and
, with His6-neurabin. Protein staining of His6-neurabin with Coomassie brilliant blue (inset). Protein markers used are myosin (206),
-galactosidase (117), BSA (89), and ovalbumin (45). G-actin was mixed
with 15 µg/ml of the Mono S sample of native neurabin and incubated for an indicate time, followed by low shear viscometry
(right).
, With neurabin; and
, without neurabin. (E) F-actin-
cross-linking activity of neurabin estimated by electron microscopy. (a and c) with neurabin; and (b and d) without neurabin.
Bars: (a and b) 200 nm; (c and d) 50 nm.
[View Larger Versions of these Images (45 + 154K GIF file)]
). The frictional ratio
of neurabin was calculated to be about 1.3. Because the
molecular mass value of neurabin estimated by SDS-PAGE was ~180 kD, and neurabin had predicted coiled-coil structures at the COOH-terminal region, these results suggest that neurabin forms a dimer. It is also possible that neurabin forms a trimer or tetramer because the molecular mass value of neurabin calculated from its predicted aa
sequence was ~120 kD.
Fig. 5.
Oligomerization
property of neurabin. (A) Superdex 200 column chromatography. Each fraction was
subjected to protein staining with silver and Western blot
analysis using the anti-
neurabin-1 antibody. The
amount of His6-neurabin of
each fraction was determined with the densitometer. Protein markers used are thyroglobulin (669 kD, Stokes' radius 85.0 Å), catalase (232 kD, Stokes' radius 52.2 Å),
and aldolase (158 kD,
Stokes' radius 48.1 Å). Estimation of the molecular mass
value of His6-neurabin (inset). (B) Sucrose density gradient ultracentrifugation. Each fraction was subjected to protein
staining with silver and Western blot analysis using the anti-
neurabin-1 antibody. The amount of His6-neurabin of each fraction was determined with the densitometer. Protein markers used are catalase (232 kD, 11.3 S), and BSA (67 kD, 4.4 S). Estimation of the S value of His6-neurabin (inset).
[View Larger Version of this Image (23K GIF file)]
Fig. 6.
Tissue distribution
of neurabin. (A) Northern
blot analysis. A RNA blot
membrane (CLONTECH,
Palo Alto, CA) was hybridized with the 32P-labeled, 2.3-kbp KpnI-XhoI fragment of
the neurabin cDNA according to the manufacturer's
protocol. (B) Western blot
analysis. The homogenates of
various rat tissues (10 µg of
protein each) were subjected
to SDS-PAGE (8% polyacrylamide gel), followed by
immunoblot using the anti-
neurabin-1 antibody. Protein
markers used are myosin
(206), -galactosidase (117),
BSA (89), and ovalbumin (47).
[View Larger Version of this Image (25K GIF file)]
). At this stage, neurabin was concentrated in the lamellipodia of the growth cone (Fig. 7 B),
where F-actin is enriched (Goslin et al., 1989
; Letourneau
and Shattuck, 1989
). Neurabin was not clearly concentrated in the filopodia. After 10 d in culture, both axons
and dendrites develop and the synapses are observed (Basarsky et al., 1994
). At this stage, neurabin showed
dotty signals along the dendrites (Fig. 7 C, a). Synaptotagmin I, known to be highly concentrated at the presynaptic
nerve terminal (Matthew et al., 1981
), showed the similar
staining pattern (Fig. 7 C, b). This result suggests that
neurabin is highly concentrated in the synapse.
Fig. 7.
Expression and localization of neurabin during the development of primary cultured rat hippocampal neurons. (A) Expression of neurabin during the development. The cultured cells
at various stages were subjected to Western blot analysis using
the anti-synapsin I, anti-synaptophysin, or anti-neurabin-1 antibodies. (B) Localization of neurabin in the hippocampal neurons
at 24 h in culture. The sample was incubated with the anti-
neurabin-1 antibody and visualized with an FITC-conjugated,
anti-rabbit antibody. High magnification of the indicated box (inset). Arrows and arrowheads indicate the growth cone and cell
body, respectively. (C) Localization of neurabin in the hippocampal neurons at 10 d in culture. The sample was doubly stained using the rabbit anti-neurabin-1 and mouse anti-synaptotagmin I
antibodies. They were visualized with FITC-conjugated, anti- rabbit and rhodamine-conjugated, anti-mouse antibodies. (a)
neurabin and (b) synaptotagmin I. Bars, 20 µm.
[View Larger Version of this Image (44K GIF file)]
). In the cerebellum, neurabin showed dotty signals in
the molecular layer and discontinuous signals along the
Purkinje cell body (Fig. 8 A). It also showed intense signals in the gromerulus of the granular layer, where the
synapses are complexed. In the hippocampus, neurabin
showed dotty signals in the stratum oriens, discontinuous
signals along the cell body in the stratum pyramidale, and
intense signals in the stratum radiatum (Fig. 8 C). These staining patterns of neurabin were similar to those of synaptophysin (Fig. 8, A-D). These results indicate that
neurabin is highly concentrated in the synapse of these tissues. In the retina, neurabin was highly concentrated in
the inner and outer plexiform layers, where the synapses
are formed (Fig. 8 E). Neurabin was also expressed in the
neuromuscular junction, where the synapses are formed
between motor neurons and muscle fibers (Fig. 8 G), and
in adrenal chromaffin cells (Fig. 8 I). In these tissues other
than brain, the staining patterns of neurabin were also similar to those of synaptophysin (Fig. 8, E-J). Similar results
were obtained with another antibody (anti-neurabin-2),
which was raised against GST-neurabin-8 (aa 890-1095)
(data not shown). These results demonstrate that neurabin is widely distributed in neural tissue and highly concentrated in the synapse of the developed neurons.
Fig. 8.
Localization of neurabin in rat neural tissue. The samples were doubly stained using the rabbit anti-neurabin-1 and
mouse anti-synaptophysin antibodies. They were visualized with
Texas red-conjugated, anti-rabbit and FITC-conjugated, anti-
mouse antibodies. (A, C, E, G, and I) neurabin, and (B, D, F, H,
and J) synaptophysin. (A and B) cerebellum. ml, molecular layer;
pl, Purkinje cell layer; and gl, granular layer. (C and D) hippocampus (CA3). so, stratum oriens; sp, stratum pyramidale; and sr,
stratum radiatum. (E and F) retina. ip, inner plexiform layer; in,
inner nuclear layer; op, outer plexiform layer; and on, outer nuclear layer. (G and H), neuromuscular junction. (I and J) adrenal
gland. am, adrenal medulla; and ac, adrenal cortex. Bars, 20 µm.
[View Larger Version of this Image (130K GIF file)]
-actinin, an F-actin-bundling protein, was not affected, suggesting that antisense-1 specifically reduced the expression of neurabin. The neurons treated with antisense-1 were
morphologically compared with those treated with sense-1
by the immunofluorescence staining of F-actin and tubulin.
In the neurons treated with sense-1, as well as in the control neurons, >90% of the cells were characterized by one
long axon and several processes (Fig. 9 C). In contrast, in
>90% of the neurons treated with antisense-1, any neurite
formation was not observed. Trypan blue exclusion test
showed that the neurons treated with antisense-1 were
alive (data not shown). Moreover, this effect of antisense-1
was reversible. When antisense-1 was washed out after the
last addition, the neurite formation of the hippocampal
neurons began to be observed 48 h later (data not shown).
Discussion
-actinin/spectrin family members have most extensively
been studied as F-actin-binding proteins having F-actin-
cross-linking activity (Hartwig and Kwiatkowski, 1991
;
Matsudaira, 1991
). They usually form oligomers by association at their rod domains and thereby show the F-actin-
cross-linking activity. Neurabin has the domains for predicted coiled-coil structures. Coiled-coil structures have
been identified in a variety of cytoskeleton proteins (Lupas et al., 1991
; Lupas, 1996
). They typically form rodlike,
homodimeric, or higher order structures. The molecular mass of neurabin estimated by SDS-PAGE and that calculated by its predicted aa sequence are ~180 and 120 kD,
respectively, whereas those estimated by gel filtration and
sucrose density gradient ultracentrifugation are ~440 and
380 kD, respectively. The molecular masses of neurabin
was calculated to be ~440 from both the Stokes' radius
and S value (Siegel and Monty, 1966
). These results together with the dimerization or oligomerization property
of the coiled-coil structures suggest that neurabin forms an
oligomer with multiple F-actin-binding heads via its predicted coiled-coil structures and thereby shows the F-actin-
cross-linking activity.
; Kistner et al., 1993
), and implicated in
protein-protein interactions (Saras and Heldin, 1996
).
This domain has been found in a variety of proteins that
are typically located at specific regions of cell-cell junctions, such as the tight junction, the synaptic junction, and
the septate junction. Recent studies have revealed that the
PDZ domain binds the unique COOH-terminal motifs of
target proteins (Doyle et al., 1996
; Songyang et al., 1997
),
which are found in a large number of transmembrane proteins, such as N-methyl-D-aspartate receptors and Shaker-type
K+ channel (Saras and Heldin, 1996
). Of many proteins
having the PDZ domain(s) thus far reported, no protein
having F-actin-binding activity has been reported. Conversely, of many F-actin-binding proteins thus far reported,
no protein having the PDZ domain(s) has been reported.
We have shown here that neurabin is highly concentrated
in the synapse. These results, together with the unique
structural and biochemical properties of neurabin, suggest that it is linked to a transmembrane protein(s) through its
PDZ domain, providing a linkage between the synaptic
junction and the actin cytoskeleton. We have recently
identified another F-actin-binding protein having one
PDZ domain, and named it afadin (Mandai et al., 1997
).
Afadin appears to provide a linkage between cadherin-based cell-cell adherens junction and the actin cytoskeleton in various epithelia. These F-actin-binding proteins
having one PDZ domain may constitute a functional family, of which members provide a linkage between cell-cell
junctions and the actin cytoskeleton.
.
Address all correspondence to Y. Takai, Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Japan. Tel.: 81-6-879-3410. Fax: 81-6-879-3419. E-mail: ytakai{at}molbio.med.osaka-u.ac.jpWe thank Dr. D.W. Russell (University of Texas Southwestern Medical
Center at Dallas, Dallas, TX) for providing us the pCMV vector, and Dr.
M. Imamura (National Institute of Neuroscience, Kodaira, Japan) for providing us the anti--actinin antibody. We also thank Dr. Sh. Tsukita
(Kyoto University, Kyoto, Japan), and Dr. Y. Hata (Takai Biotimer
Project, ERATO, Kobe, Japan) for helpful discussions.
aa, amino acid; F-actin, actin filament; G-actin, actin monomer; GST, glutathione-S-transferase; neurabin, neural tissue-specific F-actin-binding protein; PDZ, PSD-95, DlgA, ZO-1-like; PON, phosphorothioate oligonucleotide; S1, subfragment 1; SV, synaptic vesicle.
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