The Journal of Cell Biology, Volume 139, Number 2, October 20, 1997 517–528
The Rockefeller University Press, 0021-9525/97/10/517/12 $2.00
Afadin: A Novel Actin Filament–binding Protein with One PDZ Domain
Localized at Cadherin-based Cell-to-Cell Adherens Junction
Kenji Mandai,* Hiroyuki Nakanishi,* Ayako Satoh,* Hiroshi Obaishi,* Manabu Wada,* Hideo Nishioka,*
Akira Mizoguchi, Takeo Aoki, Toyoshi Fujimoto,
and Yoshimi Takai*
*Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, c/o JCR Pharmaceuticals Co., Ltd.,
Kobe 651-22, Japan;
Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Japan;
Department of Cell Biology, Department of Anatomy and Neurobiology, Faculty of Medicine, Kyoto University, Kyoto 606-01,
Japan; Department of Anatomy and Cell Biology, Gunma University School of Medicine, Maebashi 371, Japan; and
**Laboratory of Animal Genetics, School of Agricultural Sciences, Nagoya University, Nagoya 464-01, Japan
tein with a molecular mass of
was concentrated at cadherin-based cell-to-cell adherens
junction (AJ), was isolated and characterized. p205 was
purified from rat brain and its cDNA was cloned from a
rat brain cDNA library. p205 was a protein of 1,829
amino acids (aa) with a calculated molecular mass of
207,667 kD. p205 had one F-actin–binding domain at
1,631–1,829 aa residues and one PDZ domain at 1,016–
1,100 aa residues, a domain known to interact with trans-
membrane proteins. p205 was copurified from rat brain
with another protein with a molecular mass of 190 kD
(p190). p190 was a protein of 1,663 aa with a calculated
molecular mass of 188,971 kD. p190 was a splicing vari-
ant of p205 having one PDZ domain at 1,009–1,093 aa
residues but lacking the F-actin–binding domain. Ho-
mology search analysis revealed that the aa sequence of
A novel actin filament (F-actin)–binding pro-
205 kD (p205), which
p190 showed 90% identity over the entire sequence
with the product of the
be fused to the
gene, known to be involved in
acute leukemia. p190 is likely to be a rat counterpart of
protein. p205 bound along the sides of
F-actin but hardly showed the F-actin–cross-linking ac-
tivity. Northern and Western blot analyses showed that
p205 was ubiquitously expressed in all the rat tissues
examined, whereas p190 was specifically expressed in
brain. Immunofluorescence and immunoelectron mi-
croscopic studies revealed that p205 was concentrated
at cadherin-based cell-to-cell AJ of various tissues. We
named p205 l-afadin (a large splicing variant of AF-6
protein localized at adherens junction) and p190 s-afa-
din (a small splicing variant of l-afadin). These results
suggest that l-afadin serves as a linker of the actin cy-
toskeleton to the plasma membrane at cell-to-cell AJ.
gene, which was found to
Address all correspondence to Y. Takai, Department of Molecular Biol-
ogy and Biochemistry, Osaka University Medical School, Suita 565, Osaka,
Japan. Tel.: 81-6-879-3410. Fax: 81-6-879-3419. E-mail: ytakai@molbio.
F-actin, actin filament; FISH, fluorescence in situ hybridization; G-actin,
actin monomer; GST, glutathione
-transferase; His6, six histidine resi-
dues; l-afadin, a large splicing variant of AF-6 protein localized at adherens
junction; S1, subfragment 1; s-afadin, a small splicing variant of l-afadin.
Abbreviations used in this paper
: aa, amino acid; AJ, adherens junction;
teins, such as cell adhesion molecules, receptors, and
channels, and these domains are often associated with the
actin cytoskeleton (Geiger, 1983, 1989; Geiger and Ginsberg,
1991; Turner and Burridge, 1991; Luna and Hitt, 1992;
Tsukita et al., 1992, 1997; Bretscher, 1993). The linkage
between the actin cytoskeleton and the plasma membrane
plays crucial roles in these cellular events, and proteins link-
ing the actin cytoskeleton to the transmembrane proteins
have been identified. However, the molecular basis of the
various cellular events, such as cell adhesion, cell mo-
tility, and cell shape determination, specialized mem-
brane domains are formed with transmembrane pro-
linkage between the actin cytoskeleton and the plasma mem-
brane is not fully understood.
To understand this molecular linkage, the cell adhesion
sites have most extensively been studied (Geiger, 1983,
1989; Geiger and Ginsberg, 1991; Turner and Burridge,
1991; Luna and Hitt, 1992; Tsukita et al., 1992, 1997;
Bretscher, 1993). The actin filament (F-actin)
cell adhesion sites are subclassed into two types: cell-to-cell
and cell-to-matrix adherens junctions (AJ). Many linker
proteins have been identified at cell-to-cell AJ where cad-
herins interact with each other at the extracellular surface
(Takeichi, 1988; Geiger and Ginsberg, 1991; Takeichi,
The Journal of Cell Biology, Volume 139, 1997
1991; Tsukita et al., 1992). The cytoplasmic domain of cad-
herin is associated with cytoplasmic proteins such as
-catenins (Ozawa et al., 1989; Nagafuchi et al., 1991;
Takeichi, 1991; Tsukita et al., 1992).
teracts with F-actin (Rimm et al., 1995).
teracts indirectly with F-actin through
ZO-1 (Knudsen et al., 1995; Itoh et al., 1997). Vinculin, an-
other F-actin–binding protein, is concentrated at cell-to-
cell AJ, although its interacting molecule at cell-to-cell AJ
has not yet been identified (Geiger and Ginsberg, 1991;
Tsukita et al., 1992). At cell-to-matrix AJ where integrin
interacts with matrix proteins at the extracellular surface,
the cytoplasmic domain interacts directly or indirectly
with F-actin–binding proteins, including
lin, and talin (Jockusch et al., 1995). Thus, many F-actin–
binding proteins appear to serve as linkers of the actin cy-
toskeleton to the plasma membrane cadherin and integrin.
The linkage between the actin cytoskeleton and the
plasma membrane is also important for neuron-specific
events, such as growth cone pathfinding and the subse-
quent formation and maintenance of synaptic junction
(Mitchison and Kirschner, 1988; Smith, 1988; Bentley and
O’Connor, 1994; Lin et al., 1994; Tanaka and Sabry, 1995).
However, it remains to be clarified which molecules link
the actin cytoskeleton to the plasma membrane in these
neuron-specific events. To address this issue, we attempted
to isolate novel F-actin–binding proteins from the rat brain
by using a blot overlay method with
We had isolated a neural tissue-specific F-actin–binding
protein that is concentrated at synapse and named it
neurabin (Nakanishi et al., 1997). Neurabin has one F-actin–
binding domain and one PDZ domain. The PDZ domain
has been found in a variety of proteins, some of which are
localized at cell-to-cell junctions, such as PSD-95/SAP90 at
synaptic junction (Cho et al., 1992; Kistner et al., 1993),
Dlg at septate junction (Woods and Bryant, 1991), and
ZO-1 and ZO-2 at tight junction (Stevenson et al., 1986;
Gumbiner, 1991; Itoh et al., 1993; Tsukita et al., 1993; Wil-
lott et al., 1993; Jesaitis and Goodenough, 1994). Recent
studies have revealed that the PDZ domain binds to
unique COOH-terminal motifs of target proteins (Saras
and Heldin, 1996), which are found in a large number of
transmembrane proteins, such as
ceptors and Shaker-type K
channel (Kim et al., 1995; Kor-
nau et al., 1995; Niethammer et al., 1996). It is likely,
therefore, that neurabin serves as a linker of the actin cy-
toskeleton to a transmembrane protein(s) at the synapse
although we have not yet identified its interacting trans-
Along this line, we further attempted to isolate other
novel F-actin–binding proteins from rat brain. Here, we
isolated a novel F-actin–binding protein with a molecular
205 kD (p205). p205 also had one F-actin–bind-
ing domain and one PDZ domain, and was localized at
cadherin-based cell-to-cell AJ. During the purification of
p205, it was copurified with another protein with a molec-
ular mass of 190 kD (p190) that was a splicing variant of
p205 having one PDZ domain but lacking the F-actin–
binding domain. Homology search analysis revealed that
the amino acid (aa) sequence of p190 showed 90% identity
over the entire sequence with the product of the
gene, which has been found to be fused to the
-Catenin directly in-
-Catenin also in-
(Prasad et al., 1993), known to be involved in acute leuke-
mia (Cimino et al., 1991). We named p205 l-afadin (a large
splicing variant of AF-6 protein localized at adherens junc-
tion) and p190 s-afadin (a small splicing variant of l-afadin).
We describe here isolation and characterization of l-afadin.
Materials and Methods
I-labeled F-Actin Blot Overlay
Pestonjamasp et al., 1995). Briefly, purified actin monomer (G-actin) was
I-Bolton Hunter reagent.
erage specific activity = 63.3
Ci/mg) was polymerized with 18
gelsolin (molar ratio = 100:1; Sigma Chemical Co., St. Louis, MO) by incu-
bation for 10 min at 4
C in a solution containing 20 mM Pipes, pH 7.0, 50
mM KCl, and 2 mM MgCl
. Phalloidin was then added to give a final con-
centration of 40
M. The mixture was then incubated for another 15 min
at room temperature and stored at 4
to be tested was subjected to SDS-PAGE and transferred to a nitrocellu-
lose membrane. The membrane was blocked in TBS containing 5% defat-
ted powder milk. The membrane was then incubated for 1 h at room tem-
perature with 10
g/ml of I-labeled F-actin in TBS containing 5%
defatted powder milk and 4
M 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).
For competition experiments,
I-labeled F-actin was prepared as de-
scribed above, except that the concentration of gelsolin was reduced to 7.2
g/ml (molar ratio = 250:1). 20
for 30 min at room temperature with 0.42 mg/ml of myosin subfragment 1
(S1; Sigma Chemical Co.) in a solution containing 20 mM Pipes, pH 7.0, 58
mM KCl, 2 mM MgCl
dicated, 4 mM MgATP was added to the mixture. After the incubation,
the mixture was diluted with an equal volume of TBS containing 10% de-
fatted powder milk, and it was added to the blot membrane, followed by
incubation for 1 h at room temperature.
I-labeled F-actin blot overlay was done as described (Chia et al., 1991;
I-labeled G-actin (1 mg/ml, av-
I-labeled F-actin. The sample
I-labeled F-actin was incubated
, and 0.8
M phalloidin. Where in-
Purification of l-Afadin
All the purification procedures were carried out at 0–4
from 60 mother rats (17-d gestation) were homogenized with a solution
containing 10 mM Tris/Cl, pH 8.0, 2 mM EDTA, 5 mM EGTA, and a pro-
tease inhibitor cocktail (1 mM PMSF, 20
of pepstatin A). The homogenate was mildly stirred for 1 h and centri-
fuged at 200,000
for 1 h. The supernatant (600 ml, 2,070 mg of protein)
was stored at
C until use. The supernatant was applied to a Q-Seph-
arose FF column (2.6
34 cm; Pharmacia Biotech, Inc., Piscataway, NJ)
equilibrated with buffer A (20 mM Tris/Cl, pH 8.0, 0.5 mM EDTA, and
1 mM DTT). After the column was washed with 700 ml of buffer A, elu-
tion was performed with an 800-ml linear gradient of NaCl (0–0.5 M) in
buffer A. 10-ml fractions were collected. l-Afadin appeared in fractions
56–77. These fractions (220 ml, 500 mg of protein) were collected, and 1.2 M
was added to give a final concentration of 0.6 M. The sample
was centrifuged at 200,000
for 20 min, and the supernatant was applied
to a phenyl-5PW column (2.15
15 cm; Tosoh, Tokyo, Japan) equili-
brated with buffer A containing 0.6 M (NH
washed with 250 ml of the same buffer, elution was performed with a 240-ml
linear gradient of (NH
(0.6–0 M) in buffer A. 6-ml fractions were
collected. l-Afadin appeared in fractions 28–34. The active fractions (42 ml,
24 mg of protein) were collected and applied to a hydroxyapatite column
10 cm; Koken, Tokyo, Japan) equilibrated with buffer B (10 mM
potassium phosphate, pH 7.8, and 1 mM DTT). After the column was
washed with 30 ml of buffer B, elution was performed with a 150-ml linear
gradient of potassium phosphate (10–500 mM), pH 7.8, in buffer B. 2.0-ml
fractions were collected. l-Afadin appeared in fractions 39–41. The active
fractions (6 ml, 0.86 mg of protein) were collected and diluted with an
equal volume of buffer A, and half of the sample was applied to a Mono Q
HR 5/5 column (Pharmacia Biotech, Inc.) equilibrated with buffer A. After
the column was washed with 6 ml of buffer A, elution was performed with
a 30-ml linear gradient of NaCl (0.15–0.4 M) in buffer A. 0.5-ml fractions
were collected. The other half of the sample was subjected to Mono Q col-
umn chromatography in a similar manner. l-Afadin appeared in fractions
C. Fetal brains
g/ml of leupeptin, and 1
. After the column was
Mandai et al.
34–36 (see Fig. 1). The active fractions of the two Mono Q column chro-
matographies (3.0 ml, 0.2 mg of protein) were combined and stored at
C until use.
Peptide Mapping of l- and s-Afadins and Molecular
Cloning of Their cDNAs
The purified Mono Q sample (
PAGE (8% polyacrylamide gel). The protein bands corresponding to
p205 (l-afadin) and p190 (s-afadin) were separately cut out from the gel,
digested with a lysyl endopeptidase, and the digested peptides were sepa-
rated by TSKgel ODS-80Ts (4.6
150 mm; Tosoh) reverse-phase high
pressure liquid column chromatography as described (Imazumi et al.,
1994). The aa sequences of the peptides were determined with a peptide
sequencer. A rat brain cDNA library in
CA) was screened using the oligonucleotide probes designed from the
partial aa sequences. DNA sequencing was performed by the dideoxynu-
cleotide termination method using a DNA sequencer (model 373; Applied
Biosystems, Inc., Foster City, CA).
g of protein) was subjected to SDS-
ZAPII (Stratagene, La Jolla,
Chromosome Preparation and In Situ Hybridization
The fluorescence in situ hybridization (FISH) analysis was performed as
described (Matsuda et al., 1992; Matsuda and Chapman, 1995). Briefly,
the chromosomes were prepared from rat splenocytes, the R-band was
stained with Hoechst 33258, and the chromosomes were exposed to UV
light. After the cDNAs of clones 20, 84, and 94 were labeled with biotin
16-dUTP, the chromosomes were hybridized with these labeled cDNAs,
and they were incubated with an antibiotin goat antibody and then with an
FITC-labeled anti–goat IgG antibody. The chromosomes were then
stained with propidium iodide and were viewed using Nikon filter B-2A
(450–490 nm; Nikon Inc., Melville, NY).
Expression and Purification of Recombinant l-Afadin
The full-length l-afadin cDNA corresponding to bp 326–5812 (aa 1–1829)
and its fragment corresponding to bp 326–5344 (aa 1–1673) were inserted
to the pCMV-myc vector using standard molecular biology methods
(Sambrook et al., 1989). The constructs were transfected to COS7 cells
with the DEAE-dextran method (Hata and Südhof, 1995). The whole-cell
extracts were subjected to
I-labeled F-actin blot overlay and Western
blot analysis using an antibody against the myc-epitope as described be-
low. The 597-bp fragment of the l-afadin cDNA corresponding to bp
5216–5812 (aa 1631–1829) was inserted to the pGEX and pQE vectors.
These constructs were transformed into
-transferase (GST) fusion protein (GST–l-afadin-C) and the six
histidine residues (His6)–fusion protein (His6-l-afadin-C) were purified
by use of glutathione-Sepharose and Ni-agarose, respectively.
, and the glu-
F-Actin–binding and –cross-linking Activities
Low shear viscometry was performed as described (Pollard and Cooper,
1982; Kato et al., 1996). Briefly,
-actinin (recombinant chick lung type)
or the Mono Q sample of l-afadin, including s-afadin, in an indicated
amount was mixed with 0.28 mg/ml of G-actin in a solution containing 20
mM Tris/Cl, pH 8.0, 0.25 mM EDTA, 2 mM MgCl
mM ATP, and 1 mM EGTA, and the solution was sucked into a 0.1-ml mi-
cropipette. After the incubation for 1 h at 25
steel ball to fall a fixed distance in the pipette was measured.
Cosedimentation of l-afadin with F-actin was performed as follows:
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 MgCl
ATP, and 90 mM KCl). An indicated amount of GST–l-afadin-C or His6–
l-afadin-C (each aa 1631–1829) was incubated for 30 min at room temper-
ature with 0.3 mg/ml of F-actin, and the mixture (100
l cushion of 30% sucrose in the polymerization buffer. After the sam-
ple was centrifuged at 130,000
for 20 min, the supernatant was removed
from the cushion, and the pellet was brought to the original volume in an
SDS sample buffer. To quantitate cosedimentation of GST–l-afadin-C or
His6–l-afadin-C, the comparable amounts of the supernatant and pellet
fractions were subjected to SDS-PAGE (15% polyacrylamide gel), trans-
ferred to a nitrocellulose membrane, incubated with an antibody against
l-afadin as described below, and then incubated with
A. Amounts of the free and bound fusion proteins were calculated by de-
, 140 mM NaCl, 0.1
C, the time for a stainless
, 1 mM
l) was placed over a
termining the radioactivity from the supernatant and pellet fractions with
an image analyzer (Fujix BAS-2000II).
Antibodies and Immunofluorescence Microscopy
A rabbit polyclonal antibody against l-afadin, which specifically recog-
nized l-afadin, was directed against a 16-mer synthetic peptide corre-
sponding to aa 1814–1829 (KASRKLTELENELNTK). Another antibody
against l-afadin, which recognized both l- and s-afadins, was directed
against a 16-mer synthetic peptide corresponding to aa 577–592
(RLDQEQDYRRRESRTQ). Each peptide was coupled via cysteine at
-terminal residue to keyhole limpet hemocyanin and was used to
raise the antiserum. The antiserum specific to l-afadin was affinity-puri-
fied with GST–l-afadin-C covalently coupled to NHS-activated Sepharose
(Pharmacia Biotech, Inc.) and was used as the anti–l-afadin antibody. The
antiserum recognizing both l- and s-afadins was purified with the synthetic
peptide covalently coupled to EAH-Sepharose (Pharmacia Biotech, Inc.)
and was used as the anti–l- and s-afadin antibody. Monoclonal anti–ZO-1
(Itoh et al., 1991) and anti–myc epitope (Takaishi et al., 1995) antibodies
were prepared as described. Monoclonal anti–E-cadherin (Takara, Tokyo,
Japan), antivinculin (Sigma Chemical Co.), and antidesmoplakin (Progen
Biotechnik, Germany) antibodies were purchased from commercial
Immunofluorescence microscopy of frozen sections of various rat or
mouse tissues was done as described (Itoh et al., 1991). Briefly, samples of
various tissues were frozen using liquid nitrogen, and the frozen sections
m) were cut in a cryostat. The samples were mounted on glass
slides, air dried, and fixed with 95% ethanol for 30 min at 4
100% acetone for 1 min at room temperature. After being washed with
PBS containing 1% BSA, the samples were incubated with the polyclonal
anti–l-afadin antibody alone, or with a mixture of the anti–l-afadin anti-
body and the monoclonal anti–E-cadherin, anti–ZO-1, or antivinculin an-
tibody. The samples were then washed with PBS containing 1% BSA and
0.1% Triton X-100, followed by incubation with rhodamine-conjugated
donkey anti–rabbit IgG and FITC-conjugated sheep anti–mouse IgG anti-
bodies. After being washed with PBS, they were embedded and viewed
with a confocal imaging system (MRC-1024; Bio-Rad Laboratories, Her-
Immunofluorescence microscopy of EL cells was done as described
(Itoh et al., 1991). Briefly, cells were cultured on a coverglass and fixed
with 1% formaldehyde in PBS. The fixed sample was treated with 0.2%
Triton X-100 in PBS and washed three times with PBS. After the sample
was soaked with PBS containing 1% BSA, the sample was treated with
the rabbit anti–l-afadin antibody and the mouse anti–ZO-1 antibody, and
washed with PBS containing 1% BSA, followed by incubation with the
rhodamine-conjugated anti–rabbit IgG and FITC-conjugated anti–mouse
IgG antibodies. After the incubation, the sample was washed with PBS,
embedded in PBS containing 95% glycerol, and viewed with the confocal
imaging system (MRC-1024; Bio-Rad Laboratories).
Immunofluorescence microscopy of the bile canaliculi isolated from
mouse liver was done as described (Tsukita et al., 1989). Briefly, the frac-
tion rich in the bile canaliculi (Tsukita and Tsukita, 1989) was put on a
coverglass, air dried, and fixed with 1% formaldehyde in PBS. The sample
was soaked with PBS containing 1% BSA and then processed as described
C and with
Immunoelectron microscopy using the silver enhancement technique was
done as described (Mizoguchi et al., 1994). Briefly, adult rat small intes-
tine was perfused with 4% paraformaldehyde in PBS. The sample was in-
cubated with the anti–l-afadin antibody, followed by incubation with an
anti–rabbit IgG antibody coupled with 1.4-nm gold particles (Nanoprobes
Inc.). After the sample was washed, it was fixed with 2% glutaraldehyde
in PBS for 15 min, and the sample-bound gold particles were silver en-
hanced by the HQ-silver kit (Nanoprobes Inc., Stony Brook, NY) for 8
min at 18
C. The sample was again washed and postfixed with 0.5% os-
mium oxide in a buffer containing 100 mM cacodylate buffer, pH 7.3.
They were dehydrated by passage through a graded series of ethanol (50,
70, 90, and 100%) and propylene oxide, and were embedded in epoxy
resin. From this sample, ultrathin sections were cut, stained with uranyl
acetate and lead citrate, and then observed with an electron microscope
(JEM-1200EX; Jeol Ltd., Tokyo, Japan).
Immunoelectron microscopy using the ultrathin cryosection technique
was done as described (Tokuyasu, 1980). Briefly, adult rat small intestine
The Journal of Cell Biology, Volume 139, 1997
was fixed with 4% formaldehyde in 0.1 M phosphate buffer, pH 7.4, for 6 h
at room temperature. The fixed sample was infused with 1.8 M sucrose
containing 20% polyvinylpyrrolidone at 4
rapidly frozen using liquid nitrogen, and then cryosectioned at
ing a diamond knife. The ultrathin sections were collected on Formvar-
filmed grids and rinsed with PBS containing 10 mM glycine. The samples
were treated with a mixture of 3% BSA and 0.5% gelatin for 30 min, and
then incubated sequentially with the anti–l-afadin antibody and with a 5-nm
colloidal gold-conjugated goat anti–rabbit IgG antibody (Amersham
Corp., Arlington Heights, IL). The samples were fixed with 2% glutaral-
dehyde in PBS, rinsed with distilled water, incubated with 2% methylcel-
lulose and 0.5% uranyl acetate (Griffiths et al., 1984), and air dried. The
samples were observed under an electron microscope (JEM-100CX; Jeol
C overnight (Tokuyasu, 1989),
G-Actin was purified from rabbit skeletal muscle as described (Pardee and
Spudich, 1982). Protein concentrations were determined with BSA as a ref-
erence protein (Bradford, 1976).
Purification of l-Afadin and Molecular
Cloning of Its cDNA
To identify novel actin-binding proteins, we attempted to
detect actin-binding proteins using a blot overlay method
I-labeled F-actin. The soluble fraction of rat fetal
brain was subjected to SDS-PAGE followed by the blot
overlay. Several radioactive protein bands with various
molecular masses were detected. Of these F-actin–binding
proteins, the protein band with a molecular mass of
kD (p205) was highly purified by column chromatogra-
phies, including Q-Sepharose, phenyl-5PW, hydroxyapa-
tite, and Mono Q column chromatographies. On the final
Mono Q column chromatography, the F-actin–binding
protein band well coincided with a protein with a molecu-
lar mass of
205 kD that was identified by protein staining
with Coomassie brilliant blue (Fig. 1). p205 was copurified
with another protein with a molecular mass of
(p190), and they could not be separated from each other
by further column chromatographies, including Mono S
and Superdex 200 column chromatographies (data not
shown). These proteins appeared at a position correspond-
ing to a molecular mass of
The Mono Q sample including p205 and p190 was used to
study the biochemical properties of p205 as described below.
When the peptide mapping analyses of the Mono Q
sample of these two proteins, p205 and p190, were per-
formed, their maps were almost identical, whereas p205
showed the additional peptide peaks. When the aa se-
quences of the five peptide peaks common between the
two proteins were separately determined, all the aa se-
quences of the p205 and p190 peptides were identical. A
computer homology search indicated that the aa se-
quences of the five peptide peaks were identical with or
significantly homologous to those of human AF-6 protein.
When the aa sequences of the two peptide peaks specific to
p205 were determined, they were not found in the current
protein database. These results suggest that p205 and p190
are related to human AF-6 protein, and that p190 is a splic-
ing variant, a homologue, or a degradative product of p205.
A rat brain cDNA library was screened using the degen-
erated oligonucleotide probes designed from the partial aa
600 kD on the gel filtration.
sequences of the seven peptide peaks described above. We
obtained several overlapping clones (Fig. 2). Among these
clones, clone 20 contained a coding region with
but it lacked a predicted initiation codon. The deduced aa
sequence included all the aa sequences of the peptides of
p205. This aa sequence analysis indicated that the aa se-
quences of the two peptides specific to p205 were both lo-
cated in the COOH-terminal region. Clone 94 contained a
coding region with
4.5 kb and an in-frame stop codon
upstream of the stop codon of clone 20. This clone also
lacked a predicted initiation codon. The deduced aa se-
quence of the coding region included all the aa sequences
of the peptides of p190, but not those of the peptides spe-
cific to p205. Clone 84 contained a coding region with
kb and a predicted initiation codon. To confirm whether
these cDNAs were derived from the same locus, we made
the chromosomal assignment by FISH analysis using the
cDNAs as probes. All the cDNAs were localized on rat
chromosome 1q12.2 (data not shown).
Figure 1. Mono Q column chromatography. (a) Absorbance at
280 nm (A280). (b) 125I-labeled F-actin blot overlay. An aliquot (3
?l) of each fraction was subjected to 125I-labeled F-actin blot
overlay. (c) Protein staining with Coomassie brilliant blue. An al-
iquot (10 ?l) of each fraction was subjected to SDS-PAGE (8%
polyacrylamide gel), followed by protein staining with Coomassie
Mandai et al.
The full-length p205 and p190 cDNAs were constructed
from clones 84 and 20, and clones 84 and 94, respectively.
When the p205 cDNA was transfected into COS7 cells and
the cell extract was subjected to
overlay, the recombinant protein (myc–l-afadin) showed
mobility similar to that of native p205 on SDS-PAGE, as
well as 125I-labeled F-actin–binding activity (Fig. 3 a). The
deletion mutant of p205 lacking the COOH-terminal 156
aa (myc–l-afadin?C) did not show the 125I-labeled F-actin–
binding activity. A fusion protein of the COOH-terminal
region of p205 (199 aa) with GST (GST–l-afadin-C) showed
the 125I-labeled F-actin–binding activity.
Based on these observations, together with the fact that
the Mono Q sample of p205 indeed showed the F-actin–
binding activity as described below, we have concluded
that the p205 gene encodes a protein of 1,829 aa with a cal-
culated molecular mass of 207,667 kD (Gen Bank/EMBL/
DDBJ accession number U83230), of which the COOH-
terminal 199 aa contributes to its F-actin–binding activity,
and that the p190 gene encodes a protein lacking ?160
COOH-terminal aa (GenBank/EMBL/DDBJ accession
number U83231) and is a splicing variant of the p205 gene.
A computer homology search revealed that the aa se-
quence of p190 showed 90% identity over the entire se-
quence with that of human AF-6 protein (Prasad et al.,
1993), while human AF-6 protein and p190 lacked the
COOH-terminal region of p205. p190 is likely to be a rat
counterpart of human AF-6 protein. The COOH-terminal,
F-actin–binding domain showed no significant homology
to any known F-actin–binding protein. Both p205 and
p190 had one PDZ domain (Fig. 3 b). We named p205 and
p190 l- and s-afadins, respectively.
Northern blot analysis using a sequence specific to the
l-afadin cDNA as a probe detected ?7.5-kb mRNA in all
the rat tissues examined, including the heart, brain, spleen,
lung, liver, skeletal muscle, kidney, and testis (Fig. 4 a). A
similar result was obtained when a sequence common be-
tween the cDNAs of l- and s-afadins was used as a probe
(data not shown). Western blot analysis using an antibody
specific to l-afadin detected an ?205-kD protein in all the
rat tissues examined (Fig. 4 b1). When an antibody recog-
nizing both l- and s-afadins was used, however, an ?190-
kD protein was detected only in the brain (Fig. 4 b2).
I-labeled F-actin blot
These results indicate that l-afadin is expressed ubiqui-
tously while s-afadin is specifically expressed in brain. The
reason why the Northern blot analysis did not detect the
two bands in the brain may simply be because of the simi-
lar length of the mRNAs of l- and s-afadins.
Biochemical Properties of l-Afadin
It was examined by competition experiments whether the
Mono Q sample of l-afadin (including s-afadin) bound along
the sides of F-actin or at its ends. The binding of 125I-labeled
F-actin to l-afadin was completely inhibited by an exces-
sive 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. 5 a). This inhibition was re-
versed by the addition of MgATP because MgATP disso-
ciates the actin–myosin complex (Fraser et al., 1975). These
results indicate that l-afadin binds along the sides of F-actin.
The effect of the Mono Q sample of l-afadin on F-actin was
examined using the falling ball method for low shear vis-
cometry. This effect was compared to that of ?-actinin, a
well-characterized protein that shows F-actin–cross-linking
activity (Burridge and Feramisco, 1981). l-Afadin increased
the viscosity of F-actin in a dose-dependent manner, and the
viscosity became maximal at approximately threefold (Fig.
Figure 2. Schematic drawings of l-afadin (p205) and s-afadin
(p190) cDNAs. (1) alternative insertion of 21 bp; (2) alternative
insertion of 36 bp. The sequence data of the l- and s-afadin genes
are available from GenBank/EMBL/DDBJ under accession num-
bers U83230 and U83231, respectively.
Figure 3. Domain organization of l-afadin (p205) and s-afadin
(p190). (a) F-actin–binding activity of various fragments of re-
combinant l-afadin. Full-length l-afadin (aa 1–1829) (myc–l-afa-
din) and its deletion mutant (aa 1–1673) (myc–l-afadin?C) were
expressed as myc-tagged proteins in COS7 cells. The COOH-ter-
minal fragment (aa 1631–1829) (GST–l-afadin-C) was expressed
as a GST fusion protein in E. coli and was purified with glu-
tathione-Sepharose. The extracts from the COS7 cells and the
protein purified from the bacteria were subjected to a 125I-labeled
F-actin blot overlay. The extracts from the COS7 cells were sub-
jected to Western blot analysis using the myc epitope antibody to
confirm the expression amounts. (Left) 125I-labeled F-actin blot
overlay; (right) Western blot analysis using the anti-myc antibody.
(Asterisks) endogenous proteins of COS7 cells with 125I-labeled
F-actin–binding activity. (b) Schematic drawings of l- and s-afadin
The Journal of Cell Biology, Volume 139, 1997
5 b). ?-Actinin also increased the viscosity, but the viscosity
became unmeasurably high. This effect of the Mono Q sam-
ple of l-afadin on F-actin was further assessed by transmis-
sion electron microscopy of negatively stained specimens.
l-Afadin hardly caused F-actin to associate into bundles
and meshworks under conditions where ?-actinin showed a
marked F-actin–cross-linking activity (data not shown).
This result was consistent with that of the low shear vis-
cometry. The stoichiometry of the binding of l-afadin to
actin and the affinity constant were examined using His6–
l-afadin-C. The stoichiometry was calculated to be 1 His6–
l-afadin-C molecule per ?500 actin molecules (Fig. 5 c).
The kilodalton value was calculated to be the order of 10?7.
A similar result was obtained with GST–l-afadin-C (data
not shown). The effect of the Mono Q sample of l-afadin
on actin polymerization was finally examined using pyrene-
conjugated actin. Pyrene-conjugated actin is known to in-
crease its fluorescent intensity with the actin polymeriza-
tion (Cooper and Pollard, 1982). l-Afadin did not affect the
actin polymerization under the conditions where gelsolin
stimulated it (data not shown).
Localization of l-Afadin at Cadherin-based
The localization of l-afadin was first examined by immu-
nofluorescence microscopy of the frozen sections of vari-
ous rat or mouse tissues using the anti–l-afadin antibody.
In the liver, l-afadin was localized at the beltlike junctional
complex region along the bile canaliculi (Fig. 6 a). When
the frozen sections of the small intestine were doubly
stained with the monoclonal anti–E-cadherin, l-afadin was
concentrated with E-cadherin at the junctional complex
region of intestine absorptive epithelia, but l-afadin was
more highly concentrated at the junctional complex region
than E-cadherin (Fig. 6, b1–3 and c1–3). The frozen sec-
tions of the heart were doubly stained with the monoclonal
antivinculin antibody. Vinculin is known to be a marker,
not only for cell-to-cell AJ, but also for cell-to-matrix AJ
(Geiger, 1979, 1983). l-Afadin was colocalized with vincu-
lin at the intercalated disc (Fig. 6 d1–3). Vinculin was also
periodically located along the lateral borders of cardiac
muscle cells (costamere) while l-afadin was not. When the
cultured EL cells expressing E-cadherin (Nagafuchi et al.,
1987) were doubly stained with the anti–ZO-1 antibody,
l-afadin showed localization similar to that of ZO-1 (Fig. 7,
a and b). ZO-1 is known to be concentrated at cadherin-
based, spotlike cell-to-cell AJ in fibroblasts (Itoh et al.,
1991, 1993). These results suggest that l-afadin is localized
at cadherin-based cell-to-cell AJ.
To examine the precise localization of l-afadin at the junc-
tional complex region, the frozen sections of small intes-
tine were doubly stained with the monoclonal anti–ZO-1
antibody, and the bile canaliculi isolated from liver were
doubly stained with the monoclonal antidesmoplakin anti-
body. ZO-1 and desmoplakin are known to be markers for
tight junction (Stevenson et al., 1986; Itoh et al., 1993) and
desmosome (Skerrow and Matoltsy, 1974; Franke et al.,
1983; Mueller and Franke, 1983; Miller et al., 1987), re-
spectively. In the absorptive epithelia, l-afadin was local-
ized slightly more at the basal side than ZO-1 (Fig. 8 a1–a3).
In the canaliculi, the localization of l-afadin did not coin-
Figure 4. Tissue distribution
of l-afadin. (a) Northern blot
analysis. A RNA blot mem-
brane (CLONTECH, Palo
Alto, CA) was hybridized
with the 32P-labeled fragment
l-afadin cDNA according to
the manufacturer’s protocol.
In addition to the ?7.5-kb
mRNA of l-afadin, a smaller
mRNA of ?4.2 kb was de-
tected in several tissues, but
significance of this is un-
known. (b1 and b2) Western
blot analyses. Various rat tis-
sue homogenates (10 ?g of
protein each) were subjected
to SDS-PAGE (8% poly-
acrylamide gel), followed by
immunoblot using the anti–l-
afadin (b1) or the anti–l- and
–s-afadin antibody (b2). The
lower band in the heart lane
with the anti–l-afadin anti-
body was nonspecific, since
the treatment with the pep-
tide that was used to raise the
antibody did not quench this
lower band, while it quenched
the band with a molecular
mass of ?205 kD (l-afadin).
Figure 5. Biochemical proper-
ties of l-afadin. (a) Inhibition by
myosin S1 of the binding of
l-afadin to 125I-labeled F-actin.
The Mono Q sample of l-afadin
(0.1 ?g of protein) was subjected
to SDS-PAGE (8% polyacryla-
mide gel), followed by the blot
overlay with 125I-labeled F-actin
pretreated with myosin S1 in the
presence or absence of ATP. (b)
Increase by l-afadin of the vis-
cosity of F-actin. The viscosity of
F-actin was measured by low
shear viscometry. ?, With l-afa-
din; ?, with ?-actinin. (c) Bind-
ing of His6–l-afadin-C to F-actin.
The binding curve was gener-
ated by the cosedimentation of
His6–l-afadin-C with F-actin.
Mandai et al. Afadin
Figure 6. Localization of l-afadin, E-cadherin, and vinculin in various rat or mouse tissues. (a) The staining of rat liver with the anti–
l-afadin antibody. Arrow, longitudinal-section view of bile canaliculi; arrowhead, cross-section view of bile canaliculi. (b1–b3) The double
staining of mouse small intestinal epithelial cells with the anti–l-afadin and anti–E-cadherin antibodies: (b1) l-afadin; (b2) E-cadherin;
(b3) l-afadin and E-cadherin. Arrows, basal level; arrowheads, apical level. (c1–c3) The double staining of the tangential section of
mouse small intestinal cells at the level of the apical surface with the anti–l-afadin and anti–E-cadherin antibodies: (c1) l-afadin; (c2) E-cad-
herin; (c3) l-afadin and E-cadherin. (d1–d3) The double staining of mouse heart with the anti–l-afadin and antivinculin antibodies. It
should be noted that the staining of l-afadin is also associated with the blood vessels: (d1) l-afadin; (d2) vinculin; (d3) l-afadin and vincu-
lin. arrows, intercalated disc; arrowhead, costamere; double arrowheads, blood vessel. Bars: (a and b) 10 ?m; (c) 5 ?m; (d) 50 ?m.
The Journal of Cell Biology, Volume 139, 1997
cide with that of desmoplakin (Fig. 8 b1–b3). These results
indicate that l-afadin is localized at cell-to-cell AJ rather
than at tight junction or desmosome. The localization of
l-afadin at cell-to-cell AJ was finally confirmed by immu-
noelectron microscopy of intestine absorptive epithelia using
the silver enhancement and ultrathin cryosection tech-
niques. l-Afadin was associated with the undercoat of cell-
to-cell AJ (Fig. 9, a and b).
Figure 7. Localization of l-afadin and ZO-1 in the EL cells expressing E-cadherin. The EL cells expressing E-cadherin were doubly
stained with the anti–l-afadin and anti–ZO-1 antibodies. l-Afadin was concentrated at cell-to-cell AJ. There were perinuclear and nu-
clear stainings with this anti–l-afadin antibody, but its significance is not clear. (a) l-afadin; (b) ZO-1. arrows, cell-to-cell AJ. Bar, 25 ?m.
Figure 8. Different localiza-
tion of l-afadin, ZO-1, and
desmoplakin. (a1–a3) Local-
ization of l-afadin and ZO-1
in mouse small intestine: (a1)
l-afadin; (a2) ZO-1; (a3) l-afa-
din and ZO-1. arrows, the
sites where green (ZO-1),
yellow (the mixture of l-afa-
din and ZO-1), and red (l-
afadin) signals occurred in
this order from the apical
side to the basal side. (b1–b3)
Localization of l-afadin and
desmoplakin in the isolated
bile canaliculi: (b1) l-afadin;
(b2) desmoplakin; (b3) l-afa-
din and desmoplakin. Aster-
isk, the inner space of the
bile canaliculi. Bars: (a) 15
?m; (b) 5 ?m.
Mandai et al. Afadin
Here, we isolated a novel F-actin–binding protein with a
molecular mass of ?205 kD (p205). This protein was copu-
rified with another protein with a molecular mass of 190 kD
(p190) that lacked the F-actin–binding activity on various
column chromatographies. The molecular cloning of the
cDNAs of these two proteins revealed that the nucleotide
sequence of the p190 cDNA was identical to that of the
p205 cDNA, except for the two splicing regions. FISH
analysis revealed that the genes of these two proteins were
localized at the same locus. These results suggest that p205
and p190 are splicing variants derived from the same gene.
Because a computer homology search revealed that the aa
sequence of p190 was almost identical to that of human
AF-6 protein, we theorize that p190 may be a rat counter-
part of human AF-6 protein. We named p205 and p190 l- and
s-afadins, respectively. Further purification steps of l-afa-
din, including Mono S column and Superdex 200 column
chromatographies, did not separate l- and s-afadins from
each other, and their elution profiles on these column
chromatographies were similar but not identical. These
proteins appeared at a position corresponding to a molec-
ular mass of ?600 kD on the gel filtration. These results
suggest that 1- and s-afadins form a complex, but we can-
not exclude the possibility that these two proteins do not
form a complex, but instead are incidentally eluted at sim-
ilar fractions on these column chromatographies. It is,
therefore, possible that the complex is composed of a hete-
rooligomer alone, a homooligomer alone, or the mixture.
Western blot analysis indicated that the tissues other than
brain expressed l-afadin alone. It is likely that l-afadin
forms a homooligomer in these tissues.
Recently, Luna’s group (Pestonjamasp et al., 1995) has
shown that a protein with a molecular mass of ?205 kD in
bovine neutrophils has the 125I-labeled F-actin–binding ac-
tivity. When human neutrophils were subjected to SDS-
PAGE, followed either by Western blot analysis using the
antibody specific to l-afadin or by 125I-labeled F-actin blot
overlay, an immunoreactive band with a molecular mass of
?200 kD was detected, and this band showed the 125I-labeled
F-actin–binding activity (data not shown). This protein
band is likely to be l-afadin, although its molecular mass
was slightly smaller than that of rat l-afadin. The different
molecular mass values may result from the species’ differ-
ences. The exact relationship between the 205-kD protein
described by Luna’s group and l-afadin is not known, since
the primary structure of the 205-kD protein has not been
determined, but the 205-kD protein may be l-afadin.
l-Afadin showed 125I-labeled F-actin–binding activity.
Neither s-afadin nor the deletion mutant of l-afadin lack-
ing the COOH-terminal 156 aa (myc–l-afadin?C) showed
this activity, whereas GST–l-afadin-C corresponding to the
COOH-terminal 199 aa showed the 125I-labeled F-actin–
binding activity. The Mono Q sample of l-afadin increased
the viscosity of F-actin on the low shear viscometry. Fur-
thermore, His6–l-afadin-C corresponding to the COOH-
terminal 199 aa was cosedimented with F-actin. These re-
sults indicate that l-afadin shows F-actin–binding activity
and that the domain responsible for this activity is located
at the COOH-terminal 199 aa. Many actin-binding protein
families have been isolated and characterized. Of these
families, for instance, the ?-actinin/spectrin family mem-
bers have been shown to usually form oligomers, such as a
homodimer or a heterotetramer, and thereby show the
F-actin–cross-linking activity. ?-Actinin made the viscosity
of F-actin unmeasurably high on the low shear viscometry,
and it caused F-actin to associate into bundles and mesh-
works, as estimated by transmission electron microscopy.
The stoichiometry is one ?-actinin molecule per about
seven actin molecules, and the kilodalton value is the or-
der of 10 ?7 (Meyer and Aebi, 1990). In contrast, the effect
Figure 9. Ultrastructural localization of l-afadin
in rat small intestine. Intestinal epithelial cells
were labeled singly with the anti–l-afadin anti-
body. (a) silver enhancement technique; (b) ul-
trathin cryosection technique. Open arrow, tight
junction; closed arrow, cell-to-cell AJ; mv, mi-
crovilli. Bars, 0.2 ?m.
The Journal of Cell Biology, Volume 139, 1997
of the Mono Q sample of l-afadin on F-actin was small on
the low shear viscometry, and the sample hardly showed
F-actin–cross-linking activity, as estimated by transmission
electron microscopy. The stoichiometry was calculated to
be 1 His6–l-afadin-C molecule per ?500 actin molecules.
The kilodalton value was calculated to be the order of
10?7. These results indicate that l-afadin belongs to a fam-
ily different from the ?-actinin/spectrin family. The ERM
family, including ezrin, radixin, and moesin, belongs to the
band 4.1 superfamily, an F-actin–binding protein super-
family (Bretscher, 1993; Tsukita et al., 1997), and there
have been several reports that purified ezrin and moesin
are hardly cosedimented with F-actin (Bretscher, 1983;
Pestonjamasp et al., 1995; Shuster and Herman, 1995).
Luna’s group (Pestonjamasp et al., 1995) has recently
shown by the 125I-labeled F-actin blot overlay that ezrin
and moesin exhibit the F-actin–binding activity. They have
also shown by competition experiments using the 125I-labeled
F-actin blot overlay that ezrin and moesin bind along the
sides of F-actin. The biochemical properties of l-afadin are
apparently similar to those of ezrin and moesin. l-Afadin
may bind F-actin in a manner similar to those of ezrin and
An immunofluorescence microscopic study by use of the
antibody specific to l-afadin revealed that l-afadin was lo-
calized at the junctional complex region. Further microscopic
and electron microscopic studies revealed that l-afadin was
concentrated at cadherin-based cell-to-cell AJ. Taken to-
gether with the biochemical properties of l-afadin, l-afadin
is likely to be involved in the linkage between the actin cy-
toskeleton and cell-to-cell AJ. Both l- and s-afadins have
one PDZ domain. The PDZ domain has been found in a
variety of proteins that are typically localized at cell-to-cell
junctions (Saras and Heldin, 1996) and shown to bind to
the unique COOH-terminal motifs of target proteins, which
are found in a large number of transmembrane proteins
(Kim et al., 1995; Kornau et al., 1995; Niethammer et al.,
1996). l-Afadin may be recruited to cell-to-cell AJ through
its PDZ domain. To understand the mechanism by which
l-afadin is localized at cell-to-cell AJ, it is of crucial impor-
tance to identify its interacting molecule(s).
Many F-actin–binding proteins, including ?-actinin, talin,
and vinculin, have been shown to serve as linkers between
the actin cytoskeleton and integrin at cell-to-matrix AJ
(Jockusch et al., 1995). The cytoplasmic domain of the
?-subunit of integrin interacts directly with ?-actinin and
talin and indirectly with vinculin through ?-actinin and
talin (Jockusch et al., 1995). At cell-to-cell AJ, the cyto-
plasmic domain of cadherin interacts with ?-catenin through
?-catenin (Ozawa et al., 1989; Nagafuchi et al., 1991; Takei-
chi, 1991; Tsukita et al., 1992). ?-Catenin interacts directly
with F-actin (Rimm et al., 1995), ?-actinin (Knudsen et al.,
1995), and ZO-1 (Itoh et al., 1997). Vinculin is highly con-
centrated at cell-to-cell AJ, but its interacting molecule
has not yet been identified (Geiger and Ginsberg, 1991;
Tsukita et al., 1992). We showed here that l-afadin was lo-
calized at cell-to-cell AJ. Therefore, the molecular linkage
mechanism between the actin cytoskeleton and cadherin is
apparently similar to that between the actin cytoskeleton
and integrin in the sense that F-actin is linked to each trans-
membrane protein through multiple F-actin–binding pro-
The AF-6 gene has originally been identified as a fusion
partner of the ALL-1 gene (Prasad et al., 1993), which is
known to be involved in acute leukemia (Cimino et al.,
1991). It has recently been shown that the AF-6 gene maps
outside the minimal region of deletion in ovarian cancers
with similar chromosomal aberrations (Saha et al., 1995;
Saito et al., 1996). However, neither the significance of the
fusion of these two genes nor the role of s-afadin in car-
cinogenesis has been clarified. s-Afadin has recently been
shown to have significant homology to the product of the
Drosophila canoe gene (Miyamato et al., 1995). The canoe
gene interacts genetically with the Notch signal cascade in
the eye, bristle, and wing development (Miyamato et al.,
1995). Moreover, the canoe gene interacts genetically with
the armadillo (?-catenin) gene (Miyamato et al., 1995).
The products of the Notch and armadillo genes have been
implicated in adhesive cell-to-cell communications (Peifer
et al., 1993; Artavanis-Tsakonas et al., 1995). l- and s-Afa-
dins may also play a role in the development of various tis-
sues by mediating interactions between the adhesive mole-
cule–initiated signal transduction pathways.
The AF-6 protein (s-afadin) has recently been shown to
interact specifically with the GTP-bound active form of
Ras small G protein (Kuriyama et al., 1996); however, we
have not been able to reproduce this earlier finding. We
found that both the GDP- and GTP-bound forms of Ki-
Ras interacted with l- and s-afadins to similar extents and
that the stoichiometry of these interactions was negligible
(data not shown). Moreover, an immunofluorescence mi-
croscopic study of Madin-Darby canine kidney cells stably
expressing an myc-tagged dominant active mutant of Ki-
Ras (N12V) or an myc-tagged wild type of Ki-Ras showed
that these two types of Ras are not concentrated at cell-to-
cell AJ (Takaishi, K., and Y. Takai, unpublished observa-
tions). All of these results do not support the specific inter-
action of Ras with s-afadin, and the relationship between
Ras and s-afadin is currently unknown.
We thank Dr. M. Takeichi (Kyoto University, Kyoto, Japan) for providing
the EL cells expressing E-cadherin, and Dr. M. Imamura (National Insti-
tute of Neuroscience, Kodaira, Japan) for providing us the recombinant
chick lung type ?-actinin.
Received for publication 6 May 1997 and in revised from 7 June 1997.
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