A Neurally Enriched Coronin-like Protein, ClipinC, Is a Novel
Candidate for an Actin Cytoskeleton-Cortical Membrane-linking
Protein*
Takeshi
Nakamura
§¶,
Kosei
Takeuchi§
,
Sumie
Muraoka**,
Hirotaka
Takezoe
,
Naoki
Takahashi
, and
Nozomu
Mori**
§§
From the
Biomedical R & D Department, Sumitomo
Electric Industries, Sakae-ku, Yokohama 244-8588, the
Department of Cell Biology, Graduate School of Biological
Sciences, Nara Institute of Science and Technology, Ikoma, Nara
630-0101, the ** Inheritance and Variation Group, PRESTO, Science and
Technology Corporation of Japan, Keihanna Plaza, Seika-cho, Kyoto
619-02, the 
Department of Molecular
Genetic Research, National Institute for Longevity Sciences and the
§§ Program of Protecting Brain, CREST, Science
and Technology Corporation of Japan, Oobu, Aichi 474-8522, Japan
 |
ABSTRACT |
Brain-enriched human FC96 protein shows a close
sequence similarity to the Dictyostelium actin-binding
protein coronin, which has been implicated in cell motility,
cytokinesis, and phagocytosis. A phylogenetic tree analysis revealed
that FC96 and two other mammalian molecules (p57 and IR10) form a new
protein family, the coronin-like protein (Clipin) family; thus
hereafter we refer to FC96 as ClipinC. A WD domain and a succeeding
-helical region are conserved among coronin and Clipin family
members. ClipinC is predominantly expressed in the brain, and discrete
areas in the mouse brain were intensely labeled with anti-ClipinC
antibodies. ClipinC was also shown to bind directly to F-actin
in vitro. Immunocytochemical analysis revealed that ClipinC
accumulated at focal adhesions as well as at neurite tips and stress
fibers. Furthermore, ClipinC was associated with vinculin, which is a
major component of focal contacts. These results indicate that ClipinC
is also a component part of the cross-bridge between the actin
cytoskeleton and the plasma membrane. These findings and the previously
reported function of coronin suggest that ClipinC may play specific
roles in the reorganization of neuronal actin structure, a change
that has been implicated in both cell motility and growth cone advance.
 |
INTRODUCTION |
Actin filaments in neuronal cells form a cortical framework that
helps to localize membrane proteins, and F-actin dynamics has been
implicated in directing neuronal outgrowth. Rearrangement of the actin
cytoskeleton occurs in response to various stimuli such as soluble
factors or attachment to a substratum (1, 2). The regulation of F-actin
patterns involves actin polymerization and actin cross-linking. Factors
regulating these processes communicate with the small G proteins of the
Rho family (3) and the phosphatidylinositol metabolism system (4), both
of which are triggered by extracellular cues through a variety of receptors.
The Dictyostelium actin-binding protein coronin was first
purified from an actin-myosin complex and was hypothesized to transmit signals from the membrane receptors to the cortical cytoskeleton (5).
Coronin accumulates at the leading edges of moving cells and in
crown-shaped extensions on the dorsal cell surface. The involvement of
coronin in cell motility, cytokinesis, and phagocytosis, all of which
depend on cytoskeletal rearrangement, has been demonstrated by use of a
gene replacement mutant. In a mutant that lacks coronin, cell motility
is reduced to less than half of the normal speed, and cytoplasmic
cleavage in cytokinesis is impaired (6). Further, in the coronin null
(cor
) mutant, the rate of yeast uptake is reduced by
about 70% (7). However, the distribution of actin filaments in
cor
cells is similar to that in the wild-type ones
(6).
In this study, we found a novel candidate for an actin
cytoskeleton-cortical membrane linking protein; this protein, ClipinC, is also the third member of a family of mammalian homologs of Dictyostelium coronin. The ClipinC transcript was
predominantly expressed in the nervous system. The association of
ClipinC with F-actin was demonstrated in vitro.
Immunocytochemical analysis of neuronal cells showed that ClipinC
accumulated at neurite tips and focal adhesions and along stress
fibers. Immunoprecipitation experiments demonstrated that ClipinC was
associated with vinculin, which is a cytoskeletal protein implicated in
the control of adhesion or motility (8) and is a major constituent of
focal adhesions (9). Together with the recent report on the phenotype
of a coronin null mutant, the present study indicates that ClipinC may
play specific roles in the reorganization of neuronal actin structure,
a change that has been implicated in both cell motility during
neuronal development and growth cone advance leading to synapse formation.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture--
SH-SY5Y human neuroblastoma cells were
maintained in RPMI medium containing 10% fetal bovine serum. PC12 rat
pheochromocytoma cells were grown in RPMI medium containing 10% horse
serum and 5% fetal bovine serum. COS-1 and NIH3T3 cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum.
Isolation of ClipinC cDNA--
An equalized cDNA library
was previously constructed from a human forebrain cortex (10).
Individual clones from this library were sequenced and compared with
sequences in the GenBankTM data base, as described before
(11). Thereby 100 unidentified clones were collected, and their tissue
specificity was examined by RNA dot blot analysis (11). One
brain-enriched clone, FC96, was selected for further study. To obtain
the full-length clone of FC96, we screened a human frontal
cortex-derived cDNA library (Stratagene) with the 1.0-kilobase
EcoRI-XhoI fragment of clone FC96. Eight
overlapping cDNAs were obtained, and the nucleotide sequences of
these cDNAs were determined to give rise to the complete coding
sequence of FC96, i.e. of ClipinC.
Northern Blot Analysis--
Human multiple tissue blots I and II
(CLONTECH) were hybridized as described before (11)
with DNA probes: ClipinA/p57 (nucleotides 489-930) (12), ClipinB/IR10
(nucleotides 372-1333) (13), and ClipinC (nucleotides 587-1602). The
ClipinA and B probes used were the reverse transcription-polymerase
chain reaction products from human brain poly(A)+ RNA
(CLONTECH).
Antibody Production and Immunohistochemistry--
Rabbits were
immunized with the purified recombinant ClipinC protein (amino acids
297-475) expressed as a histidine-tagged form. For
immunohistochemistry, heads of mouse embryos and newborns were fixed in
ice-cold 5% acetic acid in ethanol. Immunohistochemistry was performed
on 8-µm-thick microtome sections from paraffin-embedded brains. The
sections were pretreated with 3% hydrogen peroxide, washed, and
incubated with the polyclonal antibodies against ClipinC at a dilution
of 1:5000. After having been washed, the sections were incubated with
peroxidase-conjugated anti-rabbit IgG (MBL). The immunocomplexes were
visualized in 0.05 M Tris-HCl (pH 7.4), 0.1%
diaminobenzidine tetrahydrochloride, and 0.1% hydrogen peroxide.
Cosedimentation Assays--
For actin cosedimentation, skeletal
muscle actin (Sigma) was resuspended in actin polymerization buffer (10 mM Tris-HCl, pH 8.0, 100 mM KCl, 0.5 mM dithiothreitol, 0.2 mM ATP, 1 mM
MgCl2, 0.2 mM CaCl2).
[35S]Methionine-labeled FLAG-tagged ClipinC and a control
protein (firefly luciferase) were synthesized by coupled transcription and translation by use of a TNT expression system (Promega). The FLAG-tagged 35S-ClipinC was purified by means of anti-FLAG
M2 affinity gel (Kodak). In tubes without actin,
35S-ClipinC or luciferase TNT product was diluted in the
actin polymerization buffer. In those with actin, the TNT product was
added to the resuspended actin (1 mg/ml). Mixtures were incubated for
1 h at 25 °C and then centrifuged for 30 min at 4 °C and
100,000 × g. Supernatants and pellets were resolved by
SDS-polyacrylamide gel electrophoresis and visualized by autoradiography.
Immunocytochemistry--
Cells were fixed in 1% fresh
formaldehyde and permeabilized with 0.1% Triton X-100. After having
been soaked in phosphate-buffered saline containing 1% bovine serum
albumin and 1% normal goat serum, the samples were incubated with the
anti-ClipinC antibodies at a dilution of 1:2000 to 1:5000, washed with
phosphate-buffered saline, and then incubated with fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG (Tago). Staining the
same cells for F-actin or vinculin was performed with
rhodamine-phalloidin (Molecular Probes) or 5 µg/ml of anti-vinculin
monoclonal antibody V284 (Cybus Biotechnology) and rhodamine-conjugated
sheep anti-mouse IgG (Chemicon), respectively. The samples were then
washed with phosphate-buffered saline and examined under a fluorescence
microscope (Axiophoto2; Carl Zeiss). For antigen absorption
experiments, anti-ClipinC antibody was incubated with the recombinant
ClipinC-bound beads for 30 h before cell staining.
Immunoprecipitation--
Cells were lysed on ice for 1 h in
Nonidet P-40 lysis buffer (10 mM Tris-HCl, pH 7.8, 1%
Nonidet P-40, 150 mM NaCl, 1 mM EDTA) containing protease inhibitors (Complete, Roche Molecular
Biochemicals). The lysates were then centrifuged for 30 min at
14,000 × g. Immunoprecipitation was done by incubation
with the desired primary antibodies and anti-mouse or rabbit
IgG-agarose beads (American Qualex) at 4 °C for 12 h. Immune
complexes were washed three times with 1% Nonidet P-40 lysis buffer,
eluted, and resolved on SDS-polyacrylamide gel electrophoresis.
Immunoblotting was performed as described previously (14). For
exogenous expression of FLAG-tagged ClipinC, the human ClipinC cDNA
with the FLAG peptide tag at the carboxyl terminus was subcloned in the
pSR
296 vector (15) and transfected into NIH3T3 cells by use of
LipofectAMINE Plus (Life Technologies, Inc.).
 |
RESULTS |
Isolation of Human ClipinC cDNA--
An equalized cDNA
library was previously constructed from human forebrain cortex. Using
this library, we obtained and analyzed individual clones to search for
novel genes that showed regional expression in the adult brain. In part
of this research, 100 unidentified clones were collected based on
partial DNA sequencing and comparison with a DNA data base, and then
their tissue specificity was examined by RNA dot blot analysis. Among
these clones, several genes were found to be abundantly expressed in
the brain. The sequence of one of the brain-enriched cDNAs, clone
FC96, had a remarkable similarity to that of coronin, an actin-binding
protein in Dictyostelium discoideum (Fig.
1), and was selected for further
investigation.

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 1.
Amino acid sequence of ClipinC.
A, predicted amino acid sequence of human ClipinC is shown
by the one-letter amino acid code. The WD repeats are indicated on the
top of the sequence. Amino acids conserved among
Dictyostelium coronin, human ClipinA/p57, human
ClipinB/IR10, and human ClipinC are indicated by asterisks.
Shaded boxes indicate the identical amino acids between
ClipinC and its closest member, ClipinB. B, phylogenetic tree of
coronin, Clipins, and their related molecules.
|
|
The full-length FC96 transcript was 3.6 kilobases in size (Fig.
2A) and contained one open
reading frame of 1728 base pairs. The open reading frame encoded a
putative protein of 475 amino acids with a predicted molecular mass of
54.0 kDa (Fig. 1A). Analysis of the FC96 protein sequence
with a protein data base disclosed an overall similarity to the
Dictyostelium coronin (38.5%) and its mammalian homologs,
i.e. p57 (43.9%) (12) and IR10 (61.0%) (13). An
amino-terminal domain containing five WD repeats and a succeeding
domain covering about 100 amino acids with a tendency to form an
-helical structure were well conserved among these molecules. A
phylogenetic tree analysis revealed that p57, IR10, and FC96 form a new
protein family, the Clipin (coronin-like
protein) family (Fig. 1B); thus
hereafter we refer to p57, IR10, and FC96 as Clipins A, B, and C,
respectively.

View larger version (68K):
[in this window]
[in a new window]
|
Fig. 2.
ClipinC is predominantly expressed in
brain. A, tissue distribution of human Clipin family
members. Northern blot analysis of ClipinA (top), ClipinB
(middle), and ClipinC (bottom) mRNAs from
various adult human tissues. B-D, immunohistochemical
staining for ClipinC in parasagittal (B) and coronal
(C) sections of P1 mouse brain and in a frontal coronal
section of E16 mouse embryos (D). Ob, olfactory
bulb; Ctx, cerebral cortex; Hp, hippocampus;
Th, thalamus; Cb, cerebellum; Re,
retina.
|
|
ClipinC Is Predominantly Expressed in Brain--
We examined the
expression of ClipinC mRNA in various human adult tissues and
compared this expression with that of Clipins A and B (Fig.
2A). The level of the ClipinC mRNA was extremely high in
the brain, moderate in heart and ovary, and very low or undetectable in
the other tissues examined in this study. As previously reported (12),
the ClipinA transcript was mainly detected only in immune system
tissues, i.e. spleen, thymus, and peripheral leukocytes. A
high level expression of ClipinB was restricted to some tissues,
e.g. colon, prostate, and testis, in which ClipinA and C
transcripts were nearly undetectable. It is interesting to note that
the expression profiles of Clipin members were tissue-specific and
almost mutually exclusive.
Northern blot analysis showed that ClipinC mRNA was preferentially
expressed in brain tissue. We confirmed this point at the protein level
by using polyclonal antibodies against ClipinC (data not shown). The
antibodies specifically detected ClipinC protein in human and rat
brains and neuronal cell lines of peripheral origin, such as SH-SY5Y
and PC12 cells. ClipinC protein was undetected in the other tissues and
non-neuronal cells examined.
We next examined the distribution of ClipinC protein in various brain
regions by using the ClipinC-selective antibodies (Fig. 2,
B-D). ClipinC immunostaining was detected in discrete areas in the mouse brain. In the P1 brain, immunoreactivity was observed in
the cerebral cortex, hippocampus, thalamus, olfactory bulb, and
cerebellum (Fig. 2, B and C). In the cerebellum,
the Purkinje cell layer was intensely labeled; but no immunoreactivity
was detected in the molecular layer or granule cell layer (Fig.
2B). Intense immunoreactivity was also observed in the inner
nuclear (neuroblastic) layer in the retina and in the olfactory bulb of the mouse embryo (Fig. 2D).
In Vitro Binding of ClipinC to F-actin--
The close sequence
similarity between ClipinC and coronin (Fig. 1) suggested that ClipinC
is also an actin-binding protein; thus binding of ClipinC to actin
filaments was investigated by spin-down experiments. Full-length
ClipinC was prepared in vitro, added to the actin
polymerization buffer, and incubated in the absence or presence of
actin. Thenafter, macroaggregates were isolated by centrifugation. Fig.
3A showed that ClipinC
cosedimented with F-actin. In control experiments, we also examined the
cosedimentation property of firefly luciferase protein, which was in
the soluble fraction even in the presence of actin (Fig.
3B). This confirmed that the ClipinC interaction with actin
macroaggregates is selective.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 3.
Cosedimentation of ClipinC and F-actin in
spin-down experiments. A, ClipinC was added to actin
polymerization buffer and incubated in the absence or presence of
actin. The supernatants (S) and pellets (P)
obtained by centrifugation were resolved by SDS-polyacrylamide gel
electrophoresis and then visualized by autoradiography. In the absence
of actin, ClipinC is in the soluble fraction. In the presence of actin,
ClipinC is concentrated in the pellet with F-actin. B, the
firefly luciferase protein as a control remains in the supernatant
under both conditions.
|
|
Immunofluorescence Localization of ClipinC in Focal Adhesions,
Stress Fibers, and Neurite Tips in Neuronal Cells--
The
anti-ClipinC antibodies were used for immunocytochemical studies of
neuronal cells to assess the subcellular localization of ClipinC. Shown
in Fig. 4 (A and C)
are various shapes of SH-SY5Y human neuroblastoma cells stained for
ClipinC. The site of localization of ClipinC in flattened SH-SY5Y cells
was clarified by double-labeling of ClipinC (Fig. 4A) and
F-actin (Fig. 4B). Staining for both ClipinC and F-actin was
most intense at the focal contacts (arrowhead) and stress
fibers (arrows). The accumulation of ClipinC at the focal
adhesion, i.e. cross-bridge between the actin cytoskeleton and the substrate-adherent plasma membrane, was confirmed by
double-staining for ClipinC (Fig. 4C) and vinculin, a major
constituent of focal adhesive complexes (Fig. 4D). The data
suggest that ClipinC is a component of the cross-bridge between actin
filaments and the cortical membrane. Nuclear staining in SH-SY5Y cells
stained for ClipinC (Fig. 4, A and C) was shown
to be a nonspecific artifact by antigen absorption experiments (Fig.
4I). In Fig. 4 (E and F), nerve growth
factor-treated PC12 cells showing neurite outgrowth were stained for
ClipinC. ClipinC remarkably accumulated at the tips of the neurites
(arrows). Note that ClipinC was also abundant at the
protrusions in the cellular periphery (arrowheads in Fig. 4E). In addition to the apparent accumulation of ClipinC at
neurite tips, a considerable amount of ClipinC was dispersed in the
cell body (Fig. 4G); this cytoplasmic distribution of
ClipinC overlapped with that of F-actin (Fig. 4H). These
data fit well with the previous observation that
Dictyostelium coronin is reversibly recruited from the
cytoplasm and is incorporated into the actin network of leading edges
of the slime mold (16).

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 4.
Localization of ClipinC in neuronal
cells. A and B, SH-SY5Y cells were costained
for ClipinC (A) and F-actin (B). ClipinC is seen
to be concentrated at the focal contacts (arrowhead) and
also localized along stress fibers (arrows). C
and D, double-staining for ClipinC (C) and
vinculin (D) in SH-SY5Y cells. The accumulation of ClipinC
at the focal adhesions is clearly demonstrated (arrowheads).
E-H, localization of ClipinC (E-G) and F-actin
(H) in PC12 cells treated for 3 days with nerve growth
factor. E and F, the tips of neurites
(arrows) and the protrusive regions (arrowheads)
are enriched in ClipinC. G, in addition to the enrichment at
the neurite tips, the diffuse distribution of ClipinC is evident in the
cell body. H, fluorescence of rhodamine-phalloidin staining
showing the distribution of F-actin in the same cell. I,
antigen absorption experiments with SH-SY5Y cells. The anti-ClipinC
antibodies were incubated with the recombinant ClipinC protein and then
used for staining of SH-SY5Y cells. Scale bars, 20 µm.
|
|
Physiological Interaction between ClipinC and Vinculin--
The
accumulation of ClipinC at focal adhesions was an unexpected result,
and thus we tried to identify a focal adhesive protein(s) that
specifically binds to ClipinC. [35S]Methionine-labeled
SH-SY5Y cells were lysed and subjected to immunoprecipitation with the
anti-ClipinC antibodies. We observed the binding of several proteins to
ClipinC; one of them had an approximate molecular mass of 120 kDa (data
not shown). Vinculin, which was used as a marker of focal contacts in
Fig. 4D, is a cytoskeletal protein of 117 kDa; thus we
examined whether ClipinC could interact with vinculin in a
physiological complex. In Fig. 5A, the ClipinC-selective
antibodies coprecipitated vinculin from SH-SY5Y cells. The specificity
of the immunoprecipitation was demonstrated by an antigen absorption
experiment: the amount of vinculin coprecipitated with the
antigen-absorbed anti-ClipinC antibodies (Fig. 5A,
lane 2) was significantly reduced from that of the vinculin
coprecipitated with the untreated antibodies (Fig. 5A,
lane 1). Conversely, the anti-vinculin antibody
coprecipitated ClipinC from SH-SY5Y cells (Fig. 5B,
lane 1), and the corresponding band of about 54 kDa was
absent from the anti-vinculin immunoprecipitate of the COS-1 lysate
containing no ClipinC protein (Fig. 5B, lane 2).
The FLAG-tagged ClipinC exogenously expressed in NIH3T3 cells (Fig.
5C, lane 1) was also coprecipitated with the
anti-vinculin antibody (Fig. 5C, lane 2). The
specificity was confirmed by a similar experiment in mock-transfected
cells (Fig. 5C, lane 3). In NIH3T3 cells, the
exogenously expressed ClipinC accumulated at focal adhesions in
addition to being associated with stress fibers (data not shown), as it
did in SH-SY5Y cells (Fig. 4, A-D). These results indicate
that some amount of ClipinC was present in complexes with vinculin in
focal adhesive structures.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 5.
Physiological interaction between ClipinC and
vinculin. A, SH-SY5Y cells were immunoprecipitated with
the untreated (lane 1) or the antigen-absorbed (lane
2) anti-ClipinC antibodies. The immunoprecipitates were then
immunoblotted with the anti-vinculin monoclonal antibody. B,
SH-SY5Y cells (lane 1) and COS-1 cells (lane 2)
were immunoprecipitated by the anti-vinculin antibody. The
immunoprecipitates were probed with the anti-ClipinC antibodies.
C, NIH3T3 cells transfected with FLAG-tagged ClipinC
cDNA (lane 2) or the empty vector (lane 3)
were immunoprecipitated with the anti-vinculin antibody,
followed by the immunoblot analysis with the anti-FLAG M2 monoclonal
antibody. The whole lysate of the FLAG-tagged ClipinC-transfected cells
was immunoblotted with the anti-FLAG antibody (lane
1).
|
|
 |
DISCUSSION |
In this study, we found a novel candidate for a cytoskeleton
plasma membrane connecting protein in neuronal cells. This protein, ClipinC, is also the third member of a family of mammalian homologs of
coronin, a Dictyostelium actin-binding protein implicated in cell motility, cytokinesis, and phagocytosis (6, 7). The ClipinC
transcript was predominantly expressed in the nervous system, and the
association between ClipinC and F-actin was demonstrated in
vitro. The association of ClipinC with actin filaments was observed at neurite tips and focal contacts and along stress fibers. ClipinC was shown to interact with vinculin, a cytoskeletal protein that is a major component of focal contacts (9) and is implicated in
the control of growth cone motility (8). These data suggest that
ClipinC may play specific roles in the reorganization of neuronal actin structure.
In coronin null mutants, cell locomotion, chemotaxis, and phagocytosis
are slowed down, and cytokinesis is impaired (6, 7); thus these defects
of cor
mutants strongly suggest that coronin plays a
regulatory role in actin reorganization. We expect that Clipin members
potentially share this regulatory role with coronin based on the close
conservation of their structure (Fig. 1) and shared capability of actin
binding (Fig. 3 and Refs. 5 and 12).
A recent study on Dictyostelium cells (16) has provided two
important clues for clarifying how coronin plays a regulatory role in
F-actin dynamics: (i) In chemoattractant-stimulated cells, the temporal
relationship between the coronin-green fluorescent protein accumulation
and the appearance of a protrusion at a cell front (i.e.
local actin rearrangement) was examined. Although the local
accumulation of coronin-green fluorescent protein was seen 7 s
after a protrusion became detectable on average, coronin-green fluorescent protein accumulation could precede the protrusion by 5 s at most (16). Thus coronin accumulation is not merely controlled by
binding to a newly polymerized actin but may be regulated itself, at
least in part. (ii) In coronin null mutants, the extended
organelle-free zone, which appeared as a hyaline area, was formed at
the front region, and more importantly, treatment with cytochalasin A,
an actin-depolymerizing agent, partially rescued the wild-type
phenotype (16).
A third clue for the function of coronin/Clipins is provided by our
present finding that ClipinC becomes accumulated at focal contacts.
Furthermore, ClipinC was shown to bind to vinculin, which occurs in
multimolecular complexes at focal adhesions. In collaboration with
other cytoskeleton-membrane linking proteins, the accumulated
Clipins/coronin at the cell front may possibly construct the molecular
machinery that enables the movement of organelles into the front
region. A similar mechanism elicited by ClipinC could be considered at
neurite tips, accumulation sites of ClipinC in PC12 cells, because in
neuronal growth cones, integrin clustering (17-19) and the
accumulation of some components of focal adhesive complexes including
vinculin, talin, and paxillin (8, 20) were observed following
extracellular cues. In particular, the reduction of vinculin was shown
to cause loss of growth cone stability and to reduce axonal growth in
PC12 cells (8). The involvement of ClipinC in this function of
vinculin, i.e. stabilizing the nerve growth cone, is an
intriguing possibility.
A functional linkage of coronin with heterotrimeric or small G proteins
has been considered. Mutant Dictyostelium cells devoid of
G
subunits are unable to chemotax (21) and are impaired in their uptake of yeast particles (7). The involvement of coronin in
chemoattractant-controlled cell locomotion (6) and in particle uptake
(7) suggests that coronin plays a role in signaling pathways downstream
of the heterotrimeric G proteins. In mammalian cells including neuronal
ones, G protein-coupled receptor stimulation is known to cause actin
reorganization (2, 22). Recent evidence indicates that the small G
proteins of the Rho family transmit signals from G protein-coupled
receptors to the actin cytoskeleton (3, 23). In organisms ranging from yeast to mammals, Rho subfamily members play an important role in
regulating the actin cytoskeleton in response to a broad spectrum of
stimuli such as epidermal growth factor, platelet-derived growth factor, and phorbol myristic acetate (24-26). It is tempting to assume
the linkage between Rho subfamily and coronin/Clipins in the
reorganization of the actin cytoskeleton.
The coronin and Clipin family members are composed of three domains: an
amino-terminal domain containing five WD repeats, an internal domain
with a tendency to form an
-helical structure, and a highly
divergent carboxyl-terminal domain (Fig. 1 and Refs. 5, 12, and 13).
The WD repeat motif is thought to be capable of undergoing pairwise or
multimeric interactions (27). The
subunits of G proteins, the best
known proteins with WD repeats, act in signal transduction by forming
multiprotein complexes through such repeats (28-30). Therefore,
Gerisch et al. (16) suggested that coronin binds not only to
actin but also to other proteins and in this way couples regulatory
proteins to the actin-myosin system; the same suggestion applies to the
Clipin family molecules. The presumed
-helical domain was suggested
to be important for actin binding (5). In a ClipinC deletion mutant
containing a WD domain only, in vitro F-actin binding was
reduced to one-third of that by the intact ClipinC (data not shown).
This may indicate the importance of ClipinC's internal
-helical
domain for actin-binding, although the residual WD repeats can weakly
associate with F-actin.
Attention should be paid to the tissue-specific expressions of Clipin
members, for their expression profiles were almost mutually exclusive.
In marked contrast, the majority of cytoskeleton-membrane linking
proteins, e.g. vinculin, paxillin, and ERM
(ezrin/radixin/moesin) family
proteins, are ubiquitously expressed (9, 14). Further, the expressions
of the three members of the ERM family overlap nearly completely. It is
an interesting possibility that the differential expressions of Clipin
members may reflect a member-specific function in addition to the
shared role in regulating actin organization. A recently reported
interaction between p57/ClipinA and p40phox, a cytosolic
component of the NADPH oxidase that generates microbicidal superoxide
in phagocytes, may be related to the ClipinA-specific function
(31).
Another exceptional characteristic of ClipinC as an actin-binding
protein at the periphery of neuronal cells is that the protein was also
detected along stress fibers in flattened SH-SY5Y cells (Fig.
4A), because GAP-43 (32), MARCKS (33), and ERM family proteins (34), typical actin-regulating proteins in the growth cone,
are known to be found at nerve terminals only. If ClipinC at the cell
front functions as a connector between the actin cytoskeleton and the
cortical membrane, ClipinC along stress fibers is unlikely to have the
same function. Thus we may have to consider another role for ClipinC
(or other Clipin members) residing along stress fibers. In fact, a
homolog of coronin in yeast was recently reported to modulate actin
filament assembly (35).
Guided neuronal migration in development and growth cone motility
leading to neuronal plasticity are both controlled by cytoskeletal dynamics in neurons (36, 37). In this respect, functional linkage
between the intracellular cytoskeleton and extracellular substrates is
a particularly important theme. As is important in other tissues (9),
cytoskeleton-cortical membrane linking complexes play a fundamental
role in this process at the periphery of neurons. In the developing
neocortical intermediate zone, the distribution of tangentially
migrating neurons overlapped with that of intense immunoreactivity
of ClipinC in neocortex (Fig. 2C)1; this may
mean the involvement of ClipinC in the tangential cell migration in
this area. Furthermore, we found that the overexpressed ClipinC had
an effect on the cell attachment to the substrate.1 Our
results provide a strong indication that ClipinC, a possible candidate
for a cytoskeleton-membrane connector, is implicated in the control of
cell adhesions and cell movements in neuronal cells.
 |
ACKNOWLEDGEMENTS |
We thank R. Sanokawa for technical
assistance, T. Kojima for help in the cloning of IR10 cDNA, and
Drs. Y. Sasaki, D. Ayusawa, and M. Oishi for providing us with a human
brain equalized cDNA library.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB010098.
§
These two authors contributed equally to this work.
¶
To whom correspondence should be addressed. Tel.:
81-45-853-7275; Fax: 81-45-853-3528.
1
K. Takeuchi, T. Nakamura, H. Takezoe, N. Mori,
and N. Takahashi, manuscript in preparation.
 |
REFERENCES |
-
Stossel, T. P.
(1993)
Science
260,
1086-1094[Medline]
[Order article via Infotrieve]
-
Zigmond, S. H.
(1996)
Curr. Opin. Cell Biol.
8,
66-73[CrossRef][Medline]
[Order article via Infotrieve]
-
Van Aelst, L.,
and D'Souza-Schorey, C.
(1997)
Genes Dev.
11,
2295-2322[Free Full Text]
-
Janmey, P. A.
(1994)
Annu. Rev. Physiol.
56,
169-191[CrossRef][Medline]
[Order article via Infotrieve]
-
de Hostos, E. L.,
Bradtke, B.,
Lottspeich, F.,
Guggenheim, R.,
and Gerisch, G.
(1991)
EMBO J.
10,
4097-4104[Abstract]
-
de Hostos, E. L.,
Rehfueß, C.,
Bradtke, B.,
Waddell, D. R.,
Albrecht, R.,
Murphy, J.,
and Gerisch, G.
(1993)
J. Cell Biol.
120,
163-173[Abstract]
-
Maniak, M.,
Rauchenberger, R.,
Albrecht, R.,
Murphy, J.,
and Gerisch, G.
(1995)
Cell
83,
915-924[Medline]
[Order article via Infotrieve]
-
Varnum-Finney, B.,
and Reichart, L. F.
(1994)
J. Cell Biol.
127,
1071-1084[Abstract]
-
Burridge, K.,
and Chrzanowska-Wodnicka, M.
(1996)
Annu. Rev. Cell Dev. Biol.
12,
463-519[CrossRef][Medline]
[Order article via Infotrieve]
-
Sasaki, Y. F.,
Iwasaki, T.,
Kobayashi, H.,
Tsuji, S.,
Ayusawa, D.,
and Oishi, M.
(1994)
DNA Res.
1,
91-96[Medline]
[Order article via Infotrieve]
-
Nakamura, T.,
Sanokawa, R.,
Sasaki, Y.,
Ayusawa, D.,
Oishi, M.,
and Mori, N.
(1996)
Oncogene
13,
1111-1121[Medline]
[Order article via Infotrieve]
-
Suzuki, K.,
Nishihata, J.,
Arai, Y.,
Honma, N.,
Yamamoto, K.,
Irimura, T.,
and Toyoshima, S.
(1995)
FEBS Lett.
364,
283-288[CrossRef][Medline]
[Order article via Infotrieve]
-
Zaphiropoulos, P. G., and Toftgård, R. (1996) DNA Cell
Biol. 15
-
Takeuchi, K.,
Sato, N.,
Kasahara, H.,
Funayama, N.,
Nagafuchi, A.,
Yonemura, S.,
Tsukita, S.,
and Tsukita, S.
(1994)
J. Cell Biol.
125,
1371-1384[Abstract]
-
Tanabe, Y.,
Seiki, M.,
Fujisawa, J.,
Hoy, P.,
Yokota, K.,
Akai, K.,
Yoshida, M.,
and Arai, N.
(1988)
Mol. Cell. Biol.
8,
466-472[Medline]
[Order article via Infotrieve]
-
Gerisch, G.,
Albrecht, R.,
Heizer, C.,
Hodgkinson, S.,
and Maniak, M.
(1995)
Curr. Biol.
5,
1280-1285[Medline]
[Order article via Infotrieve]
-
Schmidt, C. E.,
Dai, J.,
Lauffenburger, D. A.,
Sheetz, M. P.,
and Horwitz, A. F.
(1995)
J. Neurosci.
15,
3400-3407[Abstract]
-
Wu, D. Y.,
Wang, L. C.,
Mason, C. A.,
and Goldberg, D. J.
(1996)
J. Neurosci.
16,
1470-1478[Abstract]
-
Grabham, P. W.,
and Goldberg, D. J.
(1997)
J. Neurosci.
17,
5455-5465[Abstract/Free Full Text]
-
Leventhal, P. S.,
Shelden, E. A.,
Kim, B.,
and Feldman, E. L.
(1997)
J. Biol. Chem.
272,
5214-5218[Abstract/Free Full Text]
-
Wu, L. J.,
Valkema, R.,
Van Haastert, P. J.,
and Devreotes, P. N.
(1995)
J. Cell Biol.
129,
1667-1675[Abstract]
-
Jalink, K.,
and Moolenaar, W. H.
(1992)
J. Cell Biol.
118,
411-419[Abstract]
-
Nobes, C. D.,
and Hall, A.
(1995)
Cell
81,
53-62[Medline]
[Order article via Infotrieve]
-
Hall, A.
(1994)
Annu. Rev. Cell Biol.
10,
31-54[CrossRef]
-
Ridley, A. J.,
and Hall, A.
(1992)
Cell
70,
389-399[Medline]
[Order article via Infotrieve]
-
Qiu, R.-G.,
and Hall, A.
(1995)
Nature
374,
457-459[CrossRef][Medline]
[Order article via Infotrieve]
-
Neer, E. J.,
Schmidt, C. J.,
Nambudripad, R.,
and Smith, T. F.
(1994)
Nature
371,
297-300[CrossRef][Medline]
[Order article via Infotrieve]
-
Garritsen, A.,
and Simonds, W. F.
(1994)
J. Biol. Chem.
269,
24418-24423[Abstract/Free Full Text]
-
Wang, D. S.,
Shaw, R.,
Winkelmann, J. C.,
and Shaw, G.
(1994)
Biochem. Biophys. Res. Commun.
203,
29-35[CrossRef][Medline]
[Order article via Infotrieve]
-
Pumiglia, K. M.,
Le Vine, H.,
Haske, T.,
Habib, T.,
Jove, R.,
and Decker, S. J.
(1995)
J. Biol. Chem.
270,
14251-14254[Abstract/Free Full Text]
-
Grogan, A.,
Reeves, E.,
Keep, N.,
Wientjes, F.,
Totty, N. F.,
Burlingame, A. L.,
Hsuan, J. J.,
and Segal, A. W.
(1997)
J. Cell Sci.
110,
3071-3081[Abstract/Free Full Text]
-
Benowitz, L. I.,
and Routtenberg, A.
(1997)
Trends Neurosci.
20,
84-91[CrossRef][Medline]
[Order article via Infotrieve]
-
Ouimet, C. C.,
Wang, J. K.,
Walaas, S. I.,
Albert, K. A.,
and Greengard, P.
(1990)
J. Neurosci.
10,
1683-1689[Abstract]
-
Paglini, G.,
Kunda, P.,
Quiroga, S.,
Kosik, K.,
and Cáceres, A.
(1998)
J. Cell Biol.
143,
443-455[Abstract/Free Full Text]
-
Goode, B. L.,
Wong, J. J.,
Butty, A.-C.,
Peter, M.,
McCormack, A. L.,
Yates, J. R.,
Drubin, D. G.,
and Barnes, G.
(1999)
J. Cell Biol.
144,
83-98[Abstract/Free Full Text]
-
Rakic, P.,
Cameron, R. C.,
and Komuro, H.
(1994)
Curr. Opin. Neurobiol.
4,
63-69[Medline]
[Order article via Infotrieve]
-
Lin, C.-H.,
Thompson, C. A.,
and Forscher, P.
(1994)
Curr. Opin. Neurobiol.
4,
640-647[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.