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INTRODUCTION |
The actin cytoskeleton is central in a number of cellular
processes such as motility, morphogenesis, cytokinesis, and
endocytosis. The structure and dynamics of the actin cytoskeleton are
spatially and temporally regulated by a large number of actin-binding
proteins, whose own activities and localities are precisely regulated
by various signaling pathways (1). Sequence and structural data on
actin-binding proteins has revealed that many of these proteins interact with actin through a relatively small number of protein motifs. These include the calponin homology domain, the gelsolin homology domain, the actin-depolymerizing-factor homology domain, and
the WASP homology 2 (WH2)1
domain (for reviews see 2-5).
The WH2 domain is a small (~35 residue) protein motif that interacts
only with monomeric actin (5, 6). WH2 domains are found in many
regulators of actin dynamics, including
-thymosins and ciboulot,
which bind actin monomers and regulate filament assembly.
-thymosins
are actin monomer-sequestering proteins, whereas ciboulot promotes
actin assembly at the barbed end of the filaments (7, 8). WH2 domains
are also present in more complex proteins such as WASP/Scar,
verprolin/WIP, and Srv2/CAP. These are multifunctional regulators of
actin dynamics that link intracellular signaling pathways to actin
dynamics (5). For example, WASP and Scar mediate signals from
PIP2 and the small GTPases Cdc42 and Rac to the
actin cytoskeleton by inducing actin assembly through
activation of the Arp2/3 complex (9-11). The WH2 domain is essential
for the activity of WASP and Scar and is believed to facilitate the
assembly of actin monomers to the newly formed filament ends (12, 13).
Also verprolin/WIP proteins interact with actin monomers through their
WH2 domains, but the biological role of their actin monomer-binding
activity is still unclear (14).
We searched sequence databases for WH2 domain-containing proteins
in mammals, Drosophila melanogaster,
Caenorhabditis elegans, and
Saccharomyces cerevisiae. In addition to the
previously characterized WH2 domain proteins (
-thymosins, ciboulot,
WASP, verprolin/WIP, Scar, Srv2/CAP) we identified several previously
unknown WH2 domain-containing proteins (5). One of these proteins is
particularly interesting because it is highly homologous to a human
cDNA fragment named MIM (Missing In Metastasis) that was recently
identified in a differential screen for mRNAs specifically
expressed in non-metastatic bladder cancer cells (15).
We show that mouse MIM is an actin monomer-binding protein that
efficiently inhibits the nucleotide exchange on actin monomers and
actin filament nucleation in vitro. The strong
actin-modulating activities reside in the C-terminal WH2 domain of MIM.
We found that MIM is strongly expressed in the developing heart,
skeletal muscle, and central nervous system. In adult mouse tissues,
strong expression levels are detected in liver and in certain regions of brain and kidney. Overexpression of MIM results in the disappearance of stress fibers and formation of abnormal F-actin structures in NIH
3T3 cells, suggesting that MIM regulates the dynamics of the actin
monomer pool in specialized mammalian cells.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction and Site-directed Mutagenesis--
A DNA
fragment corresponding to the full-length mouse MIM cDNA was
amplified from I.M.A.G.E. Consortium mouse EST clone plasmid ID:5101888
(UK HGMP Resource Centre). The C-terminal half of mouse MIM was
amplified from mouse E10 cDNA and I.M.A.G.E. Consortium mouse EST
clone plasmid ID:2648606. Two different splice variants were obtained
(see Fig. 1A), from which the shorter and more abundant form
was used for the biochemical studies and is referred as MIM-CT in the
text. The oligonucleotides used in the amplification of the full-length
mouse MIM created BamHI and HindIII sites and the
oligonucleotides used in the amplification of MIM-CT created NcoI and HindIII sites at the 5' and 3' ends of
the PCR fragment. These fragments were digested and ligated into the
pGAT2 vector (16) to create plasmids pPL149 (full-length mouse MIM) and
pPL106 (MIM-CT). The site-directed mutation, which replaced lysines 746 and 747 into alanines and deleted the last 11 amino acids 749-759, was
introduced into the MIM-CT by PCR, and the amplification product was
cloned into NcoI-HindIII-digested pGAT2 vector to
create a plasmid pPL141. For generation of in situ probes
MIM-CT cDNA fragment was cloned into the BamHI
(5')-HindIII (3') sites of pBSIIKS to create a plasmid
pPL139. The plasmids for overexpressing green fluorescence protein
(GFP) fusions of MIM were constructed by cloning the full-length MIM
cDNA into the XhoI-BamHI sites of pEGFP-N1
and pEGFP-C1A (Clontech) to create plasmids pPL151
and pPL153.
Northern Blotting--
A mouse MIM cDNA probe was prepared
from the plasmid pPL106 as described for mouse twinfilin (17). The
MIM-CT cDNA probe was then hybridized to commercial mouse multiple
tissue and mouse embryo Northern blots (Clontech)
according to the manufacturer's instructions. The Northern blot
filters were exposed on a phosphorimaging screen overnight.
-Actin
controls were used to ensure equal amounts of RNA in each lane.
Radioactive in Situ Hybridizations--
The antisense probe was
obtained by linearizing the plasmid pPL139 with BamHI and
transcribing it with T3 RNA polymerase. For the sense probe
HindIII and T7 RNA polymerase were used. The in
situ hybridizations on the mouse tissue sections were performed using 35S-UTP-labeled riboprobes as previously described
(18).
Protein Expression and Purification--
Mouse MIM-CT was
expressed as a glutathione S-transferase (GST) fusion
protein in Escherichia coli BL21 (DE3) cells under the
control of T7 lac promoter. Cells were grown in 12 liters of
Luria broth medium to an optical density of 0.5 at 600 nm, and the
expression was induced with 0.2 mM
isopropyl-
-D-1-thiogalactosidase. Cells were harvested
3 h after induction, washed with 180 ml of 20 mM Tris
(pH 7.5), resuspended in 40 ml of phosphate-buffered saline, 0.2 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, lysed
by sonication, and centrifuged for 15 min at 15,000 × g. GST fusion proteins were enriched from the supernatant by
glutathione-agarose beads (Sigma). The beads were then incubated
overnight with thrombin (5 units/ml) at +4 °C to cleave MIM-CT away
from GST. The beads were washed five times with 50 mM
Tris-HCl (pH 7.5) and 150 mM NaCl2, and the
supernatant was diluted into 30 ml with 20 mM Tris-HCl (pH
7.5) and 25 mM NaCl. The solution was loaded onto an
equilibrated Q-Sepharose High Performance Anion-exchange chromatography
column (Amersham Biosciences) and eluted with a linear 0.025-1
M NaCl gradient. The peak fractions containing MIM-CT that
eluted at ~0.35 M NaCl were pooled, and the buffer was exchanged to
10 mM Tris-HCl (pH 7.5) and 50 mM NaCl with a
PD-10 desalting column (Amersham Biosciences). The protein solution was
then concentrated in 10-kDa cutoff tubes (Centricon) into a final
concentration of 100-200 µM divided into small aliquots,
frozen in liquid N2, and stored at
70 °C. Human
platelet actin and pyrene-labeled actin (from rabbit skeletal muscle)
were from Cytoskeleton Inc. Rabbit muscle actin was prepared from
acetone powder as described in Pardee and Spudich (19).
Actin Monomer-binding Assay--
We assayed the binding of
MIM-CT to actin monomers by measuring the fluorescence of NBD-labeled
G-actin. Rabbit muscle actin was labeled by NBD-Cl as described in
Detmers et al. (20) and Weeds et al. (21).
ADP-actin was prepared by incubating NBD-actin with hexokinase-agarose
beads (Sigma) and glucose for 3 h at +4 °C (22). We used 0.2 µM actin and varied the concentration of MIM-CT from 0 to
3.2 µM. The reactions were carried out at room temperature in F-buffer (2 mM Tris, pH 8.0, 0.1 mM CaCl2, 0.1 mM DTT, 0.2 mM ADP or
ATP, 0.5 mg/ml bovine serum albumin, 1 mM MgCl2, 0.1 M KCl). The normalized enhancement of fluorescence as
determined by the equation
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(Eq. 1)
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was measured with BioLogic MOS250 fluorometer at each
concentration of MIM-CT with an excitation at 482 nm and emission at 535 nm. The data were analyzed using SigmaPlot software and fitted using the following equation
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(Eq. 2)
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where z and c are described in the two following equations.
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(Eq. 3)
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(Eq. 4)
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Actin Filament Sedimentation Assays--
Forty-µl aliquots of
human platelet actin at desired concentration in G-buffer (20 mM Tris, pH 7.5, 0.2 mM ATP, 0.2 mM
DTT, 0.2 mM CaCl2) were polymerized for 30 min
by the addition of 5 µl of 10× initiation mix (20 mM
MgCl2, 10 mM ATP, 1 M KCl). Five µl of MIM-CT (0, 10, 20, or 40 µM) in G-buffer was
added to the actin filaments followed by a 30-min incubation. The actin
filaments were sedimented by centrifuging at 217,000 × g for 30 min in a Beckman Optima MAX Ultracentrifuge and a
TLA 100 rotor. All steps were performed at room temperature. Equal
proportions of supernatant and pellet fractions were loaded onto 10%
SDS-polyacrylamide gels; the gels were stained with Coomassie Blue and
scanned with FluorS-Imager (Bio-Rad, Hercules, CA).
Pyrene Actin Filament Assembly Assays--
We followed actin
polymerization in the presence of MIM by determining the increase in
the fluorescence of pyrene-labeled actin (Cytoskeleton). Three
µM actin (1:5 pyrene-labeled rabbit muscle actin:human
platelet actin) with 0, 0.75, or 3 µM MIM-CT or
MIM-CT
WH2 in G-buffer (5 mM Tris-HCl, pH 7.5, 0.2 mM ATP, 0.2 mM DTT, 0.2 mM
CaCl2) was polymerized by adding 1/10 reaction volumes of
10× initiation mix (20 mM MgCl2, 10 mM ATP, 1 M KCl). Kinetics of actin filament
assembly was followed by a BioLogic MOS-250 fluorescence
spectrophotometer at an excitation wavelength of 365 nm and an emission
wavelength of 407 nm. To examine the effect of MIM-CT for pointed end
and barbed end filament assembly, gelsolin-actin seeds (10 nM) or phalloidin-stabilized actin seeds (0.4 µM) were added to a mixture of 3 µM actin
and desired concentration (0-6 µM) of MIM-CT in
F-buffer. The phalloidin-actin seeds were prepared as described
(23),and gelsolin-actin seeds as described (24). The rate of actin
filament assembly was monitored as described above.
Nucleotide Exchange Assay--
The rate of nucleotide exchange
was measured from the fluorescent signal provided by
-ATP bound to
G-actin. Human platelet actin was dialyzed against
-ATP-G-buffer (20 mM Tris, pH 8.0, 1 mM DTT, 0.4 mM
CaCl2, 0.2 mM
-ATP) overnight at +4 °C.
An 80-µl mixture of
-ATP-actin and MIM-CT was prepared in G-buffer
with final concentrations of 0.5 µM for actin and 0, 0.5, 1, 1.5, 2, or 4 µM for MIM-CT. Twenty µl of 5 mM ATP was added to the mixture, and the reaction was
immediately monitored by a BioLogic MOS-250 fluorescence
spectrophotometer using an excitation wavelength of 360 nm and an
emission wavelength of 410 nm.
Cell Culture and Immunofluorescence--
NIH 3T3 cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum (Invitrogen), 2 mM
L-glutamine, 100 units/ml penicillin, and 100 mg/ml
streptomycin. Cells were transfected with 1.5 µg of the desired
plasmid using the FuGENE6 transfection reagent (Roche Molecular
Biochemicals) according to the manufacturer's instructions.
Immunofluorescence was carried out as described in Vartiainen et
al. (17). Tetramethylrhodamine B isothiocyanate-conjugated
phalloidin (Molecular Probes) was used at a 1:400 dilution to visualize
actin filaments.
Miscellaneous--
SDS-PAGE was carried out by using the buffer
system of Laemmli (25). Protein concentrations were determined with a
Hewlett Packard 8452A diode array spectrophotometer by using the
calculated extinction coefficients for mouse MIM-CT (
280 = 17,780 M
1 cm
1) and for actin
(
290-340 = 26,600 M
1
cm
1). Protein concentrations were also estimated from
Coomassie Blue-stained SDS-PAGE. Fluorescence-monitored urea
denaturation assays were carried out as described in (26).
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RESULTS |
Identification of a Novel Mouse WH2 Domain Protein--
In
database searches for novel WH2 domain-containing proteins, we
identified several independent mouse ESTs that encoded a putative
protein with a C-terminal WH2 domain. A full-length sequence of the
coding region of this cDNA was obtained by sequencing I.M.A.G.E. consortium mouse EST clone plasmid ID:5101888. The predicted protein is
composed of 759 residues with an N-terminal sequence (MEAVI ... ).
There is an in-frame stop codon 85 nucleotides upstream to the
methionine (AUG) codon, and the nucleotides immediately 5' to this AUG
codon fulfill the rules for translational initiation sequences (27),
suggesting that translation is initiated at this methionine. Our EST
database searches and sequencing of PCR fragments from this mouse
cDNA showed that a shorter form of this protein also exists. The
shorter form lacks amino acids 646-681 and contains Leu-to-Pro
substitutions at residues 483 and 487 (Fig.
1A). Because the nucleotide
sequences of these two isoforms are otherwise identical, they are most
likely splice variants of the same gene.

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Fig. 1.
The sequences and domain structures of mouse
and human MIMs. A, an alignment of mouse
(Mm) and human (Hs) MIM amino acid sequences. The
WH2 domains in the C termini are boxed. Two splice variants were
identified from mouse MIM. The longer variant corresponds to the
sequence shown in the figure, whereas the shorter and more abundant
variant lacks amino acids 646-681 (underlined) and contains
Leu-to-Pro substitutions in residues 483 and 487 (underlined). The Met-404 residue that was used as a start
codon for expressing the C-terminal half of MIM in E. coli
is bolded. B, a multiple sequence alignment of
the WH2 domains of mouse MIM, human thymosin- 4 (EMBL M17733), human
WASP (TrEMBL AF115549), human N-WASP (TrEMBL D88460), human Scar1 (EMBL
D87459), human WIP (TrEMBL AF031588), fly Scar (NCBI AAF53042), and
yeast verprolin (SGD YLR337W). The asterisks indicate the
residues that are essential for actin monomer-binding in thymosin- 4
(28). Furthermore, the position of an -helix in the WH2 domain of
thymosin- 4 in indicated above the sequence (37). C, a
schematic representation of the domain structure of mouse MIM. The
GenBank accession number for mouse MIM cDNA sequence is AY214918.
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Our database searches revealed a human homologue that is 96% identical
at the amino acid level. While the putative coding regions of the human
and mouse cDNA clones were nearly identical at the nucleotide
level, the regions 5' from the putative translational initiation codons
showed significantly lower homology. This provides further support that
translation starts at this AUG codon. Interestingly, a region in the
human cDNA is identical to an ~300-bp cDNA fragment that was
recently identified by differential display as a gene that was
specifically expressed in non-metastatic bladder cancer cells and
consequently named MIM (15). Both mouse and human MIM contain a WH2
domain in their C termini (Fig. 1A). The residues that are
important for actin monomer-binding in the WH2 domain of thymosin-
4
(28) are well conserved in human and murine MIM, suggesting that these
proteins interact with monomeric actin (Fig. 1B). The human
and mouse MIMs also contain a proline-rich region but otherwise do not
share any detectable sequence homology to other known proteins (Fig.
1C).
Expression of MIM in Mouse Embryos and Adult Mouse Tissues--
To
examine the expression patterns of MIM, we carried out Northern blot
and in situ hybridization analyses on embryonic and adult
mouse tissues. A multiple-tissue Northern blot analysis showed that MIM
mRNA is expressed in several adult mouse tissues at variable levels
(Fig. 2, left panel).
Particularly strong expression was detected in liver. The kidney,
heart, lung, spleen, and brain showed only moderate or weak expression
of MIM mRNA. No expression could be detected in skeletal muscle and
testis. A Northern blot analysis of mouse embryos at different stages
showed that the relative MIM expression increases during development
(Fig. 2, right panel). The MIM cDNA probe used in this
study recognized only a single ~6-kb mRNA, indicating that the
probe was specific. The two MIM splice variants are very close to each
other in size, and therefore cannot be distinguished from each other in
this Northern blot. The blots were also hybridized with a
-actin
control probe to ensure that each tissue samples contained equal
amounts of mRNA (data not shown).

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Fig. 2.
Northern blot analysis of MIM mRNA
expression in adult mouse tissues (left panel) and
embryogenesis (right panel). The ~6-kb MIM
mRNA is strongly expressed in the liver, in moderate amounts in the
kidneys and heart, and at lower levels in lung, spleen, and brain.
There is no detectable expression in skeletal muscle and testis. The
relative levels of MIM mRNA increase during embryonic
development.
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The expression pattern of MIM in developing mouse embryos was studied
in more detail by RNA in situ hybridizations. During development (E10.5-18.5) the strongest MIM expression was in the developing heart, skeletal muscle, and central nervous system (Fig.
3A and data not shown). Weak
expression was detected in the liver, kidney, and lung (Fig.
3A and data not shown). A control hybridization carried out
with the sense probe showed that the signal was specific for the MIM
mRNA (Fig. 3B). Additional hybridizations on mouse
embryo sections from various developmental stages demonstrated that MIM
is highly expressed in the postmitotic neurons of the spinal cord and
brain (Fig. 3, C and E). Strong expression could also be detected in somitic and migrating myoblasts (Fig.
3C), in the developing muscles of the limbs and the body
(Fig. 3, C and F), and in the developing cardiomyocytes
(Fig. 3D). Weak expression was detected in the developing
sensory systems including the inner ear and eye (Fig. 3E and
data not shown).

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Fig. 3.
An in situ
hybridization analysis of the MIM expression in mouse embryos and
in adult mouse brain and kidney. A, in situ
hybridization on a sagittal section of an E12.5 embryo with an
antisense probe. The expression of MIM is particularly strong in the
central nervous system and heart. Weak expression in several inner
organs can also be detected. B, hybridization of an adjacent
section with a control sense probe. Note, that blood cells give some
background activity for example in the umbilical cord. C, a
cross-section through an E11.5 embryo. MIM is expressed in postmitotic
neurons of the spinal cord (spc), somitic, and migrating
myoblasts, and in the developing muscles of the limb. D, at
E11.5 MIM is strongly expressed in the developing heart and weakly
expressed in liver. E, cross-section of the mouse hindbrain
at E12.5. Strong MIM expression is detected in the differentiating
postmitotic neurons. Weak expression can be observed in small
populations of proliferating neural precursor cells lining the
ventricle (arrows) and in the developing inner ear.
F, at E12.5 high levels of MIM mRNA can be detected in
limb and intercostal muscles (i.c.musc). No expression can
be observed in the developing rib bones. G, in the adult
mouse brain, MIM expression was detected in the Purkinje cells of the
cerebellum. H, in adult mouse kidney, MIM expression is
restricted to the medullary outer zone (moz) and cortex. MIM
expression was not detected in the medullary inner zone
(miz).
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We also examined MIM expression in adult mouse tissues by in
situ hybridizations. Whereas MIM is strongly and widely expressed in the developing central nervous system until birth, in the adult brain MIM is expressed at high levels only in one special neuronal cell
type: the Purkinje cells of the cerebellum (Fig. 3G). Weak expression was detected in other parts of the brain such as the septum,
the piriform cortex, and the ependymal cells lining the ventricles
(data not shown). In the kidney, MIM was highly expressed in the outer
medullary zone and weakly in the cortex. MIM expression could not be
detected in the medullary inner zone (Fig. 3H). Taken together, these hybridizations show that MIM is strongly expressed in
the developing neurons and muscle cells but is down-regulated during
the maturation of these cells. In adult mice MIM is highly expressed
only in kidney, liver, and in specialized neuronal cells such as
Purkinje cells.
MIM Binds ATP-Actin Monomers through Its C-terminal WH2
Domain--
The mouse and human MIM proteins contain a putative actin
monomer-binding motif, WH2 domain, in their C termini. To examine whether mouse MIM is an actin-binding protein, we carried out a
biochemical characterization of this protein. Because our attempts to
express and purify the full-length mouse MIM were unsuccessful, we
decided to express the WH2 domain containing C-terminal half (residues
404-759) of the more abundant splice variant of MIM (Fig.
1A). This fragment (MIM-CT) was produced as a GST fusion protein in E. coli. The GST was removed by thrombin
digestion, and recombinant MIM-CT was further purified by anion
exchange chromatography.
The fluorescence of the NBD-labeled actin monomers is affected upon
interaction with many proteins, including ADF/cofilin, twinfilin,
thymosin-
4, and ciboulot, thereby providing a method to determine
the affinities of these proteins for actin monomers (29-31). MIM-CT
induced a 20-25% decrease in the fluorescence of NBD-G-actin (Fig.
4). The extent of the NBD-actin
fluorescence decrease displayed a saturating behavior enabling us to
calculate the KD values for MIM-CT/actin monomer
complexes. MIM-CT bound to both ADP- (Fig. 4A) and ATP-actin
monomers (Fig. 4B) with high affinity. However, the affinity
of MIM-CT for ATP-actin monomers was 5-fold higher,
(KD = 0.06 µM) than for ADP-actin monomers (KD = 0.3 µM). The assay was
carried out in physiological ionic conditions (0.1 M KCl, 2 mM MgCl2).

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Fig. 4.
Interaction of MIM-CT and
MIM-CT WH2 with actin monomers. The change
in the fluorescence of 0.2 µM NBD-labeled MgADP-G-actin
(A) or MgATP-G-actin (B, C) was
measured at different concentrations of MIM-CT under physiological
ionic conditions at pH 8.0. Symbols are data, and
lines are the calculated binding curves. Each data point on
graphs A and B is the mean of three independent
experiments, whereas the data in graph C are from one
experiment. Dissociation constants (KD) derived from
the binding curves are indicated in the figure. The MIM-CT WH2
protein did not affect NBD-actin fluorescence in this assay, indicating
that it does not bind actin monomers with a detectable affinity.
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To examine the role of the C-terminal WH2 domain for actin
monomer-binding, we designed a mutant protein, MIM-CT
WH2, in which the WH2 domain was inactivated. The actin-binding site in the WH2
domain of thymosin-
4 has been mapped by mutational studies (28). We
constructed a mutant protein in which the corresponding residues
(Lys-746 and Lys-747) that have been shown to be most critical for
G-actin binding in thymosin-
4 were replaced by alanines, and the
last 11 amino acids (749-759) were deleted. The mutant protein was
purified as described for wild-type MIM-CT. It showed similar
stability than the wild-type MIM-CT in a fluorescence-monitored urea
denaturation assay, suggesting that these mutations do not affect the
protein stability (Fig. 5). MIM-CT
WH2
did not affect the fluorescence of NBD-G-actin, suggesting that this
mutant protein does not bind actin monomers with a detectable affinity
(Fig. 4C).

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Fig. 5.
The stability of wild-type and mutant MIM-CT
proteins measured by fluorescence monitored urea denaturation
assay. The arbitrary fluorescence units are shown on the
y-axis, and the urea concentration is shown on the
x-axis. Both the wild-type MIM-CT and MIM-CT WH2 unfold at
~4 M urea.
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Mouse MIM Inhibits the Nucleation of Actin Filaments, but Its
Complex with ATP-G-actin Can Participate in Filament Barbed End
Growth--
The effect of MIM-CT on actin dynamics was examined by
steady-state and kinetic assembly assays. The steady-state assay was an
actin filament co-sedimentation assay where the actin concentration was
2 µM and the concentration of MIM-CT was varied from 0 to 4 µM. In the absence of MIM, a majority of actin was
present in the pellet fraction, whereas in the presence of 2 and 4 µM MIM-CT ~75% of the actin was shifted to the
supernatant fraction. This shows that mouse MIM efficiently decreases
actin filament assembly (Fig.
6A, upper panel).
In a similar assay MIM-CT
WH2 did not increase the amount of
monomeric actin, demonstrating that the C-terminal WH2 domain is
essential for the ability of MIM to inhibit steady-state F-actin
assembly (Fig. 6A, lower panel). We also carried
out an actin filament co-sedimentation assay to examine the interaction
of MIM with F-actin. In this assay we used a constant concentration of
MIM-CT (2 µM) and varied the concentration of actin from
0 to 8 µM. MIM-CT did not co-sediment with actin
filaments, suggesting that MIM does not interact with F-actin with a
detectable affinity (data not shown).

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Fig. 6.
Effects of MIM-CT on actin filament
nucleation and assembly. A, MIM-CT increases the amount
of monomeric actin in an actin filament sedimentation assay
(upper panel), but MIM-CT WH2 does not affect actin
sedimentation (lower panel). Platelet actin filaments (2 µM) were incubated with 0 (lane 1), 1 (lane 2), 2 µM (lane 3), and 4 µM (lane 4) MIM-CT or MIM-CT WH2 for 30 min,
and the filaments were sedimented by centrifugation. B, MIM
prevents the nucleation of actin filaments. 3 µM pyrene
actin (1:5 pyrene actin:human platelet actin) was polymerized in the
presence of 0, 0.8, or 3.0 µM MIM-CT or 3.0 µM MIM-CT WH2 by the addition of 0.1 M KCl,
2 mM MgCl2, and 0.5 mM ATP. The
polymerization of filaments was followed by the increase in pyrene
fluorescence. Wild-type MIM-CT efficiently prevents actin filament
assembly, whereas MIM-CT WH2 does not have any detectable effect on
actin polymerization. C, MIM-CT/ATP-G-actin complex can
participate in filament barbed end growth. Phalloidin-actin seeds (0.4 µM) were added on a mixture of 3 µM
ATP-G-actin and 0/1/2/3/4/6 µM MIM-CT in F-buffer. The
polymerization of filaments was followed by the increase in pyrene
fluorescence, and the rates were normalized to the rate in the absence
of MIM-CT.
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Pyrene actin assembly assays were carried out to directly analyze the
effects of MIM on actin filament polymerization and nucleation. We
first followed the polymerization of 3 µM actin (in the
presence of 0/0.75/3 µM MIM-CT or MIM-CT
WH2) for 10 min after addition of polymerization salts. The data showed that
wild-type MIM-CT efficiently inhibits the nucleation and assembly of
the actin filaments, whereas MIM-CT
WH2 did not affect the
nucleation/assembly of actin filaments (Fig. 6B). We next
examined the effects of MIM-CT on filament pointed end and barbed end
assembly by using gelsolin-actin and phalloidin-stabilized actin seeds,
respectively. MIM-CT efficiently inhibits actin assembly on 10 nM gelsolin-actin seeds demonstrating that MIM-CT blocks
the pointed end filament growth (data not shown). MIM-CT also decreases
the rate of actin assembly on phalloidin-actin seeds, but even high
concentrations of MIM-CT (2-fold molar excess to ATP-G-actin) do not
completely block the assembly. Instead, the assembly rate of
MIM-CT/ATP-G-actin complex appears to saturate to ~40% level of that
of ATP-G-actin alone (Fig. 6C). In conclusion, these assays
demonstrate that mouse MIM efficiently inhibits the nucleation and
pointed end assembly of actin filaments, but its complex with
ATP-G-actin can participate in filament barbed end growth. The
participation of MIM-CT in barbed end elongation is also in agreement
with the co-sedimentation assay (Fig. 6A) where MIM-CT,
despite its very high affinity for ATP-G-actin (0.06 µM;
Fig. 4B), was unable to shift all actin to the monomeric
fraction even when present in 2-fold molar excess to actin.
MIM Inhibits the Nucleotide Exchange on G-actin--
Many actin
monomer-binding proteins, such as ADF/cofilin, twinfilin, ciboulot, and
thymosin-
4, decrease the rate of nucleotide exchange on actin
monomers (31-34). On the other hand small actin monomer-binding
protein, profilin, catalyzes the nucleotide exchange on actin monomers
(35). We examined the effects of mouse MIM on the nucleotide exchange
by following the rate of replacement of
-ATP by ATP on actin
monomers in the absence and presence of MIM-CT. MIM inhibits the rate
of nucleotide exchange on actin monomers in a
concentration-dependent manner (Fig.
7). The inhibition of the nucleotide
exchange of 0.5 µM actin is almost saturated already at
0.5 µM MIM-CT. This result is in good agreement with the
very high affinity of MIM-CT for ATP-G-actin (0.06 µM,
Fig. 4B).

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Fig. 7.
MIM-CT inhibits the nucleotide exchange on
actin monomer. The exchange of -ATP for ATP was monitored by
the decrease in the fluorescence at 410 nm. 0.5 µM
-ATP-G-actin was mixed with 0, 0.5, 1, 1.5, 2, or 4 µM
MIM-CT, and the decrease in the fluorescence was followed after the
addition of 1 mM ATP. The kobs rates
obtained from the data are plotted in the graph.
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Effects of MIM on Actin Dynamics in Vivo--
To study the effects
of MIM on actin dynamics in vivo, we overexpressed mouse MIM
as a GFP fusion protein in NIH 3T3 fibroblasts. In the fusion protein
constructs GFP was located either at the N terminus or at the C
terminus of MIM. In both cases the localization of MIM and the induced
phenotypes in cells were similar to each other, indicating that the GFP
did not affect the activity of MIM in cells (data not shown). MIM-GFP
fusion proteins were uniformly cytoplasmic in NIH 3T3 cells and not
present in the nucleus. Cells over-expressing MIM-GFP showed a clear
decrease in the amount of stress fibers and accumulation of abnormal
worm-like cytoplasmic actin filament structures (Fig.
8, A and B). These
abnormalities did not occur in untransfected cells or in the cells
overexpressing GFP alone (Fig. 8, C and D).
Furthermore, overexpressing MIM-GFP fusion protein in HeLa and NRK
cells induced similar abnormalities in the actin cytoskeleton (data not
shown). These results suggest that the actin monomer-binding protein
MIM is able to regulate actin dynamics also in living cells.

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Fig. 8.
Over-expression of MIM results in the
formation of abnormal actin filament structures in NIH-3T3 cells.
Cells were transfected with a vector expressing MIM-GFP fusion protein
(A, B) or GFP as a control (C,
D). Localization of GFP-MIM and GFP are shown in
panels A and C, and the actin filaments are
visualized by fluorescein-phalloidin in panels B and
D. The cells overexpressing MIM have less stress fibers than
untransfected cells and also show an accumulation of abnormal worm-like
actin structures. Scale bar, 10 µm.
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DISCUSSION |
We show here that mouse MIM is an actin monomer-binding protein
that inhibits the nucleation of actin filaments in vitro. These activities reside in the C-terminal WH2 domain of MIM. WH2 domains are also found in many other actin monomer-interacting proteins, such as WASP, Scar, thymosin-
4, ciboulot, actobindin, and
WIP/verprolin. All available data indicate that these proteins also
interact with actin monomers through their WH2 domains (for recent
review see Ref. 5). Furthermore, at least thymosin-
4, ciboulot, and
actobindin have significantly higher affinity for ATP-G-actin than for
ADP-G-actin (31, 36). Our data shows that mouse MIM affinity for
ATP-G-actin is ~5-fold higher than its affinity for ADP-G-actin,
suggesting that WH2 domain has evolved as a motif that preferentially
binds ATP-G-actin. It is important to note that mouse MIM binds
ATP-G-actin with ~20-fold higher affinity (KD = 0.06 µM, Fig. 4) than thymosin-
4, ciboulot, and
actobindin (KD = 0.7-2 µM) (31).
For that reason, we would expect MIM to more drastically affect actin
dynamics in cells than for example thymosin-
4.
Most actin monomer-binding proteins, including profilin, ADF/cofilin,
twinfilin and WH2 domain proteins bind to the same site on the actin
monomer, and thus compete with each other in actin monomer-binding (30,
37-39). Our data show that mouse MIM has a higher affinity for
ATP-G-actin (KD = 0.06 µM) than other
actin monomer-binding proteins such as profilin (KD = 0.1-0.15 µM), ADF/cofilin (KD = 4-8 µM) and twinfilin (KD = 0.5 µM) (1, 30). This suggests that in the cells where MIM is
expressed, the majority of active MIM is associated with ATP-G-actin.
On the other hand, the affinity of mouse MIM for ADP-G-actin
(KD = 0.3 µM), is significantly lower than the ones of ADF/cofilin (KD = 0.1-0.15
µM) and twinfilin (KD = 0.05 µM), indicating that MIM would not significantly affect
the localization and dynamics of the cellular ADP-actin monomer pool
(1, 31).
Some WH2 domain proteins such as thymosin-
4 inhibit actin filament
assembly, whereas others such as ciboulot, actobindin and WASP do not
sequester actin monomers when the barbed ends of filaments are
available (8, 12, 31, 36, 40). Mouse MIM increases the amount of actin
monomers in steady-state assembly assays and inhibits actin filament
nucleation in kinetic assays (Fig. 6, A and B).
MIM also blocks the pointed end filament assembly, but
MIM-CT/ATP-G-actin complexes can participate in barbed end filament
elongation (Fig. 6C). Therefore, our data suggests that MIM
has similar effects on actin assembly as previously described for small
WH2 domain proteins cibulot and actobindin (8, 31). However, because
MIM is a large, multidomain protein, it is likely to have additional
activities that could affect or regulate the actin-modulating
activities of the C-terminal WH2 domain.
Our Northern blot and in situ hybridization assays
demonstrated that MIM mRNA is strongly expressed in developing
neurons and myoblasts but absent in most mature neurons and muscle
cells. In adult mice, MIM is present in high levels in liver, kidney, and in the cerebellar Purkinje cells (Fig. 3). Similarly, the small
actin monomer-sequestering protein, thymosin-
10, is expressed in
several developing neuronal cell-types, but only in Purkinje cells in
adult animals (41). The transient expression of MIM in developing
neurons and myoblasts suggests a role in polarized growth or motility:
both processes are actin-dependent and typical for
differentiating neurons and myoblasts. Mature muscle cells and neurons
lack MIM and have a less dynamic actin cytoskeleton and consequently do
not undergo similar morphogenetic changes than their embryonic
precursors. The presence of MIM in adult Purkinje cells may be related
to the growth and refinement of Purkinje cell dendrites, a phenomenon
that probably also takes place in adult animal brains.
It is interesting to note, that the human homologue of MIM was
identified as a gene that was present in non-metastatic but absent in
metastatic bladder cancer cells (15). Our in situ hybridization data show that MIM is strongly expressed in developing neurons and myoblasts (Fig. 3). These are highly polar cell types, suggesting that MIM may help promote and maintain cell polarity. Polarity is essential for the growth and motility of myoblasts and
neurons, whereas the loss of polarity in epithelial cells may affect
adhesion and precede increased motility, invasiveness, and metastatic
capability. This may provide an explanation for the lack of MIM in
metastatic bladder cancer cell lines (15). Alternatively, MIM may
negatively regulate cytoskeletal dynamics by restricting actin
polymerization only to certain regions of migrating cells and in cells
that undergo polarized growth. This negative regulatory role is
supported by our biochemical (Fig. 6) and cell biological studies,
where overexpressing MIM disrupted stress fibers and resulted in
formation of abnormal actin filament structures (Fig. 8). In the future
it will be important to examine the localization of endogenous MIM
protein in various cell types. Unfortunately, our attempts to generate
antibodies against mouse MIM have so far been unsuccessful. We would
also like to use small interfering RNA and traditional mouse
knockout technology to examine the importance of MIM during development
and in adult tissues.
Taken together, our data show that mouse MIM binds actin monomers and
regulates actin filament assembly in specific polarized mammalian
cell-types. Because MIM is a relatively large, multidomain protein, it
may have other activities and regulate actin dynamics in a complex
fashion. Other large WH2 domain proteins (WASP, Scar, WIP/verprolin)
are involved in signaling to the actin cytoskeleton, and the activities
of these proteins are regulated by relatively complex mechanisms (1).
Therefore, MIM may also be a link between cellular signaling pathways
and actin filament assembly. In this respect, it is important to note
that MIM contains also several proline-rich motifs that may interact
with Src homology 3 or WW domains of certain signaling proteins. In the
future it will be important to identify the interaction partners of MIM
and learn how they regulate the activity and localization of MIM in
specialized mammalian cells.