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Molecular Biology of the Cell
Vol. 19, 2328–2338, May 2008
mDia2 Induces the Actin Scaffold for the Contractile Ring
and Stabilizes Its Position during Cytokinesis in NIH 3T3
Cells
Sadanori Watanabe,* Yoshikazu Ando,* Shingo Yasuda,* Hiroshi Hosoya,
†
Naoki Watanabe,* Toshimasa Ishizaki,* and Shuh Narumiya*
*Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto 606-8501, Japan; and
†
Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima
739-8526, Japan
Submitted October 29, 2007; Revised January 25, 2008; Accepted February 7, 2008
Monitoring Editor: Fred Chang
mDia proteins are mammalian homologues of Drosophila diaphanous and belong to the formin family proteins that
catalyze actin nucleation and polymerization. Although formin family proteins of nonmammalian species such as
Drosophila diaphanous are essential in cytokinesis, whether and how mDia proteins function in cytokinesis remain
unknown. Here we depleted each of the three mDia isoforms in NIH 3T3 cells by RNA interference and examined this
issue. Depletion of mDia2 selectively increased the number of binucleate cells, which was corrected by coexpression of
RNAi-resistant full-length mDia2. mDia2 accumulates in the cleavage furrow during anaphase to telophase, and concen-
trates in the midbody at the end of cytokinesis. Depletion of mDia2 induced contraction at aberrant sites of dividing cells,
where contractile ring components such as RhoA, myosin, anillin, and phosphorylated ERM accumulated. Treatment with
blebbistatin suppressed abnormal contraction, corrected localization of the above components, and revealed that the
amount of F-actin at the equatorial region during anaphase/telophase was significantly decreased with mDia2 RNAi.
These results demonstrate that mDia2 is essential in mammalian cell cytokinesis and that mDia2-induced F-actin forms
a scaffold for the contractile ring and maintains its position in the middle of a dividing cell.
INTRODUCTION
Cytokinesis is the final step in cell division that physically
separates a dividing cell into two. In a somatic cell, separa-
tion, i.e., cleavage occurs in the middle of a dividing cell
between the two spindle poles to ensure each set of segre-
gated chromosomes inherited to each daughter cell. Al-
though cytokinesis is a multistep process under coordinated
control of cell cycle progression, cytoskeletal dynamics, and
vesicle transport, the actomyosin-based constriction by the
contractile ring that is constructed in the equatorial region of
a dividing cell is recognized as a major driving force for
physical separation till abscission (Balasubramanian et al.,
2004). However, how the position of the contractile ring, i.e.,
cleavage plane, is determined and maintained through cy-
tokinesis and how and where actin filaments are produced
and assembled with myosin and other molecules into the
contractile ring in mammalian cells remain largely un-
known.
The small GTPase Rho functions in several organisms and
several lines of cultured mammalian cells as a molecular
switch linking nuclear division and cytokinesis; Rho is acti-
vated in anaphase to telophase and induces the contractile
ring in dividing cells (Mabuchi et al., 1993; Piekny et al., 2005;
Narumiya and Yasuda, 2006). In mammalian cells, the GTP-
bound, activated form of Rho acts on two downstream ef-
fectors to induce actomyosin bundles; one is ROCK/Rho-
kinase that activates myosin for cross-linking of anti-parallel
actin filaments, and the other is mammalian homolog of
Drosophila diaphanous (mDia) protein that induces actin
filaments by catalyzing actin nucleation and polymerization
(Watanabe et al., 1997; Sagot et al., 2002). mDia belongs to the
formin family of proteins and there are three isoforms,
mDia1-3 (Higgs, 2005). mDia has multiple domains, GBD
(GTPase-binding domain) in the N-terminus, FH (formin
homology) domains, FH1 and FH2, in the middle, and DAD
(diaphanous auto-regulatory domain) in the C-terminus
(Higgs, 2005; Rose et al., 2005). The FH2 domain binds to the
barbed end of an actin filament and catalyzes actin nucle-
ation and polymerization. The FH1 domain accelerates actin
elongation by the FH2 domain through binding to the actin
monomer-binding protein, profilin (Watanabe et al., 1997;
Romero et al., 2004; Kovar et al., 2006). By this action, mDia
induces long unbranched actin filaments in contrast to
Arp2/3 complex that induces actin meshwork (Goode and
Eck, 2007). In addition to the action on actin, mDia has been
reported to stabilize and orient microtubules in interphase
and mitotic cells (Ishizaki et al., 2001; Palazzo et al., 2001;
Yasuda et al., 2004). Intriguingly, although involvement of
ROCK in cytokinesis has been examined previously (Kosako
et al., 2000), whether mDia protein is involved in cytokinesis
of mammalian cells and if so, which mDia isoform functions
in this process have not yet been examined thoroughly,
though its nonmammalian orthologues such as Cdc12p in
Schizosaccharomyces pombe and Diaphanous in Drosophila
This article was published online ahead of print in MBC in Press
(http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07–10–1086)
on February 20, 2008.
Address correspondence to: Shuh Narumiya (snaru@mfour.med.kyoto-u.
ac.jp).
2328 © 2008 by The American Society for Cell Biology
melanogaster have been shown essential for cytokinesis in
each species (Castrillon and Wasserman, 1994; Tominaga et
al., 2000; Pelham and Chang, 2002; Dean et al., 2005). To
examine this issue, we have used RNA interference (RNAi)
to deplete each mDia isoform in NIH 3T3 cells and identified
that one of the mDia isoforms, mDia2, is essential for cyto-
kinesis of this cell line. We have also performed fluorescence
microscopy for contractile ring components such as RhoA,
F-actin, myosin, anillin, and phosphorylated ERM (pERM),
as well as live cell-imaging for myosin and mDia2, and
examined localization and actions of mDia2 in cytokinesis.
We now show that mDia2 localizes in the equatorial region
of a dividing cell in anaphase and induces F-actin there to
provide an actin scaffold for assembly of the contractile ring
and stabilize its position during cytokinesis.
MATERIALS AND METHODS
Materials
Short interfering double-stranded RNA oligomers (siRNAs) K2 and A6 (Ara-
kawa et al., 2003; Yamana et al., 2006) were used for RNAi for mDia1. Three
different siRNAs, siRNAmDia2#1, #2 and #3, corresponding to nucleotide
sequences of 289-313, 1889-1907, and 2150-2168, respectively, were used for
RNAi for mDia2 (NM_019670). Two different siRNAs, siRNAmDia3#1 and #2,
corresponding to nucleotide sequences of 967-991 and 1295-1319, respectively,
were used for RNAi for mDia3 (AY312280). Stealth RNAi negative control
duplexes (Invitrogen, Carlsbad, CA) were used for control RNAi. Block-iT
Alexa Fluor Red fluorescent Oligo (Invitrogen) was used for determination of
efficiency of siRNA transfection. pEGFP-MRLC was described previously
(Miyauchi et al., 2006). pDsRed2-Histone H2BK has been described previously
(Yasuda et al., 2004). pEGFP-mDia2 was prepared as follows. cDNA for
mDia2 (Yasuda et al., 2004) was subcloned into pCR-Blunt vector (Invitrogen;
pCR-Blunt-mDia2). The plasmid was digested with XhoI and KpnI and then
subcloned into pEGFP-C1 (Clontech, San Jose, CA) to generate pEGFP-C1-
mDia2 (XhoI-3⬘), lacking a fragment 5⬘of the XhoI site of the full-length
mDia2. The 5⬘-XhoI fragment of mDia2 was obtained by PCR amplification
using the pCR-Blunt-mDia2 as a template, with 5⬘-GAACTCGAGCTATG-
GAGAGGCACCGGGC-3⬘as the forward primer and 5⬘-GTCCCTCTGCTC-
GAGTTTCC-3⬘as the reverse primer. The resultant PCR fragment was di-
gested with XhoI and then subcloned into pEGFP-C1-mDia2 (XhoI-3⬘)to
generate pEGFP-C1-mDia2. To prepare mDia2-RNAi–resistant pEGFP-
mDia2, pEGFP-mDia2 r#1 and r#2, we introduced silent mutations into the
region of pEGFP-C1-mDia2 targeted by mDia2siRNA #1 using the
QuikChange Site-Directed Mutagenesis Kit II (Stratagene, La Jolla, CA) with
5⬘-GAAAACAACCCAAAGGCGCTGCCCGAAAGCGAGGTGTTGAAGCTTTT-
TGAGAAGATG-3⬘as a template for pEGFP-mDia2 r#1 and 5⬘-CCCAAAG-
GCGCTGCCAGAGTCCGAAGTCTTGAAGCTTTTTGAG-3⬘for pEGFP-mDia2
r#2. To prepare pEGFP-mDia2 I704A, we introduced mutations into the codon
of Ile 704 with 5⬘-GCTCAGAACCTTTCAGCCTTCCTGAGCTCCTTCCG-3⬘
as a template as described above.
Primary antibodies (Abs) used were mouse DM1A monoclonal Ab (mAb)
to
␣
-tubulin, fluorescein isothiocyanate (FITC)-conjugated DM1A, FITC-con-
jugated Ab to

-actin, and rabbit polyclonal Ab to myosin IIA (Sigma-Aldrich,
St. Louis, MO); rat mAb to
␣
-tubulin (Chemicon, Temecula, CA); goat Ab to
mDia3 (N15), rabbit polyclonal Ab to green fluorescent protein (GFP; FL), and
mouse 26C4 mAb to RhoA (Santa Cruz Biotechnology, Santa Cruz, CA); and
rabbit polyclonal Ab to GFP from MBL (Nagoya, Japan). Rabbit polyclonal
antibody to mDia1 was described previously (Watanabe et al., 1997). Poly-
clonal C1 Ab to mDia2 was generated in rabbits against a glutathione S-
transferase (GST) fusion protein of the C-terminal fragment of mDia2 (amino
acid residues, 1056-1171). The fragment was subcloned into pGEX-6P-1 (GE
Healthcare Life Science, Piscataway, NJ) to generate pGEX-mDia2-C, which
was then used for transformation of Escherichia coli BL21 (Novagen, Madison,
WI). After induction with 1 mM IPTG, the bacteria were lysed and the fusion
protein was purified with a GSH-Sepharose column (GE Healthcare Life
Science). The purified protein was injected into rabbits as antigen. The anti-
body to mDia2 was purified from antiserum by affinity chromatography
using the antigen coupled with NHS-activated Sepharose (GE Healthcare Life
Science). Polyclonal N1 Ab to mDia2 was also generated against a GST fusion
protein of the N-terminal fragment of mDia2 (amino acid residues, 33-411) as
described above and purified using the antigen coupled with CNBr-activated
Sepharose (GE Healthcare Life Science). Rabbit anti-anillin and rat anti-
phospho-ERM (pERM) Abs were kind gifts from Dr. Makoto Kinoshita
(Kyoto University) and Professor Sachiko Tsukita (Osaka University, Japan).
Cell Culture and Transfection
NIH 3T3 cells and C2C12 cells were maintained in DMEM (GIBCO, Rockville,
MD) supplemented with 10% fetal calf serum (FCS) at 37°C with an atmo-
sphere containing 10% CO
2
. Transfection of plasmids was performed using
Lipofectamine LTX Reagent (Invitrogen) according to the manufacturer’s
protocol. We diluted 1
g of each plasmid DNA and 2
l of PLUS reagent
(Invitrogen) in 400
l of Opti-MEM, subsequently mixed with 5
l of Lipo-
fectamine LTX. The lipofectamine solution was mixed with 2 ml of fresh
medium and added to cells of 50–60% confluency in one well of a six-well
plate. RNAi was performed using Lipofectamine RNAiMAX Reagent (In-
vitrogen) according to the manufacturer’s reverse transfection protocol. We
mixed 1.2
lof20
M siRNA duplex and 4
l of Lipofectamine RNAiMAX in
400
l of Opti-MEM. NIH 3T3 cells or C2C12 cells of semiconfluency were
washed and suspended with trypsin-EDTA. The siRNA mixture was added
to 1.0 ⫻10
5
cells in 2 ml of the culture medium, and the cell suspension was
then seeded in a well of a six-well plate. siRNA experiments in synchronized
cells were performed as follows. NIH 3T3 cells were seeded and cultured for
16 h in a 100-mm dish with the culture medium containing 2 mM thymidine.
The cells were then washed twice with phosphate buffered saline (PBS) and
subjected to RNAi transfection as described above. The cells were then seeded
and further cultured for 8 h. The medium was then replaced again with the
culture medium containing 2 mM thymidine and the cells were cultured for
another 16 h. After washing three times with PBS and once with fresh
medium, the cells were incubated in the culture medium alone or in that
containing 40 ng/ml nocodazole for 8 h. The cells in the latter procedure were
then washed free of nocodazole as described above for thymidine removal.
The cells were cultured in fresh medium for 2 h for time-lapse imaging for the
statistical analysis of the phenotype induced by mDia2 RNAi or for 20 min
either with or without 80
M blebbistatin (Tocris, Ballwin, MO) before being
fixed for immunofluorescence.
Microinjection
NIH 3T3 cells were seeded and cultured for 6 h. The cells were microinjected
with normal rabbit IgG (Santa Cruz) or affinity-purified N1 antibody to
mDia2 (0.01 mg/ml) in PBS with 0.5
g/ml dextran-Alexa-fluor 594 (Molec-
ular Probes, Eugene, OR) using a microinjection system (Eppendorf, Fremont,
CA) with maintenance pressure of 400 hPa and injection pressure of 20 hPa
for 0.1 s. The cells were then incubated for 10 h before fixation.
Fluorescence Microscopy
NIH 3T3 cells or C2C12 cells were plated onto a coverslip in a 35-mm culture
dish for fluorescence microscopy. We used three different fixation protocols.
For phalloidin staining for F-actin, cells were fixed with 4% paraformalde-
hyde in PBS at 37°C for 15 min. The cells were washed three times with PBS
and permeabilized with 0.1% Triton X-100 in PBS for 5 min on ice, followed
by three washes with PBS. For immunofluorescence for GFP, RhoA, anillin,
myosin IIA, pERM, and mDia2, cells were fixed with 10% TCA on ice for 15
min (Yonemura et al., 2004). The fixed cells were washed three times with PBS
containing 30 mM glycine (G-PBS) and permeabilized with 0.2% Triton X-100
in G-PBS for 5 min on ice, followed by three washes with G-PBS. For staining
for
␣
-tubulin and for comparison of mDia1, mDia2, or mDia3 staining, the
cells were fixed with methanol at ⫺20°C for 5 min. After fixation and perme-
abilization as described above, the cells were incubated with 3% BSA in PBS
for 1 h and were incubated at room temperature for2horat4°Covernight
with following primary antibodies: rat anti-tubulin (1:1000 dilution), anti-
pERM (1:1), anti-myosin (1:50), anti-anillin (1:200), anti-mDia1 (1:200), anti-
mDia2 (1:200), and anti-mDia3 (1:50) Abs. After three washes with Tris-
buffered saline (TBS) containing 0.1% Tween-20 (TBST), the cells were
incubated with appropriate secondary antibodies coupled to Alexa Fluor 594
(Molecular Probes), and/or either Texas-Red phalloidin (1:200) or FITC-
conjugated anti-
␣
-tubulin (1:200). The samples were washed three times with
TBST before mounting in Prolong Antifade DAPI-Gold (Molecular Probes) on
glass slides. Staining was examined with a Leica SP5 confocal imaging system
(Plan-Apo 63/1.40 NA; Deerfield, IL). Binucleate or multinucleate cells were
identified on samples stained for tubulin and DNA. The percentage of binu-
cleate or multinucleate cells was determined in a blinded manner by an
observer without information of the identity of the samples. For the images of
interphase cells in Figure 1 and Figure S1, build-up images were obtained
from a collection of 10 Z sections of 0.5-
m step intervals from the bottom to
the top of the cells. For the images of dividing cells in Figure S3D and S4C and
Figures 4–6, build-up images were obtained from a collection of 15–25 Z
sections of 0.5-
m step intervals around the middle section of the cells. The
images were analyzed by the built-in software. Quantification of F-actin
intensity in Figure 5A was performed using MetaMorph software (Universal
Imaging, West Chester, PA) as follows. Images of 20 mitotic cells each in
control and mDia2 RNAi groups were obtained from two different experi-
ments, and the F-actin intensity in each cell was calculated by dividing the
sum of Texas-Red phalloidin fluorescence intensity in each pixel by the total
pixel counts in the cell area.
Immunoblotting
At indicated times of siRNA treatment, NIH 3T3 cells or C2C12 cells were
washed twice with PBS and lysed in 100
l of Laemmli sample buffer.
Immunoblotting was performed as described previously (Ando et al., 2007).
Primary antibodies were diluted in the blocking buffer and added: 1:1000
mDia2 in Cytokinesis
Vol. 19, May 2008 2329
Figure 1. Effects of depletion of mDia isoforms in NIH 3T3 cells. (A) Depletion of mDia proteins (mDia1, mDia2 and mDia3) by siRNA
treatment. NIH 3T3 cells were treated with siRNA specific for mDia1, 2, and 3 (A6, siRNAmDia2#1 and siRNAmDia3#2, respectively) or
scrambled control siRNA. After 24 and 48 h, the cells were harvested and subjected to immunoblot for each protein. (B) Fluorescence staining
for DNA (blue), F-actin (red), and tubulin (green) in NIH 3T3 cells treated with siRNAmDia2#1 for 48 h. All images show stacks of 10 different
focal planes. Bar, 50
m. Arrows represent examples of the binucleate cells. (C) Frequency of binucleation in NIH 3T3 cells subjected to RNAi
for mDia isoforms. NIH 3T3 cells were subjected to RNAi as indicated and the number of binucleate cells in each population was determined.
The results are from three independent experiments, in each of which N ⬎200 cells were examined. Values are shown as percentage of
binucleate cells. Error bars, SD. *p ⬍0.01 versus control RNAi, mDia1 RNAi, and mDia3 RNAi cells. (D) Rescue of the binucleate phenotype
by expression of RNAi-resistant full-length mDia2 construct. NIH 3T3 cells were transfected with pEGFP or pEGFP-mDia2 r#1, and after 24 h,
the cells were subjected to control or mDia2 RNAi (siRNAmDia2#1) for 48 h. The cells were either subjected to Western blotting with
anti-mDia2 (top), GFP (middle), or tubulin (bottom, left) or subjected to fluorescence staining as described above for determining the number
of binucleate cells in each population. Values are shown as percentage of binucleate cells expressing GFP per total cells expressing GFP (right).
Error bars, SD. *p ⬍0.01. (E) Effects of microinjection of N1 antibody to mDia2. Control IgG or N1 antibody to mDia2 were microinjected
into randomly growing NIH 3T3 cells along with Alexa Fluor 594 Dextran as a marker, and after 10 h the cells were fixed and stained for
tubulin (green) and DNA (blue). Left, immunofluorescence images of control IgG- (top) or anti-mDia2 antibody- (bottom) injected NIH 3T3
cells. Bar, 50
m. Right, values are shown as percentage of binucleate cells per injected cells. Error bars, SD. *p ⬍0.01. The results are from
three independent experiments, in each of which N ⬎100 cells were examined.
S. Watanabe et al.
Molecular Biology of the Cell2330
dilution for the Abs to
␣
-tubulin, mDia1, mDia2, or GFP and 1:200 dilution for
the Ab to mDia3. After overnight incubation at 4°C with these Abs except for
incubation at room temperature for 2 h with the Abs to tubulin and GFP, the
membranes were washed three times with TBS containing 0.05% Tween-20.
The bound primary Abs were detected with corresponding horseradish per-
oxidase–conjugated secondary Abs (GE Healthcare Bio-Science, Piscataway,
NJ; 1:3000 dilution in the blocking buffer) and ECL Western Blotting Detection
System (GE Healthcare Bio-Sciences).
Time-Lapse Live Cell Imaging
NIH 3T3 cells were seeded on 35-mm glass-bottom dishes (MatTek, Ashland,
MA) with or without siRNA transfection. The medium was replaced 18–24 h
after siRNA treatment with DMEM containing 10% FCS and 300 nM Syto11
(Invitrogen), and the cells were incubated for 30 min at 37°C. The dish was
then placed on a temperature-controlled stage maintained at 37°C with 5%
CO
2
. Live cell imaging was performed on an inverted microscope (model
DMIRE2; Leica) as described previously (Oceguera-Yanez et al., 2005). Se-
quential time-lapse images were acquired every 3 min for 120–225 min. For
imaging of cells expressing either EGFP-mDia2 or MRLC-EGFP together with
DsRed-histone-H2B, the cells were transfected at 8 h after seeding with
indicated plasmid DNAs. Live cell imaging was performed on the confocal
microscope (TCS-SP5; Leica) with 63⫻/1.40 NA lenses. Sequential time-lapse
images were acquired every 30 s or 1 min for 30 min.
Statistical Analysis
Data are presented as mean ⫾SD and were analyzed by Student’s ttest. p⬍
0.01 was considered statistically significant.
RESULTS
Effects of Depletion of Each mDia Isoform on Cytokinesis
To examine whether mDia isoforms are required for cytoki-
nesis, and, if so, to identify which mDia isoform functions in
this process, we transfected NIH 3T3 cells with siRNA spe-
cific to each mDia isoform, A6 for mDia1, siRNAmDia2#1
for mDia2, and siRNAmDia3#2 for mDia3. In these experi-
ments, transfection efficiency of siRNA determined by
Block-iT Red fluorescent Oligo was almost 100% (Figure
S1A). The cells were collected 24 and 48 h after transfection
and subjected to Western blotting. The treatment with each
siRNA specifically and time-dependently suppressed the
expression of corresponding mDia isoforms. After 48 h, only
a negligible amount of each protein was detected by West-
ern blotting; the percentage of depletion of endogenous
proteins at 48 h were 75, 95, and 70% for mDia1, mDia2, and
mDia3, respectively (Figure 1A). The siRNA treatment for
each mDia isoform did not apparently change the cell
shapes. Although RNAi for mDia1 and mDia3 did not affect
cytokinesis (Figure S1, C and D), mDia2-RNAi increased the
proportion of the cells with two nuclei (binucleate cells) as
evidenced by immunofluorescence as well as flow cytom-
etry (Figure 1B and Figure S1D). Failure of cytokinesis,
consequently, increased the size of these cells (Figure S1B).
Quantitative analysis revealed that the proportion of the
binucleate cells significantly increased in the mDia2-de-
pleted cell population compared with that in the control,
mDia1-depleted, or mDia3-depleted population; the per-
centage of binucleate cells increased to 25.8 ⫾4.9 and 39.8 ⫾
1.2% 24 and 48 h after transfection with mDia2 siRNA,
respectively (Figure 1C). We confirmed these results by us-
ing additional nonoverlapping siRNAs for mDia1 and
mDia3, K2 and siRNAmDia3#1, respectively, and two addi-
tional nonoverlapping siRNAs for mDia2, siRNAmDia2#2
and #3. Although all siRNAs sufficiently depleted respective
mDia isoforms, only treatment with siRNAs specific for
mDia2 significantly increased the rates of binucleate cells
(data not shown). To further validate that binucleation is
caused by mDia2 depletion, we attempted to rescue the
phenotype by cotransfecting a RNAi-resistant full-length
construct of mDia2 fused to enhanced GFP (EGFP) (EGFP-
mDia2 r#1) with siRNAmDia2#1. Expression of EGFP was
used as a control in these experiments. Depletion of endog-
enous mDia2 and expression of EGFP-mDia2 r#1 were ver-
ified by immunoblotting (Figure 1D, left). Expression of the
EGFP-mDia2 r#1 fully rescued the effects of mDia2 RNAi on
cytokinesis; the rate of binucleate cells returned to the level
in the control cells after the expression of this construct
(Figure 1D, right). These results suggest that the generation
of binucleate cells was specifically induced by reduction of
mDia2 protein. We further microinjected the antibody N1 to
mDia2 and evaluated its effect on cytokinesis. Ten hours
after we microinjected the mDia2 antibody into randomly
growing NIH 3T3 cells, we observed significant increase in
the percentage of binucleate cells compared with control IgG
injected cells (Figure 1E). We obtained similar results when
we injected the mDia2 antibody into NIH 3T3 cells synchro-
nized in G2 by double thymidine block and observed 5 h
later (data not shown). These results that depletion of mDia2
induces cytokinesis failure demonstrate that an mDia iso-
form is required for cytokinesis of NIH 3T3 cells and that it
is mDia2 that functions as an mDia isoform essential in this
process. In contrast to these findings, Tominaga et al. (2000)
previously microinjected antibodies to mDia1 or mDia2 into
dividing NIH 3T3 cells and found that the injection of anti-
mDia1 antibody and not the antibody to mDia2 interfered
with cytokinesis. Curiously, they did not find significant
expression of mDia2 in these cells, and yet they rescued
cytokinesis failure induced by anti-mDia1 antibody by over-
expression of full-length mDia2. Although the reason for
such apparent discrepancy is currently unknown, we exam-
ined a possibility that mDia1 functions redundantly with
mDia2 in cytokinesis. We therefore depleted mDia isoforms
in every possible combination and examined cytokinesis
failure. We found significant increases in the number of
multi(bi)-nucleate cells upon depletion of mDia1 or mDia3
only combined with mDia2 depletion, i.e., combined deple-
tion of mDia1 and mDia2, that of mDia2 and mDia3 or that
of mDia1, mDia2 and mDia3, and not that of mDia1 and mDia3
(Figure S1E). Importantly, combination of mDia1 and mDia2
depletion did not enhance the rate of binucleate cells compared
with that found on mDia2 depletion alone. These results sug-
gest that at least in the NIH 3T3 cells we used, mDia2 is
primarily important in cytokinesis among mDia isoforms.
Localization of mDia2 during Cell Division
To obtain insights into action mechanism of mDia2 in cyto-
kinesis, we next investigated the localization of endogenous
mDia2 in different phases of cell division by staining for
mDia2, tubulin, and DNA in dividing NIH 3T3 cells using
the C1 antibody to mDia2. This analysis revealed that
signals of mDia2 localized around the basal part of cell
cortex of rounding cells in prometaphase to metaphase,
appeared at equatorial cell cortex in late anaphase, accu-
mulated in the cleavage furrow in telophase, and finally
concentrated in the intercellular bridge at the end of
cytokinesis (Figure 2). The specificity of these signals was
verified by their loss with mDia2 RNAi both on Western blot
analysis and immunofluorescence (Figure 1A and Figure
S2A). We observed similar localization of mDia2 by using a
different mDia2 antibody N1 (data not shown). The localiza-
tion of mDia2 described above was also confirmed by ex-
pressing pEGFP-mDia2 and monitoring the GFP signal. The
GFP signals began to localize at the equatorial surface dur-
ing late anaphase and concentrated in the cleavage furrow
during cytokinesis (Movie S1). For comparison, we exam-
ined the localization of mDia1 and mDia3 during cell divi-
sion. Weak diffuse signals for mDia1 were observed over the
cell bodies and intercellular bridge at the end of telophase
mDia2 in Cytokinesis
Vol. 19, May 2008 2331
(Figure S2B); signals for mDia3 were found in association
with the central spindle in anaphase, and the associated
mDia3 signals were concentrated in the midbody as central
spindle microtubules were bundled in cytokinesis (Figure
S2C). Staining for neither mDia1 nor mDia3 yielded signals
in the cleavage furrow as that for mDia2. These localization
patterns of mDia1 and mDia3 are consistent with our pre-
vious results (Kato et al., 2001; Yasuda et al., 2004). These
results suggest that mDia2 accumulates specifically in the
cleavage furrow during cytokinesis.
We next wondered whether this function of mDia2 in
cytokinesis is conserved in other cell lines. We observed that
mDia2 RNAi also induced cytokinesis failure in C2C12
mouse myoblast cells and mDia2 localized to the cleavage
furrow of these cells (Figure S3), supporting the conserved
localization and function of mDia2 during cytokinesis.
Abnormal Contraction in mDia2-depleted Cells
To investigate how mDia2 depletion induces cytokinesis
failure in NIH 3T3 cells, we monitored the progression of
cell division of mDia2-RNAi cells by videomicroscopy. We
first used randomly growing cells subjected to control and
mDia2 RNAi. In control cells, the cleavage furrow appeared
6 min after anaphase onset and ingressed progressively
thereafter to separate two daughter cells within several min-
utes, and the separated daughter cells linked by the inter-
cellular bridge began to spread at 18 min (Figure 3A and
Movie S2). In contrast, mDia2 RNAi cells exhibited abnor-
mal cytokinesis behavior. In one group of the cells, the
cleavage furrow appeared between two daughter cells in
anaphase and began to ingress. However, the ingression was
not properly maintained but followed by robust contraction
at aberrant sites of daughter cells that prevented the ingres-
sion at the cleavage furrow and pushed separating chromo-
somes one end to the other, resulting in fusion of the daugh-
ter cells and production of a binucleate cell (Figure 3B and
Figure 2. Localization of mDia2 during cell division. NIH 3T3 cells
in different phases of cell division were stained for mDia2 (red),
tubulin (green), and DNA (blue). Each image shows a single focal
plane; the image of a prometaphase cell represents that at the basal
plane, and those of cells in anaphase, telophase and cytokinesis
represent those at the middle plane. Bar, 5
m.
Figure 3. Cytokinesis failure in mDia2-RNAi cells.
(A–C) Selected frames from time-lapse movies of
NIH 3T3 cells subjected to control RNAi (A and
Movie S2) or RNAi for mDia2 (B and C and Movies
S3 and S4). Phase contrast and green fluorescence
images were monitored by videomicroscopy after
DNA (green) was stained by Syto11. 0 min, anaphase
onset. Insets show magnified views of the DNA in
each merged image. Arrows in B and C indicate
abnormal contraction in the mDia2-depleted cells.
Bar, 10
m.
S. Watanabe et al.
Molecular Biology of the Cell2332
Movie S3). In another group of mDia2 RNAi cells, contrac-
tion was frequently observed already at metaphase, and
such contraction at abnormal sites continued in anaphase to
telophase and apparently inhibited appearance and func-
tioning of the cleavage furrow at the prospective site, lead-
ing to formation of a binucleate cell upon spreading (Figure
3C and Movie S4).
To statistically analyze the cytokinesis failure in mDia2-
RNAi cells, we next enriched mitotic cells by using thymi-
dine and nocodazole after RNAi treatment and tracked the
cell division by videomicroscopy. We confirmed a similar
phenotype of the cytokinesis failure in mDia2-RNAi cells
(data not shown). Percentages of cells showed cytokinesis
failure were 6.1 and 49.0% for control (n ⫽33) and mDia2-
RNAi cells (n ⫽51), respectively. Percentages of cells
showed the abnormal contraction were 3.0 and 76.5% for
control (n ⫽33) and mDia2-RNAi cells (n ⫽51), respec-
tively, suggesting that there is a link between the cytokinesis
failure and the abnormal contraction induced by mDia2
RNAi. These results indicate that mDia2 is required for
maintenance and functioning of the contractile ring between
two daughter cells.
Effects of mDia2 Depletion on Distribution of the
Contractile Ring Components
The contractile ring is composed of several components
including F-actin, myosin, anillin, and pERM (Straight et al.,
2003; Yokoyama et al., 2005). To examine whether these
components accumulate and are maintained at the prospec-
tive cleavage site during cytokinesis of mDia2 RNAi cells,
we stained for these contractile ring components by using
Texas-Red phalloidin and antibodies to each molecule in
cells depleted of mDia2. We stained for myosin by using
antibody to myosin heavy chain IIA. mDia2-RNAi cells
showed aberrant shapes (Figure 4), which probably reflected
abnormal contraction observed in time-lapse imaging anal-
ysis (Movies S3 and S4). In these mDia2-RNAi cells, signals
for each of F-actin, myosin, anillin, and pERM were not
observed at the cleavage furrow as in control cells, but
aberrantly localized around the cell cortex where abnormal
cell shape change was observed (Figure 4A). To examine
dynamics of such localization of the contractile ring compo-
nents, we expressed EGFP-fusion of myosin regulatory light
chain (MRLC-EGFP; Miyauchi et al., 2006) and followed its
movement during cell division. Although myosin accumu-
lated normally at the cleavage furrow from anaphase to
telophase and concentrated there as the furrow ingressed in
cytokinesis in control-RNAi cells (Figure 4B and Movies S5
and S6), significant accumulation of MRLC-EGFP was found
at sites of abnormal contraction in the cell cortex of mDia2-
RNAi cells. Intriguingly, such abnormal contraction was
observed already in prometaphase/metaphase before chro-
mosomes began to segregate and became more robust in
anaphase to telophase, and MRLC-EGFP was found to ac-
cumulate at sites of each contraction (Figure 4C and Movies
S7 and S8). This contraction-associated localization pattern
of EGFP-MRLC appears similar to localization of other com-
ponents of the contractile ring in fixed preparation of mDia2
RNAi cells, indicating that the components of the contractile
ring in mDia2-depleted cells were not maintained at the
cleavage furrow but moved together to the site of abnormal
contraction. We next examined effects of combined deple-
tion of mDia2 with other mDia isoforms in order to examine
whether this abnormal contraction is a direct consequence of
the loss of mDia2 or due to a shift in the balance of Rho
effectors caused by the mDia2 depletion. Combined deple-
tion of either mDia1 or mDia3 or both with mDia2 did not
abolish abnormal contraction caused by the loss of mDia2
(Figure 4D). Immunofluorescence study also showed that
there was no clear accumulation of mDia1 and mDia3 at the
site of abnormal contraction (data not shown). These results
suggest that mDia2 itself is important for proper positioning
and maintenance of the contractile ring components during
cell division.
Effects of Blebbistatin on Accumulation of the Contractile
Ring Components in mDia2-depleted Cells
The above results demonstrate that the contractile ring com-
ponents accumulate at various sites of mDia2-RNAi cells
where aberrant contraction occurs. On the other hand, it has
Figure 4. Abnormal localization of contrac-
tile ring components during anaphase/telo-
phase in mDia2-depleted cells. (A) Immuno-
fluorescence. NIH 3T3 cells were transfected
with siRNA for mDia2 (siRNAmDia2#1) and
synchronized with thymidine and nocoda-
zole, and at 32 h after transfection the cells
were released into DMEM containing 10%
FCS for 20 min. The cells were fixed and
stained for indicated molecules (red), tubu-
lin (green), and DNA (blue). All images
show stacks of focal planes as described in
Materials and Methods. Bar, 5
m. Arrows
indicate abnormal accumulation of indicated
molecules. (B and C) Selected frames from
time-lapse movies of NIH 3T3 cells subjected
to control (B and Movies S5 and S6) or
mDia2 RNAi (C and Movies S7 and S8), and
labeled with DsRed-histone H2Bk (red) and
MRLC-EGFP (green). Bar, 5
m. (D) Effects
of combined depletion of mDia2 and other
mDia isoforms. NIH 3T3 cells were trans-
fected with siRNA for mDia isoforms in in-
dicated combination for 48 h, and the cells
were stained for F-actin (red), tubulin
(green), and DNA (blue). A typical example is shown out of 20 cells examined. Arrows indicate abnormal accumulation of F-actin. Bar,
5
m.
mDia2 in Cytokinesis
Vol. 19, May 2008 2333
been shown that various contractile ring components utilize
apparently different mechanisms and accumulate at the
equatorial region of the dividing cell cortex in anaphase and
are assembled there to the contractile ring (Straight et al.,
2003; Yokoyama et al., 2005). We wondered whether mDia2
depletion interfered with such initial accumulation process of
the components. We could not fully examine this issue in intact
mDia2-depleted cells because of strong contraction in these
cells. We therefore used blebbistatin, a myosin II ATPase
inhibitor, to suppress contraction and examined localization
of each component (Straight et al., 2003). Immunofluores-
cence analysis showed that the accumulation of signals for
F-actin at the equatorial region appeared weaker in mDia2-
RNAi cells compared with control-RNAi cells (Figure 5A,
Figure 4. (cont).
S. Watanabe et al.
Molecular Biology of the Cell2334
left). Decrease of F-actin in mDia2-RNAi cells was verified
by quantitative fluorescence intensity measurements of
whole cells subjected to control and mDia2 RNAi (Figure
5A, right). Given that mDia proteins have actin-nucleating
and -polymerizing activity, these results indicate that mDia2
is indeed responsible for formation of F-actin at this site.
Therefore, we next examined whether this actin-nucleating/
polymerizing activity of mDia2 is indeed required for the
cytokinesis by attempting to rescue the mDia2 RNAi-in-
duced cytokinesis failure by expressing an actin-polymer-
ization–defective GFP-mDia2 mutant. Mutation into Ile
704
of
mDia2 has been shown to render mDia2 actin polymeriza-
tion-defective (Xu et al., 2004; Harris et al., 2006). We found
that GFP full-length mDia2 I704A could not rescue the
mDia2 RNAi-induced cytokinesis failure, whereas this GFP
full-length mDia2 I704A localized to the cleavage furrow
(Figure S4, B and C). These data further support that the
actin-nucleating/polymerizing activity of mDia2 is required
for cytokinesis.
In contrast to the above findings on F-actin, we found
signals for myosin, anillin, or pERM at equatorial cortex of
both control and mDia2-RNAi cells in the presence of bleb-
bistatin, but these components showed broader localization
in mDia2-RNAi cells compared with control cells (Figure
5B). The percentages of cells showing the localization of each
components wider than the 120° sector from the center of the
cell are 20 and 62% for myosin for control and mDia2-RNAi
cells (n ⫽50 for each), 16 and 58% for anillin for control and
mDia2-RNAi cells (n ⫽50 for each), 29 and 40% for pERM
for control and mDia2-RNAi cells, respectively (n ⫽45 for
each). We also treated control-RNAi cells with latrunculin B
in the presence of blebbistatin during anaphase/telophase to
test the contribution of F-actin in this effect, and found that
latrunculin-treated cells showed dispersion of signals for
anillin and myosin around the cell cortex (data not shown).
Given that latrunculin-treated cells showed dispersed local-
ization of the contractile ring components including anillin,
the mDia2-driven F-actin production in the equatorial cortex
appeared important for concentration and maintenance of
the contractile ring components, myosin II, anillin, and
pERM in the cleavage furrow during anaphase/telophase.
Effects of mDia2 Depletion on Rho Localization during
Cytokinesis
The small GTPase Rho is activated in anaphase to telophase,
localizes in the cleavage furrow, and induces assembly of the
contractile ring there (Piekny et al., 2005). It is also believed
that Rho acts on downstream effectors such as ROCK and
citron kinase and produces contraction of the contractile ring
for cleavage (Madaule et al., 1998; Kosako et al., 2000; Ueda
et al., 2002; Yamashiro et al., 2003; Piekny et al., 2005). It is
therefore interesting to know whether aberrant contraction
seen in mDia2-depleted cells is associated with or dissoci-
ated from Rho activation. To examine this issue, we stained
for RhoA during anaphase/telophase in mDia2-depleted
cells in the presence or absence of blebbistatin (Figure 6, A
and B). Although signals for RhoA accumulated in the cleav-
age furrow during anaphase/telophase in control cells,
RhoA localized aberrantly at sites of the cell cortex of ap-
parently abnormal contraction in mDia2-RNAi cells without
blebbistatin (Figure 6A). On the other hand, in the presence
of blebbistatin, almost all signals for RhoA were restricted to
the equatorial region of mDia2-RNAi cells. The equatorial
localization of RhoA was found in 18 of 20 cells in mDia2-
Figure 5. Localization of contractile ring
components in blebbistatin-treated cells. NIH
3T3 cells were subjected to control or mDia2
RNAi and synchronized with thymidine and
nocodazole, and at 32 h after transfection the
cells were released into the medium contain-
ing 80
M blebbistatin for 20 min. The cells
were fixed and stained for indicated molecules
(red), tubulin (green), and DNA (blue). All
images show stacks of focal planes as de-
scribed in Materials and Methods. Bar, 5
m. (A)
Phalloidin staining for F-actin in blebbistatin-
treated cells. Left, phalloidin staining of con-
trol or mDia2-RNAi cells are shown. Right,
quantitative analysis of fluorescence intensity
for F-actin in control or mDia2-RNAi cells.
Fluorescence intensity of phalloidin staining
for total area of each cell was quantified using
MetaMorph in 20 cells of each group from two
different experiments as described in Materials
and Methods. *p ⬍0.01. (B) Immunofluores-
cence for MyosinIIA, anillin, and pERM in
blebbistatin-treated cells.
mDia2 in Cytokinesis
Vol. 19, May 2008 2335
RNAi cells with blebbistatin compared with 8 of 20 in
mDia2-RNAi cells without blebbistatin (Figure 6B). Given
that blebbistatin was added upon mitosis in this experi-
ment, these results taken together suggest that RhoA lo-
calizes in the prospective cleavage furrow and induces the
contractile ring complex there in a manner independent of
mDia2, but that mDia2-driven F-actin is important for
maintenance of the RhoA-containing contractile ring com-
plex at the site of the cleavage furrow.
DISCUSSION
In this work, using RNAi, we have examined the involve-
ment of three mDia isoforms in cytokinesis and identified
mDia2 as an essential mDia isoform required for cytokinesis
in NIH 3T3 cells and C2C12 cells (Figure 1 and Figure S3).
We have also analyzed localization of the mDia isoforms
and found that mDia2 localizes specifically in the cleavage
furrow during cytokinesis. We have found that depletion of
mDia2 substantially reduced the amount of F-actin accumu-
lating in the cleavage furrow but did not affect accumulation
of other contractile ring components such as RhoA, myosin,
anillin, and pERM in the equatorial region. However, with-
out mDia2, these components could not be properly inte-
grated into the contractile ring, but apparently formed in-
complete complexes, which were shifted away from the
equatorial region and induced contraction at aberrant site of
a dividing cell.
Our findings summarized above can thus not only suggest
functions of mDia2 in cytokinesis but also address to some
of the major questions regarding cytokinesis. First, given
that mDia molecules can stabilize and align microtubules
(Ishizaki et al., 2001; Palazzo et al., 2001; Yasuda et al., 2004)
and that the cleavage plane is suggested to be specified by
spindle microtubules that are stabilized in the equatorial
region (Canman et al., 2003), it is possible that mDia isoforms
are involved in determination of the cleavage plane. How-
ever, the above observation that depletion of mDia2 does not
interfere with RhoA accumulation in the equator argues
against this idea and rather suggests that RhoA accumulates
first in the cleavage plane and recruit mDia2 there. This is
consistent with the property of mDia2 to bind to members of
the Rho GTPases such as RhoA, Rac1, and Cdc42 (Alberts et
al., 1998; Yasuda et al., 2004). However, binding to RhoA
cannot explain selective localization of mDia2 to the cleav-
age furrow, because other mDia isoforms can bind to RhoA
as well. Further analysis is therefore required to elucidate a
mechanism for selective localization of mDia2 to the cleav-
age furrow.
Second, it is intriguing that mDia2 localizes in the cleav-
age furrow and that its depletion reduced the F-actin
amount there. Previously, it was argued whether F-actin is
formed in the cleavage furrow or formed elsewhere and
transported to the furrow (Wang, 2005; Eggert et al., 2006).
Given that mDia molecules are capable of catalyzing actin
nucleation and polymerization, our results strongly suggest
that the majority of F-actin is produced in situ in the cleav-
age furrow by the action of mDia2 and accumulate there.
Then, what functions do actin filaments induced by mDia2
exert in cytokinesis? Abnormal contraction at aberrant sites
apparently by the contractile ring components including
RhoA, myosin, anillin, and pERM in mDia2-depleted cells
suggests that with mDia2 depletion and/or with depletion
of mDia2-induced F-actin, the contractile ring is not prop-
erly organized and is not maintained at the prospective site
of the cleavage furrow, which indicates that mDia2 and
mDia2-induced F-actin link these components together to
form the contractile ring and stabilize its position in the
equatorial region. Formins such as mDia2 can produce long,
straight actin filaments. The structure of the contractile ring
was studied by electron microscopy in fission yeast and
newt eggs, and these studies revealed that it consists of
anti-parallel bundles of straight actin filaments that are
bound to the plasma membrane through barbed ends (Ma-
buchi et al., 1988; Kamasaki et al., 2007). Although it is
argued whether such structure is applied also to the con-
tractile ring in mammalian cells (Eggert et al., 2006), it is
tempting to speculate that mDia2 induces straight actin
filaments of opposite directionality in the prospective site of
the cleavage furrow, which provide an actin-based scaffold
encircling the equatorial region of dividing cells and facili-
tate formation of the contractile ring complex by incorporat-
ing other components of the ring such as myosin, anillin,
and pERM to this actin scaffold. By such actions, mDia2 may
restrict the movement of the contractile ring and stabilize its
position. mDia2 may also function in anchoring the actin
filaments of the contractile ring to the plasma membrane,
because it accumulates in the equatorial cell cortex and its
binds to the barbed end of actin filaments. Our results are
thus consistent with and have substantially extended the
findings by Dean et al. (2005), who examined localization of
myosin in dividing Drosophila S2 cells subjected to RNAi for
diaphanous and showed that diaphanous is required for
Figure 6. Effects of mDia2 depletion on RhoA localization during
cytokinesis. (A) Aberrant localization of RhoA in mDia2-RNAi cells.
NIH 3T3 cells were transfected and synchronized as described in the
legend for Figure 4. The cells were released into the medium with-
out blebbistatin. After 20 min, the cells were fixed and stained for
RhoA (red), tubulin (green), and DNA (blue). All images show
stacks of 15–25 focal planes. Bar, 5
m. (B) Effects of blebbistatin on
RhoA localization in mDia2-RNAi cells. NIH 3T3 cells were sub-
jected to mDia2 RNAi and synchronized with double thymidine
block. The cells were treated with DMSO or 80
M blebbistatin for
30 min 6.5 h after thymidine removal and stained for RhoA (red),
tubulin (green), and DNA (blue). All images show stacks of 15–25
focal planes. Bar, 5
m.
S. Watanabe et al.
Molecular Biology of the Cell2336
maintenance of myosin II to the cleavage furrow. It has to be
mentioned, however, that construction of the contractile ring
may not be governed solely by mDia2 but by interdependent
actions of the contractile ring components including mDia2.
Recently, anillin has been reported to bind myosin, and
RNAi of anillin induces a phenotype similar to that we have
found in mDia2-depleted cells (Straight et al., 2005).
Finally, in this study, we also noted that the mitotic cells
depleted of mDia2 exhibited mild oscillatory contractions
during prometaphase and metaphase and that the cortical
localization of myosin was not uniform as typically seen in
control cells (Movies S4, S7, and S8). Mitotic cell rounding is
the process in which a flat interphase cell becomes spherical
and is associated with rearrangement of the actin cytoskel-
eton, de-adhesion, and an increase in cortical rigidity (Mad-
dox and Burridge, 2003; Thery and Bornens, 2006). Maddox
and Burridge (2003) reported that mitotic cell rounding re-
quires activation of RhoA. In this study, we observed that
mDia2 localized to the cell margin in rounding NIH 3T3 cells
and mDia2-depleted cells exhibit impaired mitotic rounding
(Figures 2 and 3B). The oscillatory contractions of mDia2-
depleted cells mentioned above may be caused by impaired
rigidity of mitotic cells in the absence of mDia2. Eisenmann
et al. (2007) showed that expression of Dip, which they
claimed as an inhibitory binding protein for mDia2, induced
nonapoptotic blebbing in HeLa cells, which is thought to be
caused by breaks in cortical rigidity. These results suggest
that, in addition to its action in cytokinesis, mDia2 also
function in maintenance of cortical rigidity and rounding of
mitotic cells. Given that cytokinesis is now recognized as a
consequence of many events occurring globally in the cell
cortex through cell division (Maddox and Burridge, 2003;
Wang, 2005; Mukhina et al., 2007), these results may suggest
that mDia2 functions not only by inducing F-actin in the
cleavage furrow but also by regulating the cortical rigidity
globally. It may shift the balance of the contractility of
mitotic cells by shifting its accumulation in the cell depen-
dent on the phase of cell division. Elucidation of a mecha-
nism how functions of mDia2 in different phases of cell
division is regulated may unravel how cells execute mitosis
and cytokinesis properly through adjusting cell morphogen-
esis with chromosome separation.
ACKNOWLEDGMENTS
We thank K. Nonomura, Y. Kitagawa, and T. Arai for assistance and A. Fujita
(of our department) for N1 antibody to mDia2. We also thank C. Higashida,
T. Miki, F. Oceguera-Yanez, and J. Monypenny for helpful suggestions and
comments. This work was supported by a Grant-in-Aid for Specially Pro-
moted Research from the Ministry for Education, Culture, Sports, Science,
and Technology.
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