Biochem. J. (2008) 414, 261–270 (Printed in Great Britain)
261
doi:10.1042/BJ20071655
Critical roles of actin-interacting protein 1 in cytokinesis and chemotactic
migration of mammalian cells
Asuka KATO1 , Souichi KURITA1 , Aya HAYASHI, Noriko KAJI, Kazumasa OHASHI and Kensaku MIZUNO2
Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan
Cofilin regulates actin filament dynamics by stimulating actin
filament disassembly and plays a critical role in cytokinesis and
chemotactic migration. Aip1 (actin-interacting protein 1), also
called WDR1 (WD-repeat protein 1), is a highly conserved WDrepeat protein in eukaryotes and promotes cofilin-mediated actin
filament disassembly in vitro; however, little is known about
the mechanisms by which Aip1 functions in cytokinesis and
cell migration in mammalian cells. In the present study, we
investigated the roles of Aip1 in cytokinesis and chemotactic
migration of human cells by silencing the expression of Aip1 using
siRNA (small interfering RNA). Knockdown of Aip1 in HeLa
cells increased the percentage of multinucleate cells; this effect
was reversed by expression of an active form of cofilin. In Aip1knockdown cells, the cleavage furrow ingressed normally from
anaphase to early telophase; however, an excessive accumulation
of actin filaments was observed on the contractile ring in late
telophase. These results suggest that Aip1 plays a crucial role in
the completion of cytokinesis by promoting cofilin-mediated actin
filament disassembly in telophase. We have also shown that Aip1
knockdown significantly suppressed chemokine-induced chemotactic migration of Jurkat T-lymphoma cells, and this was blocked
by expression of an active form of cofilin. Whereas control cells
mostly formed a single lamellipodium in response to chemokine
stimulation, Aip1 knockdown cells abnormally exhibited multiple
protrusions around the cells before and after cell stimulation.
This indicates that Aip1 plays an important role in directional
cell migration by restricting the stimulus-induced membrane
protrusion to one direction via promoting cofilin activity.
INTRODUCTION
cofilin-mediated actin filament disassembly [5,6]. Genetic and
biochemical studies have shown that Aip1 enhances actin filament
disassembly only in the presence of cofilin, but has a negligible
effect on actin dynamics on its own [24–27]. Two distinct mechanisms have been proposed for the action of Aip1 in promoting
cofilin-mediated actin filament disassembly: Aip1 could cap the
barbed ends of cofilin-severed actin filaments to prevent elongation and reannealing of severed filaments [28–30] and/or could
actively sever cofilin-decorated actin filaments [31–35]. In addition, the cofilin-stimulating activity of Aip1 is further enhanced
by co-operation with other actin-binding proteins, such as coronin
or caspase 11 [36,37].
The cellular and biological functions of Aip1 have been investigated in several model organisms. For example, in budding
yeast, the deletion of the Aip1 gene is viable, but results in a
synthetic lethal phenotype when deleted in strains harbouring
cofilin mutations [24,26,30,33]. In Dictyostelium, Aip1 is
involved in endocytosis, cytokinesis and cell migration [27]. In
Caenorhabditis elegans, a null mutation in the Aip1 gene causes
disorganized actin filament assembly in muscles of the body
wall as well as defects in muscle contractile activity [34,38].
In Drosophila, mutations in the flare gene, which encodes
Drosophila Aip1, cause F-actin accumulation and abnormal hair
morphology [39]. The genetic studies in these organisms suggest
that Aip1 functionally interacts with cofilin and is involved in the
regulation of actin filament dynamics and actin-dependent cellular
processes in vivo by enhancing cofilin-mediated actin filament
disassembly. In vertebrates, microinjection of Aip1 in Xenopus
blastomeres causes cleavage arrest by disrupting the cortical
accumulation of actin and cofilin [25]. Recent studies have also
Actin cytoskeletal dynamics and reorganization play fundamental
roles in a variety of cellular processes, including cell migration,
cytokinesis, endocytosis and morphological change. During cytokinesis, an actomyosin-based contractile ring is formed and constricts at the equator of dividing cells before being disassembled
at the end of cytokinesis [1]. During cell migration, an F-actin-rich
lamellipodial membrane protrusion is generated at the front of the
cell and extends forward by actin filament assembly at the tip of
the leading edge [2,3]. Actin filament dynamics and reorganization are regulated by a number of actin-binding proteins. Among
them, cofilin is one of the most important regulators of actin
filament dynamics, because it functions to sever and depolymerize actin filaments [4–6]. Cofilin is localized in the regions where
actin filaments turn over rapidly, such as the cleavage furrow
of dividing cells [7,8] and the lamellipodium of migrating cells
[9,10]. Depletion or inactivation of cofilin results in a marked
decrease in the actin monomer pool and an increase in filamentous
actin structures within the cell and impairs cytokinesis and cell
migration [4–6,11–18]. This indicates that cofilin plays a crucial
role in cytokinesis and cell migration by promoting actin filament
turnover through its actin filament-disassembling activity [17,18].
Cofilin activity is controlled in several ways, including phosphorylation and dephosphorylation [19–21], changes in pH [22],
and association with phosphoinositides [23] and other regulatory
proteins such as Aip1 (actin-interacting protein-1) and cyclaseassociated protein [4–6]. Aip1, also called WDR1 (WD-repeat
protein 1) in mammals, is a WD-repeat protein that is ubiquitously expressed in eukaryotes and has the potential to promote
Key words: actin-interacting protein 1 (Aip1), cell migration,
chemotaxis, cofilin, cytokinesis, WD-repeat protein 1 (WDR1).
Abbreviations used: Aip1, actin-interacting protein-1; CFP, cyan fluorescent protein; CFP–H2B, CFP-tagged histone H2B; DAPI, 4 ,6-diamidino-2phenylindole; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; SDF-1, stromal cell-derived factor-1; siRNA, small interfering RNA;
sr-Aip1, siRNA-resistant Aip1; YFP, yellow fluorescent protein.
1
These authors contributed equally to this work.
2
To whom correspondence should be addressed (email [email protected]).
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A. Kato and others
shown that knockdown of Aip1 expression in mouse macrophagelike cells causes defects in cell migration [37], and knockdown
of Aip1 in human osteosarcoma cells causes defects in mitotic
cell rounding [40]. Furthermore, mutations in the Aip1 gene
in mice result in embryonic lethality, macrothrombocytopenia
and autoinflammatory disease, depending on the level of gene
disruption [41].
In the present study, we have examined the role of Aip1 in
cytokinesis and the chemotactic migration of human cell lines
by knocking down the expression of Aip1 using siRNA (small
interfering RNA). Knockdown of Aip1 in HeLa cells induced
the aberrant accumulation of F-actin near the contractile ring in
late telophase and impaired cytokinesis, leading to an increase
in the number of multinucleate cells. Knockdown of Aip1 in
Jurkat leukaemic T-cells suppressed their chemotactic migration
in response to SDF-1 (stromal cell-derived factor-1) and induced
multiple membrane protrusions before and after cell stimulation.
The ectopic expression of an active cofilin mutant significantly
blocked the inhibitory effects of the Aip1 knockdown on
cytokinesis and the chemotactic response. Our results indicate
that Aip1 plays crucial roles in both cytokinesis and chemotactic
migration of mammalian cells by promoting cofilin-mediated
actin filament disassembly.
EXPERIMENTAL
Materials
SDF-1 was purchased from PeproTech (London, U.K.). The rabbit
polyclonal antibody against cofilin (COF-1) was prepared as
described previously [14]. Mouse monoclonal antibodies against
the Myc epitope (9E10) and β-actin (AC-15) were purchased from
Roche Diagnostics and Sigma respectively.
Plasmids
Plasmids coding for Myc- or YFP (yellow fluorescent protein)tagged Aip1 were constructed by subcloning PCR-amplified
human Aip1 cDNA into the pMYC-C1 [14] or pEYFP-C1
(Clontech) expression vectors. Plasmids for YFP–actin, YFP–
cofilin(S3A) and CFP–H2B [CFP (cyan fluorescent protein)tagged histone H2B] were constructed as described previously
[15,16]. The siRNA plasmids targeting human Aip1 and cofilin
were generated using the pSUPER vector [42]. The 19-base targeting sequences were as follows: 5 -GGAGCACCTGTTGAAGTAT-3 (Aip1 siRNA-1), 5 -AGTGCGTCATCCTAAGGAA3 (Aip1 siRNA-2) and 5 -GGAGGATCTGGTGTTTATC-3
(cofilin). As a control, we used a non-targeting sequence, 5 -GAATGTTGTGGTGGCTGCC-3 , which does not exist in the human
genome. In order to construct expression plasmids coding for the
sr-Aip1 (siRNA-resistant Aip1), three bases in the 19-base target
sequence in the YFP–Aip1 or Myc–Aip1 plasmid were mutated
(GGAACATCTCTTGAAGTAT) by using a QuikChange® sitedirected mutagenesis kit (Stratagene).
Immunoblot analysis
Immunoblot analysis was performed as described previously [14].
Cell culture, synchronization and transfection
HeLa cells were cultured in DMEM (Dulbecco’s modified Eagle’s
medium) supplemented with 10 % (v/v) FBS (fetal bovine serum).
Cells were transfected with plasmids using LipofectamineTM 2000 (Invitrogen), according to the manufacturer’s protocol. To
analyse the distribution of YFP–Aip1, HeLa cells that had been
co-transfected with YFP–Aip1 and CFP–H2B were cultured in
c The Authors Journal compilation c 2008 Biochemical Society
DMEM containing 10 % FBS and 1 mM thymidine for 36 h, and
were then released for 12 h in fresh DMEM containing 10 % FBS.
For staining the mitotic cells, HeLa cells that had been transfected
with plasmids were initially cultured in DMEM containing
10 % FBS for 48 h and synchronized by incubating in DMEM
containing 10 % FBS and 1 mM thymidine for 36 h, followed by
fresh DMEM containing 10 % FBS for 6 h, DMEM containing
10 % FBS and 0.3 μM nocodazole for 6 h, fresh DMEM
containing 10 % FBS for 2–3 h, and finally cells were fixed and
stained. Jurkat human leukaemia T-cells were maintained in RPMI
1640 medium containing 10 % FBS. For transfection, approx.
107 cells were mixed with plasmids in 400 μl of electroporation
medium (RPMI 1640 medium containing 20 % FBS and 25 mM
Hepes, pH 7.4) before being electroporated at 280 V and 725 μF
using a Gene Pulser II (Bio-Rad). After electroporation, the cells
were cultured for 72 h in RPMI 1640 medium supplemented with
10 % FBS. The transfection efficiency of the Jurkat cells was
> 90 %, as assessed by transfection of the YFP plasmid.
Cell staining
HeLa cells were fixed in 4 % formaldehyde in PBS for
20 min and permeabilized with 0.2 % Triton X-100 in PBS for
rhodamine–phalloidin staining, or with absolute methanol for
cofilin staining for 10 min at − 20 ◦C. After blocking with 1 %
BSA in PBS, cells were stained with rhodamine–phalloidin,
DAPI (4 ,6-diamidino-2-phenylindole) or an anti-cofilin antibody
(COF-1). Rhodamine-conjugated anti-rabbit IgG (Chemicon,
Temecula, CA, U.S.A.) was used as the second antibody. After
washing with PBS, coverslips were mounted on a glass slip and
images were obtained using fluorescence microscopy (model
DMLB; Leica). Jurkat cells were suspended in RPMI 1640
medium containing 25 mM Hepes (pH 7.4) and 0.2 % BSA,
incubated for 20 min at 37 ◦C, then plated on coverslips and
allowed to attach for 5 min at 37 ◦C. Attached cells were left
unstimulated or were stimulated with 5 nM SDF-1 for 20 min and
fixed with 3.7 % (w/v) paraformaldehyde. The cells were permeabilized and stained using the HeLa cell protocol described above.
Stacked fluorescent images were obtained using a laser scanning
confocal microscope (LSM510; Carl Zeiss) equipped with a PL
Apo (NA 1.4) × 63 oil immersion objective lens (Carl Zeiss).
Time-lapse analysis
For time-lapse imaging, Jurkat cells that had been electroporated
with YFP–actin and siRNA plasmids were cultured for 72 h in
RPMI 1640 medium supplemented with 10 % FBS, and then
suspended in RPMI 1640 medium containing 25 mM Hepes
(pH 7.4) and 0.2 % BSA. Cells were plated on to a 35 mm glassbottom dish and incubated for 5 min to attach them to the dish.
Then cells were stimulated with SDF-1, and time-lapse images
of the stacked optical sections were collected every 10 s for
11 min using a laser scanning confocal microscope and objective
lens, as described above. Kymograph analyses were done with
ImageJ (http://rsb.info.nih.gov/ij/). To generate kymographs, a
0.42 μm × 14 μm (3 × 100 pixels) rectangular region was drawn
outwardly from the cell centre and the image information was
compiled sequentially from each frame of the time-lapse movie.
The frequency of lamellipodial protrusions for 10 min after SDF-1
stimulation was counted from the kymograph images. Protrusions
with the peak height over 10 pixels (1.4 μm) in the kymograph
were counted.
Chemotaxis assays
Transwell culture chambers (pore size: 5 μm; Costar) were used
for the chemotactic cell migration assays. The lower wells were
Roles of human Aip1 in cytokinesis and chemotaxis
Figure 1
Knockdown of Aip1 increases the number of multinucleate cells
(A) Suppression of Aip1 expression by siRNA plasmids. HeLa cells were co-transfected with
plasmids for Myc–Aip1 and either control or two distinct Aip1 siRNAs. After 48 h in culture,
cell lysates were analysed by immunoblotting (IB) with anti-Myc and anti-β-actin antibodies.
(B) Aip1 knockdown increases the number of multinucleate cells. HeLa cells that had been
co-transfected with YFP and control or Aip1 siRNA-1 plasmids (with a molar ratio of 1:9) were
cultured for 96 h, before being fixed and stained with DAPI. Expression of YFP was visualized
by fluorescence microscopy. Arrows indicate cells expressing YFP. Scale bar, 20 μm. (C)
Quantification of the ratio of multinucleate cells in total YFP-positive cells (at least 200 cells
in each experiment). Values represent the means +
− S.D. for three independent experiments.
∗
P < 0.01, compared with cells transfected with control siRNA plasmids. (D) sr-Aip1 rescues
the multinucleate phenotype of Aip1-knockdown cells. HeLa cells were co-transfected with Aip1
siRNA-1 plasmid and either YFP or YFP–sr-Aip1 plasmids, cultured for 96 h and analysed as
in (C). ∗ P < 0.05. (E) An active form of cofilin partially rescues the multinucleate phenotype of
Aip1-knockdown cells. HeLa cells that had been co-transfected with Aip1 siRNA-1 plasmid and
either YFP or YFP–cofilin(S3A) were cultured for 96 h and analysed as in (C). ∗ P < 0.05.
filled with 600 μl of medium (RPMI 1640 containing 0.5 % BSA
and 25 mM Hepes, pH 7.4) with or without 5 nM SDF-1. Jurkat
cells (2 × 105 cells) suspended in 100 μl of the medium were
loaded on to the upper wells. After incubation for 3 h at 37 ◦C, the
cells that had migrated into the lower wells were counted.
RESULTS
Knockdown of Aip1 increases the percentage of multinucleate
cells and this effect is significantly blocked by the overexpression
of cofilin
In order to examine the role of Aip1 in mammalian cell
cytokinesis, we constructed two distinct siRNA plasmids targeting
human Aip1 (Aip1 siRNA-1 and -2) and analysed their effects
in HeLa cells. The effect of these siRNAs on the expression of
Aip1 was analysed by co-transfecting HeLa cells with the plasmid
for Myc-tagged Aip1 as well as the plasmid for the control
or Aip1 siRNAs. Immunoblot analysis revealed that both Aip1
siRNA plasmids effectively suppressed the expression of Myc–
Aip1 (Figure 1A). To examine the effects of Aip1 knockdown on
cytokinesis, HeLa cells were co-transfected with the YFP plasmid
and the plasmid for the control or Aip1 siRNA with a molar ratio of
1:9. Cells were cultured for 96 h (a period sufficient for one or two
cycles of cell division after knockdown of Aip1) and stained with
263
DAPI (Figure 1B) and then the percentage of multinucleate cells
in YFP-positive cells was determined (Figure 1C). While only a
small percentage (< 4 %) of cells that had been transfected with
the control siRNA plasmid exhibited multinucleate phenotypes,
14–15 % of the cells that were transfected with each Aip1 siRNA
plasmid became multinucleate (Figure 1C). The plasmid coding
for YFP-tagged sr-Aip1 has three silent mutations in the siRNA
target sequence of Aip1 cDNA. Co-transfection of this YFP–
sr-Aip1 plasmid with the Aip1 siRNA significantly reduced
the percentage of multinucleate cells (Figure 1D), indicating
that the multinucleate phenotype of Aip1-knockdown cells can
be attributed to suppression of the expression of endogenous
Aip1. These results suggest that Aip1 plays a critical role in
cytokinesis in HeLa cells. To examine whether Aip1 contributes
to cytokinesis by regulating cofilin activity, HeLa cells were cotransfected with the plasmid for Aip1 siRNA and the plasmid
for YFP or YFP-tagged cofilin(S3A), a constitutively active
form of cofilin, in which the inhibitory phosphorylation residue
(Ser-3) was replaced by alanine residue. After culture for 96 h,
the number of multinucleate cells in YFP-positive cells was
determined. Co-transfection of YFP–cofilin(S3A) with Aip1
siRNA significantly decreased the percentage of multinucleate
cells, compared with cells co-transfected with Aip1 siRNA and
control YFP (Figure 1E). Thus Aip1 appears to be involved in
cytokinesis by enhancing cofilin activity.
Knockdown of Aip1 induces an excessive accumulation of actin
filaments and an abnormal contractile ring
In order to investigate the role of Aip1 in cytokinesis, we examined changes in cell morphology and actin cytoskeletal
organization in Aip1-knockdown cells. When asynchronized
HeLa cells that had been co-transfected with YFP and Aip1
siRNA were fixed and stained with rhodamine-labelled phalloidin
to detect actin filaments, Aip1-knockdown (YFP-positive) cells
exhibited thicker actin fibres, compared with the surrounding
non-transfected (YFP-negative) cells (Figure 2A, top panels). In
addition, Aip1-knockdown cells occasionally induced membrane
blebbing (Figure 2A, bottom panels). Quantitative analyses
revealed that knockdown of Aip1 significantly increased the
percentages of the cells with thick stress fibres and membrane
blebs, compared with control siRNA cells (Figure 2A, right-hand
panels). Similar phenotypes were also observed in cofilin-depleted
cells [11,13,17] or LIM-kinase (LIM motif-containing protein
kinase, where LIM is an acronym of the three gene products
Lin-11, Isl-1 and Mec-3)-overexpressing cells, in which cofilin is
excessively phosphorylated and inactivated [43,44]. These results
indicate that Aip1 functions to promote cofilin activity in HeLa
cells and that knockdown of Aip1 decreases the actin filamentdisassembling activity of cofilin. We next examined the effects
of Aip1 knockdown on the actin cytoskeletal organization in
mitotic HeLa cells. HeLa cells that had been co-transfected with
YFP and the control or Aip1 siRNA were synchronized with nocodazole, co-stained with rhodamine–phalloidin and DAPI, and
the morphology and actin cytoskeletal organization of YFPpositive cells were analysed. In contrast with the reported
phenotypes for U2OS osteosarcoma cells [40], Aip1-knockdown
cells, similar to the results observed in control siRNA cells,
normally rounded up from the substratum and became spherical in
metaphase (Figures 2Ba and 2Bd), and then the cleavage furrow
formed and constricted in anaphase and early telophase (Figures 2Bb and 2Be). However, in late telophase, actin filaments at
the cleavage furrow accumulated excessively and asymmetrically
in Aip1-knockdown cells, compared with those in control cells
(Figures 2Bc and 2Bf), and the cleavage furrow subsequently
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A. Kato and others
Aip1 is diffusely localized in the cytoplasm during mitosis
In order to examine the subcellular localization of Aip1 in HeLa
cells during mitosis, YFP–Aip1 was co-expressed with CFP–
H2B, and the localization of YFP–Aip1 was analysed using
fluorescence microscopy. Similar to our observations with control
YFP, YFP–Aip1 was diffusely distributed in the cytoplasm in both
metaphase and telophase, and no accumulation was observed near
the cleavage furrow (Figure 3A).
Localization of cofilin in Aip1-knockdown cells
Figure 2 Knockdown of Aip1 induces an excessive actin filament assembly
and an abnormal contractile ring
(A) Effects of Aip1 knockdown on actin filament assembly in asynchronized interphase cells.
HeLa cells that had been co-transfected with YFP and Aip1 siRNA plasmids were cultured
for 96 h and then fixed and stained with rhodamine–phalloidin. Arrows indicate YFP-positive
cells. Scale bar, 20 μm. Right-hand panels show the quantitative data of the percentages of
the cells with thick stress fibres and membrane blebs in total YFP-positive cells (at least 200 cells
in each experiment). Values represent the means +
− S.E.M. for five independent experiments.
∗
P < 0.005; ∗∗ P < 0.001. (B) Effects of Aip1 knockdown on actin filament assembly in mitotic
cells. HeLa cells that had been co-transfected with YFP and control or Aip1 siRNA plasmids
were synchronized with nocodazole, fixed and co-stained with rhodamine–phalloidin and DAPI.
Images of YFP-positive cells were obtained by fluorescence microscopy. Scale bar, 10 μm. The
histogram shows the quantitative data of the percentages of the cells with aberrantly asymmetric
cleavage furrow in a total of 111 control siRNA cells and 156 Aip1 siRNA cells. Values represent
∗
the means +
− S.D. for four independent experiments. P < 0.002.
regressed to produce binucleate cells (Figure 2Bg). Quantitative
analysis showed that Aip1-knockdown cells exhibited the
increased ratio of the cells with asymmetric cleavage furrow,
compared with control siRNA cells (Figure 2B, histogram).
These observations suggest that Aip1 plays a critical role
in the completion of cytokinesis by regulating actin filament
disassembly in the contractile ring in the final stage of cytokinesis.
c The Authors Journal compilation c 2008 Biochemical Society
To investigate further the role of Aip1 in cytokinesis, we
examined the distribution of cofilin in Aip1-knockdown cells.
When asynchronized HeLa cells were co-transfected with YFP
and the control or Aip1 siRNA plasmids and stained with an
anti-cofilin antibody, cofilin aberrantly accumulated on fibrous
structures in Aip1-knockdown cells but localized diffusely in the
cytoplasm in control siRNA cells (Figure 3B, upper panels). To
determine whether the fibrous structures decorated with cofilin
are actin fibres, HeLa cells were co-transfected with CFP–
actin and Aip1 siRNA and the localization of actin and cofilin
was analysed by CFP fluorescence and immunostaining with
an anti-cofilin antibody. As shown in the lower panels in Figure 3(B), cofilin co-localized well with CFP–actin in Aip1 siRNA
cells, thus indicating that knockdown of Aip1 causes aberrant
localization of cofilin on actin stress fibres and cortical actin
filaments. We next examined the effects of Aip1 siRNA on the
localization of cofilin in mitotic HeLa cells. HeLa cells that
had been co-transfected with CFP–H2B and the control or Aip1
siRNA plasmids were synchronized with nocodazole and the
cofilin distribution was analysed with an anti-cofilin antibody
(Figure 3C). Cofilin was diffusely distributed in the cytoplasm of
both control and Aip1 siRNA cells in metaphase and accumulated
on the contractile ring in late telophase; however, the localization
of cofilin in late telophase differed between control and Aip1
siRNA cells. In control siRNA cells, cofilin was localized both
in the cytoplasm and on the contractile ring. In contrast, in Aip1
siRNA cells, cofilin concentrated on the contractile ring and the
cell cortex (Figure 3C). The line scan analyses across the control
and Aip1-knockdown cells clearly showed the increase in the
intensity of cofilin immunofluorescence in the cortical region of
Aip1 siRNA cells, compared with control siRNA cells (Figure 3C,
bottom panels). Thus the Aip1 knockdown appears to increase the
localization of cofilin on cortical actin filaments.
Knockdown of Aip1 suppresses SDF-1-induced chemotaxis of
Jurkat cells and this effect is significantly blocked by the
overexpression of cofilin
To examine the role of Aip1 in the chemotactic response of
mammalian cells, we analysed the effect of the Aip1 knockdown
on SDF-1-induced chemotactic migration of Jurkat cells. Jurkat
cells are known to express the SDF-1 receptor [CXCR4 (CXC
chemokine receptor 4)] and exhibit chemotactic migration
towards SDF-1 [16,45]. Jurkat cells were transfected with the
siRNA plasmid targeting Aip1 or cofilin and cultured for 72 h
before their chemotactic migration towards SDF-1 was analysed
using Transwell culture chambers. SDF-1 was added to the
lower chamber to analyse the chemotactic response, whereas it
was not added for control experiments. As previously reported
[16], knockdown of cofilin markedly reduced the chemotactic
response of Jurkat cells towards SDF-1, compared with the
cells that were transfected with the control siRNA plasmid
(Figure 4A). Knockdown of Aip1 by either of two distinct
siRNA constructs also suppressed chemotactic cell migration
Roles of human Aip1 in cytokinesis and chemotaxis
265
Figure 4 Knockdown of Aip1 suppresses the chemotactic migration of
Jurkat cells towards SDF-1
Figure 3 Diffuse distribution of Aip1 and the effects of Aip1 knockdown on
cofilin localization in HeLa cells
(A) Localization of Aip1. HeLa cells expressing YFP or YFP–Aip1 with CFP–H2B were
synchronized by thymidine block and released for 12 h. The localization of YFP or YFP–Aip1 was
analysed by fluorescence microscopy. Scale bar, 10 μm. (B) Effects of Aip1 knockdown on cofilin
distribution in interphase cells. In the upper panels, HeLa cells that had been co-transfected
with YFP and control or Aip1 siRNA plasmids were cultured for 96 h, before being fixed and
stained with an anti-cofilin antibody. Arrows indicate YFP-expressing cells. In the lower panels,
HeLa cells were co-transfected with CFP–actin and Aip1 siRNA plasmids, cultured for 96 h
and analysed by CFP fluorescence and immunostaining with an anti-cofilin antibody. Scale bar,
20 μm. (C) Effects of Aip1 knockdown on cofilin localization in mitotic cells. HeLa cells that had
been co-transfected with CFP–H2B and control or Aip1 siRNA plasmids were synchronized with
nocodazole, fixed and stained with an anti-cofilin antibody. Images of CFP–H2B were obtained
by fluorescence microscopy. Scale bar, 10 μm. The lower panels show the line scan analyses
of the intensity of cofilin immunofluorescence in control and Aip1 siRNA cells.
significantly, compared with control siRNA cells (Figure 4A).
These results suggest that Aip1 is involved in the SDF-1-induced
chemotactic response of Jurkat cells. Inhibition of the chemotactic
(A) Suppression of chemotaxis by Aip1 or cofilin knockdown. Jurkat cells were transfected with
control, Aip1 or cofilin siRNA plasmids, cultured for 72 h and then the migration of these cells
towards SDF-1 was analysed using a Transwell culture chamber. Jurkat cells (2 × 105 cells)
were loaded into the upper chamber in the presence (grey bars) or absence (white bars) of
5 nM SDF-1 in the lower chamber. After incubation for 3 h, the relative number of cells that had
migrated into the lower chambers was quantified. Results are expressed as the means +
− S.D. for
four independent assays. ∗ P < 0.01, compared with control siRNA cells. (B) sr-Aip1 rescues the
inhibitory effects of Aip1 knockdown on Jurkat cell chemotaxis. Jurkat cells were co-transfected
with siRNA plasmids and either mock or Myc–sr-Aip1 plasmids. After 72 h in culture, cells
were subjected to Transwell culture chamber assays. Results are expressed as the means +
− S.D.
for three independent assays. ∗ P < 0.05. (C) An active form of cofilin partially rescues the
inhibitory effects of Aip1 knockdown on Jurkat cell chemotaxis. Jurkat cells were co-transfected
with siRNA plasmids and either YFP or YFP–cofilin(S3A) plasmids. After 72 h in culture, cells
were subjected to Transwell culture chamber assays. Results are expressed as the means +
− S.D.
for three independent assays. ∗ P < 0.05. (D) The combination of cofilin and Aip1 knockdowns
has no additive effect. Jurkat cells were co-transfected with cofilin siRNA plasmids and either
control or Aip1 siRNA plasmids. After 72 h in culture, cells were subjected to Transwell culture
chamber assays. Results are expressed as the means +
− S.D. for three independent assays.
∗
P < 0.05, compared with control siRNA cells.
response by Aip1 knockdown was significantly blocked by cotransfection with the Myc-tagged sr-Aip1 plasmid (Figure 4B),
thus indicating that Aip1 siRNA inhibited the chemotactic
response by suppressing endogenous Aip1 expression. To examine whether Aip1 knockdown inhibits chemotactic migration
by regulating cofilin activity, Jurkat cells were co-transfected
with Aip1 siRNA and YFP–cofilin(S3A), cultured for 72 h and
subjected to the chemotaxis assay. Expression of cofilin(S3A)
significantly blocked the inhibitory effect of Aip1 knockdown
on the chemotactic response of Jurkat cells, which suggests
that cofilin acts downstream of Aip1 (Figure 4C). To further
examine whether cofilin and Aip1 function in the same pathway,
we analysed the combined effects of cofilin and Aip1 double
knockdowns on Jurkat cell migration. As shown in Figure 4(D),
the combination of cofilin and Aip1 knockdowns did not
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A. Kato and others
respectively (Figure 5B). Furthermore, the percentages of the
cells with single and multiple lamellipodial protrusion(s) after
SDF-1 stimulation were 71 and 21 % for control siRNA cells,
46 and 44 % for Aip1 siRNA cells and 24 and 64 % for cofilin
siRNA cells (Figure 5C). Thus the number of the multipolar cells
significantly increased by the knockdown of Aip1 or cofilin.
These results suggest that both Aip1 and cofilin play critical
roles in maintaining the round morphology of Jurkat cells before
SDF-1 stimulation and in restricting the lamellipodial membrane
protrusion to one direction after SDF-1 stimulation.
Time-lapse analysis of the lamellipodial membrane protrusions in
Aip1-knockdown cells
Figure 5 Aip1- or cofilin-knockdown cells aberrantly induce multiple
membrane protrusions before and after SDF-1 stimulation
(A) Jurkat cells that had been transfected with siRNA plasmids were left unstimulated or were
stimulated for 20 min with SDF-1, fixed and stained with rhodamine–phalloidin to visualize
actin filaments. Images were obtained with a laser scanning confocal microscope. Scale bar,
10 μm. (B, C) Quantification of the percentages of round cells and cells with single and multiple
lamellipodia (at least 200 cells were counted in each experiment) before SDF-1 stimulation
(B) and after SDF-1 stimulation for 20 min (C). Values represent the means +
− S.D. for three
independent assays.
additively decrease the number of migrating cells. Together,
these results suggest that Aip1 contributes to the SDF-1-induced
chemotactic migration of Jurkat cells by promoting cofilin
activity.
Knockdown of Aip1 induces multiple membrane protrusions in
Jurkat cells before and after SDF-1 stimulation
To investigate the mechanisms by which Aip1 siRNA impaired
Jurkat cell chemotaxis, alterations in actin filament assembly and
cell morphology were analysed by rhodamine–phalloidin staining
before and after SDF-1 stimulation. Jurkat cells that had been
transfected with the plasmids for control, Aip1 or cofilin siRNA
were left unstimulated or were stimulated for 20 min with SDF-1.
Cells were then fixed and stained with rhodamine–phalloidin to
visualize actin filaments. Most of the control siRNA-transfected
cells exhibited a round and symmetrical morphology before
SDF-1 stimulation and generated a single lamellipodial membrane
protrusion on one side of the cell after SDF-1 stimulation
(Figure 5A). In contrast, the cells that were transfected with Aip1
or cofilin siRNA exhibited aberrant F-actin assembly and multiple
membrane protrusions around the cells both before and after
SDF-1 stimulation (Figure 5A). Quantitative analyses revealed
that the total numbers of the cells with single and multiple
lamellipodial membrane protrusion(s) before SDF-1 stimulation
were 20, 48 and 68 %, for control, Aip1 and cofilin siRNA cells
c The Authors Journal compilation c 2008 Biochemical Society
Knockdown of cofilin generates multiple lamellipodial membrane
protrusions and remarkably suppresses the motion of the protrusions even after SDF-1 stimulation [16]. We therefore
examined the effect of Aip1 knockdown on the motility of the
lamellipodial membrane protrusions. Jurkat cells that had been
co-transfected with the plasmid for control, Aip1 or cofilin siRNA
and the plasmid for YFP–actin were analysed by time-lapse
fluorescence microscopy (Figure 6A and Supplementary Movies
S1–S3 at http://www.BiochemJ.org/bj/414/bj4140261add.htm).
Control siRNA cells maintained a round morphology before
SDF-1 stimulation, but after SDF-1 stimulation YFP–actin
quickly accumulated in the cell periphery and multiple membrane
protrusions were generated around the cell (Figure 6A and
Supplementary Movie S1). These protrusions moved rapidly and
were then converted into a single lamellipodial protrusion by
10 min after stimulation. In contrast, cofilin-knockdown cells
generated multiple large protrusions before and after SDF-1
stimulation, and these protrusions moved only slowly, compared
with those in control cells (Figure 6A and Supplementary
Movie S3). These observations suggest that cofilin-mediated
actin remodelling plays a critical role in the dynamic motion of
the lamellipodial membrane protrusion(s) and the formation
of a single protrusion in response to SDF-1 stimulation. Aip1knockdown cells also produced membrane protrusions in multiple
directions before and after SDF-1 stimulation; however, these
protrusions moved more dynamically compared with those in
cofilin-knockdown cells (Figure 6A and Supplementary Movie
S2). To quantify the effects of cofilin and Aip1 knockdowns on
lamellipodial dynamics, we used kymograph analysis (Figure 6B).
Kymographs showed that the lamellipodium rapidly protruded
and retracted in control or Aip1-knockdown cells, but it moved
slowly in cofilin-knockdown cells. When we calculated the
average frequency of protrusions for 10 min after SDF-1 stimulation, cofilin-knockdown cells showed significantly less frequent
protrusions than control or Aip1-knockdown cells (Figure 6C).
Collectively, these results suggest that the knockdown of Aip1 is
less effective than the knockdown of cofilin in the inhibition of
lamellipodial dynamics.
Aip1 is diffusely localized in Jurkat cells before and after SDF-1
stimulation
We examined the subcellular localization of Aip1 in Jurkat cells
before and after SDF-1 stimulation by expressing YFP–Aip1 and
analysing its distribution by confocal fluorescence microscopy
(Figure 7A). YFP–Aip1 was diffusely distributed throughout the
cytoplasm in Jurkat cells in a pattern similar to that observed
with YFP alone. No accumulation of YFP–Aip1 was detected in
the SDF-1-induced lamellipodial membrane protrusions in Jurkat
cells.
Roles of human Aip1 in cytokinesis and chemotaxis
267
Figure 7 Diffuse distribution of Aip1 and the effects of Aip1 knockdown on
cofilin distribution in Jurkat cells
(A) Jurkat cells expressing YFP or YFP–Aip1 were left untreated or were stimulated with SDF-1
for 20 min and fixed. Localization of YFP or YFP–Aip1 was analysed by YFP fluorescence. Images
were obtained with a confocal microscope. Scale bar, 10 μm. (B) Effects of Aip1 knockdown on
cofilin distribution. Jurkat cells were transfected with control or Aip1 siRNA plasmids, cultured
for 72 h, and then left untreated or were stimulated with SDF-1 for 20 min. Cells were fixed and
stained with an anti-cofilin antibody. Images were obtained with a confocal microscope. Scale
bar, 10 μm.
Localization of cofilin in Aip1-knockdown cells before and after
SDF-1 stimulation
Figure 6 Time-lapse analyses of the lamellipodial membrane protrusions
in Aip1- or cofilin-knockdown cells
(A) Time-lapse analyses. Jurkat cells were electroporated with YFP–actin and the indicated
siRNA plasmids and cultured for 72 h. After suspension for 5 mins in RPMI 1640 medium
containing 25 mM Hepes (pH 7.4) and 0.2 % BSA, cells were stimulated with SDF-1, and
analysed by time-lapse fluorescence microscopy, making use of the YFP fluorescence. The
times after SDF-1 stimulation are indicated. Scale bar, 10 μm. See also Supplementary Movies
S1–S3 (http://www.BiochemJ.org/bj/414/bj4140261add.htm). (B) Kymograph analyses from
time-lapse movies of the cells shown in (A). The time-lapse images (every 10 s) of the rectangular
region in (A) were compiled to generate the kymographs. SDF-1 was added at the time indicated
by black arrowheads. Lamellipodial protrusions are indicated by white arrowheads. (C) Frequency
of protrusions. The frequency of the lamellipodial protrusions was counted, as shown in (B).
Results represent the means +
− S.D. from 20 control siRNA cells, 16 Aip1 siRNA cells and 19
cofilin siRNA cells in two independent experiments. ∗ P < 0.05; ∗∗ P < 0.001.
We next examined the localization of cofilin in Aip1-knockdown
Jurkat cells. Cells that had been transfected with the control or
Aip1 siRNA plasmid were left unstimulated or were stimulated for
20 min with SDF-1, before being fixed and stained with an anticofilin antibody. In control siRNA cells, cofilin accumulated in
the lamellipodial membrane protrusion that was induced by SDF1 stimulation (Figure 7B, upper panels). In Aip1 siRNA cells,
multiple lamellipodial protrusions were generated both before
and after SDF-1 stimulation, and cofilin accumulated in these
membrane protrusions, in a similar manner to that observed in
control siRNA cells (Figure 7B, lower panels). These results
suggest that cofilin accumulates in the lamellipodial membrane
protrusions, independently of Aip1.
DISCUSSION
In the present study, we provided evidence that Aip1 plays an
important role in cytokinesis by positively regulating cofilin
activity. We also showed that in Aip1-silenced HeLa cells, the
cleavage furrow ingressed normally from anaphase to early
telophase, but an excessive and asymmetric accumulation of
c The Authors Journal compilation c 2008 Biochemical Society
268
A. Kato and others
actin filaments was observed near the contractile ring in late
telophase. Similar phenotypes were observed in cofilin-depleted
cells from several model organisms. For example, depletion of
cofilin in Drosophila cells induced the aberrant accumulation of
actin filaments and formation of a misshaped contractile ring in
telophase, leading to frequent failure of cytokinesis [11,13]. In
mammalian cells, the knockdown of cofilin or overexpression
of a phosphatase-dead form of Slingshot (a cofilin-phosphatase
that activates cofilin) induced the accumulation of F-actin on
the contractile ring and the production of multinucleate cells
[14,15,17]. These results suggest that cofilin activity is required
for the accurate disassembly of actin filaments in the contractile
ring in late telophase. The aberrant accumulation of actin filaments
in cofilin- or Aip1-depleted cells probably inhibits the ring
contraction and the abscission of the cell into two daughter
cells, thus leading to the failure of cytokinesis. Consequently,
Aip1 appears to play a critical role in cytokinesis, particularly
in the completion of cytokinesis, by promoting the cofilinmediated disassembly of actin filaments in the contractile ring
in the final stage of cell division. A recent study showed that
Aip1 knockdown in U2OS human osteosarcoma cells suppressed
mitotic cell rounding, resulting in the cell flattening during mitosis
[40]. In contrast, we found that mitotic cell rounding was normal
by Aip1 knockdown in HeLa cells.
We also provided evidence that Aip1 contributes to the chemotaxis of Jurkat cells through enhancing cofilin activity.
Inhibition of the chemotactic response by knocking down Aip1
was also reported during fMLP (N-formylmethionyl-leucylphenylalanine)-mediated chemotaxis of J774 macrophage-like
cells [37]. In this study, we have shown that knocking down
Aip1 induced changes in the actin filament organization and
cell morphology in Jurkat cells. Phalloidin staining revealed that
while most control cells have a round and symmetric morphology
in the absence of SDF-1, Aip1-knockdown cells abnormally
produced multiple lamellipodial membrane protrusions even
before SDF-1 stimulation. When exposed to SDF-1, control
cells produced multiple F-actin-rich lamellipodial protrusions
around the cell in the initial stages of the cell response (1–
2 min after stimulation), which then converted into a single
lamellipodium by 10 min. In contrast, Aip1-knockdown cells
retained multiple lamellipodial protrusions for up to 20 min
after SDF-1 stimulation. These results suggest that Aip1 is
required for maintaining the round morphology of the nonstimulated Jurkat cells and producing the single lamellipodium
and polarized cell morphology after cell stimulation. As described
previously [16,46] and shown in the present study, knockdown of
cofilin also induced multiple lamellipodial protrusions around
the cells before and after stimulation of Jurkat cells and MTC
mammary adenocarcinoma cells. It is therefore likely that the
Aip1 knockdown causes a decrease in cofilin-mediated actin
filament disassembly in the cells and, as a result, leads to an
excessive actin filament assembly, a decrease in actin filament
turnover in the lamellipodia and a failure in the remodelling
of multiple lamellipodia into a single lamellipodium. The
inability to form a single lamellipodium in the direction of
cell migration probably causes the inhibition of the chemotactic
migration of Jurkat cells. Although both Aip1- and cofilinknockdown cells generated multiple membrane protrusions, the
cofilin knockdown exhibited more severe phenotypes in F-actin
accumulation in the lamellipodia, the motility of lamellipodia
and chemotactic migration towards SDF-1. Time-lapse analyses and kymographs showed that the dynamic movement of the
lamellipodial protrusions was markedly lost in cofilin-knockdown
cells, whereas it was almost retained in Aip1-knockdown cells.
These differences in phenotype are probably due to the fact that
c The Authors Journal compilation c 2008 Biochemical Society
cofilin still exists in Aip1 siRNA cells and has its own basal
activity to promote actin filament disassembly, whereas in cofilin
siRNA cells, cofilin expression is directly suppressed. Taken
together, our results suggest that Aip1 plays a crucial role in
chemokine-induced directional cell migration by enhancing actin
filament dynamics and the formation of a single lamellipodium
in the direction of cell migration via its action to promote cofilin
activity.
We showed that Aip1 knockdown caused the aberrant accumulation of F-actin and unusual localization of cofilin on thick
actin fibres in interphase HeLa cells. These observations are
consistent with the previously reported observations in yeast
and C. elegans where mutations in the Aip1 gene induced
the formation of thick actin fibres and the alteration of cofilin
localization on these F-actin structures [30,34]. It is likely that
the actin filament-disassembling activity of cofilin is weakened in
Aip1-knockdown cells; therefore actin filaments are stabilized
and the cofilin that is present on the actin filaments is retained for an extended period of time. In HeLa cells, cofilin
is localized diffusely throughout the cytoplasm in metaphase in
both control and Aip1-knockdown cells; this is probably because
cofilin is highly phosphorylated and loses its F-actin binding
ability in metaphase of the cell cycle [14,47]. In late telophase,
cofilin is dephosphorylated and concentrated on to the contractile
ring in both control and Aip1-knockdown cells; however, while
cofilin is localized in the cytoplasm and on the contractile ring in
control cells, it accumulates in both the contractile ring and the
cell cortex in Aip1-knockdown cells. Again, this could be due to
the decrease in cofilin activity in Aip1-knockdown cells, which
may lead to the stabilization of cofilin-decorated actin filaments
in the cell cortex. Aip1 was distributed diffusely in the cytoplasm
and its co-localization with F-actin or cofilin was not observed in
either HeLa or Jurkat cells, although Aip1 has the potential to bind
to F-actin and cofilin in biochemical studies [25,26]. The diffuse
distribution of Aip1 may suggest that Aip1 can disassemble actin
filaments very rapidly, once it associates with cofilin-bound
actin filaments.
Whereas cofilin promotes actin filament disassembly by severing and depolymerizing actin filaments in vitro, it is also required
for actin filament assembly in the cell. Two distinct models
have been proposed to explain the role of cofilin in actin filament
assembly in vivo. The first model proposes that cofilin contributes
to actin filament assembly by disrupting actin filaments and
thereby supplying actin monomers for polymerization [3,17,18].
We and other investigators have shown that cofilin is involved in
producing an abundant pool of actin monomers in the cytoplasm
and that these monomers are used for actin polymerization in the
cell [17,18], hence supporting this model. An alternative model
is that cofilin can promote actin filament assembly by severing
actin filaments and creating free barbed ends that are used as
nucleation sites for actin polymerization [48,49]. This model is
possible under the conditions that actin monomers are sufficiently
available and the cofilin-created barbed ends remain uncapped. As
Aip1 has the potential to cap the barbed ends of cofilin-severed
actin filaments, it can block the elongation or reannealing of
cofilin-severed actin filaments. Thus, in cells that express high
levels of Aip1, it is implausible that cofilin mediates actin filament
assembly by creating the nucleation sites for polymerization.
We have also shown that the severing activity, rather than the
depolymerizing activity, of cofilin plays a dominant role in
increasing the actin monomer pool in the cell [18]. The ability
of Aip1 to cap the barbed ends of cofilin-severed actin filaments
could ensure the cofilin-induced actin filament disruption (by
severing of actin filaments and subsequent breakdown of F-actin
fragments to actin monomers) and seems to play an important role
Roles of human Aip1 in cytokinesis and chemotaxis
in the regulation of the balance of cofilin-mediated actin filament
disassembly and assembly in the cell.
In conclusion, we have provided evidence that Aip1 plays
critical roles in mammalian cell cytokinesis and chemotactic
migration by promoting cofilin activity. Aip1 regulates the actin
filament dynamics of the contractile ring and is crucial for
completing cell abscission at the final stage of cytokinesis. Aip1 is
also required for maintaining the round cell morphology of nonstimulated Jurkat cells and contributes to SDF-1-induced directional cell migration by restricting the lamellipodial protrusion
to one side of the cell. Further studies on the mechanisms
regulating Aip1 activity will be important to understand the
spatiotemporal control of cofilin activity and actin cytoskeletal
dynamics during cytokinesis and polarized cell migration.
This study was supported by a grant-in-aid for scientific research from the Ministry of
Education, Culture, Sports, Science and Technology of Japan.
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