2246
Research Article
SCAR/WAVE is activated at mitosis and drives
myosin-independent cytokinesis
Jason S. King1,*, Douwe M. Veltman1, Marios Georgiou2, Buzz Baum2 and Robert H. Insall1
1
Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD, UK
MRC-LMCB, University College London, Gower Street, London, WC1E 6BT, UK
2
*Author for correspondence ([email protected])
Journal of Cell Science
Accepted 7 April 2010
Journal of Cell Science 123, 2246-2255
© 2010. Published by The Company of Biologists Ltd
doi:10.1242/jcs.063735
Summary
Cell division requires the tight coordination of multiple cytoskeletal pathways. The best understood of these involves myosin-IIdependent constriction around the cell equator, but both Dictyostelium and mammalian cells also use a parallel, adhesion-dependent
mechanism to generate furrows. We show that the actin nucleation factor SCAR/WAVE is strongly activated during Dictyostelium
cytokinesis. This activation localises to large polar protrusions, driving separation of the daughter cells. This continues for 10 minutes
after division before the daughter cells revert to normal random motility, indicating that this is a tightly regulated process. We
demonstrate that SCAR activity is essential to drive myosin-II-independent cytokinesis, and stabilises the furrow, ensuring symmetrical
division. SCAR is also responsible for the generation of MiDASes, mitosis-specific actin-rich adhesions. Loss of SCAR in both
Dictyostelium and Drosophila leads to a similar mitotic phenotype, with severe mitotic blebbing, indicating conserved functionality.
We also find that the microtubule end-binding protein EB1 is required to restrict SCAR localisation and direct migration. EB1-null
cells also exhibit decreased adhesion during mitosis. Our data reveal a spindle-directed signalling pathway that regulates SCAR
activity, migration and adhesion at mitosis.
Key words: Dictyostelium, SCAR/WAVE, Cytokinesis
Introduction
Cell division is highly complex, involving the coordination of a
number of cellular processes. At mitosis, in addition to the
duplication, condensation and sorting of chromosomes, a cell must
also undergo a dramatic series of morphological changes, unique
to mitosis, which results in the production of two independent
daughter cells (Zhang and Robinson, 2005). This is known as
cytokinesis.
In eukaryotes, entry into mitosis coincides with the silencing of
the actin cytoskeleton and retraction of cellular protrusions resulting
in a round cell. These rounded cells then rapidly pinch in two, by
the formation of an equatorial furrow. This is a complex process
that is still not completely understood, but requires both myosin II
contraction, and exocytosis (De Lozanne and Spudich, 1987; Lecuit
and Wieschaus, 2000; Mabuchi and Okuno, 1977).
Although equatorial constriction appears to be the dominant
mechanism for cell division in metazoa, other parallel mechanisms
for splitting the cell in two also exist. The first indication of this
was in studies using the social amoeba Dictyostelium discoideum,
which were still able to divide with fairly normal speed and
efficiency after genetic disruption of myosin heavy chain (mhcA)
– provided they were attached to a surface (De Lozanne and
Spudich, 1987; Neujahr et al., 1997). Cytokinesis is therefore so
important that Dictyostelium cells possess at least two mechanisms
for cell division, working in parallel to ensure success. These have
been termed cytokinesis A (myosin-II dependent) and cytokinesis
B (myosin-II independent, adhesion dependent) (Uyeda et al.,
2000; Zang et al., 1997). Even if both these pathways fail, and a
multinucleate cell is produced, Dictyostelium remains able to
recover single cells by tearing the enlarged cell apart, in a cellcycle-independent manner, known as traction-mediated cytofission
or cytokinesis C (De Lozanne and Spudich, 1987; Nagasaki et al.,
2002).
Dictyostelium is a unicellular organism but there is evidence
that a pathway similar to cytokinesis B is conserved in metazoans.
In a number of cultured mammalian cell lines, mitotic cells become
only weakly attached to the substratum, although others remain
adherent. For example, HeLa cells are unable to complete
cytokinesis in the presence of myosin II inhibitors (Straight et al.,
2003), but more adherent cell lines such as NRK (normal rat
kidney) and HT1080 fibrosarcoma cells are still able to make a
furrow in an adhesion-dependent manner (although cytokinesis
ultimately fails) (Kanada et al., 2005). In addition, disruption of
Rho, which lies upstream of myosin II, by either treatment with the
Rho-kinase inhibitor Y27632, or microinjection with the Rho
inhibitor C3 ribosyltransferase is only able to block the division of
weakly adherent cells, whereas strongly attached cells are able to
complete cytokinesis (O’Connell et al., 1999; Kanada et al., 2005).
Like the work with Dictyostelium, these studies demonstrate a
similar dependency on substrate adhesion, and therefore
mammalian cells are likely to share a common mechanism for
generating a furrow, independently of myosin II.
However, little is known about the mechanism of myosin-IIindependent cytokinesis, and Dictyostelium remains the only
organism where cytokinesis A and B can be clearly separated. We
know it requires adhesion, and is partially blocked by the disruption
of adhesion molecules such as talin, vinculin and paxillin (Hibi
et al., 2004; Nagasaki et al., 2009) but the mechanical process and
the signalling pathways responsible for its regulation remain
unknown. One clue, however, is provided by the observation that
the combined disruption of the actin-binding protein coronin and
myosin II leads to a much more severe cytokinesis defect than
Journal of Cell Science
Activation of SCAR/WAVE at mitosis
each individual mutation, even when cells are attached to a surface
(Nagasaki et al., 2002). Coronin strongly localises to the poles of
mitotic cells (de Hostos et al., 1993) indicating that myosin-IIindependent cytokinesis might be driven by actin-dependent
processes at the cell extremities. This implies that actin plays a
key, but undefined role at the poles as well as the furrow of
dividing cells.
During Dictyostelium cytokinesis, a number of regulators of
the cytoskeleton such as racE and phosphatidylinositol 3,4,5trisphosphate [PtdIns(3,4,5)P3] localise to the poles of dividing
cells (Larochelle et al., 1996; Janetopoulos et al., 2005). Others
have shown that in NRK cells, the localised application of
cytochalasin D (an inhibitor of actin polymerisation) to the cell
poles is catastrophic to cytokinesis, but has little effect at the
furrow (O’Connell et al., 2001) and thus polar actin
polymerisation appears to be crucial in mammalian cells too. It
is therefore important to understand both the functional role and
the nature of the pathways regulating the production of actin at
the cortex of dividing cells.
Recent work has implicated the SCAR/WAVE complex, a potent
activator of the ARP2/3 complex (Machesky and Insall, 1998), in
regulating the cytoskeleton of mitotic cells. SCAR function is not
essential for Dictyostelium cytokinesis (Blagg et al., 2003), but
disruption of the Abi subunit of the complex leads to inappropriate
SCAR activation (Pollitt and Insall, 2008), which can disrupt
normal cell division. Consistent with this, others showed that cells
overexpressing an N-terminal truncation of SCAR also rapidly
become multinucleate (Caracino et al., 2007) indicating that SCAR
activity must be tightly regulated for normal cytokinesis.
In this study we identify a defined period at the end of mitosis
where Dictyostelium cells rapidly migrate apart. We show that
during this time the SCAR/WAVE complex is strongly activated
and constitutively localised to the polar cortex of dividing cells.
This activation is essential in driving the migration of the daughter
cells. We further demonstrate that this is crucial for successful
myosin-II-independent division and show that the localisation of
active SCAR is regulated by astral microtubules.
2247
Results
Enhanced migration and activation of SCAR at cytokinesis
Mitosis involves a number of sequential rearrangements to the
cytoskeleton. As previously described, the Dictyostelium mitotic
cycle starts with an initial rounding up and loss of cellular polarity
(Kitanishi-Yumura and Fukui, 1989). The first external sign of the
internal polarity formed by the spindle is the formation of ruffles
on opposing sides of the otherwise round cell. Subsequently, as the
furrow starts to form and ingress, the cells simultaneously start to
spread and the polar ruffles change into large, lamellipodia-type
protrusions. At this stage, the daughter cells move away from the
furrow (and each other) with high directionality (Fig. 1A; Fig. 7C).
Interestingly, when we examined this process in more detail, we
noticed that the period in which cells maintained a spread, lamella
morphology and moved in a directional manner lasted almost 12
minutes from the initiation of furrowing (as indicated by the sudden
change in gradient in Fig. 1A). During this hypermotile phase,
wild-type cells maintained their directionality away from the furrow
and moved much faster (6.1 mm/minute) than randomly moving
interphase cells (3.2 mm/minute; Fig. 7B). Importantly, this phase
continued for almost 10 minutes after the cells had completed
division (Fig. 1A), and therefore cannot be merely explained
mechanically by the repression of pseudopod extension at the
furrow by myosin II assembly. In addition, the end of this phase
occurs as a sudden ‘switch’ back to normal interphase morphology
and motility rather than a slow dissipation of polarity. We conclude
that the mitotic activation of motility is a highly regulated process,
specifically activated for a defined period as the cells begin to
separate.
Previous work has shown that in Dictyostelium, misregulation
of the SCAR complex by disruption of the Abi subunit leads to
aberrant mitosis (Pollitt and Insall, 2008). The only in vivo assay
for SCAR activity is to image the localisation of the complex,
which coincides with areas of polymerised actin (Weiner et al.,
2007). The dynamics of the SCAR/WAVE complex can be observed
in live neutrophils by imaging GFP-tagged fusions of complex
members with TIRF microscopy (Weiner et al., 2007). We
Fig. 1. Enhanced migration and SCAR localisation at cytokinesis. (A)Dividing wild-type (Ax3) cells were observed by video microscopy and the movement of
each daughter cell away from the furrow was analysed. Values are the means of 10 divisions; error bars represent standard deviation. (B)Cytokinesis of Ax3 cells
coexpressing HSPC300-GFP (green) and mRFPmars-actin (red) was observed by TIRF microscopy, showing a strong localisation and activation of SCAR at the
end of mitosis (see Movie 2 in supplementary material for the full sequence). Scale bar: 10mm.
Journal of Cell Science
2248
Journal of Cell Science 123 (13)
expressed a number of different subunits of the SCAR complex
fused to GFP in Dictyostelium. Using TIRF microscopy, HSCP300GFP, GFP-NapA (not shown) and SCAR-GFP all had a highly
dynamic localisation, and were clearly recruited to the very leading
edge of cellular protrusions (Fig. 1B; supplementary material Fig.
S1) (D.M.V. and R.H.I., unpublished). Of all the probes, HSPC300GFP gave the highest contrast images and was used for all further
experiments. The localisation of HSPC300-GFP was always closely
followed by both a narrower band of ARP2/3 (indicated by mRFPARPC4; supplementary material Fig. S1B and Movie 1) and a
broad band of mRFP-actin (Fig. 1B; supplementary material Movie
2). Therefore, although it is currently impossible to formally prove
that SCAR complex localisation is equivalent to its activity, this
appears to be the case. Expression of HSPC300-GFP is able to
rescue both morphology and cytokinesis defects of hspc300-null
cells (data not shown and supplementary material Fig. S3) and we
conclude that this probe gives an authentic representation of SCAR
localisation. This enabled us for the first time to image SCAR
dynamics in live Dictyostelium, giving a similar but much clearer
localisation than previous images of fixed cells using a SCARspecific antibody (Pollitt et al., 2006) and with temporal resolution.
During interphase, Ax3 cells only have weak SCAR-complex
localisation (Pollitt et al., 2006) and HSPC300-GFP only
transiently localised to the small pseudopodia that vegetative
Dictyostelium cells make (data not shown). However, during
cytokinesis, the SCAR complex became strongly localised to the
large lamellipodial protrusions of each daughter cell, just ahead
of a broad band of actin and ARPC4 and was excluded from the
furrow (Fig. 1B; supplementary material Movies 1 and 2). This
strongly suggests that SCAR is highly activated at the poles,
leading to local ARP2/3 activity and actin polymerisation. The
localisation of both HSPC300-GFP and mRFP-actin is much
stronger at cytokinesis than in interphase. Therefore SCAR
activity is strongly activated at mitosis, and is specifically
localised to the cell poles.
SCAR is responsible for generating MiDASes
In addition to its localisation at the mitotic cortex, we frequently
observed that mRFP-actin localised to small patches beneath the
nuclei of dividing cells (Fig. 2A). These actin patches have been
described previously and termed MiDASes (mitosis-specific
dynamic actin structures) (Itoh and Yumura, 2007). MiDASes are
highly dynamic structures formed underneath the mitotic spindle
and appear to be the major sites of cell attachment to the substratum
at cytokinesis. This and the observation that MiDASes become
much more stable in myosin-II-deficient cells led to the hypothesis
that they play an important role in adhesion-dependent cytokinesis
as points through which the cell can exert a mechanical force (Itoh
and Yumura, 2007).
When we coexpressed mRFP-actin and HSPC300-GFP, we
observed that MiDASes always colocalised with the SCAR
complex as well as mRFP-ARPC4 (a subunit of the ARP2/3
complex) (Fig. 2A; supplementary material Fig. S1B). Furthermore,
as MiDASes were transient – often appearing and disappearing
several times over the course of the mitosis – we were able to
investigate the causal relationship between the two. When we
simultaneously imaged HSPC300-GFP and mRFP-actin we saw
that the HSPC300-GFP localisation always preceded the actin by
Fig. 2. SCAR localisation precedes MiDAS formation.
(A)HSPC300-GFP and mRFPmars-actin were imaged
simultaneously by TIRF microscopy in dividing Ax3 cells
(see Movie 2 in supplementary material). Scale bar: 10mm.
Both MiDAS and SCAR localisation appeared and
disappeared over the course of the movie; the intensity of
both the red and the green channels within the circled
regions (labelled L and R) are plotted in B. Not all cells
produced MIDASes, and when Ax3 cells expressing GFPactin were observed at mitosis (in widefield), cells that
produced MiDASes (at any point) had a significantly more
spread morphology than those that did not. (C)Cells
dividing either with (top) or without (bottom) MiDASes.
Scale bar: 10mm. The area of these cells just before
abscission was measured and is plotted in D. Error bars
indicate the standard deviation.
Activation of SCAR/WAVE at mitosis
2249
~5 seconds (Fig. 2; supplementary material Movie 3). In addition,
MiDASes were present in 67% of mitoses in control cells, but
were never observed in any of the 36 scar-null mitoses that we
observed (for example see Fig. 3B) so we conclude that MiDASes
are generated by localised ARP2/3 activity driven by SCAR.
Dictyostelium cells have two parallel mechanisms for
cytokinesis, cytokinesis A that is myosin-II dependent and
cytokinesis B that is adhesion dependent. MiDASes are much
more frequent and stable in cells lacking myosin-II (Itoh and
Yumura, 2007), and therefore it is probable that they are upregulated
as part of a switch to cytokinesis B. When we measured the area
of dividing wild-type cells, we noticed that they split into two clear
populations, and cells that produced MiDASes had almost twofold
the area of cells without (Fig. 2C,D). Cells that had MiDASes also
had more cortical actin, and therefore it appears that all SCARgenerated structures are coordinately upregulated. Furthermore,
the fact that the two populations are distinct and not part of a
continuum indicates that there is a switch that determines whether
a cell divides using predominantly SCAR or myosin II as the
primary driver of cytokinesis.
Journal of Cell Science
Cells lacking SCAR have abnormal mitosis
The mitotic activation of SCAR at the cortex and MiDASes implies
an important role for SCAR in cell division. To investigate this
further, we looked at mitosis in cells in which SCAR had been
disrupted. Previous work had shown that the loss of SCAR has a
surprisingly mild effects on the growth, motility and development
of Dictyostelium (Bear et al., 1998). Indeed, although
hyperactivation of SCAR by disruption of the Abi subunit leads to
defects in cytokinesis, cells lacking SCAR remain almost entirely
mononucleate in both adherent and suspension culture (Pollitt and
Insall, 2008).
When we looked more closely by high-resolution DIC
microscopy we observed that although scar-null cells were able to
efficiently complete cytokinesis, they did so with a dramatically
altered morphology. Unlike wild-type cells, which appeared
strongly adhered and formed flat, lamella structures (Fig. 3A;
supplementary material Movie 4) scar-null cells produced
numerous large bulbous projections (Fig. 3A; supplementary
material Movie 5). As these projections do not contain actin (Fig.
3B) we conclude that they are membrane blebs, caused by
hydrostatic pressure. Similar to the activation of SCAR, these
blebs only appeared as the furrow formed and lasted almost 30
minutes, long after division had completed, and twice as long as
the hypermotile phase of wild-type cells (Fig. 3E). Similar to the
hypermotile phase of wild-type cells, scar-null cells reverted to a
normal vegetative morphology with a sudden, rapid change,
indicating a specific change in upstream signalling.
In addition, although control cells remained adherent throughout
mitosis (100% of over 50 divisions), scar-null cells frequently
detached with at least one daughter cell released from the substrate,
indicating reduced adhesion, consistent with the loss of MiDASes
(see supplementary material Table S1). In many divisions, it also
appeared that the furrow of scar-null cells was formed in the wrong
orientation, pinching off the top of the cell, rather than forming
perpendicular to the substratum. When we directly observed the
mitotic spindle in three dimensions using GFP-tubulin, we observed
that in scar-null cells, the spindle appeared less constrained and
more mobile, frequently elongating at an angle to the substratum,
whereas in wild-type cells, the spindle always remained parallel to
the surface (see supplementary material Fig. S2).
Fig. 3. Abnormal mitosis of Dictyostelium cells lacking SCAR. (A)DIC
images of control (Ax3) and scar-null (IR46) cells dividing on a glass surface.
(B)Wide-field fluorescence images of dividing cells expressing GFP-actin.
Scale bar: 10mm. Note the lack of MiDASes and cortical actin in IR46 cells.
(C)Plot indicating the paths of cells at cytokinesis. Movies of dividing cells
were orientated such that the furrow was formed perpendicular to the x-axis
and the centroids of the daughter cells were tracked manually from the first
frame at which the furrow could be seen. Ten divisions of each cell line are
superimposed; the paths of each pair of daughter cells are same colour.
(D)Directionality of the cell paths plotted in C. (E)The duration of the motile
phase, from the initiation of furrowing to a return to interphase morphology
and behaviour (i.e. random migration and the cessation of blebbing). Values
are the means ± standard deviation of 20 cells from 10 divisions. *P<0.005,
Students t-test.
From these experiments it is impossible to disseminate whether
reduced adhesion is responsible for aberrant spindle extension, or
caused by it, but it is an attractive hypothesis that interactions
between the spindle and the actin cytoskeleton act as a scaffold to
hold the spindle in place to orientate the plane of division (see
supplementary material Fig. S2C). Nonetheless, the defects in
adhesion and furrow orientation, combined with a loss of actinrich lamellipodial protrusions, lead to a complete loss of directional
migration, and scar-null cells moved randomly at cytokinesis (Fig.
3C,D). Therefore SCAR is essential for directed migration at
mitosis.
In order to investigate the evolutionary conservation of the role
SCAR in metazoa, we looked at cell division within columnar
epithelial cells in the dorsal thorax of developing Drosophila
melanogaster pupae. In this organism, we were able to develop a
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Journal of Cell Science 123 (13)
genetic system where a small, random population of scar-null cells
grow and divide within a wild-type tissue. We observed cell division
in real time by using Neuralized-Gal4 (Reddy and Rodrigues,
1999) to drive expression of UAS-GFP:Moe, which labels actin
filaments, specifically within well-spaced sensory organ precursor
cells. The MARCM system (Lee and Luo, 2001) was used to
positively label scar mutant cells within this tissue and observe
their behaviour over time. When scar-null cells divided, they also
produced numerous blebs – strikingly similar to the phenotype of
Dictyostelium SCAR mutants (Fig. 4; supplementary material
Movies 6 and 7). Wild-type controls did not do this, indicating that
SCAR is active at mitosis and suppresses blebbing. Therefore,
although dividing cells are unable to migrate apart within a tissue,
SCAR is important to maintain cell morphology and form proper
F-actin structures.
We also tested mutants of other members of the SCAR complex;
consistent with their loss of SCAR function phenotype (Ibarra et
al., 2006; Pollitt and Insall, 2009), disruption of either hspc300 or
napA led to multinucleate cells when grown in the presence of
blebbistatin (supplementary material Fig. S3). This was rescued by
expression of the HSPC300-GFP probe in hspc300-null cells. Loss
of the PIR121 subunit leads to increased pseudopod activity (Blagg
et al., 2003) and in this mutant, treatment with blebbistatin had no
effect on the number of multinucleate cells. Abi-null cells have a
specific defect of increased SCAR activity at cytokinesis (Pollitt
and Insall, 2008) and have an intermediate phenotype, becoming
only moderately more multinucleate upon blebbistatin treatment
(supplementary material Fig. S3).
Journal of Cell Science
SCAR drives myosin-II-independent cytokinesis
Loss of SCAR radically changes the morphology of cells at mitosis,
yet both Dictyostelium and Drosophila mutants are still able to
complete division. Previous work has shown that both
Dictyostelium and some mammalian cell lines in tissue culture are
able to form a cleavage furrow independently of myosin II, in an
adhesion-dependent manner (Kanada et al., 2005) and Dictyostelium
cells lacking myosin II are still able to divide efficiently, using this
alternative pathway (Uyeda et al., 2000; Zang et al., 1997).
As SCAR is a key regulator of directed migration during
cytokinesis, we asked whether SCAR is important for myosin-IIindependent division. To test this, we grew both wild-type and
scar-null Dictyostelium cells in the presence of the myosin-IIspecific inhibitor blebbistatin (Straight et al., 2003). Whereas wildtype (Ax3) cells were able to grow and divide normally by
adhesion-dependent cytokinesis, scar-null cells rapidly became
multinucleate, such that after 3 days 85% of cells were
multinucleate, with the majority having four or more nuclei (Fig.
5). Previously it has been observed that in Dictyostelium, different
genetic backgrounds use SCAR to a greater or lesser extent during
vegetative growth (Pollitt et al., 2006). When we repeated these
experiments with scar-null cells in an Ax2 genetic background [a
kind gift from Jan Faix, Hannover Medical School, Hannover,
Germany (Steffen et al., 2006)] we saw the same effect, with a
large increase in multinucleate cells upon blebbistatin treatment as
well as a disorganised, blebby morphology as the cells divided
(supplementary material Fig. S3 and data not shown).
Fig. 4. Abnormal mitosis of Drosophila columnar epithelial cells lacking
SCAR. Images of (A) control and (B) scar mutant sensory organ precursor
cells undergoing cell division during pupal development. Cells are labelled
with GFP-Moe. Scale bar: 5mm. Optical Z-sections through the tissue were
taken every 2 minutes on a confocal microscope and representative slices
through a dividing cell are shown. Arrows indicate blebs produced when cells
lacking SCAR divide. The full sequences can be viewed in Movies 6 and 7 in
supplementary material.
Fig. 5. SCAR is essential for efficient myosin-II-independent cytokinesis.
Control (Ax3) and scar-null cells were grown in glass dishes in the presence
of 40mM blebbistatin. JK107 cells grown in the presence of 10mg/ml
doxycycline were transferred to medium lacking it (i.e. scar expression was
repressed). (A)Every 24 hours cells were fixed, stained with DAPI and the
number of nuclei per cell were counted . At least 100 cells were counted for
each condition and the values plotted are the means ± standard deviation of
three independent experiments. (B)Representative images of DAPI stained,
fixed cells after 3 days growth in the conditions indicated. Scale bar: 50mm.
(C)JK107 cells were grown in medium lacking doxycycline for 24 hours and
cytokinesis was imaged by DIC microscopy. Scale bar: 10mm. Mitosis
frequently resulted in a failed (bottom), or asymmetric (middle) division, with
one daughter cell significantly larger than the other. (D)Frequency of
symmetrical, asymmetric and failed cytokinesis in JK107 cells grown in either
the presence or absence of 10mg/ml doxycycline.
Journal of Cell Science
Activation of SCAR/WAVE at mitosis
Although blebbistatin appears to be a highly specific inhibitor
of myosin II, others have shown that in Dictyostelium, the
inhibited myosin II molecules form aggregates, leading to
additional effects on normally myosin-independent processes
(Shu et al., 2005). To ensure that this is not responsible for the
effects we see upon disruption of scar we developed a genetic
system, in which myosin heavy chain (mhcA) was constitutively
disrupted, but we were able to inducibly switch off SCAR
expression using the Tet-On promoter (supplementary material
Fig. S4). These cells (named JK107) divided efficiently and
remained mononucleate in the presence of doxycycline (i.e. with
SCAR expressed), however, they also rapidly became
multinucleate when doxycycline was removed and scar expression
was repressed (Fig. 5A,B). Therefore SCAR is a major effector
for myosin-II-independent cytokinesis.
This inducible system allowed us to study in detail the division
of mononucleate cells as they try to divide without myosin II or
SCAR. When cells lacking myosin II but expressing SCAR
divide they migrate apart, generating a symmetrical furrow and
split into two cells of equal size (Fig. 5D). This process is
completed in approximately the same time in wild-type, scarnull and mhcA-null cells (Fig. 6C). However, in the absence of
both myosin II and SCAR, furrow progression is extremely slow
and unstable, leading to an asymmetric (where one daughter cell
is much larger than the other) or failed division in 60% of cases
(Figs 5C,D). Frequently however, mhcA–/scar– double mutants
did succeed in splitting into two cells, but this was very slow,
taking four times longer to divide than when SCAR was
expressed (Fig. 6C).
When we looked at actin and SCAR dynamics in JK107 cells,
we saw that when SCAR was induced, HSPC300-GFP localised to
the initial polar ruffles, as well as large lamellae (supplementary
material Movie 8). When SCAR expression was repressed,
HSPC300-GFP became uniformly distributed, and the flat lamella
structures were lost (Fig. 6; supplementary material Movie 9).
Cells were still able to produce actin-rich ruffles, indicating that
these can be generated independently of SCAR, but these ruffles
were not sufficient to drive migration. Therefore dividing cells
exhibit two overlapping actin processes at the cell poles, SCARindependent polar ruffling, and a superimposed activation of SCAR
at the cortex and under the spindle, driving cell spreading and
migration (supplementary material Fig. S2C).
The mitotic activation of SCAR may therefore serve two
purposes: first, by causing daughter cells to migrate apart, and
increasing adhesion via MiDASes, SCAR provides a secondary
mechanism for cytokinesis in the absence of myosin II, improving
the robustness of mitosis; second, activation of SCAR appears to
stabilise the mitotic furrow, ensuring it is correctly positioned for
an equal division. This agrees with previous studies that
demonstrated that it is the mechanical properties of both the
cortex and the furrow that determine the morphology of the
mitotic cell and ensure a symmetrical division (Girard et al.,
2004).
It is interesting that disruption of Abi, which leads to
hyperactive SCAR, or coronin, which also regulates polar actin,
result in cytokinesis defects in suspension where migration is
impossible (Pollitt and Insall, 2008; de Hostos et al., 1993).
Therefore, whereas migration-assisted division may be limited to
unicellular organisms or a specific subset of metazoan cells,
SCAR-mediated stabilisation of the furrow may be more generally
applicable to cell division when movement is restricted.
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Fig. 6. Cytokinesis of cells lacking scar and mhcA. JK107 cells coexpressing
HSPC300-GFP and mRFPmars-actin were grown overnight in the presence
(A) or absence (B) of 10mg/ml doxycycline and imaged by TIRF microscopy.
Scale bar: 10mm. Note the frame interval in B is twice as long as in A.
(C)Cells lacking both SCAR and myosin II take significantly longer to divide.
Movies were recorded of dividing cells and the time from furrow formation to
abscission was measured for control (Ax3), mhcA-null (JK103), scar-null
(IR46) and inducible scar/mhcA-null cells (JK107). Values plotted are the
mean ± standard deviation of at least five cells. *P<0.005.
SCAR is also essential for traction-mediated cytofission
Dictyostelium cells are extremely robust at dividing, and in addition
to myosin-II- and adhesion-dependent cytokinesis, a number of
cytokinetic mutants remain able to maintain a mononucleate
population by cytokinesis C (also known as traction-mediated
cytofission) (De Lozanne and Spudich, 1987; Vithalani et al.,
1996). In this process, multinucleate cells are able to tear themselves
into individual cells in a cell-cycle-independent but adhesiondependent manner.
Although cells lacking both SCAR and myosin II have severely
defective cytokinesis, they do frequently succeed (70% end up as
two cells; Fig. 5D). However after just six generations, over 85%
of scar-null cells grown in blebbistatin have become multinucleate
(Fig. 5A). We therefore investigated whether SCAR was also
important for traction-mediated cytofission. To do this, we
generated multinucleate cells by growing them in suspension with
blebbistatin for 2 days, then washed out the drug and let them
attach and divide on a surface. In this assay, both wild-type and
scar-null cells were initially large and multinucleate, but whereas
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Journal of Cell Science 123 (13)
wild-type cells rapidly tore themselves into smaller fragments,
cells lacking SCAR were completely unable to do this, and
remained multinucleate throughout the assay unless they divided
by a cell-cycle-dependent multinucleate mitosis, resulting in
multiple daughter cells from a single mitosis (supplementary
material Fig. S5). Interestingly, although vegetative scar-null cells
are still able to move (Pollitt et al., 2006) multinucleate scar-null
cells are stationary and did not significantly move over several
hours. SCAR is thus more important for multinucleate cell motility
and is essential for traction-mediated cytofission.
Journal of Cell Science
Localised SCAR activation and motility are regulated by
astral microtubules
We have demonstrated that at mitosis, SCAR is strongly localised
to the distal poles of dividing cells leading to an extended period
of rapid, directional migration. What then is responsible for
localising and activating SCAR? Unlike chemotaxing cells, which
respond to an extracellular gradient of chemoattractant, the
directional migration of a dividing cell must be determined by an
intrinsic, intracellular stimulus. The mitotic spindle is an obvious
candidate for the regulation of the direction of migration, and it is
the astral microtubules that provide the mitotic cell with an intrinsic
polarity (Carminati and Stearns, 1997; Pearson and Bloom, 2004;
Shaw et al., 1997). Therefore, we looked to see if the astral
microtubules were responsible for directing migration and SCAR
activation at mitosis.
EB1 is one of a number of proteins that bind to the growing ends
of microtubules and localises to the astral microtubules, centrosomes
and kinetochores at mitosis (Berrueta et al., 1998; Rehberg and Graf,
2002). Disruption of Dictyostelium EB1 has previously been reported
to delay prometaphase progression, but subsequent spindle elongation
and division occurs as normal and eb1-null cells are able to efficiently
complete cytokinesis (Rehberg and Graf, 2002). However, when we
observed the motility of eb1-null cells (a kind gift from Ralf Gräf,
University of Potsdam, Germany), we saw that whereas interphase
cells were indistinguishable from wild type, at mitosis their directional
migration was lost, and they moved randomly (Fig. 7A,C). Like
scar-null cells, they also exhibited reduced adhesion, with at least
one cell detaching in 73% of divisions (supplementary material
Table S1).
When we examined the localisation of HSPC300-GFP in eb1null cells, similar to control cells, SCAR complex localisation was
enhanced at mitosis, and cells had an overall increase in speed
compared with interphase cells, and the mitotic activation of SCAR
appeared to be intact (Fig. 7B,E). However, whereas wild-type
cells have a highly organised SCAR localisation, producing wide
protrusions exclusively at the cell extremities, cells lacking EB1
were much less organised producing smaller, incoherent protrusions
which frequently extended from the rear of the cell and the furrow
(Fig. 7E; supplementary material Movie 10).
We have shown that EB1 is required for the proper localisation
of SCAR at mitosis, and subsequent directed migration of the
daughter cells. Consistent with this, when treated with blebbistatin,
furrow ingression was severely retarded and eb1-null cells took
50% longer to divide than control cells (P<0.01, Student’s t-test;
Fig. 7D). Unlike scar-null cells, eb1-null cells did eventually
succeed in dividing, and did not become multinucleate (data not
shown), showing that although SCAR activity is uncoordinated in
these cells, it is sufficient to drive an inefficient cytokinesis.
Previous work has also shown that MiDASes colocalise with
the microtubule tips underneath the spindle, and that they are
Fig. 7. EB1 directs post-mitotic migration. (A)Paths of control (Ax2) and
eb1-null cells at cytokinesis. Ten divisions are superimposed and the plots for
each pairs of daughter cells are same colour. (B)Speed of migration of both
dividing and interphase cells and (C) Cytokinetic directionality (defined as the
distance moved perpendicular to the furrow divided by the total path length) of
mutant cells. (D)Time to complete division, from the initiation of furrowing to
abscission of control (Ax2) and eb1-null cells after treatment for 1 hour with
40mM blebbistatin. *P<0.01; **P<0.05, Student’s t-test. (E)TIRF images of
cells coexpressing HSPC300-GFP (green) and mRFPmars-actin (red). Scale
bar: 10mm. Arrows indicate regions where SCAR is inappropriately localised
in eb1-null cells (for the full sequence see Movie 10 in supplementary
material).
disrupted by treatment with microtubule depolymerising drugs
(Itoh and Yumura, 2007). Consistent with this, MiDASes were
never observed in eb1-null cells (n15) and in 73% of mitoses one
of the daughter cells lost contact with the substratum before division
was complete. Therefore, we conclude that microtubule plus ends
direct the localised activity of SCAR and regulate the adhesion and
furrow orientation of dividing cells.
Whether the regulation of SCAR by EB1 is constitutive, present
in interphase and chemotaxing cells, or is specific to mitosis,
requires further investigation. However, Dictyostelium EB1 has
been shown to localise to the tips of pseudopodia in migrating cells
(Rehberg and Graf, 2002). In addition, depletion of EB1 in mouse
melanoma cells inhibits the formation of lamellae and migration
(Schober et al., 2009) implying a more general role of EB1 and
microtubule plus ends in the regulation of SCAR.
Discussion
In this study, we have shown that at the end of mitosis, cells initiate
a highly motile phase, which continues for a defined period of
time, ending suddenly. The switch-like characteristics of this motile
Journal of Cell Science
Activation of SCAR/WAVE at mitosis
phase indicate that it is a tightly regulated process. Migration is
activated simultaneously with furrow formation and it is probable
that it shares a common master-regulator with myosin II contraction.
Although the primary mitotic signal is unknown, we show that one
of the major effectors of this pathway is the SCAR/WAVE complex,
which drives both migration and adhesion through the localised
activation of the ARP2/3 complex.
The activation of SCAR at mitosis appears to serve a number of
functions. Firstly, we demonstrate that SCAR activity and resultant
migration is crucial for cytokinesis B and the completion of cell
division when myosin II is compromised. Secondly, the dramatic
mitotic blebbing seen upon disruption of SCAR indicates a role in
maintaining the cell cortex and shape in order to cope with increased
hydrostatic pressure. Thirdly, the demonstration that SCAR directly
regulates the formation of MiDASes implies a role in regulating
cell adhesion at mitosis. This is consistent with the observation
that SCAR-null cells frequently detach when they divide. In
addition, others have recently demonstrated that dividing cells
possess a robust mechanosensing system, whereby the cell
redistributes myosin II and cortexillin I to the cortex in order to
dynamically correct for any defects in cell shape that may occur
(Ren et al., 2009). We show that cells lacking both SCAR and
myosin II are unable to correct asymmetric divisions as they
progress, implying that SCAR may be an effector of this
mechanosensory pathway.
Are these roles for SCAR conserved in higher eukaryotes?
Although cells within a tissue cannot migrate when they divide,
there is good evidence for the presence of myosin-independent,
adhesion-mediated mechanism in mammalian cells. Normal rat
kidney (NRK) epithelial cells are highly adherent and when treated
with blebbistatin, are still able to generate a furrow (albeit irregular)
provided they have a surface to adhere to (Kanada et al., 2005).
Others have shown that both NRK cells and 3T3 fibroblasts are
able to divide after microinjection of the Rho-inhibitor C3
ribosyltransferase, which also blocks the activity of myosin II
(O’Connell et al., 1999). This evidence clearly indicates that
metazoan cells also possess a secondary, adhesion-dependent
pathway that reinforces the predominant myosin-II-dependent
furrow formation. Indeed, it has been suggested that migrationdependent division is the more ancient mechanism, with myosin II
contraction superimposed later as an adaptation to multicellularity
(Gerisch and Weber, 2000).
The activation of SCAR and cell migration is clearly beneficial
for the division of independent, single cells, but its importance
within a tissue, where migration is obstructed by the surrounding
cells is less obvious. Our observation that loss of SCAR results in
aberrant mitotic blebbing in both Dictyostelium and Drosophila
tissue indicates conserved function. At a basic level, SCARgenerated cortical actin is important to contain the increased
hydrostatic pressure generated by myosin contraction at the furrow.
There is also some evidence to suggest an additional, more specific
role for SCAR at cytokinesis. When the Abi subunit of the SCAR
complex is disrupted, cells extend aberrant protrusions and become
multinucleate (Pollitt and Insall, 2008); as this also occurs when
the cells are grown in suspension where migration is impossible,
the defect in cytokinesis must be more complex than simple
disruption of directional movement. Others have also observed
that excessive, or disregulated cortical actin disrupts cell division,
and null mutants of coronin, as well as overexpression of a truncated
form of SCAR also leading to cytokinesis defects (Caracino et al.,
2007; de Hostos et al., 1993). There is also evidence that it is the
2253
balance between the forces and mechanical properties at the cortex
and the furrow which determines the shape of the dividing cell
(Girard et al., 2004). As SCAR is a major regulator of the cortical
cytoskeleton, it is probable that its tight regulation will be important
for cell shape and symmetry independently of any role in migration.
In addition, mutants that lack or have uncoordinated SCAR
activity and migration share a common defect in spindle and furrow
orientation: whereas wild-type cells always produce a furrow
perpendicular to the substratum, scar and eb1-null cells do not, and
frequently produce furrows towards the top of the mitotic cell such
that one daughter cell is detached (supplementary material Table
S1). As MiDASes are the major points of adhesion within mitotic
cells (Itoh and Yumura, 2007), and are generated by SCAR, this
may be simply due to a loss of adhesion, but it is clear that in
Dictyostelium at least, SCAR is important for maintaining
the orientation of the division plane. In addition, in scar-null cells,
the spindle appears to be more motile and we postulate that by
pushing out the cell cortex, SCAR is able to increase tension along
astral microtubules and stabilise the spindle and furrow
(supplementary material Fig. S2C).
Indeed, it has been shown that in Drosophila neuroblasts, the
repression of asymmetric division relies upon the presence of
adherence junctions and is disrupted by depletion of either APC or
EB1 (Lu et al., 2001). Others have recently shown that formation
of cell-cell junctions is inhibited by depletion of SCAR (Yamazaki
et al., 2007). Therefore the localised activation of SCAR and
stabilisation of cell-cell junctions provides a plausible mechanism
for orientating the mitotic spindle and organising asymmetric cell
division.
In conclusion, the actin cytoskeleton plays a number of distinct
roles during cell division, and its proper regulation is crucial. In
this study we have demonstrated that SCAR is essential for the
proper morphology of both Dictyostelium and Drosophila cells
during mitosis, and is the major regulator of both migration and
adhesion. Further studies are required to investigate the potential
implications of this within a whole organism context, or during
embryogenesis but it seems probably that, in at least a subset of
cell divisions, SCAR is crucial.
Materials and Methods
Dictyostelium strains and cell culture
The genotypes of all the strains of Dictyostelium used are listed in supplementary
material Table S1. When mutant strains were generated in Ax2 (Watts and Ashworth,
1970) or Ax3 (Sussman and Sussman, 1967) backgrounds the appropriate control
was used. Unless otherwise stated, all strains were cultured at 22°C adhered to
plastic dishes and maintained at low density. Cells were grown in HL-5 medium
(Formedium), which was replaced with low-fluorescent medium (Formedium)
2 hours prior to fluorescence microscopy. Cells were transformed by electroporation
and extrachromosomal vectors were selected and maintained in 50 mg/ml hygromycin
(Invitrogen). Cells inducibly expressing SCAR were maintained in 10 mg/ml
doxycycline (Sigma). The pure (–) enantiomer of blebbistatin (Tocris) was used in
all experiments.
Generation of mutant strains and expression plasmids
An inducible knockout of SCAR was generated using the previously published scarnull cell line IR46 (Blagg et al., 2003), and re-expressing SCAR under the control of
the Tet-On promoter. The full coding sequence of scar was amplified by adding 5⬘
BamHI and 3⬘ SpeI sites by PCR and cloning into the BglII-SpeI sites of the Tet-On
expression vector pDM310 (Veltman et al., 2009b). The Dictyostelium origin of
replication was then removed by MluI-HindIII digest, blunting with Klenow and religation (giving pJSK321). This was transformed into IR46 cells by REMI using XhoI
(Kuspa and Loomis, 1992) and selection with 10 mg/ml G418. Several independent
transformants were isolated and the regulation of SCAR expression tested by western
blotting using a rabbit anti-SCAR antibody (Blagg et al., 2003) of whole cell lysates
after growth in various concentrations of doxycycline (supplementary material
Fig. S4). The strain with the most similar expression to control cells at 10 mg/ml
doxycycline was used for further experiments (named SIKO2).
2254
Journal of Cell Science 123 (13)
The mhcA gene was disrupted using the following construct. Briefly, 5⬘ and 3⬘
fragments of mhcA were amplified by PCR from genomic DNA, adding BglII and
SalI sites at the 5⬘ and 3⬘ internal ends, respectively. The PCR products were then
mixed and used as template for a secondary PCR giving a 2.4 kb fragment of the
mhcA gene with a 105 bp deletion in the middle replaced by BglII and SalI sites.
This was cloned into TOPO blunt II (Invitrogen). The blasticidin-selectable marker
was then inserted as a BamHI-SalI fragment from pLPBLP (Faix et al., 2004).
Clones transformed with this construct were screened for disruption of the mhcA
locus by PCR and further validated by inability to divide in suspension culture (data
not shown). mhcA was disrupted in both Ax3 (giving JK103) and the inducible
SCAR knockout strain described above (giving JK107).
The full-length actin gene from Dictyostelium was amplified from genomic DNA
and cloned into pDONR221 using the Gateway system (Invitrogen). The actin gene
was subsequently cloned into pDM448 (Veltman et al., 2009a) for fusion to GFP
(resulting in pDM478) and into pDM449 for fusion to mRFPmars (resulting in
pDM463). The full open reading frame for HSPC300 was amplified by PCR from
cDNA and cloned into the expression vector pDM377 (yielding pDM459) and
shuttle vector pDM412 (yielding pDM557) (Veltman et al., 2009a) placing it Nterminal to GFP. The HSPC300-GFP expression cassette was excised from pDM557
with NgoMIV and ligated into the NgoMIV site of pDM463 to obtain a single vector
expressing both HSPC300-GFP and mRFPmars-actin (pJSK352). The full-length
coding sequences for -tubulin, NapA, SCAR and ARPC4 were also amplified from
cDNA by PCR and cloned into the vectors pDM449, pDM448, 450 and 579,
respectively, to yield expression vectors pJSK336 (GFP-tubulin), pDM479 (GFPNapA), pDM523 (SCAR-GFP) and the shuttle vector pDM601 (mRFPmars2-ArpC4).
The mRFPmars2-ArpC4 expression cassette was then excised from pDM601 by
NgoMIV digest and ligated into the NgoMIV site of pDM459 to yield pDM604, a
HSPC300-GFP–mRFPmars2-ArpC4 coexpression vector.
Journal of Cell Science
Microscopy and image analysis
Cell morphology and migration were imaged using differential interference contrast
(DIC) microscopy. Cells were seeded in glass-bottomed dishes (MatTek) and left for
2 hours prior to imaging using an inverted microscope fitted with a 100⫻/1.40 NA
oil objective (Nikon). For analysis of migration, images were acquired every 10
seconds. The paths of daughter cells at cytokinesis were generated by first orientating
each movie such that the furrow ingressed along the vertical axis. The centroids of
each daughter cell were manually tracked using the MtrackJ plugin for ImageJ
(http://rsb.info.nih.gov/ij) from the start of furrow formation for 5 minutes.
Cytokinetic directionality was calculated as the distance moved directly away from
the furrow (i.e. along the x-axis) divided by the total path length.
Total internal reflection fluorescence (TIRF) experiments were performed on a
Nikon Eclipse TE 2000-U microscope equipped with a 100⫻/1.45 NA Nikon TIRF
oil immersion objective. The microscope was equipped with a custom condenser in
which laser light was introduced into the illumination pathway directly from an
optical fibre output oriented parallel to the optical axis of the microscope. The light
source for evanescent wave illumination was a 473 nm diode laser or 561 laser
(Omicron), independently coupled into the condenser separately to allow individual
TIRF angle adjustment. A green-red dual filterblock (ET-GFP/mcherry; AHF
Analysentechnik, Germany) was used for 473/561 coexcitation. A Multi-Spec dual
emission splitter (Optical Insights, NM) with a 595 nm dichroic and two bandpass
filters (510-565 for green and 605-655nm for red) was used to separate both
emissions. Images were acquired every 5 seconds with a 50 msecond exposure using
a Cascade 512F EMCCD camera (Photometrics UK).
Wide-field images of live and fixed cells were obtained using a 60⫻/1.42 NA oil
immersion objective (Nikon) on an Olympus IX81 inverted microscope. Cell area
was measured by manually outlining each daughter cell at the point of abscission.
Cells were scored as having MiDASes if at least one MiDAS was observed at any
point in mitosis. To image the mitotic spindle in three dimensions, a Z-series (0.5
mm spacing) was taken at each time point and either extended focus (XY) images,
or Z-axis maximum intensity projections produced using Volocity imaging software
(Improvision).
Drosophila genetics and live-cell imaging
Live-cell imaging was performed with Neuralized GAL4:UAS GFP-Moesin flies.
GFP-Moe is the actin-binding domain of Drosophila Moesin (C-terminal 137
residues) fused to GFP (Edwards et al., 1997). Positively marked clones for scar37
(Zallen et al., 2002) were induced with Ubx-Flp. For live-cell imaging, animals were
prepared, at approximately 14 hours after puparium formation, by cutting a window
in the pupal case, attached to a slide with double-sided sticky tape. A coverslip with
a drop of injection oil was then placed over the notum, supported by coverslips at
either end to allow imaging from both upright and inverted Leica SP2 or SP5
microscopes.
Counting nuclei
Cells were grown without subculture for the duration of the experiment on glassbottomed dishes (MatTek). Cells were fixed by replacing the medium with –20°C
methanol and left for 5 minutes. Cells were then washed twice in KK2 buffer (16.5
mM KH2PO4, 3.8 mM K2HPO4 pH 6.2) and placed in KK2 with 0.5 mg/ml Hoechst
(Sigma) to stain DNA. Cells were imaged on a fluorescence microscope and the
number of nuclei were counted in at least 100 cells for each experiment.
The authors are grateful to Ralph Gräf for EB1-null cells and Jan
Faix for Ax2-derived SCAR mutants. We also thank Dave Gillespie
for critical reading of the manuscript and the Medical Research Council
for a Senior Fellowship (G117/537) to R.H.I. and project grant support
(G0600249) to J.K. Deposited in PMC for release after 6 months.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/123/13/2246/DC1
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