T H E J O U R N A L O F C E L L B I O L O G Y
© The Rockefeller University Press $30.00
The Journal of Cell Biology, Vol. 180, No. 2, January 28, 2008 285–294
Correspondence to Gilles Hickson: firstname.lastname@example.org
Abbreviations used in this paper: Dia, Diaphanous; GEF, guanine nucleotide
exchange factor; LatA, Latrunculin A; MRLC, myosin regulatory light chain;
MT, microtubule; Rok, Rho kinase.
The online version of this paper contains supplemental material .
Cytokinesis follows a complex spatiotemporal program initiated
at anaphase onset. It proceeds through several steps, includ-
ing cleavage furrow formation, ingression, midbody formation,
and abscission ( Glotzer, 2005 ; Eggert et al., 2006 ). Many con-
served proteins, including regulators of F-actin and myosin II,
localize to the cytokinetic apparatus and are required for cyto-
kinesis, but how they localize there and function together
remains unclear. A conserved Rho guanine nucleotide exchange
factor (GEF), encoded by pebble in Drosophila melanogaster
( Prokopenko et al., 1999 ), guides furrowing, in part through
stimulation of F-actin assembly via the formin Diaphanous (Dia;
Castrillon and Wasserman, 1994 ) as well as through activation of
Rho kinase (Rok) and myosin II ( Dean et al., 2005 ; Matsumura,
2005 ; Dean and Spudich, 2006 ; Hickson et al., 2006 ).
Another conserved furrow component is anillin, a putative
scaffolding protein that can bind F-actin, myosin II, and septins,
although the signifi cance of these interactions is unclear ( Field
and Alberts, 1995 ; Oegema et al., 2000 ; Paoletti and Chang, 2005 ;
Straight et al., 2005 ). Anillin localizes to the furrow early, but
its essential requirement appears to be for furrow stability and
midbody formation later in cytokinesis ( Somma et al., 2002 ;
Echard et al., 2004 ; Straight et al., 2005 ; Zhao and Fang, 2005 ).
It is not understood how anillin localizes to the furrow, al-
though it appears to require RhoGEF Pbl ( Prokopenko et al., 1999 )
and can occur independently of myosin II function ( Straight
et al., 2003, 2005 ).
Using high-resolution microscopic assays, we have ana-
lyzed the behavior of anillin at the time of cytokinesis. We fi nd
that a unique and previously unrecognized Rho-dependent input
can, independently of F-actin, promote the association of anillin
with septins, the plasma membrane, and microtubules (MTs), thus
providing insight into how anillin operates during cytokinesis.
Results and discussion
We generated D. melanogaster S2 cell lines expressing anillin-
GFP. The anillin-GFP fusion rescued loss of endogenous
anillin (Fig. S1, available at http://www.jcb.org/cgi/content/
full/jcb.200709005/DC1) and its localization paralleled that of
endogenous anillin. In interphase it was nuclear, at metaphase
it was uniformly cortical, and in anaphase it accumulated at
the equator while being lost from the poles ( Fig. 1, A and A ? ;
and Video 1, available at http://www.jcb.org/cgi/content/full/
jcb.200709005/DC1; Field and Alberts, 1995 ; Echard et al., 2004 ).
In some highly expressing cells, nuclear anillin-GFP formed
fi laments not ordinarily seen with anillin immunofl uorescence
nine nucleotide exchange factor (GEF) Pbl – dependent but
may also be mediated by known anillin interactions with
F-actin and myosin II, which are under RhoGEF Pbl -dependent
control themselves. Microscopy of Drosophila melanogaster
S2 cells reveal here that although myosin II and F-actin
do contribute, equatorial anillin localization persists in
their absence. Using latrunculin A, the inhibitor of F-actin
assembly, we uncovered a separate RhoGEF Pbl -dependent
nillin is a conserved protein required for cyto-
kinesis but its molecular function is unclear. Anillin
accumulation at the cleavage furrow is Rho gua-
pathway that, at the normal time of furrowing, allows
stable fi lamentous structures containing anillin, Rho1,
and septins to form directly at the equatorial plasma
membrane. These structures associate with microtubule (MT)
ends and can still form after MT depolymerization, al-
though they are delocalized under such conditions. Thus,
a novel RhoGEF Pbl -dependent input promotes the simul-
taneous association of anillin with the plasma membrane,
septins, and MTs, independently of F-actin. We propose
that such interactions occur dynamically and transiently
to promote furrow stability.
Rho-dependent control of anillin behavior during
Gilles R.X. Hickson and Patrick H. O ’ Farrell
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158
JCB • VOLUME 180 • NUMBER 2 • 2008 286
Figure 1. RhoGEF Pbl controls anillin-GFP localization during anaphase via F-actin – and myosin II – dependent and – independent mechanisms. (A – H) Frames
from time-lapse sequences of anillin-GFP cells progressing through anaphase/telophase (single Z sections, except for D, G, and H, which are projec-
tions of fi ve sections). (A) A control cell showing the normal redistribution of anillin-GFP (see Video 1, available at http://www.jcb.org/cgi/content/
full/jcb.200709005/DC1). (B) A cell after 3 d of RhoGEF Pbl RNAi (see Video 2). (C) A cell after 1- ? g/ml LatA treatment (surface Z section; see Video 3).
287 ANILLIN IN CYTOKINESIS • HICKSON AND O ’ FARRELL
(D) The same as C but the LatA was washed out at 10 min, soon after the formation of anillin-GFP structures (see Video 4). (E) A cell after 3 d of RhoGEF Pbl
RNAi and LatA combined. (F) A cell after 3 d of MRLC Sqh RNAi (see Video 5). (G) A cell after MRLC Sqh RNAi and LatA combined. (H) A cell after 4 d of Dia
and Rok RNAi and LatA treatment combined. (I) A LatA-treated anillin-GFP cell fi xed during anaphase/telophase and labeled with a Rho1 antibody (red
in merged; max intensity projection). (A ’ , B ’ , and F ’ ) semiquantitative measurements of the mean anillin-GFP intensities at the equator ( ? ) and poles ( ◊ ),
expressed as arbitrary units (AU) relative to time 0 (metaphase/anaphase [M/A]) in control, Pbl, and Sqh RNAi cells, shown for three cells each (different
colors represent different cells). Times are h:min:s from anaphase onset. Bars, 5 ? m.
(unpublished data), but these disassembled upon nuclear enve-
lope breakdown and the overexpression had no appreciable ef-
fect on the progress or success of cytokinesis.
Equatorial localization of anillin-GFP during
anaphase occurs via multiple RhoGEF
We tested whether RhoGEF Pbl contributed to anillin localiza-
tion during cytokinesis. After 3 d of RhoGEF Pbl RNAi or Rho1
RNAi (unpublished data), anillin-GFP localized to the cortex in
metaphase but did not relocalize to the equator during anaphase
( Fig. 1, B and B ? ; and Video 2, available at http://www.jcb.org/
cgi/content/full/jcb.200709005/DC1), indicating a requirement
for RhoGEF Pbl that is consistent with prior analysis of fi xed
RhoGEF Pbl mutant embryos ( Prokopenko et al., 1999 ). Because
anillin can bind F-actin ( Field and Alberts, 1995 ) and phosphor-
ylated myosin regulatory light chain (MRLC; Straight et al., 2005 ),
RhoGEF Pbl might regulate anillin indirectly through its control
of F-actin and myosin II.
We used latrunculin A (LatA) to test whether F-actin was
required for anillin-GFP localization. A 30 – 60-min incubation
of 1 μ g/ml LatA abolished cortical anillin-GFP localization in
metaphase ( Fig. 1 C ), indicating an F-actin requirement at this
phase. However, when anillin normally relocalizes to the equator
( ? 3 – 4 min after anaphase onset), anillin-GFP formed punctate
structures that became progressively more fi lamentous over the
next few minutes, reaching up to several micrometers in length
and having a thickness of ? 0.3 μ m ( Fig. 1 C and Video 3, available
These linear anillin-containing structures contained barely
detectable levels of F-actin (Fig. S2) and formed specifi cally
at the plasma membrane and preferentially at the equator, al-
though subsequent lateral movement often led to a more ran-
dom distribution ( Fig. 1 C and Video 3). Thus, anillin responds
to spatiotemporal cytokinetic cues even after major disruption
of the F-actin cytoskeleton. A substantial (albeit incomplete) re-
acquisition of cortical phalloidin staining was observed in cells
fi xed after washing out the drug for a few minutes (unpublished
data). In live cells, LatA washout immediately after formation
of the anillin structures allowed the preformed structures to mi-
grate from a broad to a compact equatorial zone as the cells
attempted to complete cytokinesis ( Fig. 1 D and Video 4). This
movement indicates that an F-actin – dependent process can con-
tribute to the equatorial focusing of anillin.
We tested the infl uence of RhoGEF Pbl on anillin behav-
ior in LatA. After RNAi of RhoGEF Pbl ( Fig. 1 E ) or Rho1 (not
depicted), anillin-GFP remained cytoplasmic through ana-
phase. Thus RhoGEF Pbl and Rho1 are required for anaphase
anillin behavior, whether the cortex is intact or disrupted by
We tested whether myosin II impacted anillin-GFP local-
ization. Compared with controls ( Fig. 1, A and A ? ), RNAi of
the gene encoding MRLC spaghetti squash (MRLC Sqh ; Karess
et al., 1991 ) inhibited cell elongation during anaphase ( Hickson
et al., 2006 ), slowed furrow formation, and delayed and dimin-
ished the equatorial localization of anillin-GFP ( Fig. 1, F and F ? ;
and Video 5, available at http://www.jcb.org/cgi/content/full/
jcb.200709005/DC1). However, unlike after RhoGEF Pbl RNAi,
equatorial accumulation of anillin-GFP was not altogether
blocked. It was still recruited but in a broad zone ( Fig. 1 F ). Further-
more, in the presence of LatA, MRLC Sqh RNAi did not affect the
formation of the anillin-GFP structures ( Fig. 1 G ). We conclude
that myosin II contributes to the equatorial focusing of anillin
when the F-actin cortex is unperturbed but that myosin II is dis-
pensable for anillin behavior in LatA.
Collectively, these data suggest that multiple RhoGEF Pbl -
dependent inputs control anillin localization. The slowed equa-
torial accumulation of anillin when myosin II function was
impaired indicates a myosin II – dependent input. That re-
assembly of the cortical F-actin network (after washout of LatA)
allowed preformed anillin structures to move toward the cell
equator indicates an F-actin – dependent input. This is consistent
with the concerted actions of myosin II and F-actin driving corti-
cal fl ow, as observed in other cells ( Koppel et al., 1982 ; Cao and
Wang, 1990 ; Wang et al., 1994 ; DeBiasio et al., 1996 ; Fishkind
et al., 1996 ), and is reminiscent of the coalescence of cortical
nodes during contractile ring assembly in Schizosaccharomyces
pombe ( Wu et al., 2006 ). However, the F-actin – and myosin II –
independent behavior of anillin in LatA indicates an additional
RhoGEF Pbl -dependent input. We tested possible involvement of
Dia or Rok, the Rho effectors responsible for cytokinetic F-actin
assembly and myosin II activation, respectively. Neither Dia
nor Rok RNAi, alone or combined ( Fig. 1 H and Fig. S3, avail-
able at http://www.jcb.org/cgi/content/full/jcb.200709005/DC1),
prevented formation of the anillin structures in LatA, indicating
that they too were dispensable. Thus, RhoGEF Pbl can control
anillin behavior in anaphase via a previously unrecognized route.
Immunofl uorescence analysis revealed extensive colocalization
between endogenous Rho1 and anillin-GFP in LatA, indicating
that Rho1 was itself a component of these structures ( Fig. 1 I ).
Although other Rho effectors could be involved, our fi ndings
are consistent with prior indications that Rho1 and anillin may
directly interact ( Suzuki et al., 2005 ).
Equatorial accumulation of myosin II
during anaphase can occur independently
of F-actin and anillin, but stable furrow
positioning requires anillin
We examined myosin II localization ( Fig. 2 A ), as it can bind
anillin and is controlled by RhoGEF Pbl . As previously reported
JCB • VOLUME 180 • NUMBER 2 • 2008 288
Figure 2. Myosin II localization can occur independently of F-actin and anillin but stable furrow positioning requires anillin. (A and B) Frames from time-
lapse sequences of cells expressing MRLC Sqh -GFP progressing through anaphase. (A) Control cell expressing MRLC Sqh -GFP. (B) Cell treated with LatA.
A projection of fi ve 2- ? m sections is shown (Video 6, available at http://www.jcb.org/cgi/content/full/jcb.200709005/DC1). (C and D) Cells expressing
anillin-GFP (green in merged) fi xed during anaphase/telophase in the presence of 1 ? g/ml LatA, labeled with a Ser21 phospho-MRLC Sqh antibody (red in
merged; maximum intensity projections of multiple 0.25- ? m deconvolved sections). (E) Frames from a time-lapse sequence of a MRLC Sqh -GFP cell attempting
cytokinesis after 3 d of anillin RNAi (see Video 7). (F) Cell expressing MRLC Sqh -GFP (green in merged), fi xed during anaphase in the presence of LatA after
anillin RNAi and labeled with an anillin antibody (red in merged; maximum intensity projection of multiple 0.25- ? m deconvolved sections). (G) Frames
from a time-lapse sequence of a cell expressing MRLC Sqh -GFP attempting cytokinesis after 3 d of anillin RNAi and in the presence of LatA (projection of fi ve
2- ? m sections; see Video 8). Times are h:min:s from anaphase onset. Bars, 5 ? m.
( Dean et al., 2005 ), MRLC Sqh -GFP was able to localize to the
equatorial membrane independently of F-actin, and in doing so
formed fi lamentous structures resembling those observed with
anillin-GFP ( Fig. 2 B and Video 6, available at http://www.jcb
.org/cgi/content/full/jcb.200709005/DC1). Indeed, anillin and
MRLC Sqh (detected as either MRLC Sqh -GFP or with an antibody
to serine 21 – phosphorylated pMRLC Sqh ) colocalized ( Fig. 2 C ),
although they were often offset as if labeling different regions of
the same structures ( Fig. 2 D ).
We tested the effects of anillin RNAi on MRLC Sqh -GFP
localization. MRLC Sqh -GFP recruitment and furrow initiation
appeared normal, but within a few minutes of initiation, furrows
289 ANILLIN IN CYTOKINESIS • HICKSON AND O ’ FARRELL
We analyzed anillin behavior after septin Pnut RNAi.
Although unable to fully deplete septin Pnut , we found that anillin
could localize to the equatorial cortex in regions devoid of de-
tectable septin Pnut (Fig. S3), which is consistent with fi ndings in
Caenorhabditis elegans ( Maddox et al., 2005 ). Importantly, in
septin Pnut -depleted cells, anillin-GFP still localized to the plasma
membrane in LatA but no longer appeared fi lamentous ( Fig. 3 D
and Video 9, available at http://www.jcb.org/cgi/content/full/
jcb.200709005/DC1), indicating that septin Pnut is essential for
the fi lamentous nature of the structures and that Rho1 can pro-
mote the association of anillin with the plasma membrane
independently of septin Pnut . However, in this case the plasma
membrane to which anillin-GFP localized subsequently exhib-
ited unusual behavior. It was internalized in large vesicular
structures, apparently in association with midzone MTs (Fig. S3
and Video 9). Although we do not understand this phenomenon,
it may be related to events induced by point mutations in the
septin-interacting region of anillin that give rise to abnormal
vesicularized plasma membranes during D. melanogaster
cellularization ( Field et al., 2005 ).
We tested the effects of anillin RNAi on the localization of
septin Pnut . Using Dia as a furrow marker ( Fig. 3 E ), 3 d of anillin
RNAi prevented the furrow recruitment of septin Pnut ( Fig. 3 F ).
In LatA-treated cells, anillin RNAi did not affect the formation
of septin Pnut rings in interphase cells (not depicted), but it greatly
reduced the formation of septin Pnut -containing structures during
anaphase/telophase ( Fig. 3 G ). Thus, anillin is required for
the furrow recruitment of septin Pnut and for the formation of
septin Pnut -containing structures in 1 μ g/ml LatA. In contrast, Dia
could still localize to the equatorial plasma membrane after
combined anillin RNAi and LatA treatment ( Fig. 3 G ), indicat-
ing that it can localize independently of both anillin and F-actin.
Thus, although Dia partially colocalized with anillin in LatA
(Fig. S3), this likely refl ected independent targeting to the same
location rather than an association between anillin and Dia.
Our data argue that Rho1, anillin, septins, and the plasma
membrane participate independently of F-actin in the formation
of a complex. However, anillin, septins and F-actin can also
form a different complex in vitro , independently of Rho ( Kinoshita
et al., 2002 ). Perhaps two such complexes dynamically inter-
change in vivo.
A potential role for anillin in anchoring the
cleavage furrow to midzone MTs
We tested the involvement of MTs in anillin behavior in LatA.
Overnight incubation with 25 μ M colchicine effectively depoly-
merized all MTs in mitotic cells and promoted mitotic arrest,
as expected (unpublished data). Using Mad2 RNAi to bypass
the arrest ( Logarinho et al., 2004 ), we observed anillin-GFP
during mitotic exit in the absence of MTs and in the presence of
LatA ( Fig. 4 A ). Under such conditions, anillin-GFP formed
fi lamentous structures very similar to those formed when MTs
were present, indicating that MTs were dispensable for their for-
mation. However, the structures appeared uniformly around
the plasma membrane rather than restricted to the equatorial re-
gion ( Fig. 4 A ), which is consistent with the role MTs play in
the spatial control of Rho activation ( Somers and Saint, 2003 ;
became laterally unstable and oscillated back and forth across
the cell cortex, parallel to the spindle axis, in repeated cycles,
each lasting ? 1 – 2 min and eventually subsiding to yield bi-
nucleate cells after ? 20 min ( Fig. 2 E and Video 7, available
The pheno type was very similar to that reported for anillin
RNAi in HeLa cells ( Straight et al., 2005 ; Zhao and Fang, 2005 )
and represents a requirement for anillin at an earlier stage than
previously noted in D. melanogaster ( Somma et al., 2002 ; Echard
et al., 2004 ). Thus, a conserved function of anillin is to maintain
furrow positioning during ingression. A potential mechanism is
proposed in the following paragraph.
In LatA, anillin RNAi did not prevent equatorial MRLC Sqh -
GFP recruitment, but instead of appearing as persistent linear
structures distorting the cell surface, a more reticular and dy-
namic structure lacking cell surface protrusions was observed
(compare Fig. 2 G and Video 8 [available at http://www.jcb.org/
cgi/content/full/jcb.200709005/DC1] with Fig. 2 B and Video 6;
and compare Fig. 2 F with Fig. 2 D ). Thus myosin II can localize
independently of both anillin and F-actin but the fi lamentous
appearance of myosin II in the presence of LatA requires anillin,
indicating that anillin can infl uence myosin II behavior in the
absence of F-actin, whereas myosin II appeared capable of infl u-
encing anillin behavior only in the presence of F-actin ( Fig. 1 ).
Septins are required for the fi lamentous
structures in LatA and septin recruitment
depends on anillin
Septins are multimeric fi lament-forming proteins that can bind
anillin in vitro and function with anillin in vivo ( Kinoshita et al.,
2002 ; Field et al., 2005 ; Maddox et al., 2005, 2007 ). Using an
antibody to the septin Peanut ( Neufeld and Rubin, 1994 ), we
found that in nontransfected S2 cells, septin Pnut localized to the
cleavage furrow and midbody where it colocalized with anillin
( Fig. 3, A and B ). Unexpectedly, the septin Pnut antibody also
strongly labeled bundles of cytoplasmic ordered cylindrical
structures, each ? 0.6 μ m in diameter and of variable length (up
to several micrometers; Fig. 3 B and Fig. S3). These staining
patterns could be greatly reduced by septin Pnut RNAi and were
thus specifi c (unpublished data). The cylindrical structures did
not appear to be cell cycle regulated, as they were apparent in
interphase, mitotic, and postmitotic cells. They also did not co-
localize with anillin ( Fig. 3, A and B ), nor did their stability rely on
anillin (unpublished data). Incubation with 1 μ g/ml LatA before
fi xation inevitably led to disassembly of most of these large
structures; however, the resulting distribution of septin Pnut de-
pended on the cell cycle phase. In LatA-treated interphase cells,
when anillin is nuclear, septin Pnut formed cytoplasmic rings,
? 0.6 μ m in diameter, which are similar to the Septin2 rings
seen in interphase mammalian cells treated with F-actin drugs
or in the cell body of unperturbed ruffl ing cells ( Kinoshita et al.,
2002 ; Schmidt and Nichols, 2004 ). In LatA-treated mitotic cells,
septin Pnut was diffusely cytoplasmic (or barely detectable) in
early mitosis, whereas in anaphase/telophase, it localized to the
same plasma membrane – associated anillin-containing fi lamentous
structures described in Fig. 1 , whether marked by anillin-GFP
( Fig. 3 C ) or antibody staining of endogenous anillin (Fig. S3).
JCB • VOLUME 180 • NUMBER 2 • 2008 290
GFP revealed bundles of MTs associating with the fi lamentous
anillin-GFP structures as they formed ( Fig. 4, C and D ; and
Video 10, available at http://www.jcb.org/cgi/content/full/jcb
.200709005/DC1). Colocalization between anillin-GFP struc-
tures and MT ends persisted over many minutes, even after con-
siderable lateral movement at the membrane. Thus, although
Bement et al., 2005 ; Yuce et al., 2005 ; Piekny et al., 2005 ; Kamijo
et al., 2006 ).
Shown by immunofl uorescence, the LatA-induced anil-
lin structures localized to the ends of nonoverlapping astral
MTs directed toward the equator ( Fig. 4 B , endogenous anillin).
Live imaging of cells coexpressing cherry-tubulin and anillin-
Figure 3. Anillin recruits septin to the cleavage furrow and to fi lamentous structures in LatA. (A – C) Nontransfected (A and B) and anillin-GFP – expressing
(C) S2 cells fi xed during anaphase/telophase and labeled with antibodies to septin Pnut (middle; red in merged) and anillin (A and B, left; green in merged)
and the DNA stain HOECHST (blue in merged; maximum intensity projections of deconvolved 0.25- μ m sections). C ’ is a 90 ° rotation about the y axis of the
projected stack within the boxed region in C. (D) Time-lapse sequence of a cell expressing anillin-GFP attempting cytokinesis in the presence of LatA after
6 d of septin Pnut RNAi. (E – G) Nontransfected S2 cells fi xed during anaphase/telophase and labeled with antibodies to septin Pnut (middle; red in merged) and
Dia (left; green in merged) and the DNA stain HOECHST (blue in merged; maximum intensity projections of deconvolved 0.25- μ m sections). (E) Control
cell. (F) Cell after 3 d of anillin RNAi. (G) Cell after 3 d of anillin RNAi and LatA treatment combined. Bars, 5 μ m.
291 ANILLIN IN CYTOKINESIS • HICKSON AND O ’ FARRELL
side to appear perpendicular to the cell surface while remaining
anchored at their base by MTs ( Fig. 4, C and D ; and Video 10).
We interpret this reorientation as refl ecting avid binding to and
subsequent envelopment by the plasma membrane. Although
intrinsically stable, the structures exhibited dynamic move-
ment within the plane of the plasma membrane and were ca-
pable of sticking to one another, via their ends, giving rise to
branched structures ( Fig. 5 B , Y shape) that were also capable
of breaking apart. Anillin has a pleckstrin homology domain
within its septin-interacting region and a membrane-anchoring
role of anillin has long been postulated ( Field and Alberts, 1995 ).
Our data support such a role and suggest that it is controlled
The relationship between events in LatA
and the normal events of cytokinesis
Our data highlight the complexity of RhoGEF Pbl signaling and
lead to a model in which multiple Rho-dependent inputs syner-
gize to control anillin behavior during cytokinesis ( Fig. 5 ).
the anillin structures formed independently of MTs, they stably
associated with MTs. These fi ndings support prior biochemical
evidence for interactions of MTs with both anillin and septins
( Sisson et al., 2000 ) and reveal a potential positive-feedback
loop in which MTs directed where Rho1 – anillin – septin formed
linear structures at the plasma membrane, whereas the structures
in turn associated with the MT ends. An MT plus end – binding
ability of anillin – septin could explain the furrow instability pheno-
type elicited by anillin RNAi ( Fig. 2 ). Accordingly, anillin may
physically link Rho1 to MT plus ends during furrow ingression,
thereby promoting the focusing and retention of active Rho1,
thus stabilizing the furrow at the equator ( Fig. 5 ).
Rho-dependent association of anillin with
the plasma membrane
These live-cell analyses highlight an unusual behavior of the
Rho-dependent anillin-containing structures at the plasma mem-
brane. Initially forming beneath and parallel to the plasma mem-
brane ( Fig. 4, C and D ), the structures then often lifted on one
Figure 4. Anillin-containing structures in LatA form independently of MTs but associate with MT ends at the plasma membrane. (A) Time-lapse sequence
of a cell expressing anillin-GFP and mcherry-tubulin exiting mitosis in the presence of colchicine (16 h) and LatA (1 h) after 3 d of Mad2 RNAi (max in-
tensity projections of deconvolved 2- μ m sections). (B) A nontransfected S2 cell treated with LatA and labeled with antibodies to anillin (green in merged)
and ? -tubulin (red in merged; max intensity projection of multiple deconvolved 0.25- μ m sections). (C and D) Frames from time-lapse sequences of cells
coexpressing anillin-GFP and mcherry-tubulin progressing through anaphase in the presence of 1 μ g/ml LatA (see Video 10, available at http://www.jcb
.org/cgi/content/full/jcb.200709005/DC1). Although the anillin-GFP channel was contrasted identically for all time points in each time-lapse series, the
cherry-tubulin channel in C and D was contrasted for each time point individually to counter the effects of photobleaching. Times are h:min:s from anaphase
onset. Bars, 2 μ m.
JCB • VOLUME 180 • NUMBER 2 • 2008 292
Drosophila Genomics Resource Center) using primers 5 ? -CACCAT-
GGACCCGTTTACTCAGCACA-3 ? (sense) and 5 ? -GTGGGTGGTTCCCCA-
GGC-3 ? (antisense) and cloned into the Gateway system pENTR-D-TOPO
vector (Invitrogen) according to the manufacturer ’ s protocols. The clone
was sequenced and in vitro recombination reactions were performed to
transfer the ORF to pMT-WG (inducible metallothionein promoter driving
C-terminal GFP fusion) and pAc-WG (constitutive Act05C promoter) from
the D. melanogaster Gateway collection (from the T. Murphy laboratory,
Carnegie Institute of Washington, Baltimore, MD). The plasmid expressing
MRLC Sqh -GFP under the control of the endogenous Sqh promoter was
described in Rogers et al. (2004) ; provided by S. Rogers, University of
North Carolina, Chapel Hill, NC) and cherry-tubulin in pAc-CW was provided
by the R. Vale laboratory (Howard Hughes Medical Institute, University of
California, San Francisco, San Francisco, CA).
Cell lines, cell culture, and RNAi
Stable S2 cell lines were generated by cotransfection of plasmids with
pCoHygro using Cellfectin reagent followed by selection with hygromycin,
all according to the manufacturer ’ s protocols (Invitrogen). Cells were cultured
in Schneider ’ s medium supplemented with 10% heat-inactivated FCS and
penicillin/streptomycin and passaged every 4 – 6 d. double-stranded RNAs
were synthesized as described in Echard et al. (2004) using Ribomax large-
scale transcription kits (Promega) with cDNA templates amplifi ed from genomic
whole-fl y DNA. Anillin, RhoGEF Pbl , Rok, Dia, and MRLC Sqh double-stranded
RNAs were the same as those described in Echard et al. (2004) and Hickson
et al. (2006) . Others were generated using the following primer pairs:
anillin 5 ? UTR, 5 ? -CCACCCTGCCAAATACGAC-3 ? and 5 ? -GGGTCCATT-
GTTGTTGCTGC-3 ? ; anillin 3 ? UTR, 5 ? -GGAACCACCCACTGACCCGCT-3 ?
and 5 ? -GCGAGTCATCCTAAATTAAATG-3 ? ; and Mad2, 5 ? -TGCTGGGACTT-
TAATATGCAGG-3 ? and 5 ? -GCTCATCTTGTAGTTGACCACG-3 ? . RNAi de-
pletions (performed as in Hickson et al.  ), drug treatments, fi xations,
and imaging were all performed in untreated 96-well glass-bottomed plates
(Greiner Bio-One or Whatman). LatA was purchased from EMD and colchi-
cine was purchased from Sigma-Aldrich. LatA (1 ? g/ml fi nal concentration)
was added for at least 1 h before fi xation or imaging. For LatA washout ex-
periments, the LatA-containing medium was removed and replaced three to
four times over the course of ? 2 min with fresh medium lacking LatA while
the cells were still on the microscope and without pausing the acquisition.
Cells were fi xed for 5 min in PBS containing 4% formaldehyde, permeabi-
lized, and blocked for 1 h in PBS containing 0.1% Triton X-100 (PTX) and
5% normal goat serum. Primary antibodies were incubated on the cells
overnight at 4 ° C using the following dilutions: anti-anillin, 1:1,000
(provided by C. Field, Harvard Medical School, Boston, MA; Field and
Alberts, 1995 ); anti-Dia, 1:2,500 (provided by S. Wasserman, University
of California, San Diego, La Jolla, CA; Castrillon and Wasserman, 1994 );
anti – phospho – myosin Light Chain 2 (Ser19, analogous to D. melanogaster
At the appropriate time and location of normal cytokinesis,
several proteins, including Rho1, MRLC Sqh , Dia, anillin, and
Septin Pnut , localized to the equatorial membrane in the presence of
LatA. Of these (and apart from Rho1 itself), anillin and Septin Pnut
were uniquely and specifi cally required for the formation of the
linear fi lamentous structures that we describe. The behaviors of
these structures are consistent with prior studies of ordered assem-
blies of septins and anillin ( Oegema et al., 2000 ; Kinoshita et al.,
2002 ) and of interaction between anillin and MTs ( Sisson et al.,
2000 ). Although such structures are not normally seen in furrowing
cells, anillin localizes to remarkably similar fi lamentous structures
in the cleavage furrows of HeLa cells arrested with the myosin II
inhibitor blebbistatin (see Fig. 5 in Straight et al., 2005 ).
It seems unlikely that LatA induced the structures we de-
scribe through nonspecifi c aggregation of proteins. Rather, we
propose that LatA blocked a normally dynamic disassembly of
Rho1 – anillin – septin complexes (by blocking an F-actin – dependent
process required for the event) and that continued assembly pro-
moted formation of the linear structures ( Fig. 5 ). Because bleb-
bistatin slows F-actin turnover ( Guha et al., 2005 ; Murthy and
Wadsworth, 2005 ), blebbistatin ( Straight et al., 2005 ) and LatA
(this study) may have induced fi lamentous anillin-containing
structures via a common mechanism. A dynamic assembly/
disassembly cycle involving anillin could promote transient asso-
ciations between the plasma membrane and elements of the
contractile ring and MTs, properties that could contribute to fur-
row stability and plasticity. Finally, because local loss of F-actin
accompanies and may indeed trigger midbody formation
( Schroeder, 1972 ), LatA treatment may have stabilized events
in a manner analogous to midbody biogenesis and could therefore
be useful in understanding this enigmatic process.
Materials and methods
The anillin (CG2092-RB) ORF, lacking its stop codon, was amplifi ed using
Platinum Taq polymerase (Invitrogen) from clone LD23793 (Gold collection;
Figure 5. A model for anillin regulation and function during cytokinesis. Pathways controlled by RhoGEF Pbl , including well-documented inputs into F-actin
and myosin II (black) and a novel, separate input (pink) via anillin and septins (A). Through equatorial stimulation (1), MTs spatially control Rho activation
and the independent recruitment of Dia, Rok, and anillin to the equatorial cortex. F-actin and Rok/myosin II contribute to equatorial focusing of anillin (and
other cortical components) via cortical fl ow (2), whereas Rho – anillin – Septin complexes dynamically link contractile elements within the furrow to the plasma
membrane (PM) and to MT plus ends, thus preventing the lateral instability of the furrow seen upon anillin RNAi. (B) LatA may stabilize Rho – anillin – Septin
complexes by preventing their normal F-actin – dependent disassembly (A, blue). Continued assembly with blocked disassembly gives rise to the fi lamentous
structures in LatA, perhaps mimicking midbody biogenesis that normally accompanies local loss of F-actin at the close of furrowing.
293 ANILLIN IN CYTOKINESIS • HICKSON AND O ’ FARRELL
complement our fi ndings of the Rho-dependent control of anillin and the associ-
ation of anillin with MTs.
Submitted: 4 September 2007
Accepted: 20 December 2007
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MRLC Sqh Ser21) mouse mAb (pMRLC; Cell Signaling Technology), 1:100;
anti-tubulin mAb (Sigma-Aldrich), 1:1,000; anti-Peanut (concentrated
mAb 4C9H4; Developmental Studies Hybridoma Bank), 1:300; and anti-
Rho1 (monoclonal p1D9 supernatant; Developmental Studies Hybridoma
Bank), 1:25. Cells were washed several times in PTX before a 1-h incuba-
tion with Alexa 488 – and 546 – conjugated secondary antibodies (1:500;
Invitrogen) and HOECHST 33258 (1:500). Cells were washed again in PTX
and mounted in Fluoromount-G (SouthernBiotech) before imaging.
Image acquisition and processing
Imaging of fi xed cells was performed using a deconvolution system (Delta-
vision RT; Applied Precision) using an inverted microscope (1 × 70;
Olympus) with a 100 × 1.4 NA oil-immersion objective and a cooled charge-
coupled device camera (Coolsnap HQ; Photometrics), and Z sections were
taken 0.25 μ m apart. The resulting datasets were deconvolved using Soft-
worx software (Applied Precision) and maximum intensity projections were
saved as TIFF fi les for export to Photoshop 8.0 (Adobe). Live imaging of
cells coexpressing anillin-GFP and cherry-tubulin was also performed using
the same system. Images were acquired at room temperature every 20 to
30 s, deconvolved, and exported as mpg or TIFF fi les for manipulation
in Quicktime (Apple) and Photoshop, respectively. Live-cell imaging of
cells expressing GFP alone was performed using an inverted microscope
(DMIRB; Leica) equipped with 100 × 1.3 NA (Plan Fluotar) or 63 × 1.4 NA
(PlanApo) oil-immersion objectives, a spinning disc (csu 10; Yokogawa),
a camera (Orca AG; Hamamatsu), and Volocity v 4 acquisition software
(Improvision). Resulting fi les were exported as Quicktime movies. Frames
for the fi gures were manipulated in Photoshop. Semiquantitative measure-
ments of anillin-GFP fl uorescence were performed using Volocity software
as follows: for a given cell, the cortex in late metaphase was selected as
an object based on a fl uorescence intensity value of > 0.5 – 2 standard de-
viations above the mean intensity for the whole cell. The mean intensity
of this object was then used to normalize mean intensity measurements
from subsequent time points where regions of the equatorial or polar corti-
ces were similarly selected as objects. Relative differences in mean inten-
sity were calculated from the initial time point in metaphase (set to 100
Online supplemental material
Fig. S1 shows rescue of loss of endogenous anillin by expression of anillin-
GFP. Fig. S2 shows F-actin staining after treatment with 1 ? g/ml LatA. Fig. S3
shows more detailed analysis of the relationship between anillin and septin Pnut
or Dia. Video 1 shows a control anillin-GFP – expressing cell progressing
through anaphase and cytokinesis and corresponds to Fig. 1 A . Video 2
shows a cell expressing anillin-GFP progressing through anaphase after 3 d
of RhoGEF Pbl RNAi and corresponds to Fig. 1 B . Video 3 shows an anillin-
GFP – expressing cell progressing through anaphase in the presence of 1 ? g/ml
LatA and corresponds to Fig. 1 C . Video 4 shows a cell expressing anillin-
GFP progressing through anaphase in the presence of 1 ? g/ml LatA to allow
anillin fi lament formation, after which the LatA was immediately washed out
(over 2 min starting at 10 min from anaphase onset). This corresponds to
Fig. 1 D . Video 5 shows a cell expressing anillin-GFP progressing through
anaphase after 4 d of MRLC Sqh RNAi and corresponds to Fig. 1 F . Video 6
shows a cell expressing MRLC Sqh -GFP progressing through mitosis in the pres-
ence of 1 ? g/ml LatA and corresponds to Fig. 2 B . Video 7 shows a cell ex-
pressing MRLC Sqh -GFP progressing through anaphase and cytokinesis in the
absence of LatA after 3 d of anillin RNAi. This corresponds to Fig. 2 E . Video 8
shows a cell expressing MRLC Sqh -GFP progressing through anaphase and
cytokinesis in the presence of 1 ? g/ml LatA after 3 d of anillin RNAi. This
corresponds to Fig. 2 G . Video 9 shows a cell expressing anillin-GFP pro-
gressing through anaphase in the presence of 1 ? g/ml LatA after 6 d of
Septin Pnut RNAi. This corresponds to Fig. 3 D . Video 10 shows a cell expressing
anillin-GFP and cherry-tubulin progressing through anaphase in the presence
of 1 ? g/ml LatA and corresponds to Fig. 4 C . Online supplemental material is
available at http://www.jcb.org/cgi/content/full/jcb.200709005/DC1.
We thank members of the O ’ Farrell laboratory, A. Echard, A. Paoletti, D. Toczyski,
and R. Vale, for discussions and C. Sheppard for comments on the manuscript.
This research was supported by a postdoctoral fellowship from Susan
G. Komen for the Cure (to G. Hickson), by a Special Fellowship from the
Leukemia & Lymphoma Society (to G. Hickson), and by National Institutes of
Health grants GM37193 and GM60988 (to P. O ’ Farrell).
Note added in proof. While this paper was in fi nal review, two indepen-
dent studies were published demonstrating direct interactions between human
anillin and Rho ( Piekny and Glotzer, 2008 ) and between D. melanogaster anillin
and the MT-bound RhoGAP, RacGAP50C ( Gregory et al., 2008 ). These studies
JCB • VOLUME 180 • NUMBER 2 • 2008 294 Download full-text
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