1

A complex of p190RhoGAP and anillin modulates RhoGTP and the

cytokinetic furrow in human cells

Arkadi Manukyan �,1,5, Kirsten Ludwig �,2,4,5 , Sergio Sanchez-Manchinelly3,4,5, Sarah J.

Parsons4,5,% & P. Todd Stukenberg1,5,%,*

� = These authors contributed equally to this work

%The work was performed equally in both laboratories

1=Department of Biochemistry and Molecular Genetics, University of Virginia Health

System

2= Department of Psychiatry and Behavioral Sciences and Jonsson Cancer Center,

UCLA, Los Angeles,

3=Deallus, Inc., Los Angeles

4=Department of Microbiology, Immunology, and Cancer Biology, University of Virginia

Health System

5=Cancer Center, University of Virginia Health System

PO Box 800733, Charlottesville, VA 22908, USA

*Corresponding Author

TEL: 434-924-5253

FAX: 434-243-7244

Email: pts7h@virginia.edu

Keywords: p190RhoGAP, Anillin, Myosin II, Cytokinesis, Rho

© 2014. Published by The Company of Biologists Ltd.Jo

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JCS Advance Online Article. Posted on 29 October 2014

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ABSTRACT

The cytokinetic furrow (CF) is organized by the RhoA GTPase, which recruits actin and

myosin II to the furrow and drives contractility. Here we show a role for the RhoGAP, p190, in

cytokinesis and its involvement in regulating Rho GTP levels and contractility. Cells depleted of

p190RhoGAP (p190) accumulate high levels of RhoGTP and markers of high Rho activity in the

furrow, resulting in failure of the CF to progress to abscission. The loss of p190 can be rescued

by a low dose of the myosin II inhibitor blebbistatin, suggesting that cells fail cytokinesis

because they have too much myosin activity. p190RhoGAP binds the cytokinetic organizer

anillin, and mutants of p190 that are unable to bind anillin or unable to inactivate Rho fail to

rescue cytokinesis defects in p190-depleted cells. Together these data demonstrate that a

complex of p190RhoGAP and anillin modulates RhoGTP levels in the CF to ensure robust

cytokinesis.

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INTRODUCTION

Cytokinesis is the final step in cell division where formation and ingression of the

cytokinetic furrow (CF) results in separation of two daughter cells. The process initiates in

anaphase, continues throughout telophase when membrane invagination at the equatorial cell

cortex occurs, and is completed upon membrane abscission (Glotzer, 2001). The contractile

forces required for furrow ingression are provided by a ring of filamentous actin and myosin II

that are juxtaposed to the cell membrane at the equator of the dividing cell. Assembly and

regulation of this contractile ring is critical for achieving proper cell division and is under the

control of the small GTPase RhoA (Piekny et al., 2005).

Early in cytokinesis RhoA localizes to the site of the nascent CF (Nishimura et al., 1998;

Takaishi et al., 1995), where its activity is required for CF formation and contraction, as well as

progression through cytokinesis (Bement et al., 2006). During early anaphase, RhoGTP levels

increase at the contractile ring (Kimura et al., 2000; Maddox and Burridge, 2003; Yoshizaki et

al., 2004), and active RhoA mediates contractile ring assembly through its downstream effectors,

mDia2, and ROCK Kinase (Piekny et al., 2005). mDia2 functions as an actin nucleator that

induces the polymerization of long, unbranched actin filaments (Watanabe et al., 2008), along

with microtubule alignment and stabilization (Narumiya and Yasuda, 2006). ROCK kinase

activates the contractile mechanism of myosin II by phosphorylating Thr18/Ser19 of the

regulatory light chain of myosin II (MLC II) (Matsumura, 2005). Phosphorylation of these sites

on MLC II triggers the motor (ATPase) activity of myosin II, which in turn promotes

crosslinking with newly created actin filaments to form a fully functional contractile ring

(Vavylonis et al., 2008; Wu et al., 2006) that provides the mechanical force required for furrow

contraction and ingression (Bresnick, 1999).

Actomyosin filaments are assembled on a network of cytoskeletal proteins at the cell

cortex, which connect the filaments to the plasma membrane. Anillin, an actin-binding protein, is

a crucial component of this scaffold that is required for cytokinesis (Oegema et al., 2000; Piekny

and Glotzer, 2008; Liu et al., 2012). Anillin has been shown to interact with actin, myosin,

microtubules, septin, mDia, Ect2, MgcRacGAP, Citron Kinase, and RhoA (Gai et al., 2011;

Piekny and Maddox, 2010). Additionally, overexpression of anillin increases Rho activation,

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suggesting that anillin functions in the regulation of Rho during cytokinesis (Suzuki et al., 2005).

Together, these findings support the model that anillin plays a vital role in linking the structural

components of the contractile ring to Rho-regulated signaling proteins, which govern

cytokinesis.

RhoA is a molecular switch that cycles between active (GTP-bound) and inactive (GDP-

bound) states. Transitions between activation states are facilitated by guanine nucleotide

exchange factors (GEFs) (activators), GTPase activating proteins (GAPs) (inactivators), and

guanine nucleotide dissociation inhibitors (GDIs) (inactivators) (Jaffe and Hall, 2005).

MgcRacGAP/Cyk4/RacGAP1 is a major GAP known to function in cytokinesis (Zhao and Fang,

2005; Loria et al. 2012). It binds with the mitotic kinesin like protein, MKLP1/Zen4, to form the

heterotetrameric centralspindlin complex, a key structural component of the spindle midzone

(Mishima et al., 2002). Centralspindlin carries out several critical functions during cell division,

including bundling of antiparallel microtubules to form the central spindle (Jantsch-Plunger et

al., 2000), positioning of the cell division plane, and promoting cycles of RhoA GTPase activity

(D'Avino et al., 2005). The GAP domain of MgcRacGAP limits the amount of RhoGTP at the

furrow and is required for robust cytokinesis (Miller and Bement 2009). In early cytokinesis

MgcRacGAP also activates Rho through complex formation with the guanine nucleotide

exchange factor Ect2 (Somers and Saint, 2003, Yüce et al., 2005, Glotzer, 2009). Recently it has

been shown that MgcRacGAP can bind the plasma membrane through its C1 domain and

promotes cortical contractility and the anchoring of the CF to the midbody (Lekomtsev et al.,

2012). While it is undisputed that MgcRacGAP is a critical cytokinesis regulator it is not clear if

it acts as a RhoGAP or a RacGAP or both, since there is also evidence that MgcRacGAP inhibits

Rac activity during cytokinesis (D’avino et al., 2004, Canman et al., 2008, Bastos et al., 2012).

A second RhoGAP, MP-GAP, has been shown to repress “blebbing” in mitotic cells, and cells

depleted of MPGAP fail in cytokinesis about 18% of the time (Zanin et al., 2013).

Our previous findings suggest that mammalian cells may have an additional level of

RhoA regulation during CF progression, namely p190RhoGAP (p190) (Su et al, 2003; Mikawa

et al, 2008). P190 localizes to the CF (Su et al, 2003), and cells ectopically expressing the protein

exhibit reduced RhoGTP levels in the furrow and become multinucleated, supporting the idea

that proper cycling of RhoA between active and inactive states is required for completion of

cytokinesis (Su et al., 2009). Moreover, in the absence of ectopic expression, it has been

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demonstrated that a transient, proteasome-mediated partial reduction (50%) of endogenous p190

levels during anaphase is required for completion of cytokinesis (Manchinelly et al., 2010; Su et

al., 2003), suggesting that p190 levels and activities are also critical for cytokinesis. During the

current study we overcame a technical challenge that has allowed us to characterize the loss of

function phenotypes of p190RhoGAP in cytokinesis. We report that cells depleted of p190 have

increased levels of RhoGTP in the CF and often fail to divide. Low concentrations of the myosin

II inhibitor blebbistatin can rescue loss of p190 suggesting that cells fail cytokinesis because they

have increased contractility. Additionally, p190 physically associates with anillin, and mutants

of p190 that are unable to interact with anillin fail in cytokinesis. Evidence is presented to

indicate that the interaction between p190 and anillin is regulated by contractile forces, as the

binding is disrupted by the addition of the myosin II inhibitor blebbistatin. Together, these data

demonstrate that p190 acts as a RhoGAP during cytokinesis in mammalian cells and provide

evidence that MLC II, anillin, and p190 act in a feedback loop that modulates Rho GTPase

activity in the CF.

RESULTS

p190RhoGAP is required for proper cytokinesis in HeLa cells.

To determine if depletion of p190 causes failure of cytokinesis, HeLa cells were

transfected with p190-specific small interfering RNAs (siRNAs) for 48 hours, and the number of

multinucleated cells were quantified (Fig. 1A, B). Twelve to fifteen percent of p190 depleted

cells were multinucleated, which is significantly higher compared to control siRNA-treated cells

(n>200). Figure 1, panel C shows the level of p190 depletion after 48 hours of siRNA treatment.

To determine whether exogenous p190 expression could restore normal cell division, we

generated a stable, wild type p190 doxycycline-inducible HeLa cell line that is resistant to

siRNA depletion. Replacement of endogenous p190 with doxycycline-inducible wild type p190

rescued the multinucleation phenotype, thereby addressing concerns about off target siRNA

effects and demonstrating the requirement of p190 for productive cytokinesis (Fig. 1B-C, Fig.

S1A).

To further characterize the cytokinetic defect caused by p190, we followed the behavior

of p190-depleted HeLa cells by live cell differential interference contrast microscopy (DIC) (Fig.

1D, Movie S1/S2). p190 depletion did not affect CF formation and initial ingression, processes

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that are regulated by MgcRacGAP (Zhao and Fang, 2005). However, p190-depleted cells often

failed in abscission, and cells became multinucleated. The most striking phenotype of p190-

depleted cells in telophase was an inability to complete furrow contraction. In addition,

telophase control cells usually migrated in opposite directions and this movement was lost in

p190 depleted cells. Quantification of live cell imaging showed that eight percent of p190

siRNA-treated cells (12/195) failed cytokinesis compared to one percent of control siRNA-

treated cells (1/135), which is similar to the failure rate measured in fixed cells. We conclude that

p190 is required for optimal cytokinesis.

p190RhoGAP regulates RhoGTP during cytokinesis in HeLa cells.

p190 is a RhoGAP that down-regulates RhoGTP levels in interphase cells (Ridley et al.,

1993); however, the effect of loss of p190 on RhoGTP levels in cells undergoing cytokinesis has

not been measured. To address this question p190-depleted HeLa cells were synchronized in

nocodazole for 14-16 hours and released into drug-free medium for 40 minutes to enrich for cells

in late mitosis and undergoing cytokinesis. Lysates were subjected to a RhoA G-LISA assay, a

highly quantitative colorometric assay to measure the amount of RhoAGTP. Figure 2A shows

that p190-depleted cells in cytokinesis exhibited significantly elevated RhoGTP levels compared

to those of siRNA untreated (cytokinetic) cells or cells treated with a control Luciferase siRNA

or with C3, a toxin that efficiently inhibits RhoA activity. Moreover, RhoGTP levels in p190-

depleted cytokinetic cells were similar to those of cells treated with LPA, a potent activator of

RhoA. To determine if RhoA levels are increased at the cytokinetic furrow in p190 depleted cells

we fixed cells with TCA and immunostained for RhoA. TCA precipitation is a validated method

to preserve Rho bound to membranes for immunofluorescence (Yonemura et al., 2004). We

found significantly higher concentrations of RhoA at the CF in late cytokinesing cells after p190

depletion (Fig. 2B-B’). Thus, silencing of p190 effected both localization of Rho and the levels

of RhoGTP in cytokinesing cells. This increase is similar to the level of RhoGTP measured in

the furrow by FRET following ectopic expression of a dominant negative form of p190 in HeLa

cells (Su et al., 2009).

An independent measure of RhoGTP levels is the phosphorylation of myosin II light

chain (MLC II) on Thr18/Ser19, which we assessed by using both anti-phospho-Ser19 MLC II

and anti-phospho-Thr18/Ser19 (pMLC II) antibodies. Both antibodies recognized cytokinetic

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furrows, and have different staining patterns as anti-phospho-Ser19 MLC II antibody stains

pMLC localized only to the contracting membrane, while anti-phospho-Thr18/Ser19 (pMLC II)

antibody also stains pMLC localized to the midzone (Kondo et al., 2012). Cells were stained

with anti-phospho-Ser19 MLC antibody, the intensity of staining at cytokinetic furrows was

quantified (areas quantified are outlined in Fig. S1C), and the ratio of pMLC II at the CF and

outside of the furrow area was determined. We observed an approximate 2-fold increase in

specific MLC II Ser-19 phosphorylation in the furrows of p190-depleted cells as compared to

controls (Fig. 2C-C’). To confirm that the increase in pMLC at the CF is not due to an increase

in total myosin, cells were co-stained with antibodies to anti-phospho-Thr18/Ser19 MLC II and

MLC II, the intensity of staining of both antibodies at the cytokinetic furrow was quantified, and

the ratio of pMLC II/MLC II was determined (Fig. S1D-D’). We see an increase in Rho activity

measured in this manner using pMLC II antibodies against MLC II pSer19 or MLC II pThr18,

pSer19 (Fig. 2C, C’, S1D). Moreover, the increase in phosphorylation of MLC II at the CF due

to p190 depletion could be rescued by exogenously expressing siRNA resistant wild type p190

(Fig. 2C-C’), ruling out siRNA off target effects.

To further investigate if p190 down-regulates RhoGTP levels through its GAP activity,

we generated a stable doxycycline-inducible HeLa cell line that expresses a p190 (R1283A)

point mutant that is defective in GAP activity (Li et al., 1997) (Fig. S1B). Cells expressing this

mutant were analyzed for their ability to rescue the cytokinesis defect associated with depletion

of endogenous p190. The number of multinucleated cells was significantly decreased after rescue

by exogenous wild type (Fig. 1B) but not the GAP mutant p190 (Fig. 2D). The expression level

of p190 (R1283A) was similar to that of the endogenous protein (Fig. 2E). We conclude that

p190 acts as a RhoGAP to down-regulate RhoGTP in the cytokinetic furrow.

Overexpression of p190 decreases RhoGTP at the furrow.

Overexpression of p190 had the opposite effect as depletion on RhoGTP levels in the

furrow. HeLa cells were transiently transfected with GFP-p190 or vector control plasmid,

immuno-stained with Thr18/Ser19 pMLC and MLC antibodies 24 hours later, and examined by

fluorescent confocal microscopy. Approximately 90% of cells showed reduced phosphorylation

of MLC II in their CFs as compared to vector-transfected controls (Fig. 3A-B, Fig. S1E).

Moreover, the ratio of pMLC II/MLC II was approximately 2-fold lower (Fig. 3C-D). These

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effects were similar to those seen after depletion of anillin or addition of C3 toxin, both of which

have been previously shown to affect Rho activation at the CF (Aktories et al., 1989; Piekny and

Glotzer, 2008). Neither p190 overexpression nor C3 treatment affected the levels of total Rho

protein (Fig. S1F), nor did p190 overexpression affect the localization of RhoA, actin, Aurora B,

or microtubules (Fig. S1G), suggesting that p190 specifically affected RhoA GTPase activity

during cytokinesis.

We tested whether constitutively active-MLC II (MLCII (T18D, S19D); CA-MLC-II)

could rescue the multinucleation phenotype generated by p190 overexpression to determine

whether reduced activation of myosin II caused the failure in cytokinesis. Co-transfection of

p190 and CA MLC II significantly decreased the amount of p190-induced multinucleation, while

transfection of CA-MLC II alone had little effect (Fig. 3E). Together, these data demonstrate that

p190 is required for the completion of cytokinesis in HeLa cells, where it acts as a RhoGAP to

down-modulate RhoGTP and pMLC levels to modulate proper CF contraction.

Low doses of Blebbistatin can rescue loss of p190RhoGAP

Since we find higher levels of Rho activity at the CF we tested whether high contractility

caused the cytokinesis failure associated with p190 depletion. Specifically, we asked if low

concentrations of the myosin II inhibitor blebbistatin, which weakens contractility, could rescue

the multinucleation phenotype generated by p190 depletion. HeLa cells were synchronized using

double thymidine block, and endogenous p190 was depleted by siRNA for 48 hours. Eight hours

after release from thymidine HeLa cells were treated for 6 hours with concentrations of

blebbistatin below the amount used to fully inhibit myosin activity. Cells were then fixed and

prepared for confocal microscopy analysis and scored for multinucleation (Fig. 4A). Low

concentrations of blebbistatin rescued p190 depletion in a concentration dependent manner with

0.25 uM giving the best rescue. Higher concentrations of blebbistatin generated multinucleated

cells, as one would expect for full inhibition of myosin. To complement this experiment we also

inhibited ROCK kinase, which is a Rho dependent activator of myosin, with the inhibitor

Y27632. A dose of Y27632 that was below the concentration used to fully inhibit ROCK activity

also partially rescued the generation of multinucleated cells after p190 depletion (Fig S2A). The

fact that low doses of blebbistatin and Y27632 both rescue p190 depletion argues that cells

depleted of p190 fail cytokinesis because of too much Rho stimulated myosin activity.

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p190RhoGAP binds with anillin during cytokinesis.

Anillin is a key regulator of cytokinesis in metazoans, thought to function as a scaffold

via its ability to bind Rho, Ect2, MgcRacGAP, actin, and MLC II (Piekny and Maddox, 2010;

Kechad et al., 2012). Anillin localizes at the CF during cytokinesis. It has been shown that p190

also binds Rho and Ect2 (Ludwig et al., 2009; Mikawa et al., 2008) and localizes to the CF

during all stage of cytokinesis (Su et al., 2003). Therefore we tested whether these two proteins

interact with each other. Immunoprecipitation of endogenous p190 co-precipitated endogenous

anillin, and vice versa. The interaction was enriched in lysates from cytokinesing cells (Fig. 4B (-

bleb)).

It has been shown that anillin homology domain (AHD) interacts with RhoA (Piekny and

Glotzer, 2008; Zhaho and Fang, 2005) and Cyk4/MgcRacGAP (D’Avino et al., 2008; Gregory et

al., 2008). We determined whether this same region of Anillin could directly interact with p190.

We expressed and purified His-tagged N-terminal regions of p190 (1-763) (which contains the

anillin binding region that we will identify in the next section) and MBP-tagged C-terminal

region of Anillin (608-1087) from bacteria (Fig. 4C). The purified proteins migrated as

monomers by gel filtration suggesting they are properly folded (Fig. S2B). We bound either

MBP or MBPAnillin(608-1087) protein to amylose beads, which were incubated with p190 (1-

763). The beads were washed and the bound proteins detected by immunoblot. p190 (1-763)

was specifically pulled by beads containing MBP-Anillin(608-1087) (Fig. 4C). We estimate that

between 4 and 0.4% of the input p190 (1-763) protein is pulled down suggesting that the direct

interaction is weak and that additional factors or protein modifications are required for the strong

interaction that we detect in IPs.

Anillin localizes p190RhoGAP to the cleavage furrow.

The interaction of p190 with anillin raised the question of whether anillin was required to

localize p190 to the cleavage furrow during cytokinesis. To test this hypothesis HeLa cells were

depleted of anillin by siRNA, and the subcellular location of p190 was assessed by confocal

microscopy. p190 was no longer enriched at the CF following anillin depletion, while

localization of MLC II and actin were unaffected (Fig. S3A, Fig. S3B). However, MLC II

phosphorylation was markedly decreased and Rho was mislocalized (Fig. S3C), consistent with

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published reports (Piekney and Glotzer, 2008). Together these data suggest that p190 and anillin

interact during cytokinesis and that this interaction enriches p190 at the CF.

The interaction between p190RhoGAP and anillin is required for efficient cytokinesis.

We transfected a series of plasmid constructs expressing different regions of p190 into

HeLa cells to map the anillin interacting domain on p190 (Fig. 5A and Fig. S4A). Constructs

expressing the central area of p190 known as the GBD/S1 and MD regions bound endogenous

anillin (Fig. S4B). The common region of these two constructs contains section 1 (S1) of the

middle domain. An isolated S1 domain of p190 also bound anillin after transient transfection,

defining this domain as an interactive region (Fig. S4C). We then generated constructs of full-

length p190 that contained three similarly sized deletions of sequences within section 1 (Fig.

5A). We assayed their ability to interact with endogenous anillin by transient transfection. p190

lacking the C-terminal third of section 1 (ΔS1C) was poorly precipitated by anillin (Fig. 5B).

p190 mutant proteins lacking the middle section (ΔS1B) also did not bind as well as wild type or

protein lacking the first section of section 1 (ΔS1A), suggesting that the C-terminal portion of the

domain as well as some of S1B mediated anillin binding.

To examine whether the interaction between p190 and anillin is critical for proper

cytokinesis, we generated a stable, doxycycline-inducible HeLa cell line that expresses the p190

mutant ΔS1C (Fig. S2C), which is defective in binding anillin (Fig. 5C). This mutant was then

analyzed for its ability to rescue the increase in phosphorylation of MLC II at the CF and the

cytokinesis defect associated with depletion of endogenous p190. Mutant p190 Dox-inducible

HeLa cell lines were treated with p190 siRNA for forty-eight hours to deplete endogenous

protein, and the siRNA-resistant anillin-binding mutant of p190 was induced to endogenous

levels. Cells were then prepared for confocal microscopy analysis for measuring intensity of

pMLC II at the CF and scoring multinucleation. Replacement of endogenous p190 with similar

levels of the anillin-binding mutant of p190 (Fig. 5D) did not reduce MLC II phosphorylation

(Fig. 5E-E’) at the CF and did not rescue the multinucleation phenotype generated by p190

depletion (Fig. 5F), whereas wild type p190 did (Fig. 1C and 2B’). Together, these results

demonstrate that p190 must interact with anillin to regulate proper Rho levels at the CF for

faithful cytokinesis in HeLa cells

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To exclude the possibility that p190ΔS1C was a null mutant we determined if it

maintained two other identified functions of p190. The cell lines expressing the p190 or

p190ΔS1C were treated with doxycycline to express GFP tagged p190 proteins, lysates were

immunoprecipitated with anti-GFP and blotted for the p190 interacting protein, p120 RasGAP

(Bryant et al. 1995). P190 is also an important substrate of the Src tyrosine kinase so we also

immunoblotted the IPs with anti-phosphotyrosine antibodies (Bouton et al. 1991). Figure 5E

shows that immunoprecipitated p190ΔS1C was tyrosine phosphorylated and bound p120

RasGAP to a similar extent as wild type p190. These results indicate that p190ΔS1C protein

retains two traditional p190 functions and is not a null mutant.

p190 and anillin associate during cytokinesis in a contractility-dependent manner.

HeLa cells in cytokinesis were treated for 40 minutes with 50 uM blebbistatin, a

nonmuscle myosin II inhibitor, which produces a state of weak contractility and blocks

cytokinesis (Straight et al., 2003). The association between p190 and anillin was lost after

blebbistatin treatment (Fig. 4B). This result suggests that the interaction between p190 and

anillin is regulated. The finding that blebbistatin blocks CF progression suggests that the

p190/anillin interaction either requires contractile forces or is a late CF event.

To determine whether high levels of blebbistatin affected RhoGTP levels in a manner

similar to p190 depletion, we visualized MLC II phosphorylation by using a anti-phospho-

Thr18/Ser19 (pMLC II) antibody and confocal microscopy. Blebbistatin treatment resulted in an

almost 2-fold increase in the pMLC II/MLC II ratio, indicating an increase in RhoGTP levels

(Fig. 6A-A’). These changes in Rho activity are similar to those seen after depletion of p190

(Fig. 2A-B’).

DISCUSSION

The small GTPase Rho is a critical regulator of cellular contractility. We suggest that

p190 is required to maintain the proper amount of RhoGTP at the cytokinetic furrow in HeLa

cells. Three independent experiments support this conclusions: in vivo silencing of p190 during

cytokinesis (1) increases the amount of RhoGTP, (Fig. 2A; Fig. 2B-B’); (2) increases the

phosphorylation of MLC II at furrows (Fig. 2C-C’); and (3) causes failure of cells to progress to

abscission, ultimately terminating in multinucleation (Fig. 1). We were also able to rescue the

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loss of p190 by adding low doses of blebbistatin, which suggests that cells fail in cytokinesis

because they have too much myosin II activity. Moreover, a p190 GAP point mutant (p190

R1283A) failed to rescue the multinucleation phenotype.

p190 is one of a growing number of RhoGAPs required for cytokinesis.

MgcRacGAP/Cyk4 is required at an early stage to establish a cytokinetic furrow (Minoshima et

al., 2003; Zhao and Fang, 2005). MgcRacGAP has additional roles including the localization of

the RhoGEF Ect2 to furrow, and it is a matter of debate whether MgcRacGAP acts as a RhoGAP

or RacGAP during CF formation (Bastos et al., 2012; Maddox and Oegema, 2003; Glotzer,

2009; Davies and Canman, 2012). In contrast, p190 action does not appear to be critical for

formation of the furrow, suggesting possible temporal separation of these RhoGAPs. It has also

been proposed that MgcRacGAP/Cyk4 functions in late stages of cytokinesis by linking midzone

microtubules to the plasma membrane (Lekomtsev et al., 2012). MP-GAP also limits RhoA

activity throughout mitosis to stabilize the cortex and limit the Rho zone during cytokinesis

(Zanin et al. 2013). Similar to p190 depletion, Zanin et al. found that depletion of MP-GAP

results in partial cytokinesis failure (15-18% of cells). Thus, multiple RhoGAPs are required to

assure faithful completion of cytokinesis and further defining the roles of the three cytokinetic

GAPs is an important line of future experimentation.

We favor models where MgcRacGAP/Cyk4 establishes furrows and p190 functions to

maintain proper forces during contraction. Consistent with this model are our observations that

all cells initiate furrow formation after p190 depletion that higher levels of MLC phosphorylation

are seen at furrows in cells depleted of p190, and these elevated levels could be rescued by

exogenous expression of wild type p190, or low levels of blebbistatin. We also show that an

interaction between p190 and anillin is required for cytokinesis. The critical experiment is the

replacement of endogenous p190 with a mutant p190 that does not bind anillin. These cells were

not able to decrease MLC phosphorylation at the CF and failed cytokinesis, while wild type p190

was able to rescue these phenotypes. In addition, the interaction between p190 and anillin is

inhibited by blebbistatin, which suggests that contractile forces regulate the action of p190 at the

furrow. Our current experiments could not measure a significant change in contraction rate, and

thus we cannot rule out roles of p190 in abscission. However we prefer a role for p190 in the

furrow, because we measure higher pMLC at furrows in p190 depleted cells.

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That RhoGTP levels need to be exquisitely controlled during cytokinesis is underscored,

not only by the identification of three RhoGAPs involved in the process [(MgcRacGAP – (Zhao

and Fang, 2005), MP-GAP (Zanin et al., 2013), and p190RhoGAP (Su et al., 2003)], but also by

our previous finding that levels of p190RhoGAP are reduced approximately 50% in late

cytokinesis (Su et al., 2003) and our current findings that contraction appears to regulate the

proper positioning of p190 (through association with anillin) to reduce levels of activated Rho at

the appropriate intervals. Rho function, like other small GTPases, is highly dynamic, switching

between activated and inactivated states to maintain the proper tension on the myosin-actin

network (Fidyk et al., 2006 Biochemistry 45: 7750-62; Vavylonis et al, 2008 Science 319:97-

100). How the three identified RhoGAPs coordinate with one another and how reduced levels of

p190 and its regulated association with anillin accomplish this, in conjunction with Ect2, is a

major unanswered question requiring further investigation.

Our finding that the interaction between p190 and anillin is inhibited by treatment of the

cells with blebbistatin is an exciting result, but one whose importance is still unclear.

Blebbistatin inhibits the action of class II myosin by inhibiting actin-activated ATPase activity

(Straight et al., 2003). The simplest interpretation of our result is that the interaction between

p190 and anillin is initiated by actin- and myosin-dependent contractility, although we cannot

rule out more complicated interpretations at this time. Our ability to rescue p190 depletion by

partial inhibition of myosin II activity by blebbistatin or Y27632 argues that cells fail cytokinesis

because contractile forces are too high (Fig. 4A, Fig. S2A). Consistent with this interpretation we

see an increase in Rho activity after treatment with higher doses of blebbistatin as measured by

MLC II phosphorylation (Fig. 6A-A’). That MLC II is downstream of p190 is further

emphasized by the fact that the inability to complete cytokinesis due to p190 overexpression (and

decreased RhoGTP levels) (Mikawa et al., 2008; Su et al., 2003; Su et al., 2009) can be rescued

with a constitutively active MLC II.

Figure 7 depicts a speculative model that is consistent with our data. Optimal RhoGTP

levels are ensured by a negative feedback loop induced by the contractility-sensing nature of the

p190-anillin complex. During cytokinesis, RhoGTP is enriched at the CF by specific localization

of Ect2 and MgcRacGAP resulting in activation of ROCK and other effectors of Rho. This in

turn leads to myosin II activation and actin polymerization, resulting in CF contraction. Once

contractility forces reach a threshold, p190 productively associates with anillin at the CF and

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lowers RhoGTP levels. This event in turn results in a decrease in myosin II activation, reduced

actin polymerization, and p190 release, completing the cycle. Since anillin also binds the

RhoGEF Ect2, this model can accommodate reactivation of RhoGTP after release of p190.

Overall, this feedback mechanism would maintain contractility forces at an appropriate level for

completion of cytokinesis. To test this model, it will be important to determine whether and how

contractile forces drive a functional interaction between anillin and p190.

MATERIALS AND METHODS

Cell culture and synchronization:

HeLa cells were obtained from American Tissue Culture Collection (Manassas, VA) and

maintained by serial passage in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad,

CA) containing 10% fetal calf serum (Gibco) and 1% penicillin/streptomycin (Gibco) at 37ºC in

a 5% CO2, humidified environment. Cells were synchronized for arrest in mitosis by treatment

with 50 ng/mL nocodazole (Cat. Number: M1404, Sigma, St. Louis, MO) for 14-16 hours,

released from the nocodazole block for 40 mins, and harvested by mitotic shake off. Where

indicated, 50µM blebbistatin (Cat. Number: 203390 Calbiochem, San Diego, CA) was added at

the time of nocodazole release. Cells were also synchronized by treatment with 2mM thymidine

for 24 hours, released into fresh media for 12 hours, arrested again in 2mM thymidine for 24

hours, and released for 8-16 hours. 8 hours after release from thymidine, either blebbistatin or

ROCK inhibitor Y27632 was added at the indicated concentrations for an additional 6 hours at

which time cells were fixed for immunofluorescence (IF) analysis.

Mutant constructs:

The p190 deletion mutants were constructed using primers purchased from Integrated

DNA Technologies, Inc. pkH3 triple-HA tagged full-length p190RhoGAP plasmid served as the

template for PCR amplification. Fragments of p190 were amplified and the desired portion of

protein was deleted. Deletion fragments of p190 were replaced using BamH1 and Bmt1

restrictions sites. Amplified deletion mutants were ligated into the pkH3 vector using the T4

DNA Ligase I following standard ligation protocols. All mutants obtained by PCR were

confirmed by sequencing prior to use. Verified plasmids were purified from competent DH5α

E.coli using Maxi-prep (Qiagen) according to manufacturer’s protocol.

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Transient transfection:

HeLa cells were transfected with Polyfect Transfection Reagent (Qiagen Inc., Valencia,

CA) according to the manufacturer’s protocol. Plasmids used included the pKH3 plasmid

encoding triple HA-tagged, wild type p190 (gift from Dr. Ian Macara), all mutants of p190

derived from this plasmid, and the GFP plasmid expressing GFP-p190 or GFP-constiutively

active (CA) MLC II (phosphomimetic T18D, S19D) (gift from Dr. Rick Horwitz). Cells were

plated in either 100mm or 6-well dishes (Corning). Mock-transfected cells were treated with

transfection reagents alone. When two or more plasmids were co-transfected, total plasmid levels

were equalized with empty vector. Vector control experiments were performed with empty

pKH3 HA-vectors or empty GFP vectors with equivalent amounts of vector per sample.

siRNA transfection:

Human p190RhoGAP-A was silenced using double-stranded p190 RNAi oligos custom

made by Dharmacon that targeted a unique sequence at the N-terminus of the protein. The

sequence of the double stranded oligos was 5’ AAG AUG CAC AUU GUG GAG CAG 3’.

Human anillin was silenced with the Dharmacon ANLN ON-TARGETplus SMARTpool

(GCAAACAACUAGAAACCAA, GGCGAUGCCUCUUUGAAUA,

GAUCAAGCAUUAGCAGAAA, ACGCAACACUUUUGAAUUA). Glass coverslips were

coated with 1mg/ml poly-L-lysine (Sigma), and cells were treated with p190 oligos (50 pmol)

using the Lipofectamine RNAiMax reagent (Invitrogen), as per the manufacturer’s instructions.

After 24 hrs, cells were synchronized in mitosis by treatment with nocodazole as described

above.

Generation of HeLa Tet-on p190-inducible cell lines and knock down rescue experiments.

The Tet-on expression system obtained from CLONTECH Laboratories, Inc. was used

according to the manufacturer's directions. pcDNA5⁄FRT⁄TO-p190, pcDNA5⁄FRT⁄TO-p190

(R1283A), pcDNA5⁄FRT⁄TO-p190ΔS1C Flp-In™ expression vectors and the Flp recombinase

vector, pOG44, were co-transfected into Flp-In™ HeLa T-REx cells using Lipofectamine 2000

(Invitrogen). Cells were selected in the presence of 0.2mg/ml Hygromycin B and screened for

p190 expression after Dox treatmant. Dox inducible HeLa cell lines were plated at 30% density

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and after 16 hrs, cells were transfected with 75 nM p190 siRNA. 1ug/ml doxycycline was used to

induce expression of full length, the GAP mutant p190 (R1283A) or the anillin binding mutant of

p190. Culture medium was changed after 24 hrs, and the cells were allowed to incubate for 48

hrs post-transfection in the presence of 1ug/ml doxycycline.

RhoGTPase assay:

HeLa cells were treated with control (Luciferase) or p190-siRNA for 24 hrs as described

above, then serum starved for an additional 24 hours. Remaining plates were serum starved for

24 hours, and all plates were then synchronized with nocodazole and released from the block as

described above to capture cells in cytokinesis. Individual plates were then treated with either

10µM LPA (Cat. Number: L7260 Sigma) for 40 mins or 0.5 ug/ml C3 toxin (Cat. Number:

CT04-A Cytoskeleton Inc., Denver, CO) for 2 hours before harvest. RhoGTP levels were

measured at 4°C using the RhoA G-LISA assay from Cytoskeleton Inc. according to

manufacturer’s protocol.

Live-cell imaging and Confocal Microscopy:

To perform live cell imaging, HeLa cells were plated onto poly-L-lysine-coated 1.5

Borosilicate 2-well chambered coverglass dishes (LabTex), and images were acquired using a

20x dry objective lens (numerical aperture = 0.75; Olympus) on a deconvolution microscope

(DeltaVision). Single-plane multipoint acquisitions were captured on a CoolSNAP HQ2 camera

(Photometrics) every 5 minutes within a 37°C chamber. Images of cells treated with control or

p190 siRNAs were collected with identical exposure times and scaled equally. The acquisition

software used was SoftWoRX from Applied Precision.

Cell fixation, Immunofluorescence Microscopy and Quantification:

HeLa cells were grown on poly-L-lysine-coated coverslips, depleted of p190 or anillin,

and synchronized in mitosis as described above for 48 hrs. Cells were fixed for 20 min with 4%

paraformaldehyden and processed for immunofluorescence as described (Bolton et al 2002). For

RhoA immunostaning, cells were fixed with 10% ice cold trichloroacetic acid (TCA) on ice for

15 minutes, than were rinsed three times with 1X PBS containing 30 mM glycine (G-PBS).

(Hayashi et al., 1999; Yonemura et al., 2004)

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Cells were analyzed on a Zeiss Axiovert 200 wide-field fluorescence microscope, fitted

with a confocal scanner using a krypton/argon laser (Perkin Elmer), Hamamatsu EMCCD

camera, a NanoScanZ motor (Prior) and a 63X oil Plan-Apochromat objective. An acousto- optic

tunable filter (AOTF) was used for detection of light at 488, 568 and 647 nm. Photographs were

taken as z-series with 0.4 μm Z- steps. Exposure times were consistent for each channel

throughout individual experiments, and no images were altered after capture. Quantification was

performed using Volocity®5.5 (Perkin Elmer) or ImageJ (NIH) softwares. The areas (region of

interest-ROI) around the CF at two sites were outlined using lasso tool (mask) on a single z-

section to determine the average pixel intensity per square micron, which was then averaged per

cell. This was background corrected by subtracting the average pixel intensity per square micron

measured using the same mask. We measure pixel intensity at regions outside of the CF

similarly. The ratio between these two values was then determined to define the specific

phosphorylation of myosin II light chain using phosphomyosin (Ser19) / (Thr18/Ser19) light

chain II) and Myosin light chain II antibodies (Cell Signaling) (Fig. S1B). Fifteen-thirty cells per

treatment were analyzed for each experiment.

Protein purification, gel filtration and In vitro pull down assay:

6His-tagged p190 (1-763) and MBP-tagged Anillin (608-1087) were bacterially

expressed and purified using amylose beads (NEB) for MBP fusion Anillin and Ni-NTA agarose

beads (Qiagen) for His-tagged p190. The beads were washed, and the 6xHis tagged p190 was

eluted from Ni-NTA agarose with 300 mM imidazole and MBP-tagged Anillin was eluteds with

100mM maltose at 4ºC. Proteins are concentrated and fractionated on the Superdex 200 gel

filtration column in 50 mM Tris (pH=7.6), 150 mM NaCl, 5 mM MgCl2, 1mM DTT buffer.

Protein concentration was determined by Bradford protein assay. To investigate the direct

interaction of the p190 and anillin in vitro binding assay was performed using recombinant 6His-

p190(1-763) and MBP-Anillin(608-1087) proteins in 200 mL 50 mM Tris (pH=7.6), 150 mM

NaCl, 5 mM MgCl2, 0.5% Triton X-100 buffer. 5 ug of p190(1-763) were added to the buffer

containing 5 ug of MBP or MBP-Anillin and proteins were incubated with amylose beads by

rotating at room temperature for 1 hour. Beads where washed 4-5 times with buffer before

adding SDS sample buffer and bound proteins were detected by Western. Blots were probed by

1:250 mouse anti-His (Santa Cruz) and 1:10000 mouse anti-MBP (NEB) antibodies.

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Immunoprecipitation:

Cell lysates were prepared as described for Western blotting and incubated with 5 μg of

the indicated immunoprecipitating antibody overnight. After primary antibody incubation,

immunecomplexes were precipitated by protein G agarose (Invitrogen) and separated by

centrifugation at 5,000 rpm for 1 min. After cold PBS washes, SDS sample buffer was added to

each sample, boiled for 5 mins, and subjected to Western blotting analysis, as described above.

ACKNOWLEDGEMENTS

We thank the SJP lab for the p190RhoGAP deletion constructs and Piekny lab for MBP-

Anillin plasmid. We are grateful to J. Pritchard and M. Skalski for technical help and the

Cytokinesis Group for helpful discussions.

The authors declare no potential conflicts of interest with respect to the authorship and/or

publication of this article.

This work was supported by the National Cancer Institute [grant numbers CA39438

(SJP), CA009109 (Institutional T32)], GM063045 for AKM and PTS and UVA Cancer Training

grant [grant number 5 32T CA9109] for AM.

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FIGURE LEGENDS Figure 1: p190 is required for cytokinesis.

A: Depletion of p190RhoGAP results in multinucleation because of cytokinesis failure. 48 hrs

after LacZ (control) or p190RhoGAP siRNA treatment HeLa cells were processed for IF for

actin staining with phalloidin (green) and DNA staining with DAPI (blue). Arrows indicate

multinucleated cells. Scale bars, 10 µm. B: Quantification of multinucleation in HeLa cells

depleted of p190 by siRNA. Cells were depleted of p190 and simultaneously were indicated also

treated with 1μg/ml of doxycycline to induce expression of wild type p190. 48 hrs post-

transfection and doxycycline treatment, cells were fixed and stained with phalloidin (actin) or

DAPI (DNA) and scored blindly for bi- or multinucleation (n≥300). Error bars represent standard

deviation from three independent experiments. *=p<0.001 (Student t test (t test)) for both p190

siRNA-treated cells compared to control, and p190 siRNA-treated cells compared to cells

rescued by wild type p190. C: Immunoblot to measure relative p190 levels. D: DIC live-cell

imaging of LacZ or p190 siRNA treated HeLa cells. Recording started after 24 hours transfection

of either siRNA. The top two panels represent LacZ siRNA treated cells, and the bottom two

panels represent p190 siRNA treated cells. Arrows indicate single cell divisions after treatment

with either siRNA. 195 cells treated with p190 siRNA and 135 cells treated with control siRNA

were scored, and the number of multinucleated cells/total number of cells was quantified. Scale

bars, 10 µm.

Figure 2: p190 acts as a RhoGAP in the cytokinesis furrow.

A: p190 depleted cells increase RhoGTP levels in the cytokinetic furrow. RhoGTP levels were

measured with the colorimetric RhoA G-LISA assay. Active RhoGTP protein was used as a

positive control and set at 100%. Results are expressed as the mean percent of RhoGTP +/-

standard error of the mean (SEM), n=4. *=p<0.05 (t test) as compared to cytokinetic cells. B:

Confocal images HeLa cells during cytokinesis stained for RhoA and tubulin 48 hrs after

transfection of siRNA targeted to LacZ (control siRNA) or p190RhoGAP. Images shown are

representative of n>20. Scale bars, 7 µm. B’: Quantification of RhoA intensities at the CF n>20,

*=p=0.0002 (t test). C: Confocal images of pMLC II in wild type p190 doxycycline inducible

HeLa cells during cytokinesis 48 hrs after control (LacZ), p190RhoGAP siRNA and p190 siRNA

with doxycycline treatment. Fixed cells were stained with anti-pMLC II (S19) antibody. Images

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shown are representative of n>20. Scale bars, 4.5 µm. C’: Quantitation of pMLC at the CF. Box

and whisker plot quantifying the ratio of intensity of pMLC II at the CF and outside region of

CF. n>2 with >20 cells/experiment. *, ** =p<0.005 as compared to LacZ siRNA treated cells.

D: A GAP mutant of p190 does not rescue p190-silencing-induced multinucleation. Cells were

depleted of p190 and simultaneously were treated with 1μg/ml of doxycycline to re-express

doxycycline-inducible GAP mutant of p190. Failed cytokinesis was scored as the percentage of

bi- or multinucleated interphase cells (n≥300). Error bars represent standard deviation from three

independent experiments. *=p<0.0001 for both p190 siRNA-treated cells and cells rescued by

p190 GAP mutant (p190 R1283A) compared to control. Note that the experiment in Figure 1B

was performed under identical conditions and at the same time and acts as a control for rescue

with wild type protein. E: The GAP activity of p190 is required for cytokinesis. HeLa cells were

depleted of endogenous p190 and a GAP mutant of p190 (p190 R1283A) was expressed by the

addition of doxycyclin where indicated. Cell lysates were prepared for immunoblot analysis 48

hours post-transfection.

Figure 3: Overexpression of p190 increases RhoGTP levels.

A: Confocal images of total-MLC II and pMLC II in HeLa cells during cytokinesis. HeLa cells

were mock-treated or transiently transfected with empty plasmid or p190 plasmid. Separate

cultures were treated with anillin siRNA or C3 toxin. Cells were then fixed and stained for total

MLC II (red), pMLC II (Thr18/Ser19) (green), and DNA (blue). Images shown are

representative of n>30. Scale bars, 5 µm. B: Quantification of the number of HeLa cells with

enriched pMLC II at the CF during cytokinesis. Results are expressed as the mean percent of

total cells in cytokinesis or cytokinetic cells positive for GFP-p190 exhibiting localization of

pMLC II at the CF +/- SEM, n>3, with >15 cells/experiment. *=p<0.005 (t-test) as compared to

mock. C: HeLa cells were either Mock-treated or transiently transfected with HA-p190 plasmid.

Cells were synchronized by nocodazole block and release and the levels of total MLC II, pMLC

II, and HA-p190 were assessed by Western blot analysis. D: Overexpression of p190 decreases

Rho activity as measured by pMLC II. Quantification of C. Levels were normalized to actin, and

the ratio of pMLC II/total MLC II was then determined and normalized to the ratio in vector-

transfected (VC) cells, which was set to 1. Results are expressed as the mean fold ratio over that

of VC cells +/- SEM, n>3. *=p<0.05 as compared to VC. E: Constitutively active (CA) MLC II

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rescues multinucleation induced by p190 overexpression. HeLa cells were mock-treated or

transiently transfected with HA-p190 or CA MLC II plasmids singly or in combination and

scored for multinucleation. Results are expressed as the mean percent total cells (Mock) or cells

positive for HA-p190 and or CA MLC II that were multinucleated +/- SEM, n>3, with >90

cells/experiment. *=p<0.005 as compared to mock.

Figure 4: p190 and anillin interact during cytokinesis.

A: p190-dependent multinucleation phenotype was rescued by the myosin II inhibitor

blebbistatin. Failed cytokinesis events were measured by the percentage of bi- or multinucleated

interphase cells relative to the total population of cells (n≥300). Error bars represent standard

deviation from three independent experiments. *=p<0.005 for p190 siRNA treated cells compare

to Luc siRNA treated cells, and *=p<0.03 for p190 siRNA compared to p190 siRNA treated cells

in 0.25μM Blebbistatin.

B: Coimmunoprecipitation of anillin and p190 in asynchronous or cytokinetic HeLa cells. HeLa

cells were either untreated (Asyn) or synchronized with nocodazole (Cyto). Cells were then

washed out of nocodazole and either mock-treated or treated with 50 µM blebbistatin for 40 min

and cell lysates generated for immunoprecipitations (IPs). Western blots shown are

representative of n>3. WCL=Whole cell lysate. C: Diagrams of p190 and Anillin constructs

used for recombinant protein expression. MBP and MBP tagged C-terminal anillin was bound to

amylose beads, which were incubated with equal amount of 6His tagged p190(1-763).

Interaction was detected by Western blotting with anti-His and anti-MBP antibodies.

Figure 5: The interaction between p190 and anillin is necessary for proper cytokinesis.

A: Diagram of the p190RhoGAP mutants: Full-length, ΔS1A (Δ379-467), ΔS1B (Δ467-563),

and ΔS1C (Δ563-646). B: Mapping of anillin binding site in p190. HeLa cells were transiently

transfected with HA-tagged variants of p190 (as described in Panel A) or empty vector,

synchronized with nocodazole, and released into mitosis. Extracts were immunoprecipitated as

described in Experimental Procedures and subjected to Western blotting analysis to detect

association of exogenous HA-p190 with anillin. C: Cells expressing p190ΔS1C poorly interact

with Anillin. Cell lines were treated with doxycycline to express either GFP-p190 or GFP-

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p190ΔS1C, lysates were immunoprecipitated with anti-Anillin and blotted for GFP to detect the

interaction between Anillin and p190.

D-G Cytokinesis is not rescued by expression of the anillin-binding mutant of p190. D. p190

protein levels in cells depleted of p190 or after doxycycline expression of p190ΔS1C. E:

RhoGTP activity was indirectly measured by anti-pMLC II (Ser19) antibody. Images shown are

representative of n>30. Scale bars, 4 µm. E’: Quantification of pMLC II at the cleavage furrow.

Box and whisker plot quantifying the ratio of pMLC II intensity at CF and outside region of CF.

n>15 with >20 cells/experiment. *=p<0.005 as compared to Luc siRNA treated cells (t test), NS

= non significance. F: p190ΔS1C does not rescue the multinucletion induced by depletion of

p190 (n≥300). Error bars represent standard deviation from three independent experiments.

*=p<0.005 (t test) for both p190 siRNA-treated cells and cells rescued by anillin binding mutant

of p190 (p190 ΔS1C) compared to LacZ siRNA-treated cells. G: The p190ΔS1C protein retains

two traditional p190 functions. Cell lines were treated with doxycycline to express GFP tagged

p190 or the Anillin binding mutant of p190 (p190 ΔS1C), lysates were immunoprecipitated with

anti-GFP and blotted for p190 interacting protein p120 and pTyr.

Figure 6: p190 and anillin associate during cytokinesis in a contractility-dependent manner

to regulate MLC II activation.

A: Confocal images of total and pMLC in HeLa cells during cytokinesis. HeLa cells were either

mock-treated or treated with 50µM blebbistatin for 40 min and stained for MLC or pMLC

(Thr18/Ser19). The area outlined in red defines the CF (region of interest -ROI), which was

analyzed for pixel intensity. Images shown are representative of n>30. Scale bars, 5 µm. A’:

Quantitation of specific MLC II activity. The ratio of pMLC II to MLC II intensities was

calculated, and results are expressed as the mean pixel intensity ratio +/- SEM, n>3, with >15

cells/experiment. *=p<0.005 (t test) as compared to mock, which was set to 1.

Figure 7. Model for regulation of RhoGTP through p190RhoGAP/anillin complex.

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