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 $15.00
The Journal of Cell Biology, Vol. 176, No. 5, February 26, 2007 573–580
Cell migration is a highly regulated and coordinated process.
It is comprised of several coupled steps that include polarization,
protrusion, adhesion formation and turnover at the cell front,
and adhesion disassembly and tail retraction at the cell rear.
Many of the major regulatory pathways that control these pro-
cesses are known (Zamir and Geiger, 2001; Ridley et al., 2003;
Carragher and Frame, 2004; Webb et al., 2004). Most converge
on Rho family GTPases, which in turn activate kinases like
PAK or ROCK (Bokoch, 2003; Riento and Ridley, 2003).
Recent studies point to other, analogous pathways that control
protrusion, adhesion dynamics, and cell polarity. Cdc42 acting
on MRCK, which is a kinase that phosphorylates MLC, regulates
nuclear positioning in migrating cells (Gomes et al., 2005). In
addition, PAK localizes to the centrosome, where it plays an
essential role in MTOC positioning (Zhao et al., 2005). MII
is a common effector for all of these pathways, and thus it is
implicated as a key regulator of cell migration.
MII is a bipolar, contractile protein composed of two
myosin heavy chains (MHCs), two regulatory myosin light
chains (MLCs), and two essential MLCs. Each MHC contains
an N-terminal globular motor domain that moves actin as it
hydrolyzes ATP and a C-terminal tail region that binds to the other
MHC (Landsverk and Epstein, 2005). MLC phosphorylation
regulates the ATPase activity of MHC (Adelstein and Conti,
1975; Scholey et al., 1980). In addition to its contractile prop-
erties, MII also cross-links, and thus stabilizes, actin through its
bivalent binding to actin fi laments (Siddique et al., 2005).
In fi broblasts, two major isoforms of MHC-II have been
described, MHC-IIA and -IIB. It is likely that they serve dif-
ferent roles in the regulation of the actin cytoskeleton because of
their different ATPase activities, contraction rates, and subcellular
localization (Kolega, 1998; Kim et al., 2005). Both MIIA and
MIIB mediate stress fi ber formation (Wei and Adelstein, 2000;
Bao et al., 2005). MIIB contributes to cell migration by control-
ling protrusion stability (Lo et al., 2004), and MIIA is implicated
in the regulation of actin retrograde fl ow (Cai et al., 2006).
Although these reports point to the participation of MII
and its isoforms in migration, the mechanisms by which it controls
and integrates its component processes are unclear. In this report,
we reveal the integrative role of MII in migration and parse its
isoform-dependent and contraction-independent activities. From
these studies, MII emerges as a central, regulatory molecule that
serves to integrate and coordinate diverse migration-related
phenomena that comprise migration.
Results and discussion
MIIA and MIIB exert differential effects
on polarity and tail retraction
Previous observations have shown the differential cellular locali-
zation of MII isoforms. In general, MIIA is present in regions
Regulation of protrusion, adhesion dynamics, and
polarity by myosins IIA and IIB in migrating cells
Miguel Vicente-Manzanares, Jessica Zareno, Leanna Whitmore, Colin K. Choi, and Alan F . Horwitz
Department of Cell Biology, University of Virginia, Charlottesville, VA 22908
processes that drive cell migration. Both isoforms reside
outside of protrusions and act at a distance to regulate
cell protrusion, signaling, and maturation of nascent
adhesions. MIIA also controls the dynamics and size of
adhesions in central regions of the cell and contributes
to retraction and adhesion disassembly at the rear.
In contrast, MIIB establishes front–back polarity and
e have used isoform-specifi c RNA inter-
ference knockdowns to investigate the roles of
myosin IIA (MIIA) and MIIB in the component
centrosome, Golgi, and nuclear orientation. Using ATPase-
and contraction-defi cient mutants of both MIIA and
MIIB, we show a role for MIIB-dependent actin cross-
linking in establishing front–back polarity. From these
studies, MII emerges as a master regulator and integrator
of cell migration. It mediates each of the major compo-
nent processes that drive migration, e.g., polarization,
protrusion, adhesion assembly and turnover, polarity,
signaling, and tail retraction, and it integrates spatially
Correspondence to Miguel Vicente-Manzanares: email@example.com
Abbreviations used in this paper: MHC, myosin heavy chain; MII, myosin II; MLC,
myosin light chain.
The online version of this article contains supplemental material.
JCB • VOLUME 176 • NUMBER 5 • 2007 574
distal to MIIB, and MII is largely absent from the lamellipodium
of epithelial cells (Kolega, 1998; Gupton and Waterman-Storer,
2006). We have confi rmed these observations using migrating
CHO.K1 cells and reveal novel details (Fig. S1, available
at http://www.jcb.org/cgi/content/full/jcb.200612043/DC1), as
follows: (a) the two isoforms often decorate the same actin fi la-
ments in a stippled manner, suggesting that some functions
might result from additive activities; (b) MIIA and MIIB likely
mediate distinct functions because the two isoforms also occupy
distinct areas, and therefore do not readily form cofi laments;
and (c) MII resides well away from nascent adhesions; therefore,
any effect on adhesion dynamics would result from an indirect
rather than a local effect.
To determine whether the spatial segregation of MIIA and
MIIB results in different roles during cell migration, we generated
knockdown vectors that inhibit MIIA and MIIB expression with
high specifi city (Fig. 1, A and B). For both isoforms, down-
regulation was comparable and maximal 96 h after transfection,
where it averaged 75–95% by immunoblotting, depending on
transfection effi ciency (Fig. 1 A). Immunofl uorescence revealed
>95% knockdown in individual cells (Fig. 1 B).
When plated using migration-promoting conditions (see
Materials and methods), MIIA-defi cient cells exhibited broader
lamellipodia than control cells and did not retract their trailing
edge (Fig. 1 C and Video 1, available at http://www.jcb.org/cgi/
content/full/jcb.200612043/DC1). This resulted in cells with
extended tails (Fig. 1 C, arrowheads). This phenotype is remi-
niscent of the effect of Rho-kinase inhibitors in macrophages
(Worthylake et al., 2001) and over expression of a paxillin mu-
tant with the LD4 domain deleted (West et al., 2001).
In contrast, MIIB-defi cient cells were round and occupied
a large area without distinguishable leading and trailing edges
(Fig. 1, D and F–H; and Videos 2 and 4, available at http://www
.jcb.org/cgi/content/full/jcb.200612043/DC1), e.g., front–back
polarity. Depletion of MIIA or MIIB in Rat2 fi broblasts yielded
similar results (Fig. S2). This phenotype differs somewhat from
that reported for MEFs from MIIB−/− knockout mice (Lo et al.,
2004). Although both showed inhibited migration, the MIIB−/−
MEFs also showed long, unstable protrusions. This could arise
either from incomplete ablation of MIIB by the RNAi knockdown
or an uncharacterized adaptation. Off-target effects of the RNAi
seem unlikely because our phenotypes were rescued by ectopic
expression of RNAi-insensitive MIIA or MIIB, respectively
(Fig. 2 E and not depicted).
In addition to the round morphology, the MIIB-defi cient
cells also showed a defect in nuclear, centrosomal, and Golgi
anchoring. More than 95% of the nuclei in the knockdown
cells rotated clockwise (?2 h/cycle; Fig. 1 H and Video 4).
Figure 1. Knockdown of MIIA or MIIB differentially alters cell polarity. (A) CHO.K1 cells were transfected with pSUPER-GFP vector or pSUPER-GFP-
RNAi against MIIA or MIIB, and blotted for MIIA or MIIB. GIT1 is a loading control. (B) Representative images of MIIA- (top) and MIIB-depleted
cells (bottom) stained for MIIA or MIIB, respectively. Arrows point to transfected cells. (C–E) Time-lapse series of MIIA-depleted (C; Video 1), MIIB-
depleted (D; Video 2), or control cells (E; Video 3). In C, arrowheads point to the defect in tail retraction. (D) Arrows point to transfected, unpolarized
cells. Videos are representative of >25 cells in 6 independent experiments. (F) Fluorescence images depicting the enlargement of MIIB-defi cient cells.
Images are representative of >300 cells. (G; top) Effect of MIIB knockdown on cell area. Data are the mean ± the SD of 4 independent experi-
ments comprising >300 cells/experiment. (bottom) Cell areas in control and MIIB-depleted cells. (H) MIIB-defi cient cell showing clockwise rotation of
the nucleus. Arrowhead points to nucleolus; top-right indicator, clockwise angular displacement (Video 4). (I) Depolarization of the Golgi is observed
in MIIB-defi cient cells (arrows) and not in nontransfected cells (arrowheads). Bars: (C) 40 μm; (D–F and H) 50 μm. Videos 1–4 are available at
MYOSIN II IN PROTRUSION, POLARITY, AND ADHESION • VICENTE-MANZANARES ET AL.575
The centrosome accompanied this rotation, and the Golgi appa-
ratus was distributed around the nucleus rather than polarized,
as observed in nontransfected, migrating cells (Fig. 1 I and
not depicted), suggesting a more general role of MIIB in cell
polarization. Although the origin of this nuclear rotation is not
known, it suggests that MIIB is part of a balanced mechanism
of nuclear anchoring.
MII depletion increases cell protrusion and
inhibits maturation of nascent adhesions
at the leading edge
The increased area of MIIB-defi cient cells and the broader
lamellipodia in MIIA-defi cient cells pointed to alterations in
protrusion. By kymography, MIIA- and MIIB-defi cient cells
exhibited 2–3-fold increased rates of protrusion (Fig. 2, A–B).
In addition, the protrusion was continuous, resulting in kymograms
that showed a near linear, uninterrupted slope (Fig. 2 A, bottom).
In contrast, wild-type cells often showed an interrupted, step-
wise pattern, as previously reported (Giannone et al., 2004).
Interestingly, during these periodic interruptions in protrusion, the
adhesions stabilized and grew as MII began to localize in the pre-
viously protrusive region (Video 5, available at http://www.jcb
.org/cgi/content/full/jcb.200612043/DC1), suggesting a causal
link. Finally, when the protrusions in MIIA- and MIIB-defi cient
cells stopped advancing, they did not retract effi ciently (Videos
6 and 8), suggesting that both MII isoforms regulate retraction
of the lamellipodium. Thus, both MII isoforms control the speed,
stepwise pattern of extension, and retraction of protrusions.
To determine whether the abnormal protrusion is accom-
panied by alterations in adhesion dynamics, we knocked down
MIIA or MIIB in paxillin-GFP– (Fig. 2 C and Videos 6–8, avail-
able at http://www.jcb.org/cgi/content/full/jcb.200612043/DC1)
or vinculin-GFP–expressing cells (not depicted). Control cells
showed numerous well-defi ned adhesions in protrusions, as
well as some small adhesions near the leading edge (Fig. 2 C, top;
and Video 7) that assembled and turned over as described previ-
ously (Webb et al., 2004). In contrast, MIIA- and MIIB-defi cient
cells showed few discrete adhesions in the protrusions; instead,
Figure 2. MIIA and IIB regulate protrusion and differentially control adhesion turnover. (A; top) Kymographs from control (pSUPER), MIIA-depleted (pSUP-IIA),
and MIIB-depleted (pSUP-IIB) cells. (bottom) Overlay of periodicity and slope from the kymographs. (B) Protrusion rates from A. At least 12 cells (3–5
protrusions/cell) from four independent experiments were analyzed. Data is presented as the mean ± the SEM. (C) Image sequence of control (top; Video 7)
and MIIA- (middle; Video 6) and MIIB-depleted cells (bottom; Video 8) cotransfected with paxillin-GFP. (D) Color-inverted image sequence of MIIA- depleted
cell expressing paxillin-GFP (Video 9). Time is shown in seconds. (E) Image sequence of paxillin-GFP–expressing, MIIA- (top) or MIIB-depleted (bottom) cells
cotransfected with mChe-MIIA or mChe-MIIB, respectively (not depicted). (F) Image sequence of paxillin-GFP–expressing, MIIA-depleted cell. Arrowheads
point to central adhesions; arrows point to impaired disassembly at the trailing edge (Video 10). (G) Image sequence of paxillin-GFP in a MIIB-depleted
cell (left) expressing mChe-IIA (not depicted) and a MIIA-depleted cell (right) expressing mChe-IIB (not depicted). Bars: (C) 5 μm; (D) 3 μm; (E) 5 μm;
(F) 20 μm; (G) 5 μm. Videos 6–10 are available at http://www.jcb.org/cgi/content/full/jcb.200612043/DC1.
JCB • VOLUME 176 • NUMBER 5 • 2007 576
Figure 3. Contractility-defi cient mutants of MIIA and MIIB exhibit differential rescue of actin bundling, protrusion, and adhesion dynamics in MIIA- and
MIIB-defi cient cells. Inhibited FRAP of GFP-MIIA N93K (A) and GFP-MIIB R709C (B) in actin bundles. Data are the mean ± the SD of >20 individual measure-
ments from four independent experiments. (C) Differential FRAP of GFP-MIIA-WT and GFP-MIIB-WT in actin bundles. (D) A MIIB-depleted cell cotrans-
fected with paxillin-GFP and mChe-MIIB R709C (not depicted). (E) Localization of GFP-MIIB and GFP-MIIB R709C. Arrows show the direction of migration.
(F) Polarity index of migrating CHO.K1 cells. Data are the mean ± the SD of >100 cells analyzed/condition. (G) Time-lapse series of a MIIA-depleted cell
expressing paxillin-GFP and mChe-MIIA (not depicted). Bars: (D) 3 μm; (G) 15 μm.
MYOSIN II IN PROTRUSION, POLARITY, AND ADHESION • VICENTE-MANZANARES ET AL. 577
there was a nearly continuous band of adhesions very close to
the leading edge (Fig. 2 C, and Videos 7 and 8). The small indi-
vidual adhesions that comprise this band were readily apparent
at higher magnifi cation (Fig. 2 D, Fig. 4, and Video 9). These
adhesions disassembled and reformed rapidly (t1/2 < 15 s) as
the leading edge progressed (Fig. 2, C and D; and Videos 7–9).
More importantly, they did not evolve into larger adhesions when
lamellipodial growth halted. The defects were rescued when
RNAi-insensitive mCherry (mChe)-MIIA or -MIIB were ex-
pressed in MIIA- or MIIB-defi cient cells, respectively (Fig. 2 E).
It is interesting that the phenotypes of MIIA- and MIIB-
defi cient cells on protrusion and the dynamics of adhesions in
protrusions were almost indistinguishable because MIIA and
MIIB occupy different regions of the cell, and neither is present
in protrusions. This suggests that MII “acts at a distance”; that
is, MII activity at the base of the lamellipodium or in central
regions is transmitted to the leading edge, presumably through
actin fi laments, and generates periodic contractions that coincide
with cessation of protrusion and retraction, adhesion maturation,
and the movement (sliding) of larger adhesions toward the
center of the cell. It also suggests that myosin activity regulates
the behavior of adhesions at the leading edge, regardless of the
isoform. Finally, these observations support the notion that
protrusion and adhesion turnover are coupled.
MIIA promotes the growth of adhesions
in central regions and disassembly
at the trailing edge
Because both isoforms of MII regulate adhesion dynamics at
the leading edge, but only MIIA inhibits rear retraction, we
investigated the effect of MIIA and MIIB knockdowns on adhe-
sions in other cellular regions. MIIB-defi cient cells exhibit cen-
tral adhesions comparable to those in control cells (unpublished
data). In contrast, MIIA-defi cient cells showed abnormally
small, but static, adhesions in the central region of the cell (Fig.
2 F and Video 10, available at http://www.jcb.org/cgi/content/
full/jcb.200612043/DC1). At the cell rear, where MIIA inhibits
retraction, adhesion disassembly is greatly inhibited, e.g., the
adhesions slide slowly and do not disassemble (Fig. 2 F and
Video 10), thereby showing that adhesion sliding and disassembly
are coordinately regulated by MIIA.
MIIA is required for the effects of MIIB
on nascent, but not central, adhesions
Because both MIIA and MIIB mediate contraction and actin
bundling (Kelley et al., 1996), we used cross-rescue experi-
ments to determine whether their functions were overlapping in
the regulation of adhesion assembly and disassembly. First,
mChe-MIIA was expressed in MIIB-depleted cells coexpressing
paxillin-GFP. mChe-MIIA localized in regions very similar
to those in unperturbed cells (not depicted), and it restored the
maturation of nascent adhesions (Fig. 2 G, left). However, the po-
larity defects and the appearance of multiple protrusions around
the cell periphery remained (not depicted).
We then expressed mChe-IIB in MIIA-defi cient cells.
mChe-MIIB localized in the central areas of the cell, as it does
in unperturbed cells. However, it did not rescue the inhibited
maturation of nascent adhesions induced by the MIIA knockdown.
Instead, a band of small, dynamic adhesions remained near
the leading edge, as in MIIB knockdowns (Fig. 2 G, right). In
contrast, mChe-MIIB rescued the effect on the central adhesions,
i.e., they were larger (Fig. 2 G, top right).
Thus, increased MIIB in central areas (where endogenous
MIIB resides) of MIIA knockdown cells rescues the maturation
of adhesions in the central regions of the cell, but not the nascent
adhesions at the cells periphery. In contrast, increased MIIA in
MIIB knockdown cells rescues the maturation of adhesions at
the leading edge. This points to a mechanism in which the cen-
tral MIIB activity requires MIIA for its translation to the periphery,
perhaps by organizing the actin so that tension produced in the
middle of the cell propagates into protrusions. This suggestion
is supported by our ob servation that overexpressed MIIA in wild-
type cells localizes primarily in actin bundles and produces
more and larger adhesions (Fig. S3, available at http://www.jcb
.org/cgi/content/full/jcb.200612043/DC1), indicating that MII
activity can dial up or down adhesion assembly, depending on
its expression level.
Adhesion maturation at the leading edge
depends on the ATPase activity of MIIA
To separate the bundling from the contractile functions of MII
on cell migration, we produced ATPase-inhibited mutants of
MIIA and MIIB fused to GFP and mChe. The ATPase activity
of N93K-MIIA and R709C-MIIB are inhibited 80 and 75%,
respectively, in vitro (Heath et al., 2001; Ma et al., 2004). However,
both mutants bind and cross-link, but do not move, actin fi la-
ments in vitro (Kim et al., 2005).
We used FRAP to show that the mutants exhibit increased
time in the actin-bound state, as expected from their inhibited
ATPase cycling. Both MIIA N93K and MIIB R709C showed
decreased rates and fractional recoveries (Fig. 3, A–B). The
fractional recovery observed for both mutants was the same,
suggesting that the two isoforms bind actin similarly. Interest-
ingly, wild-type MIIA exhibited faster and higher fractional
recovery than MIIB (Fig. 3 C). This suggests that MIIA is more
active than MIIB, and therefore binds actin strongly in a smaller
fraction of time, as shown previously in vitro (Kelley et al.,
1996). It also points to the use of FRAP as a method to determine
MII activity in living cells.
When expressed in MIIB-defi cient cells, MIIB R709C did
not effectively restore adhesion maturation (Fig. 3 D). However,
MIIB R709C partially restored the front–back polarity and
localized at the back of the cell (Fig. 3, E and F). Thus, maturation
of adhesions at the leading edge requires MIIB activity; but
its role in determining front– back polarity suggests a cross-
In contrast, MIIA N93K localized like its wild-type counter-
part (unpublished data), rather than the rearward localization of
MIIB R709C, and did not rescue the increased protrusiveness
observed in MIIA-defi cient cells. However, it did restore
leading edge retraction and the concomitant growth of adhesions
in protrusions pointing to its actin-binding function in these
activities (Fig. 3 G).
JCB • VOLUME 176 • NUMBER 5 • 2007 578
MII regulates adhesive signaling
Adhesive signaling through integrin receptors both stimulates
and responds to tension through Rho GTPases, thus constituting
a feedback loop connecting adhesion and contraction through
MII regulation (Chrzanowska-Wodnicka and Burridge, 1996).
The phosphorylation of paxillin on Y31, Y118, and S273 and the
phosphorylation of FAK on Y397 are part of this signaling mecha-
nism and serve as markers for the activation of this pathway (Katz
et al., 2003; Zaidel-Bar et al., 2003; Nayal et al., 2006).
The small dynamic adhesions near the leading edge of
MIIA- and MIIB-depleted cells were prominently phosphoryl-
ated on tyrosine (Fig. 4, A–C). They also stained positively for
Y397-FAK and Y31-paxillin, defi ning an almost continuous
band of adhesions (Fig. 4, B and C). The staining of these
phospho markers decreased in the stable adhesions that reside
in regions removed from the leading edge (Fig. 4 B). Thus,
depletion of myosin function at the lamellipodium enhances an
adhesive signaling pathway that regulates adhesion turnover
and MII activity, providing a mechanistic link between myosin-
generated tension in the control of adhesion maturation at the
The complex interplay between myosin-mediated contraction,
protrusion, adhesion, and polarization underscores the central
role of MII and its integrative properties in cell migration.
Although the protrusion rate is determined by factors that
regulate actin polymerization (Pollard and Borisy, 2003), it is
also infl uenced by the rate of retrograde fl ow, which, in turn, is
regulated by MII activity and serves to counterbalance actin
polymerization (Lin and Forscher, 1995; Mitchison and Cramer,
1996). The retrograde fl ow rate is also infl uenced by adhesion,
through a clutch-like mechanism, which links actin fi laments
to the substratum and can inhibit retrograde fl ow (Mitchison
and Kirschner, 1988; Lin et al., 1994; Smilenov et al., 1999).
The net protrusion rate is also infl uenced by cycles of retrac-
tion and adhesion maturation at the leading edge. Highly mo-
tile cells protrude and move nearly continuously (Bear et al.,
2002; Jurado et al., 2005), whereas other cells can show cycles
of protrusion and retraction (Giannone et al., 2004).
MII is also involved in a feedback loop that links adhesion,
protrusion, and tension. Adhesion initiates signaling through
Rho family GTPases that leads to the formation of adhesions
and protrusions and generates tension. Tension also acts on
adhesions to promote their maturation and the formation of actin
fi lament bundles (Bershadsky et al., 2006). Highly motile cells
tend to have small, highly dynamic adhesions that turnover rapidly,
whereas the adhesions in slower moving cells stabilize and grow
in response to increased tension before turning over (Nayal et al.,
2006). Signaling components, such as phosphorylated paxillin
and PAK, localize in the small, dynamic adhesions near the
leading edge, where they function in a signaling pathway that
inhibits adhesion maturation and promote protrusion (Nayal
et al., 2006). Interestingly, in retracting regions, MII mediates
the disassembly, rather than the assembly, of adhesions.
Finally, MII polarizes and connects spatially segregated
activities. Myosin acts “at a distance” in regulating protrusion
and adhesion. It also contributes to the overall polarity of the
migrating cell and establishes front and rear. The former is through
the role of MII in orienting microtubules, Golgi, and the nucleus,
and the latter is through actin bundling at the rear and sides
(Xu et al., 2003).
Thus, MII functions as a master regulator of cell migration.
It can integrate spatially separated processes, and it is a key
effector of signaling pathways that regulate each of the major
component processes that drive migration.
Materials and methods
To generate MIIA and MIIB siRNA, the oligonucleotides G A T C T G A A C T C C-
T T C G A G C (IIA) and G G A T C G C T A C T A T T C A G G A (IIB) were inserted into
the appropriate pSUPER cassette according to the vector manufacturer’s
instructions (OligoEngine). The siRNA sequences correspond to nt 1,396–
1,414 and 506–524 of rat MIIA (NM_013194) and MIIB (NM_031520),
GFP-MIIA and GFP-MIIB were gifts from R.S. Adelstein (National
Institutes of Health, Bethesda, MD; Wei and Adelstein, 2000). Where indi-
cated, GFP was replaced by mChe, which was obtained from R. Tsien
(University of California, San Diego, La Jolla, CA; Shaner et al., 2004). siRNA-
insensitive MIIB was generated by site-directed mutagenesis (Quick Change
kit; Stratagene) introducing a silent mutation (TCA → AGC = Ser → Ser)
in the RNAi target region of human MIIB. The MIIB R709C and MIIA
N93K mutants were generated by site-directed mutagenesis using the
Antibodies and reagents
The following antibodies were used: MIIA, MIIB, and GIT1 (rabbit, pAb)
were purchased from Covance; paxillin (mouse, IgG1) was obtained from
BD Biosciences; α-actinin (mouse, IgM) was purchased from Sigma-
Aldrich; phosphotyrosine 4G10 (mouse, Ig2b) was obtained from Millipore;
phosphoTyr31-paxillin (rabbit, pAb) was purchased from BioSource; and
phosphoTyr397-FAK (rabbit, pAb) was obtained from CHEMICON Inter-
national, Inc. Bodipy FL C5-ceramide (for Golgi detection) was obtained
from Invitrogen and used as described by the manufacturer.
Figure 4. Adhesive signaling near the leading edge of MII-depleted cells.
MIIA- or MIIB-depleted or control cells were plated on fi bronectin and then
fi xed and stained for phosphotyrosine (A), phosphoTyr397-FAK (B), and
phosphoTyr31-paxillin (C). Bar, 10 μm.
MYOSIN II IN PROTRUSION, POLARITY, AND ADHESION • VICENTE-MANZANARES ET AL. 579
Cell culture and transfection
CHO-K1 cells and Rat2 cells were cultured in standard conditions and
transfected using Lipofectamine (Invitrogen). For cotransfection experi-
ments, plasmids containing the siRNA sequences were used in 10:1 excess
to GFP or mChe-containing plasmids to ensure knockdown in fl uorescence-
Cells were allowed to adhere to 2 μg/ml fi bronectin-coated coverslips for
60 min, fi xed using 4% paraformaldehyde, and permeabilized with either
0.5% Triton X-100 for 5 min or ice-cold methanol for 10 min. Coverslips
were incubated with primary antibodies and a species-appropriate sec-
ondary antibody coupled to either Alexa Fluor 488 or 568 (Invitrogen).
Microscopy and image processing
Cells were plated on 2 μg/ml fi bronectin–coated glass-bottomed dishes
(migration-promoting conditions) in CCM1 for 1 h and maintained at 37°C
at pH 7.4. For phase analyses, time-lapse images were captured at 10 min
(NA 0.50; Nikon) with a charge-coupled device camera (Orca II; Hamamatsu)
attached to an inverted microscope (TE-300; Nikon) using Metamorph
software (Universal Imaging Corp.). Time is in minutes unless otherwise
indicated. For assessment of cell polarity, the polarity index was calculated
as the length of the major migration axis (parallel to the direction of movement)
divided by the length of the perpendicular axis that intersects the center of
the cell nucleus.
Confocal images were collected on a FluoView 300 system
(60×/1.45 NA [oil] PlanApo 60× OTIRFM objective [all Olympus]). GFP
and RFP were excited using the 488-nm laser line of an Ar ion laser and
the 543-nm laser line of a He-Ne laser (Melles Griot), respectively.
A Q500LP dichroic mirror (Chroma Technology Corp.) was used for GFP-
labeled cells. For dual-color GFP-RFP imaging, a green–red cube
(488/543/633) with a DM570 dichroic mirror (Chroma Technology
Corp.) was used. Fluorescence and differential interference contrast
images were acquired using FluoView software (Olympus).
TIRF images were acquired in an inverted microscope (IX70; Olympus).
The excitation laser lines used were as described for confocal microscopy.
A dichroic mirror (HQ485/30) was used for GFP-labeled cells. For
dual GFP-RFP, a dual-emission fi lter (z488/543) was used. Images were
acquired with a charge-coupled device camera (Retiga Exi; Qimaging)
and analyzed using Metamorph.
Protrusion parameters were quantifi ed using kymography (Hinz et al.,
1999). Images were captured every 5 s for 3 min. Kymographs were gen-
erated using Metamorph software along 1-pixel-wide regions oriented in
the protrusion direction and perpendicular to the lamellipodial edge.
Confocal images for FRAP analysis were acquired using the FluoView
system. Initially, a cellular area (34.72 μm2) that contained GFP-MII–
decorated stress fi bers was scanned 3 times and bleached using 15 scans
at 100% laser power. To image the FRAP, we did 15 scans every 0.1 s,
15 scans every 3 s, 14 scans every 5 s, and 2 scans every 10 s. Background
subtraction and normalization were calculated, and normalized intensity
versus times were fi tted by a single exponential equation (R2 > 0.98).
Online supplemental material
Fig. S1 shows the spatial localization of MIIA and MIIB in a migrating cell.
Fig. S2 shows the migratory phenotypes of MIIA-depleted, MIIB-depleted,
and control Rat2 fi broblasts. Fig. S3 shows that MIIA expression levels
affect the number of adhesions. Video 1 (corresponding to Fig. 1 C) is a
time-lapse video of MIIA-defi cient CHO.K1 cells. Video 2 (corresponding
to Fig. 1 D) is a time-lapse video of MIIB-defi cient CHO.K1 cells. Video 3
(corresponding to Fig. 1 E) is a time-lapse video of pSUPER-transfected
(control) CHO.K1 cells. Video 4 (corresponding to Fig. 1 H) is a time-lapse
video of a MIIB-defi cient CHO.K1 cell, highlighting nuclear spinning.
Video 5 is a dual-color TIRF time-lapse video of a protrusion of a
MIIB-defi cient CHO.K1 cell cotransfected with mChe-MIIB (magenta) and
paxillin-GFP (green). Video 6 (corresponding to Fig. 2 C) is a TIRF time-
lapse video of a protrusion of a MIIA-defi cient CHO.K1 cell cotransfected
with paxillin-GFP. Video 7 (corresponding to Fig. 2 C) is a TIRF time-lapse
video of a protrusion of a pSUPER-transfected control CHO.K1 cell
cotransfected with paxillin-GFP. Video 8 (corresponding to Fig. 2 C) is a TIRF
time-lapse video of a protrusion of a MIIB-defi cient CHO.K1 cell cotrans-
fected with paxillin-GFP. Video 9 (corresponding to Fig. 2 D) is a TIRF
time-lapse video of a protrusion of a MIIA-defi cient CHO.K1 cell cotrans-
fected with paxillin-GFP. Video 10 (corresponding to Fig. 2 F) is a TIRF
time-lapse video of a MIIA-defi cient CHO.K1 cell cotransfected with paxillin-
We thank Bob Adelstein and Roger Tsien for providing reagents, and Bob
Adelstein, Meg Titus, and Rex Chisholm for useful discussions. We also thank
Bob Adelstein for valuable comments on the manuscript.
This work was supported by National Institutes of Health grant
Submitted: 7 December 2006
Accepted: 22 January 2007
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