2076 | Q. Wan et al. Molecular Biology of the Cell
MBoC | ARTICLE
Regulation of myosin activation during cell–cell
contact formation by Par3-Lgl antagonism:
entosis without matrix detachment
Qingwen Wana,b,*, Jing Liua,b,c,*, Zhen Zhenga,b, Huabin Zhua,b, Xiaogang Chua,b, Zheng Dongd,
Shuang Huange, and Quansheng Dua,b
aInstitute of Molecular Medicine and Genetics and bDepartment of Neurology, Medical College of Georgia, Georgia
Health Sciences University, Augusta, GA 30912; cMinistry of Education Laboratory of Combinatorial Biosynthesis and
Drug Discovery, Wuhan University School of Pharmaceutical Science, Wuhan 430072, China; dDepartment of Cell
Biology and Anatomy and eDepartment of Biochemistry and Molecular Biology, Medical College of Georgia, Georgia
Health Sciences University, Augusta, GA 30912
ABSTRACT Cell–cell contact formation following cadherin engagement requires actomyosin
contraction along the periphery of cell–cell contact. The molecular mechanisms that regulate
myosin activation during this process are not clear. In this paper, we show that two polarity
proteins, partitioning defective 3 homologue (Par3) and mammalian homologues of Droso-
phila Lethal (2) Giant Larvae (Lgl1/2), antagonize each other in modulating myosin II activa-
tion during cell–cell contact formation in Madin-Darby canine kidney cells. While overexpres-
sion of Lgl1/2 or depletion of endogenous Par3 leads to enhanced myosin II activation,
knockdown of Lgl1/2 does the opposite. Intriguingly, altering the counteraction between
Par3 and Lgl1/2 induces cell–cell internalization during early cell–cell contact formation, which
involves active invasion of the lateral cell–cell contact underneath the apical-junctional com-
plexes and requires activation of the Rho–Rho-associated, coiled-coil containing protein ki-
nase (ROCK)–myosin pathway. This is followed by predominantly nonapoptotic cell-in-cell
death of the internalized cells and frequent aneuploidy of the host cells. Such effects are
reminiscent of entosis, a recently described process observed when mammary gland epithe-
lial cells were cultured in suspension. We propose that entosis could occur without matrix
detachment and that overactivation of myosin or unbalanced myosin activation between
contacting cells may be the driving force for entosis in epithelial cells.
Cell–cell contact formation is initiated by the contact of exploratory
membrane protrusions, which is followed by the formation of cad-
herin clusters through homophilic cadherin interactions (Adams
et al., 1998; Krendel and Bonder, 1999; Nakagawa et al., 2001;
Ehrlich et al., 2002; Vaezi et al., 2002; Yamada and Nelson, 2007).
Cadherin engagement then triggers actin cytoskeleton rearrange-
ment (Krendel and Bonder, 1999; Vasioukhin et al., 2000; Hansen
et al., 2002; Kovacs et al., 2002; Green et al., 2010). Subsequently,
actin polymerization and myosin II–driven contraction of actin bun-
dles along the peripheral cell cortex of cadherin clusters lead to
cell–cell contact expansion (Krendel et al., 1999; Ivanov et al., 2004,
2005; Shewan et al., 2005; Zhang et al., 2005; Yamada and Nelson,
2007; Cavey et al., 2008). The intricacies of the molecular mecha-
nisms that modulate actomyosin contraction during cell–cell contact
formation are poorly understood.
University of Queensland
Received: Nov 22, 2011
Revised: Mar 16, 2012
Accepted: Apr 5, 2012
This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E11-11-0940) on April 11, 2012.
*These authors contributed equally to this work.
Address correspondence to: Quansheng Du (firstname.lastname@example.org).
Abbreviations used: 3-MA, 3-methyladenine; aPKC, atypical protein kinase C;
BSA, bovine serum albumin; CCD, charge-coupled device; DIC, differential inter-
ference contrast; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; GFP, green
fluorescent protein; HBSS, Hank’s balanced salt solution; KD, knockdown; Lgl1/2,
mammalian homologues of Drosophila lethal(2)giant larvae; MDCK, Madin-Darby
canine kidney; MLC2, myosin regulatory light chain 2; MLCK, myosin light chain
kinase; mRFP, monomeric red fluorescent protein; N.A., numerical aperture; Par3,
partitioning defective 3 homologue (Caenorhabditis elegans); Par6, partitioning
defective 6 homologue; PBS, phosphate-buffered saline; ROCK, Rho-associated,
coiled-coil containing protein kinase; shRNA, short hairpin RNA; TEM, transmis-
sion electron microscopy.
© 2012 Wan et al. This article is distributed by The American Society for Cell Biol-
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to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
“ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of
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Volume 23 June 1, 2012 Cell–cell contact and internalization | 2077
of fixed Venus-Lgl2 cells with anti–β-catenin antibody indicated that
those structures were also surrounded by β-catenin (Figure 1B). It is
possible that the observed phenomenon is caused by one cell sit-
ting on top of or being wrapped by the protrusions of another cell.
Alternatively, it could also reflect one cell being internalized by an-
other—a cell-in-cell structure. To explore these possibilities, we per-
formed confocal three-dimensional reconstruction of Venus-Lgl2
cells stained with anti–β-catenin antibody (Figure 1C). Clearly, cells
with brighter Venus-Lgl2/β-catenin signals are inside other cells,
suggesting cell–cell internalization. To determine whether there was
complete cell internalization, we performed cell surface biotinyla-
tion analysis. Indeed, many Venus-Lgl2–surrounded cells were pro-
tected from biotin labeling, suggesting complete internalization
We noticed that Venus-Lgl2–induced cell–cell internalization ap-
peared to happen more frequently between two contacting cells
during the early stages after cells were plated on substrate. To quan-
titate such events, we seeded the cells at a density that favored
paired cell–cell contact and monitored the progress of cell–cell in-
ternalization. Such an assay is referred as a “paired analysis.” We
counted those paired cells that exhibited more than half of one cell
body to be inside the other as internalizing cells, and those that
showed one continuous Venus-Lgl2/β-catenin circle residing in an-
other as completely internalized cells. As shown in Figure 1E, the
cell–cell internalization between paired Venus-Lgl2 cells started as
early as 2 h after plating, peaked at 6–8 h, and gradually dropped
thereafter, suggesting that such cell–cell internalization involves
early cell–cell contact formation. We performed identical paired
analysis for parental MDCK T23 cells and MDCK II cells. While
<20% of paired control cells appeared to be internalizing each
other, complete internalization was rarely observed (Figure 1E). A
similar degree of cell–cell internalization was observed in multiple
independent Venus-Lgl2 cell lines; most importantly, when Venus-
Lgl2 cells were cultured in the presence of doxycycline (+Dox) to
suppress the ectopic expression of Venus-Lgl2, the internalization
rates were inhibited to control levels (Figure 1E), indicating that the
observed cell–cell internalization was caused by ectopic expression
When cells were seeded at a high density, we were not able to
quantitate incomplete internalization, because one cell was usually
in contact with multiple cells, and cell–cell contacts appeared to
be constantly remodeling. However, at 6–8 h after plating, ∼5–7%
(5.5 ± 0.6% at 6 h; 7.2 ± 1.1% at 8 h, n = 2000) of the cells appeared
to be completely internalized by other cells (Supplemental Figure
S1A). This internalization rate was gradually reduced to ∼3% (3.1 ±
0.6%) at 12 h, when some of the internalized cells started to be sur-
rounded by large vacuoles (described in the following section).
Complete cell–cell internalization was not observed when control
MDCK cells or Venus-Lgl2 cells cultured in the presence of doxycy-
cline were plated at high cell density (unpublished data).
Cell–cell internalization was also observed in stable cell lines
overexpressing Lgl1 (Figure S1, B and C), the other mammalian ho-
mologue of Lgl, suggesting that this is a common feature for the Lgl
family of proteins. Furthermore, we could reproduce Lgl-induced
cell–cell internalization by transiently overexpressing Lgl2 in MDCK
cells (Figure S3).
We performed live-cell, time-lapse analysis of paired Venus-
Lgl2 cells after they were plated on culture dishes. As shown in
Figure 1F and Supplemental Movie S1, cell–cell internalization ap-
peared to be achieved by active invasion of one cell starting at the
cell–cell contact region, and complete internalization could occur
within 1 h.
Following initial cell–cell contact formation, polarized epithelial
cells specify cell contact regions into apical and basal-lateral do-
mains through groups of evolutionarily conserved proteins that are
called polarity proteins. These include the apical-junctional parti-
tioning defective 3 homologue/partitioning defective 6 homologue/
atypical protein kinase C (Par3/Par6/aPKC) and Crumbs3/Pals1/Patj
and the basal-lateral Scribble/Lgl/Dlg complexes (Macara, 2004;
Martin-Belmonte and Mostov, 2008). These core polarity complexes
do not function independently, but interact with one another in es-
tablishing and maintaining cell polarization (Macara, 2004). Par3, Lgl
(Lgl1 and Lgl2 in mammals), and Pals1 all bind directly to Par6
(Joberty et al., 2000; Lin et al., 2000; Betschinger et al., 2003; Hurd
et al., 2003; Plant et al., 2003; Yamanaka et al., 2003). Like Par3, Lgl
interacts with and is phosphorylated by aPKC (Izumi et al., 1998;
Joberty et al., 2000; Lin et al., 2000; Betschinger et al., 2003;
Yamanaka et al., 2003). The binding of Par3 and Lgl to Par6 appears
to be mutually exclusive (Plant et al., 2003; Yamanaka et al., 2003,
2006). It is not known whether these polarity proteins are also in-
volved in regulating early cell–cell contact formation.
Cell-in-cell structures, in which an intact cell resides in another
host cell, have been described for decades and are commonly ob-
served in different types of tumors (Overholtzer and Brugge, 2008).
Molecular mechanisms underlying the formation of cell-in-cell struc-
tures are poorly understood. Entosis is a recently described process
that accounts for homotypic cell-in-cell structures—the target cell is
internalized by a host cell of the same type (Overholtzer et al.,
2007). This process was observed in a limited number of normal and
tumor epithelial cell lines grown exclusively in suspension. It was
originally proposed as a tumor-suppressive mechanism, because
entosis frequently results in the death of internalized target cells in
a nonapoptotic pathway (Overholtzer et al., 2007). More recently,
entosis was described as a nongenetic means of generating aneu-
ploidy, which is caused by frequent cytokinesis defects in the host
cells (Krajcovic et al., 2011). Entosis appears to require cadherin-
mediated cell–cell adhesion and the activation of the Rho–Rho-as-
sociated, coiled-coil containing protein kinase (ROCK)–myosin II
pathway (Overholtzer et al., 2007). Whether entosis could occur un-
der adhesive culture conditions and what could drive entosis are
In this paper, we show that entosis can be effectively induced
under adhesive culture conditions in Madin-Darby canine kidney
(MDCK) cells. We identified Lgl and Par3 as critical, antagonistic
regulators of actomyosin contractility during early stages of cell–cell
Overexpression of Lgl led to cell–cell internalization
in MDCK cells
To study the function of mammalian Lgl, we established stable Tet-
Off MDCK cell lines expressing N-terminally Venus-tagged human
Lgl2. As previously reported (Musch et al., 2002; Yamanaka et al.,
2003), ectopically expressed Venus-Lgl2 localizes predominantly to
the cell membrane in unpolarized cells and is restricted to the lateral
cell–cell contact in polarized confluent cells (Figure 1A). Interest-
ingly, when monitoring live cells under a fluorescence microscope,
we frequently observed a bright, circled Venus-Lgl2 signal that ap-
peared to be contained in a cell (Figure 1A). This was seen when
cells were seeded at high density, but more frequently when cells
were plated sparsely (Figure 1A). Differential interference contrast
(DIC) imaging revealed that the Venus-Lgl2–surrounded structures
appeared to contain nuclei. This was confirmed by DNA staining,
suggesting that they were intact cells (Figure 1A). Immunostaining
2078 | Q. Wan et al. Molecular Biology of the Cell
FIGURE 1: Overexpression of Lgl2 led to cell–cell internalization of MDCK cells. (A) Images of MDCK cells stably
expressing Venus-Lgl2 (green). DNA was stained with Hoechst 33342. DIC images are shown on the right. Scale bar:
10 μm. (B) Images of Venus-Lgl2–expressing cells. Fixed cells were stained with anti–β-catenin antibody (red). DNA was
stained with Hoechst 33342. Scale bar: 10 μm. (C) Confocal images of control (left) and internalizing Venus-Lgl2–
expressing cells. Reconstituted y,z (left) and x,z (bottom) views along the indicated lines within each image are
presented. Fixed cells were stained with anti–β-catenin antibody (red). DNA was stained with Hoechst 33342. (D) Surface
biotinylation of Venus-Lgl2 cells. Cells were incubated with biotin (0.5 mg/ml), fixed, and stained with streptavidin–Alexa
Fluor 568 (red). DNA was stained with Hoechst 33342. Scale bar: 10 μm. (E) Quantification of cell–cell internalization.
Paired analysis of control MDCK T23, Venus-Lgl2 cells in the presence of doxycycline (V-Lgl2 (+Dox)) or in the absence
of doxycycline (V-Lgl2 (−Dox)). Cells were fixed at indicated time points after plating on coverglass and were stained
with anti–β-catenin antibody. Paired cells were analyzed. Percentages of half (more than half of one cell body was inside
the other) and complete (the whole cell body of one cell was inside the other) internalization between paired cells were
quantified and represented as different colors in the columns. Data were from three independent experiments (n > 200
for each set of data). Error bars represent SD. (F) Representative images of Venus-Lgl2 cells from a live-cell, time-lapse
analysis. Live cells were stained with Hoechst 33342 (blue). Merged images from green and blue channels are presented.
Time points are presented as minutes:seconds.
2090 | Q. Wan et al. Molecular Biology of the Cell
Proteins were transferred to nitrocellulose membrane and de-
tected using anti-Par3, anti-aPKC, and anti-Par6B antibodies.
MDCK cells grown on poly-l-lysine–coated coverglass were fixed
with 4% paraformaldehyde in PBS and permeabilized with 0.5%
Triton X-100 in PBS. Fixed cells were blocked with 10% normal goat
serum/1% BSA in PBS for 1 h, and then incubated for 1 h with the
primary antibodies. Cells were then washed and incubated for 1 h with
DNA stain Hoechst 33342 and goat anti-mouse or anti-rabbit second-
ary antibodies coupled to Alexa Fluor 488 or Alexa Fluor 594 (Invitro-
gen). Epifluorescent images were taken on a Nikon TE2000 inverted
microscope using a CFI PLAN FLUOR 60×/1.4 numerical aperture
(N.A.) oil-immersion objective and MetaMorph software (Molecular
Devices, Sunnyvale, CA). Confocal images were captured on a Zeiss
510 LSM confocal microscope using a Plan Apochromat 63×/1.4 N.A.
oil-immersion objective (Carl Zeiss, Oberkochen, Germany) and ana-
lyzed using the LSM Image Examiner (Carl Zeiss) and Adobe Photo-
shop software (San Jose, CA).
Quantification of focal adhesions and phosphorylated MLC2
For quantification analysis, all images were taken using identical mi-
croscopic settings. For quantification of focal adhesions, 20 cells in
each group were randomly selected to quantify the number and
average size of focal adhesions using ImageJ software. A particle
analysis was performed on images to select focal adhesions based
on anti-paxillin staining, and the number and size of the particles
were quantified. Similarly, ImageJ software was used to quantify the
fluorescence intensities of cortical pMLC2 (S19) staining.
E-cadherin trypsin protection assay
Cells (4 × 106) were seeded on a 100-mm culture dish. Six hours later
cells were treated with crystalline trypsin (0.05%wt/vol) in Hank’s bal-
anced salt solution (HBSS) in the presence of either 2 mM CaCl2 or
2 mM ethylene glycol tetraacetic acid for 30 min at 37°C before
adding HBSS-Ca2+-fetal bovine serum (FBS; 0.05%) to stop the ac-
tion of trypsin. Cells were collected and lysed directly with 2X sam-
ple buffer. Equal volumes of samples were analyzed by SDS–PAGE,
whiich was followed by Western blot analysis with an antibody di-
rected against the ecto-domain of E-cadherin (DECMA-1). α-Tubulin
was used as a sample loading control.
Live-cell, time-lapse analysis
Live-cell, time-lapse analyses were performed as previously de-
scribed (Du and Macara, 2004). Cells were grown on Delta T dishes
(Bioptechs, Butler, PA) in F10 medium supplemented with 10% FBS
and antibiotics. For visualizing nuclei, 2 μg/ml of Hoechst 33342 was
added to the medium, and the cells were incubated for 5 min. After
several washes, the dish was filled with F10 medium and sealed with
a 40-mm coverslip. The dish was then placed in a temperature con-
trol system (Bioptechs) that maintained a temperature of 37°C.
Time-lapse sequences were collected on a Nikon TE2000 micro-
scope using a CFI Plan Fluor 40×/1.3 N.A. oil-immersion objective,
a CoolSnap CCD camera, and MetaMorph software.
We thank Patrick Brennwald of the University of North Carolina for
the anti-Lgl1 antibody. We also thank Robert Smith of Medical
College of Georgia Electron Microscopy Core for helping with elec-
tron microscopy analysis. This work was supported by grants from
the National Institutes of Health (GM079560) and the American
Cancer Society (RSG0717601CSM) to Q.D.
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