7/31/2019 yaap

1/11

O R I G I N A L P A P E R

Phosphatidylinositol 30-kinase signalling supports cell heightin established epithelial monolayers

Angela Jeanes Michael Smutny

Joanne M. Leerberg Alpha S. Yap

Received: 17 December 2009/ Accepted: 31 January 2010/ Published online: 16 February 2010

Springer Science+Business Media B.V. 2010

Abstract Cellcell interactions influence epithelial mor-

phogenesis through an interplay between cell adhesion,trafficking and the cytoskeleton. These cellular processes

are coordinated, often by cell signals found at cellcell

contacts. One such contact-based signal is the phosphati-

dylinositol 30-kinase (PI3-kinase; PI3K) pathway. PI3-ki-

nase is best understood for its role in mitogenic signalling,

where it regulates cell survival, proliferation and differ-

entiation. Its precise morphogenetic impacts in epithelia

are, in contrast, less well-understood. Using phosphoino-

sitide-specific biosensors we confirmed that E-cadherin-

based cellcell contacts are enriched in PIP3, the principal

product of PI3-kinase. We then used pharmacologic

inhibitors to assess the morphogenetic impact of PI3-kinase

in MDCK and MCF7 monolayers. We found that inhibiting

PI3-kinase caused a reduction in epithelial cell height that

was reversible upon removal of the drugs. This was not

attributable to changes in E-cadherin expression or homo-

philic adhesion. Nor were there detectable changes in cell

polarity. While Myosin II has been implicated in regulating

keratinocyte height, we found no effect of PI3-kinase

inhibition on apparent Myosin II activity; nor did direct

inhibition of Myosin II alter epithelial height. Instead, in

pursuing signalling pathways downstream of PI3-kinase we

found that blocking Rac signalling, but not mTOR, reduced

epithelial cell height, as did PI3-kinase inhibition. Overall,

our findings suggest that PI3-kinase exerts a major mor-

phogenetic impact in simple cultured epithelia through

preservation of cell height. This is independent of potential

effects on adhesion or polarity, but may occur through PI3-kinase-stimulated Rac signaling.

Keywords Epithelia PI3-kinase Cell height

E-cadherin

Introduction

Epithelial cells come in many different shapes and sizes:

their precise morphologies have wide-reaching implica-

tions for tissue physiology and pathology. In simple

transporting epithelia, such as those that line many mucosal

barriers of the body, cells seal their paracellular pathways

by assembling specialized cellcell junctions and establish

polarized apical and basolateral membrane domains that

are necessary to support vectorial transport (Diamond

1977; Rodriguez-Boulan and Nelson 1989). Such surface

specialization is complemented by reorganization of the

cytoskeleton and organelles within the cells (Rodriguez-

Boulan and Nelson 1989).

Analysis in cell culture has identified cellcell contact as

an important step that triggers the biogenesis of many

facets of the definitive epithelial phenotype (Vega-Salas

et al. 1987a, b; Fleming et al. 2000). Epithelial cell struc-

ture is induced and maintained by interplay between cell

adhesion, the cytoskeleton, membrane traffic and cell

polarity (Yeaman et al. 1999; OBrien et al. 2002). In turn,

these cellular processes are coordinated by a range of

signalling pathways, including signals that are active at

cellcell contacts themselves (Wheelock and Johnson

2003; Yap and Kovacs 2003).

Phosphoinositides have recently emerged as major reg-

ulators of cell polarity, morphology and the cytoskeleton

A. Jeanes M. Smutny J. M. Leerberg A. S. Yap (&)

Division of Molecular Cell Biology, Institute for Molecular

Bioscience, University of Queensland, Brisbane, QLD 4072,

Australia

e-mail: a.yap@uq.edu.au; a.yap@imb.uq.edu.au

123

J Mol Hist (2009) 40:395405

DOI 10.1007/s10735-010-9253-y

7/31/2019 yaap

2/11

(Weiner et al. 1999, 2002; Wang et al. 2002; Gassama-

Diagne et al. 2006; Martin-Belmonte et al. 2007). Polarized

epithelial cells characteristically show domain-specific

differences in their distribution of specific phosphoinosi-

tides. For example, phosphatidylinositol-4,5-P2 (PIP2) is

reported to accumulate most prominently at the apical

membranes of MDCK cells grown in 3-dimensional cysts

(Martin-Belmonte et al. 2007), whereas phosphatidylino-sitol-3,4,5-P3 (PIP3) concentrates at cellcell contacts

(Watton and Downward 1999; Gassama-Diagne et al.

2006). Moreover, some of the enzymes responsible for

either the generation (phosphorylation) or metabolic turn-

over (dephosphorylation) of these signals are recruited to

the cell surface in a fashion that would place them well to

stringently control the morphogenetic expression of spe-

cific phosphoinositides (Watton and Downward 1999). Of

note, Type 1A Phosphatidylinositol 30-kinase (PI3-kinase;

PI3K) can associate with E-cadherin and be activated by

cadherin homophilic adhesion (Pece et al. 1999; Kovacs

et al. 2002a), suggesting that it may be one key signal thatis activated by cellcell adhesion to influence epithelial

morphogenesis.

PI3-kinase mediates signal transduction in response to

a wide range of cell surface receptors (Rameh and

Cantley 1999; Vanhaesebroeck et al. 2001). It is best

understood for its role in mitogenic signalling down-

stream of growth factor receptors, where it is implicated

in cell survival, proliferation and differentiation (Fruman

et al. 1999; Calautti et al. 2005; Halet et al. 2008).

However, it is becoming increasingly clear that PI3-

kinase also influences cellular morphogenesis in many

different tissues. In migrating cells the localized genera-

tion of PIP3 by PI3-kinase serves to regulate the actin

cytoskeleton and contributes to anterior-posterior polari-

zation necessary for productive translocation (Weiner

et al. 1999, 2002; Chung et al. 2001). Modulation of the

PI3K-PIP3-PTEN signalling pathway has previously been

linked to the control of cell size and shape in myocytes of

hypertrophic hearts (Luo et al. 2005) and during Dro-

sophila development (Goberdhan et al. 1999). Finally,

PI3-kinase can affect cadherin adhesion (Kovacs et al.

2002a), and its major lipid product, PIP3, was also shown

to specify basolateral membrane identity in MDCK cells

(Gassama-Diagne et al. 2006), both important processes

in controlling cell morphology.

In this study we sought to further analyse the morpho-

genetic impact of PI3-kinase signalling in simple polarized

epithelia. We report that acute inhibition of PI3-kinase

signalling in established epithelial monolayers caused

reduced cell height, which was independent of junctional

integrity, cadherin adhesion and epithelial cell polarity. We

also identify Rac signalling as a further signal implicated in

the maintenance of epithelial cell height.

Materials and methods

Cell culture

MDCK and MCF7 cells were maintained in DMEM, CHO

cells were maintained in F12 medium. CHO cells stably

expressing human E-cadherin were described previously

(Kovacs et al. 2002a, b). All media were supplementedwith 10% FBS, 1% non-essential amino acids, L-glutamine,

and Penicillin/Streptomycin. Cells treated with inhibitor

drugs were given fresh medium 1 day before treatment,

and inhibitors were added directly to the medium. Tran-

sient transfection of MDCK cells was carried out with

Lipofectamine2000 (Invitrogen), according to the manu-

facturers instructions, except that cells were between 25

and 35% confluent at the time of transfection. Cells were

grown to 100% confluence before being fixed for indirect

immunofluorescence.

Reagents

The expression plasmids pEGFP-N1-PHGrp1 and pEGFP-

N1-PHPLCd, were a kind gift from Dr Mark Lemmon and

have been described previously (Kovacs et al. 2002a) and

pEGFP-C1 was from Clontech. Inhibitors: LY294002 (final

concentration of 50 lM), wortmannin (100 nM), blebbist-

atin (100 lM), Y-27632 (50 lM) and rapamycin (1

100 nM) were purchased from Calbiochem, NSC-23766

(20200 lM) was purchased from Tocris.

Antibodies were as follows. For immunofluorescence

microscopy: E-cadherin mAB (Transduction Laboratories);

E-cadherin (rabbit polyclonal, (Helwani et al. 2004)); ZO-1

(clone 1A12, Zymed); anti-GFP (Molecular probes); Par3

(Upstate); aPKC (clone C-20, Santa Cruz); Myosin IIA and

Myosin IIB (Sigma); desmoplakin (clone NW6, a gift from

Dr. Kathy Green, Northwestern University Medical

School); Scribble (clone 7C6.D10) and Dlg1 (both gifts

from Dr Patrick Humbert, Peter MacCallum Cancer Cen-

ter); Lgl1 (a gift from Dr. Patrick Brennwald, UNC Chapel

Hill); ppMLC (a gift from Dr. James M. Staddon). DAPI

(Sigma), fluorescence-conjugated secondary antibodies and

phalloidin were purchased from Molecular Probes. For

western analysis: E-cadherin (DECMA-1, Sigma); pMLC

(pS-19, Cell signalling Technology); MLC (clone MY21,

Abcam); b-tubulin (clone Tub2.1, Sigma) and HRP-con-

jugated anti-mouse and anti-rabbit antibodies (BioRad).

Immunofluorescence microscopy

Cells were either fixed in chilled (-20C) MeOH for 5 min

or in 4% PFA/CSK stabilization buffer (100 mM KCl,

300 mM sucrose, 2 mM EGTA, 2 mM MgCl2,10 mM

PIPES) for 2060 min at RT. MDCK cells to be processed

396 J Mol Hist (2009) 40:395405

123

7/31/2019 yaap

3/11

for electron microscopy were fixed in 2.5% gluteraldehyde

for 1 h at RT. PFA-fixed cells were permeabilised in 0.5%

TritonX-100/PBS (PBTx) for 5 min at RT. Cells were

blocked in 3% BSA/PBS for 12 h at RT or overnight at

4C, incubated with primary antibodies, diluted in blocking

buffer, for 12 h at RT in a humidified chamber, followed

by five washes in blocking buffer over 30 min. Fluores-

cence-conjugated secondary antibodies and DAPI stainswere carried out for 1 h at RT. Coverslips were mounted on

Superfrost slides (Lombe Scientific) with N-propyl-gallate

and sealed with nail polish.

Images were captured on a Zeiss LSM510 laser scan-

ning confocal microscope, or an Olympus IX-81 inverted

epifluorescent microscope fitted with a Perkin Elmer Ul-

traView scanhead, KrAr laser, and Hamamatsu Orca ER

1.3Mp monochrome camera. Cell height was calculated

from XZ images of Phalloidin-stained MDCK cells, with

the Ziess LSM510 software. Cells were selected for anal-

ysis only if they were in interphase (i.e., mitotic cells were

excluded), and if the XZ image had been taken through themiddle of the nucleus (as judged from the complementary

XY image). Arrows were drawn from the base of a cell to

the top at its highest point, at a perpendicular angle to the

coverslip. The scale function of the program was used to

calculate the height of the arrow in micrometres (lm).

Western blotting

Cells were lysed directly in SDS sample buffer (1 mg/ml

bromophenol blue, 200 mM Tris pH 6.8, 4% SDS, 20% v/v

glycerol, 100 mM DTT, protease inhibitor cocktail

[Roche]), boiled for 5 min at 98C then separated by SDS

Polyacrylamide gel electrophoresis. Proteins were trans-

ferred onto nitrocellulose membrane, blocked in either 5%

skim milk powder in PBS-Tween (0.1%), or 3% BSA and

5% fish gelatin in TBS-Tween (0.5%). Membranes were

blotted with a primary antibody for 2 h at room tempera-

ture or overnight at 4C. Membranes were washed, blocked

and blotted with a horseradish peroxidase-conjugated sec-

ondary antibody, developed with Super Signal West Pico

chemiluminescent substrate (Pierce) and visualised with

Fuji medical X-ray film. Protein bands were analysed by

densitometry with ImageJ software.

E-cadherin adhesion assay

Adhesion assays were preformed as previously described

(Verma et al. 2004; Shewan et al. 2005). Briefly, nitro-

cellulose-coated six-well plates were incubated with hE/Fc

in Hanks Balanced Salt Solution, containing 2 mM CaCl2(HBSS-Ca2?), or with just HBSS-Ca2?, overnight at 4C.

The plates were blocked with BSA (10 mg/ml in HBSS-

Ca2?) for 2 h at 4C. Cells were isolated by incubation in

5 mM EDTA in HBSS for 2 min, followed by trypsinisa-

tion in 0.01% crystalline trypsin diluted in HBSS-Ca2? for

10 min (or 5 min for CHO cells). Cells were pelleted,

resuspended in 0.05% FBS in HBSS-Ca2?, allowed to

adhere to the hE/Fc- or BSA-coated substrata for 90 min at

37C, and then subjected to systematic pipetting in ten

areas of each well. Detached cells were removed from the

wells with PBS washes and remaining cells were incubatedwith MTT for 2 h at 37C, followed by treatment of cells

with dimethyl sulfoxide (DMSO) to release the colour in

solution. Lysates were centrifuged to remove cell debris,

and the supernatant read at OD595. Final index of cell

adhesion was calculated as the percentage of cells adherent

to hE/Fc compared with the starting number of cells, cor-

rected for background binding to BSA. All data points were

normalised to the average adhesion index value obtained

for the controls.

E-cadherin surface trypsinisation assay

Cells were grown to confluence and then treated with

HBSS-Ca2?, HBSS-Ca2? plus trypsin, or HBSS-EGTA

[2 mM] plus trypsin. Cells were incubated for 30 min at

37C before adding HBSS-Ca2?-FBS (0.05%) to stop the

action of the trypsin. Samples containing trypsin were

isolated by centrifugation and then lysed in 29 sample

buffer. The control cells (incubated in HBSS-Ca2? alone)

were lysed with 29 sample buffer directly on the tissue

culture plate. Samples were analysed by SDSPAGE and

immunoblotted for E-cadherin with an antibody directed

against the ectodomain (DECMA-1). b-tubulin was used asa sample loading control.

Statistical analysis

Statistical analyses were performed using GraphPad Prism

software, www.graphpad.com.

Results and discussion

PIP3 is found at epithelial cellcell contacts

The major lipid product of PI3K is phosphatidylinositol-

3,4,5-trisphosphate (PI(3,4,5)P3, or PIP3) (Rameh and

Cantley 1999). Accordingly, we began by examining the

subcellular localization of PIP3 and its principal precursor,

PI(4,5)P2 (PIP2), in polarized MDCK epithelial cells. These

phosphoinositides were identified by transient expression

of GFP-tagged fusion proteins bearing the PH domain of

Grp1 or PLCd, which bind specifically to PIP3 and PIP2,

respectively.

J Mol Hist (2009) 40:395405 397

123

http://www.graphpad.com/http://www.graphpad.com/

7/31/2019 yaap

4/11

As shown in Fig. 1a, PIP3 was present at E-cadherin-based cellcell contacts as well as on the basal plasma

membrane. PIP2 was found in all plasma membrane

domains (Fig. 1b), including at the apical surface, as pre-

viously reported (Martin-Belmonte et al. 2007). GFP

expressed alone as a control distributed diffusely in the

cytoplasm and did not co-localise with E-cadherin at the

plasma membrane (Fig. 1c). This confirmed that PIP3 was

localised to epithelial cellcell contacts in established

epithelial monolayers (Watton and Downward 1999;

Gassama-Diagne et al. 2006) and raised the question of

what role PIP3 might play in epithelial cellcell

interactions.

PI3K-PIP3 signalling maintains epithelial cell height

To test the impact of PIP3 on epithelial organization,

MDCK monolayers were treated with the PI3K inhibitors

LY294002 (50 lM) or wortmannin (100 nM). Both these

drugs readily depleted PIP3 from cell contacts in our cells

(data not shown).

We first assessed overall cellular organization of epi-

thelial junctions by immunofluorescence analysis. E-cad-

herin concentrated at cellcell contacts in control MDCK

cells and we found that its distribution was not materiallyaffected by LY294002 (Fig. 2a). Nor was the organization

of E-cadherin affected in human mammary MCF7 cells

(Fig. 2a), which also displayed junctional accumulation of

PIP3 (not shown). Similarly, ZO-1 and desmoplakin,

markers for tight junctions and desmosomes (Fig. 2b, c),

respectively, were unchanged in MDCK cells after inhi-

bition of PI3-kinase. The persistence of junctions was

confirmed by transmission electron microscopy (Fig. 2d),

which showed that both tight junctions and desmosomes

remained intact despite LY294002. Strikingly, however,cell height appeared consistently reduced in cultures after

inhibition of PI3-kinase.

To confirm this, we measured cell height by confocal

imaging in xz sections of MDCK cells, which were cho-

sen because they form columnar monolayers in culture

(Fig. 2d). As shown in Fig. 3a, both control and

LY294002-treated cells showed a characteristic domed

morphology in xz profile, with apices at the centre of the

cells. Measuring maximal cell height at these apices, we

found that LY294002-treated cells were consistently

*20% shorter than control cells treated with DMSO alone

(Fig. 2b). Wortmannin reduced cell height to a similardegree (Fig. 2b). The effect of LY294002 was reversed

upon wash-out of the drug (Fig. 2c), confirming that its

effect on cell height was not due to irreversible cellular

toxicity. This suggested that PI3-kinase signalling sup-

ported cell height in simple epithelial monolayers without

an apparent impact on the apical junctional complex.

Impact of PI3K inhibition on E-cadherin adhesion

In order to explore the cellular mechanisms that might

allow PI3-kinase to regulate cell height, we first focused onE-cadherin function. E-cadherin is important for epithelial

biogenesis and differentiation and cadherins can activate

PI3-kinase signalling (Pece et al. 1999; Kovacs et al.

2002b; Gavard et al. 2004), consistent with the observed

accumulation of PIP3 at E-cadherin contacts (Fig. 1a).

Moreover, PI3-kinase inhibition perturbed cadherin adhe-

sion (Kovacs et al. 2002a) and assembly of adhesive

junctions during epithelial biogenesis (Laprise et al. 2002).

This suggested that, whilst junctional cadherin staining

Fig. 1 Junctional localization of PIP3 in established MDCK epithe-

lial monolayers. MDCK cells were transiently transfected with GFP-

tagged biosensors that identify PI-3,4,5-P3 (PH-Grp1, a), PI-4,5-P2(PH-PLCd, b) or with GFP alone (c). Samples were co-stained for

E-cadherin and also with DAPI to identify the nuclei. The confocal

optical planes shown were taken from the apical region, at mid-height

through the cells, and from the basal region in contact with the

substrate

398 J Mol Hist (2009) 40:395405

123

7/31/2019 yaap

5/11

remained intact (Fig. 2), PI3-kinase inhibition might affectmore subtle aspects of cadherin function.

To probe cadherin biology further, we used biochemical

approaches to assess the total cellular and surface

expression of E-cadherin (Fig. 4a, b). Surface expression

was assessed by testing the proportion of total cellular

cadherin that was susceptible to surface trypsinization in

the absence of calcium (Fig. 4b) (Yap et al. 1997). Full-

length E-cadherin is protected from trypsin digestion in

the presence of extracellular calcium, but degraded when

Ca2? is chelated (Takeichi 1977); this differential sensi-

tivity of cadherin thus gives a measure of the amount of

cadherin that is present on the cell surface. We found thattotal cellular levels of E-cadherin were unaffected by

LY294002 when characterized by western blotting of cell

lysates (Fig. 4a). Furthermore, all the cellular E-cadherin

was degraded by trypsinization in the absence of calcium,

both in control as well as in LY294002- or wortmannin-

treated cells (Fig. 4b). This indicated that the vast majority

of cellular cadherin was found on the cell surface, and

surface expression was not perturbed by blocking PI3-

kinase.

Earlier we reported that cadherin adhesion in CHO cellsexpressing E-cadherin (hE-CHO cells) was supported by

PI3-kinase signalling (Kovacs et al. 2002a). To test whe-

ther this also occurred in epithelial cells we measured the

adhesion of MCF7 cells to substrata coated with hE/Fc, a

recombinant ligand that bears the complete ectodomain of

E-cadherin. MCF7 cells were chosen for these experiments

because hE/Fc derives from human E-cadherin. Consistent

with our earlier experience, adhesion of hE-CHO cells to

hE/Fc was significantly reduced by LY294002 (Fig. 4c).

However, the adhesion of MCF7 cells was unchanged upon

treatment with LY294002 (Fig. 4d). Overall, these findings

indicate that the impact of PI3-kinase on cell height inepithelial cells was not due to a demonstrable change in

cadherin function. They further suggest that the impact of

PI3-kinase on cadherin function may be critically influ-

enced by cell type and context. This is consistent with the

observation that PI3-kinase inhibition had a more pro-

nounced effect on junctional integrity and epithelial dif-

ferentiation as Caco-2 cells grew to form monolayers, than

in already-established monolayers (Laprise et al. 2002).

Overall, these findings suggested that alterations in

Fig. 2 Impact of PI3-kinase

inhibition on junctional

organization in MDCK and

MCF7 epithelial cells.

Confluent MDCK or MCF7

cells were treated with

LY294002 (50 lM, 8 h) or

DMSO carrier as a control.

ac Cells were fixed and

immuno-stained for E-cadherin

(a); ZO-1, marking tight

junctions (b); or desmoplakin

marking desmosomes (c).

Apical views of E-cadherin

staining are shown for both

MDCK and MCF7 cells (a).

ZO-1 and desmoplakin staining

is shown for MDCK cells, with

representative views at the

apical plane and at mid-height

through the cells. Bars are

10 lm. d Transmission electron

micrographs of control or

LY294002-treated MDCK cells.

Tight junctions (arrows) and

desmosomes (arrowheads) are

identified in both specimens.

Images of identical

magnification are shown; bar is

2 lm

J Mol Hist (2009) 40:395405 399

123

7/31/2019 yaap

6/11

E-cadherin function were unlikely to account for theimpact of PI3-kinase on epithelial cell height.

PI3-kinase and cell polarity

PI3-kinase and its principal lipid product, PIP3, have been

implicated in various forms of epithelial polarization,

including apico-basal polarization in simple epithelia

(Gassama-Diagne et al. 2006; Martin-Belmonte et al. 2007)

and anterior-posterior polarization in a variety of migrating

cells (Weiner et al. 1999; Chung et al. 2001). Moreover,

increased epithelial height has often been interpreted to

reflect apico-basal cell polarization (Gassama-Diagne et al.

2006). This prompted us to examine whether changes in

epithelial polarity accompanied the loss of cell height in

PI3-kinase-inhibited MDCK cells.

We assessed epithelial polarity by examining the sub-

cellular distribution of a range of polarity determinants by

immunofluorescence analysis (Fig. 5). The apical deter-

minants Par3 and atypical PKC predominantly stained in

the region of the apical junctional complex, as previously

described. Conversely, the basolateral determinants Lgl1,

Dlg and Scribble localized to the lateral membrane at cellcell contacts. However, neither the distribution of apical

nor basolateral determinants was altered in LY-treated

cells. Overall, then, these findings indicate that the reduced

cell height that occurs upon PI3-kinase inhibition is not

accompanied by any overt disruption of apical-basal

polarity.

Myosin II activity and cell height

We then turned to assess cytoskeletal molecules that can

influence cell height. Notably, the actin-based motor, non-

muscle Myosin II, facilitates changes in cell shape that

accompany cell movement, division and adhesion (Conti

and Adelstein 2008; Vicente-Manzanares et al. 2009).

Myosin II activity also supported cell height as keratino-

cytes differentiated in culture: lateral cell surfaces failed to

grow when Myosin II was blocked with blebbistatin

(Zhang et al. 2005). Moreover, the Myosin regulatory light

chain can be phosphorylated by PI3-kinase signalling

pathways (Huang et al. 2006), potentially leading to acti-

vation of the motor. These data made Myosin II an

Fig. 3 Impact of PI3-kinase

inhibition on epithelial cell

height. Confluent MDCK

monolayers were treated with

LY294002 (50 lM),

wortmannin (100 nM) or

DMSO carrier alone for 8 h,

then fixed and stained for F-

actin (phalloidin, green) or with

DAPI (blue). a Representative

XZ images are shown. Arrows

indicate the maximal apical

dimensions used to calculate

cell height. Horizontal and

vertical bars are 5 lm. b

Quantification of cell height in

control and LY294002- or

wortmannin-treated cells. c

Impact of LY294002 on cell

height is reversible. Cell heights

in control cells, cells treated

with LY294002 (50 lM, 8 h),

or 2 h after removal of

LY294002 (LY?WO).

** P\ 0.01; *** P\ 0.001,

Students t-test

400 J Mol Hist (2009) 40:395405

123

7/31/2019 yaap

7/11

attractive candidate to test as a possible downstream

effector of PI3-kinase activity for the maintenance of cell

height (Fig. 6).

We first examined the impact of PI3-kinase on the

subcellular localization of Myosin II in MDCK cells by

indirect immunofluorescence microscopy. Of the three

Myosin II isoforms found in mammalian cells (Vicente-

Manzanares et al. 2009), both Myosin IIA and Myosin IIB

were found to concentrate in perijunctional apical rings

(Fig. 6a) as well as in actin-rich pools at the basal poles ofour cells (not shown). LY294002 did not affect the apical

localization of Myosin IIB, but did appear to alter the

precise perijunctional distribution of Myosin IIA. Instead

of the intense band found in control cells, LY-treated cells

showed a more loosely-organized perijunctional ring of

Myosin IIA. To characterize this further, we examined the

localization of active Myosin II, identified using an anti-

body directed against the active phosphorylated state of the

regulatory Myosin light chain (ppMLC). As reported

previously, ppMLC stains in an apical junctional ring in

control cells (Shewan et al. 2005; Stehbens et al. 2006), but

this was not substantively affected in LY-treated cells

(Fig. 6a). Similarly, total cellular levels of active phos-

phorylated MLC were unchanged by LY294002 (Fig. 6b),

whereas they were clearly reduced when ROCK was

blocked with Y27632. This suggested that, despite some

redistribution of perijunctional Myosin IIA organization,

the active pool of Myosin II was not significantly altered by

inhibiting PI3-kinase.Then, we directly assessed the potential relevance of

Myosin II by testing the impact of its inhibition on cell

height in our MDCK cell system. We used both Y27632,

which blocks Myosin II activation by upstream ROCK

signaling, as well as blebbistatin, a direct inhibitor of

Myosin II (Fig. 6c). In contrast to inhibiting PI3K, neither

Y27632 nor blebbistatin affected MDCK cell height. This

suggests that, in contrast to the keratinocyte system,

Myosin II was unlikely to substantively regulate cell height

Fig. 4 Impact of PI3-kinase inhibition on E-cadherin expression and

function. a Effect on cellular expression of E-cadherin examined by

Western analysis in lysates of confluent MDCK or MCF7 monolayer

cultures treated with LY294002 (50 lM, 8 h) or DMSO carrier.

b-tubulin was used as a loading control. b Effect of PI3-kinase

inhibition on surface expression of E-cadherin. Confluent MDCK

cultures were treated with LY294002 (50 lM, 8 h), wortmannin

(100 nM, 8 h) or DMSO control. Parallel samples were lysed directly

(WCE), after surface trypsinization in the presence of extracellular

calcium (?Ca), or after surface trypsinization in the presence of

EGTA to chelate extracellular calcium. E-cadherin levels were

assessed by Western analysis; b-tubulin was used as a loading control.

c, d Effect on E-cadherin homophilic adhesion. Cell adhesion to hE/

Fc-coated substrata was measured as described in Methods. Adhesion

was measured in CHO cells stably expressing E-cadherin (hE-CHO,

c) or MCF7 cells (d). Cells were treated with LY294002 (50 lM) or

DMSO carrier. Cadherin-deficient parental CHO cells (pCHO) were

used as negative controls. *P\ 0.05; n.s.: non-significant

J Mol Hist (2009) 40:395405 401

123

7/31/2019 yaap

8/11

in established MDCK cell monolayers. Of note, blebbist-

atin prevented growth in keratinocyte height when the drug

was added to cells in the process of differentiation (Zhang

et al. 2005). It is possible that the impact of Myosin II on

cell height is greatest during epithelial biogenesis, rather

than for the process of maintaining height in established

monolayers that we studied.

Analysis of the PI3K effectors mTor and Rac1

in epithelial cell height

We then chose to pursue potential signals downstream of

PI3-kinase that might mediate its impact on epithelial cell

height. The mammalian Target of Rapamycin (mTOR)

pathway is a master regulator of cell size and is known to

act downstream of PI3-kinase signalling (Penuel and

Martin 1999; Ramalingam and Khuri 2009). This sug-

gested that our observed reductions in cell height might

reflect an overall decrease in cell size. To test this, we

incubated cells with the mTOR inhibitor, rapamycin, in a

range of concentrations over the same period where PI3-kinase inhibitors clearly reduced cell height. In contrast to

the impact of LY294002, rapamycin had no statistically-

significant effect on MDCK cell height during this period

(Fig. 7a).

Finally, the small GTPase, Rac1, is commonly identified

as a downstream mediator of PI3 Kinase signalling (Reif

et al. 1996), and is well-known to affect the organisation of

the actin cytoskeleton (Hall 1998; Machesky and Insall

1999; Insall and Machesky 2009). Moreover, Rac signal-

ling is activated by E-cadherin homophilic ligation through

a pathway that is partially sensitive to PI3-kinase, placing it

potentially downstream of PI3-kinase in cadherin signal-ling (Kovacs et al. 2002a). If Rac is a downstream mediator

of PI3-kinase signalling in the regulation of MDCK cell

height, we predicted that blocking Rac signalling should

phenocopy the effects of PI3-kinase inhibition. We pursued

this by directly inhibiting Rac with the small molecule

inhibitor NSC23766 (Gao et al. 2004), using a range of

concentrations incubated for periods where LY294002

clearly reduced cell height. As shown in Fig. 7b,

NCS23766 caused a statistically-significant reduction in

cell height, with a trend towards a dose-dependent effect at

higher concentrations. This is consistent with earlier evi-

dence that dominant negative Rac1N17 decreased MDCK

cell height (Bruewer et al. 2004) and therefore identifies

Rac as a potential downstream target of PI3-kinase in the

regulation of epithelial cell height.

Conclusion

Overall, our findings identify a role for PI3-kinase in

supporting epithelial cell height in established polarized

monolayers. This potentially involves the well-documented

capacity for PI3-kinase to signal through Rac, with

downstream effects on cytoskeletal organization and

membrane trafficking. Our current data are consistent with,

and complement the results of, two earlier reports. Laprise

et al. (2002) reported that chronic incubation of Caco-2

intestinal epithelial cells with LY294002 prevented the

characteristic differentiation that occurs as these cells

mature in culture. Consequently, LY-treated Caco-2 cells

were flatter and less-polarized than control cells (Laprise

et al. 2002). In contrast, MDCK cell polarity was not

overtly affected by the shorter exposure to LY2094002

Fig. 5 PI3-kinase inhibition and epithelial cell polarity. Confluent

MDCK cell monolayers were immunostained for Par3, aPKC, Lgl1,

Dlg1 and Scribble (Scrib). Representative apical (Par3, aPKC) or

basolateral (Lgl1, Dlg1, Scrib) confocal sections are shown

402 J Mol Hist (2009) 40:395405

123

7/31/2019 yaap

9/11

Fig. 6 Impact of PI3-kinase on non-muscle Myosin II in established

epithelial monolayers. a Impact on junctional localization of Myosin

II isoforms. Confluent MDCK monolayers were treated with

LY294002 (50 lM, 8 h) or DMSO alone, then fixed and immuno-

stained for Myosin IIA (MIIA), Myosin IIB (MIIB) or active,

phosphorylated Myosin regulatory light chain (ppMLC). Confocalsections from apical junctional planes are shown. Bar is 10 lm. b

Lysates from MDCK cultures treated with LY294002, Y27632

(50 lM) or DMSO controls were immunoprobed for active phos-

phorylated Myosin regulatory light chain (pMLC) by Western

analysis. b-tubulin was used as a loading control. c Effect of Myosin

II inhibition on cell height. Confluent MDCK monolayers were

treated with LY294002 (50 lM), blebbistatin (100 lM) or Y27632

(50 lM) for 8 h. Cell height was measured by confocal microscopy asdescribed in Fig. 3. * P\ 0.05; n.s.: non-significant

Fig. 7 Impact of mTOR and

Rac on epithelial cell height.

Cell height in confluent MDCK

monolayers was measured from

xz images as described in

Fig. 3. a Effect of mTOR was

compared by treatment with

either LY294002 (50 lM) or

rapamycin (1100 nM) for 8 h.

b Effect of Rac was tested by

treatment with LY294002

(50 lM) or NSC 23766 (20200 lM) for 8 h

J Mol Hist (2009) 40:395405 403

123

7/31/2019 yaap

10/11

used in our experiments, suggesting that the changes in cell

height that we observed were not due to alterations in

global cell differentiation. Alternatively, PIP3 may serve to

specify basolateral membrane identity (Gassama-Diagne

et al. 2006). Acute exposure to PIP3 itself induced the

formation of basolateral membrane protrusions at the api-

cal surfaces of MDCK cells (Gassama-Diagne et al. 2006),

suggesting that a PIP3 signal induced the conversion ofapical identity to a basolateral identity, potentially via

transcytosis. Such support of basolateral identity is con-

sistent with the preservation of epithelial polarity found in

our experiments, although our cells were exposed to

LY294002 for shorter periods than in the study of Gass-

ama-Diagne et al. (2006). How PIP3 and Rac signalling

may coordinate the cytoskeleton and membrane trafficking

will be an important issue for further investigation.

Acknowledgments We thank all our laboratory colleagues for all

their advice, technical assistance and moral support. Additional spe-

cial thanks go to Rob Parton and Nicole Schieber for their assistance

with electron microscopy, and Markus Kerr for many helpful dis-

cussions. Confocal microscopy was performed at the ACRF/IMB

Dynamic Imaging Facility for Cancer Biology, established with the

generous support of the Australian Cancer Research Foundation. This

work was funded by the National Health and Medical Research

Council of Australia. AJ was a Dora Lush Scholar of the NHMRC;

MS was an Erwin Schroedinger postdoctoral fellow of the Austrian

Science Fund (FWF); JML is funded by an Australian Postgraduate

Award and ASY is a research fellow of the NHMRC.

References

Bruewer M, Hopkins AM, Hobert ME, Nusrat A, Madara JL (2004)

RhoA, Rac1, Cdc42 exert distinct effects on the epithelial barrier

via selective structural and biochemical modulation of junctional

proteins and F-actin. Am J Physiol Cell Physiol 287:C327C335

Calautti E, Li J, Saoncella S, Brissette JL, Goetinck PF (2005)

Phosphoinositide 3-kinase signaling to Akt promotes keratino-

cyte differentiation versus death. J Biol Chem 280:3285632865

Chung CY, Funamoto S, Firtel RA (2001) Signaling pathways

controlling cell polarity and chemotaxis. Trends Biochem Sci

26:557566

Conti MA, Adelstein RS (2008) Nonmuscle myosin II moves in new

directions. J Cell Sci 121:1118

Diamond J (1977) The epithelial junction: bridge, gate, and fence.

Physiologist 20:1018

Fleming TP, Papenbrock T, Fesenko I, Hausen P, Sheth B (2000)

Assembly of tight junctions during early vertebrate development.Semin Cell Dev Biol 11:291299

Fruman DA, Snapper SB, Yballe CM, Davidson L, Yu JY, Alt FW,

Cantley LC (1999) Impaired B cell development and prolifer-

ation in absence of phosphoinositide 3-kinase p85alpha. Science

283:393397

Gao Y, Dickerson JB, Guo F, Zheng J, Zheng Y (2004) Rational

design and characterization of a Rac GTPase-specific small

molecule inhibitor. Proc Natl Acad Sci USA 101:76187623

Gassama-Diagne A, Yu W, Ter Beest M, Martin-Belmonte F, Kierbel

A, Engel J, Mostov K (2006) Phosphatidylinositol-3,4,5-tris-

phosphate regulates the formation of the basolateral plasma

membrane in epithelial cells. Nat Cell Biol 8:963970

Gavard J, Lambert M, Grosheva I, Marthiens V, Irinopoulou T, Riou

J-F, Bershadsky A, Mege RM (2004) Lamellipodium extension

and cadherin adhesion: two cell responses to cadherin activation

relying on distinct signalling pathways. J Cell Sci 117:257270

Goberdhan DC, Paricio N, Goodman EC, Mlodzik M, Wilson C

(1999) Drosophila tumor suppressor PTEN controls cell size and

number by antagonizing the Chico/PI3-kinase signaling path-

way. Genes Dev 13:32443258

Halet G, Viard P, Carroll J (2008) Constitutive PtdIns(3,4,5)P3

synthesis promotes the development and survival of early

mammalian embryos. Development 135:425429

Hall A (1998) Rho GTPases and the actin cytoskeleton. Science

279:509514

Helwani FM, Kovacs EM, Paterson AD, Verma S, Ali RG, Fanning

AS, Weed SA, Yap AS (2004) Cortactin is necessary for E-

cadherin-mediated contact formation and actin reorganization. J

Cell Biol 164:899910

Huang J, Mahavadi S, Sriwai W, Hu W, Murthy KS (2006) Gi-

coupled receptors mediate phosphorylation of CPI-17 and

MLC20 via preferential activation of the PI3K/ILK pathway.

Biochem J 396:193200

Insall RH, Machesky LM (2009) Actin dynamics at the leading edge:

from simple machinery to complex networks. Dev Cell 17:

310322

Kovacs EM, Ali RG, McCormack AJ, Yap AS (2002a) E-cadherin

homophilic ligation directly signals through Rac and PI3-kinase

to regulate adhesive contacts. J Biol Chem 277:67086718

Kovacs EM, Goodwin M, Ali RG, Paterson AD, Yap AS (2002b)

Cadherin-directed actin assembly: E-cadherin physically associ-

ates with the Arp2/3 complex to direct actin assembly in nascent

adhesive contacts. Curr Biol 12:379382

Laprise P, Chailler P, Houde M, Beaulieu JF, Boucher MJ, Rivard N

(2002) Phosphatidylinositol 3-kinase controls human intestinal

epithelial cell differentiation by promoting adherens junction

assembly and p38 MAPK activation. J Biol Chem 277:8226

8234

Luo J, McMullen JR, Sobkiw CL, Zhang L, Dorfman AL, Sherwood

MC, Logsdon MN, Horner JW, DePinho RA, Izumo S, Cantley

LC (2005) Class IA phosphoinositide 3-kinase regulates heart

size and physiological cardiac hypertrophy. Mol Cell Biol

25:94919502

Machesky LM, Insall RH (1999) Signaling to actin dynamics. J Cell

Biol 146:267272

Martin-Belmonte F, Gassama A, Datta A, Yu W, Rescher U, Gerke V,

Mostov K (2007) PTEN-mediated apical segregation of phos-

phoinositides controls epithelial morphogenesis through Cdc42.

Cell 128:383397

OBrien LE, Zegers MMP, Mostov KE (2002) Building epithelial

architecture: insights from three-dimensional models. Nat Rev

Mol Cell Biol 3:531537

Pece S, Chiariello M, Murga C, Gutkind JS (1999) Activation of the

protein kinase Akt/PKB by the formation of E-cadherin-medi-

ated cell-cell junctions. J Biol Chem 274:1934719351

Penuel E, Martin GS (1999) Transformation by v-Src: ras-MAPK andPI3K-mTOR mediate parallel pathways. Mol Biol Cell 10:1693

1703

Ramalingam SS, Khuri FR (2009) PI3 kinase, mTOR, and AKT

pathways. J Thorac Oncol 4:S1059S1060

Rameh LE, Cantley LC (1999) The role of phosphoinositide 3-kinase

lipid products in cell function. J Biol Chem 274:83478350

Reif K, Nobes CD, Thomas G, Hall A, Cantrell DA (1996)

Phosphatidylinositol 3-kinase signals activate a selective subset

of Rac/Rho-dependent effector pathways. Curr Biol 6:1445

1455

Rodriguez-Boulan E, Nelson WJ (1989) Morphogenesis of the

polarized epithelial cell phenotype. Science 245:718725

404 J Mol Hist (2009) 40:395405

123

7/31/2019 yaap

11/11

Shewan AM, Maddugoda M, Kraemer A, Stehbens SJ, Verma S,

Kovacs EM, Yap AS (2005) Myosin 2 is a key rho kinase target

necessary for the local concentration of E-cadherin at cell-cell

contacts. Mol Biol Cell 16:45314532

Stehbens SJ, Paterson AD, Crampton MS, Shewan AM, Ferguson C,

Akhmanova A, Parton RG, Yap AS (2006) Dynamic microtu-

bules regulate the local concentration of E-cadherin at cell-cell

contacts. J Cell Sci 119:18011811

Takeichi M (1977) Functional correlation between cell adhesive

properties and some cell surface proteins. J Cell Biol 75:464474

Vanhaesebroeck B, Leevers SJ, Ahmadi K, Timms J, Katso R,

Driscoll PC, Woscholski R, Parker PJ, Waterfield MD (2001)

Synthesis and function of 3-phosphorylated inositol lipids. Annu

Rev Biochem 70:535602

Vega-Salas DE, Salas PJ, Gundersen D, Rodriguez-Boulan E (1987a)

Formation of the apical pole of epithelial (Madin-Darby canine

kidney) cells: polarity of an apical protein is independent of tight

junctions while segregation of a basolateral marker requires cell-

cell interactions. J Cell Biol 104:905916

Vega-Salas DE, Salas PJI, Rodriguez-Boulan E (1987b) Exocytosis of

vacuolar apical compartment (VAC): a cell-cell contact con-

trolled mechanism for the establishment of the apical membrane

domain in epithelial cells. J Cell Biol 107:17171728

Verma S, Shewan AM, Scott JA, Helwani FM, Elzen NR, Miki H,

Takenawa T, Yap AS (2004) Arp2/3 activity is necessary for

efficient formation of E-cadherin adhesive contacts. J Biol Chem

279:3406234070

Vicente-Manzanares M, Ma X, Adelstein RS, Horwitz AR (2009)

Non-muscle myosin II takes centre stage in cell adhesion and

migration. Nat Rev Mol Cell Biol 10:778790

Wang F, Herzmark P, Weiner OD, Srinivasan S, Servant G, Bourne

HR (2002) Lipid products of PI(3)Ks maintain persistent cell

polarity and directed motility in neutrophils. Nat Cell Biol

4:513518

Watton SJ, Downward J (1999) Akt/PKB localisation and 30-

phosphoinositide generation at sites of epithelial cell-matrix

and cell-cell interaction. Curr Biol 9:433436

Weiner OD, Servant G, Welch MD, Mitchison TJ, Sedat JW, Bourne

HR (1999) Spatial control of actin polymerization during

neutrophil chemotaxis. Nat Cell Biol 1:7481

Weiner OD, Neilsen PO, Prestwich GD, Kirschner MW, Cantley LC,

Bourne HR (2002) A PtdInsP(3)- and Rho GTPase-mediated

positive feedback loop regulates neutrophil polarity. Nat Cell

Biol 4:509513

Wheelock MJ, Johnson KR (2003) Cadherin-mediated cellular

signaling. Curr Opin Cell Biol 15:509514

Yap AS, Kovacs EM (2003) Direct cadherin-activated cell signaling:

a view from the plasma membrane. J Cell Biol 160:1116

Yap AS, Brieher WM, Pruschy M, Gumbiner BM (1997) Lateral

clustering of the adhesive ectodomain: a fundamental determi-

nant of cadherin function. Curr Biol 7:308315

Yeaman C, Grindstaff KK, Nelson WJ (1999) New perspectives on

mechanisms involved in generating epithelial cell polarity.

Physiol Rev 79:7398

Zhang J, Betson M, Erasmus J, Zeikos K, Bailly M, Cramer LP, Braga

VM (2005) Actin at cell-cell junctions is composed of two

dynamic and functional populations. J Cell Sci 118:55495562

J Mol Hist (2009) 40:395405 405

123