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Phosphatidylinositol 30 -kinase signalling supports cell height in 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 Ó Springer Science+Business Media B.V. 2010
Abstract Cell–cell interactions inﬂuence epithelial morphogenesis through an interplay between cell adhesion, trafﬁcking and the cytoskeleton. These cellular processes are coordinated, often by cell signals found at cell–cell contacts. One such contact-based signal is the phosphatidylinositol 30 -kinase (PI3-kinase; PI3K) pathway. PI3-kinase is best understood for its role in mitogenic signalling, where it regulates cell survival, proliferation and differentiation. Its precise morphogenetic impacts in epithelia are, in contrast, less well-understood. Using phosphoinositide-speciﬁc biosensors we conﬁrmed that E-cadherinbased cell–cell 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 homophilic 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 morphogenetic impact in simple cultured epithelia through
preservation of cell height. This is independent of potential effects on adhesion or polarity, but may occur through PI3kinase-stimulated Rac signaling. Keywords Epithelia Á PI3-kinase Á Cell height Á E-cadherin
Introduction Epithelial cells come in many different shapes and sizes: their precise morphologies have wide-reaching implications 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 cell–cell 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 (RodriguezBoulan and Nelson 1989). Analysis in cell culture has identiﬁed cell–cell 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 structure is induced and maintained by interplay between cell adhesion, the cytoskeleton, membrane trafﬁc and cell polarity (Yeaman et al. 1999; O’Brien et al. 2002). In turn, these cellular processes are coordinated by a range of signalling pathways, including signals that are active at cell–cell contacts themselves (Wheelock and Johnson 2003; Yap and Kovacs 2003). Phosphoinositides have recently emerged as major regulators of cell polarity, morphology and the cytoskeleton
A. Jeanes Á M. Smutny Á J. M. Leerberg Á A. S. Yap (&) Division of Molecular Cell Biology, Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia e-mail: firstname.lastname@example.org; email@example.com
2001). 2006). Moreover. 2002a). was also shown to specify basolateral membrane identity in MDCK cells (Gassama-Diagne et al. 1999). desmoplakin (clone NW6. Halet et al. Sigma). For immunoﬂuorescence microscopy: E-cadherin mAB (Transduction Laboratories). phosphatidylinositol-4. Gassama-Diagne et al. Immunoﬂuorescence microscopy Cells were either ﬁxed in chilled (-20°C) MeOH for 5 min or in 4% PFA/CSK stabilization buffer (100 mM KCl. In migrating cells the localized generation of PIP3 by PI3-kinase serves to regulate the actin cytoskeleton and contributes to anterior-posterior polarization necessary for productive translocation (Weiner et al. Martin-Belmonte et al. Finally.396 J Mol Hist (2009) 40:395–405 (Weiner et al. Zymed). 2007). pMLC (pS-19. were a kind gift from Dr Mark Lemmon and have been described previously (Kovacs et al. Vanhaesebroeck et al. which was independent of junctional integrity. 1999. All media were supplemented with 10% FBS. a gift from Dr. it is becoming increasingly clear that PI3kinase also inﬂuences cellular morphogenesis in many different tissues. Northwestern University Medical School). 2002a) and pEGFP-C1 was from Clontech. Chung et al. Lgl1 (a gift from Dr. 2002. L-glutamine. PI3-kinase can affect cadherin adhesion (Kovacs et al. Cell signalling Technology). 2 mM EGTA. 2007). ZO-1 (clone 1A12. Patrick Brennwald. MLC (clone MY21. Antibodies were as follows. E-cadherin (rabbit polyclonal. CHO cells stably expressing human E-cadherin were described previously (Kovacs et al. 1999. Kathy Green. 2001). Cells treated with inhibitor drugs were given fresh medium 1 day before treatment. 2002a. We also identify Rac signalling as a further signal implicated in the maintenance of epithelial cell height. For western analysis: E-cadherin (DECMA-1. Y-27632 (50 lM) and rapamycin (1– 100 nM) were purchased from Calbiochem. according to the manufacturer’s instructions. blebbistatin (100 lM). Staddon). 2002. James M. where it is implicated in cell survival. cadherin adhesion and epithelial cell polarity. 2 mM MgCl2. GassamaDiagne et al. whereas phosphatidylinositol-3. 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. ppMLC (a gift from Dr. both important processes in controlling cell morphology. Materials and methods Cell culture MDCK and MCF7 cells were maintained in DMEM. Transient transfection of MDCK cells was carried out with Lipofectamine2000 (Invitrogen). 2002. ﬂuorescence-conjugated secondary antibodies and phalloidin were purchased from Molecular Probes. 2008). 2002a). some of the enzymes responsible for either the generation (phosphorylation) or metabolic turnover (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 speciﬁc phosphoinositides (Watton and Downward 1999). PIP3. Cells were grown to 100% conﬂuence before being ﬁxed for indirect immunoﬂuorescence. Type 1A Phosphatidylinositol 30 -kinase (PI3-kinase. 2004)). Reagents The expression plasmids pEGFP-N1-PHGrp1 and pEGFPN1-PHPLCd.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. MDCK cells to be processed 123 .4. Kovacs et al. anti-GFP (Molecular probes). proliferation and differentiation (Fruman et al. b). aPKC (clone C-20. Sigma) and HRP-conjugated anti-mouse and anti-rabbit antibodies (BioRad). PI3-kinase mediates signal transduction in response to a wide range of cell surface receptors (Rameh and Cantley 1999. except that cells were between 25 and 35% conﬂuent at the time of transfection. Myosin IIA and Myosin IIB (Sigma).D10) and Dlg1 (both gifts from Dr Patrick Humbert. 2006). 1% non-essential amino acids. wortmannin (100 nM). Calautti et al.1.10 mM PIPES) for 20–60 min at RT. and Penicillin/Streptomycin. Par3 (Upstate). Inhibitors: LY294002 (ﬁnal concentration of 50 lM). 1999. b-tubulin (clone Tub2. However. 1999. 2006. DAPI (Sigma). NSC-23766 (20–200 lM) was purchased from Tocris. (Helwani et al. In this study we sought to further analyse the morphogenetic impact of PI3-kinase signalling in simple polarized epithelia. It is best understood for its role in mitogenic signalling downstream of growth factor receptors. Santa Cruz). 300 mM sucrose. CHO cells were maintained in F12 medium. UNC Chapel Hill). Abcam). suggesting that it may be one key signal that is activated by cell–cell adhesion to inﬂuence epithelial morphogenesis. For example. 2005. We report that acute inhibition of PI3-kinase signalling in established epithelial monolayers caused reduced cell height. and inhibitors were added directly to the medium. and its major lipid product. Of note. Scribble (clone 7C6. 2005) and during Drosophila development (Goberdhan et al.5-P3 (PIP3) concentrates at cell–cell contacts (Watton and Downward 1999. PI3K) can associate with E-cadherin and be activated by cadherin homophilic adhesion (Pece et al. Wang et al. Polarized epithelial cells characteristically show domain-speciﬁc differences in their distribution of speciﬁc phosphoinositides. Peter MacCallum Cancer Center).
www. Membranes were washed. Cells were isolated by incubation in 5 mM EDTA in HBSS for 2 min. blocked in either 5% skim milk powder in PBS-Tween (0. for 1–2 h at RT in a humidiﬁed chamber. blocked and blotted with a horseradish peroxidase-conjugated secondary antibody. or an Olympus IX-81 inverted epiﬂuorescent microscope ﬁtted with a Perkin Elmer UltraView scanhead.5% gluteraldehyde for 1 h at RT. boiled for 5 min at 98°C then separated by SDS– Polyacrylamide gel electrophoresis. PI(4.or BSA-coated substrata for 90 min at 37°C. Final index of cell adhesion was calculated as the percentage of cells adherent to hE/Fc compared with the starting number of cells. containing 2 mM CaCl2 (HBSS-Ca2?). 4% SDS. Results and discussion PIP3 is found at epithelial cell–cell contacts The major lipid product of PI3K is phosphatidylinositol3. or with just HBSS-Ca2?.com. Cell height was calculated from XZ images of Phalloidin-stained MDCK cells. The scale function of the program was used to calculate the height of the arrow in micrometres (lm). Fluorescence-conjugated secondary antibodies and DAPI stains were carried out for 1 h at RT. b-tubulin was used as a sample loading control. incubated with primary antibodies. Accordingly. protease inhibitor cocktail [Roche]).05%) to stop the action of the trypsin. Protein bands were analysed by densitometry with ImageJ software. corrected for background binding to BSA. Shewan et al. respectively. The control cells (incubated in HBSS-Ca2? alone) were lysed with 29 sample buffer directly on the tissue culture plate. and the supernatant read at OD595. we began by examining the subcellular localization of PIP3 and its principal precursor. E-cadherin adhesion assay Adhesion assays were preformed as previously described (Verma et al.05% FBS in HBSS-Ca2?. Samples containing trypsin were isolated by centrifugation and then lysed in 29 sample buffer. These phosphoinositides were identiﬁed by transient expression of GFP-tagged fusion proteins bearing the PH domain of Grp1 or PLCd. nitrocellulose-coated six-well plates were incubated with hE/Fc in Hanks Balanced Salt Solution. in polarized MDCK epithelial cells. PFA-ﬁxed cells were permeabilised in 0. Cells were pelleted. or PIP3) (Rameh and Cantley 1999). and if the XZ image had been taken through the middle of the nucleus (as judged from the complementary XY image).1%). or HBSS-EGTA [2 mM] plus trypsin. allowed to adhere to the hE/Fc. Detached cells were removed from the wells with PBS washes and remaining cells were incubated with MTT for 2 h at 37°C.5-trisphosphate (PI(3.. with the Ziess LSM510 software. 123 . Statistical analysis Statistical analyses were performed using GraphPad Prism software. mitotic cells were excluded).graphpad. Lysates were centrifuged to remove cell debris. diluted in blocking buffer. 20% v/v glycerol.3Mp monochrome camera. followed by trypsinisation in 0. All data points were normalised to the average adhesion index value obtained for the controls.5% TritonX-100/PBS (PBTx) for 5 min at RT. Cells were blocked in 3% BSA/PBS for 1–2 h at RT or overnight at 4°C. KrAr laser. E-cadherin surface trypsinisation assay Cells were grown to conﬂuence and then treated with HBSS-Ca2?.4. Cells were selected for analysis only if they were in interphase (i. 100 mM DTT.5)P2 (PIP2). followed by ﬁve washes in blocking buffer over 30 min.01% crystalline trypsin diluted in HBSS-Ca2? for 10 min (or 5 min for CHO cells). Images were captured on a Zeiss LSM510 laser scanning confocal microscope. Arrows were drawn from the base of a cell to the top at its highest point. Samples were analysed by SDS–PAGE and immunoblotted for E-cadherin with an antibody directed against the ectodomain (DECMA-1). Cells were incubated for 30 min at 37°C before adding HBSS-Ca2?-FBS (0. overnight at 4°C.e. Proteins were transferred onto nitrocellulose membrane. 2004. 2005). 200 mM Tris pH 6. Brieﬂy.5)P3. The plates were blocked with BSA (10 mg/ml in HBSSCa2?) for 2 h at 4°C.8. Membranes were blotted with a primary antibody for 2 h at room temperature or overnight at 4°C. followed by treatment of cells with dimethyl sulfoxide (DMSO) to release the colour in solution.J Mol Hist (2009) 40:395–405 397 for electron microscopy were ﬁxed in 2. which bind speciﬁcally to PIP3 and PIP2. or 3% BSA and 5% ﬁsh gelatin in TBS-Tween (0. at a perpendicular angle to the coverslip. Western blotting Cells were lysed directly in SDS sample buffer (1 mg/ml bromophenol blue. and Hamamatsu Orca ER 1. resuspended in 0. developed with Super Signal West Pico chemiluminescent substrate (Pierce) and visualised with Fuji medical X-ray ﬁlm. HBSS-Ca2? plus trypsin.4. Coverslips were mounted on Superfrost slides (Lombe Scientiﬁc) with N-propyl-gallate and sealed with nail polish. and then subjected to systematic pipetting in ten areas of each well.5%).
we measured cell height by confocal imaging in x–z sections of MDCK cells. The persistence of junctions was conﬁrmed by transmission electron microscopy (Fig. a). 2b). PI3-kinase inhibition perturbed cadherin adhesion (Kovacs et al. PI3K-PIP3 signalling maintains epithelial cell height To test the impact of PIP3 on epithelial organization. which were chosen because they form columnar monolayers in culture (Fig. with apices at the centre of the cells. 2d). which showed that both tight junctions and desmosomes remained intact despite LY294002. Gavard et al.5-P3 (PH-Grp1. This conﬁrmed that PIP3 was localised to epithelial cell–cell contacts in established epithelial monolayers (Watton and Downward 1999. c). Samples were co-stained for E-cadherin and also with DAPI to identify the nuclei. which also displayed junctional accumulation of PIP3 (not shown). This suggested that PI3-kinase signalling supported cell height in simple epithelial monolayers without an apparent impact on the apical junctional complex. at mid-height through the cells.4. however. both control and LY294002-treated cells showed a characteristic domed morphology in x–z proﬁle. 1999. 2002). whilst junctional cadherin staining 123 . ZO-1 and desmoplakin. and from the basal region in contact with the substrate As shown in Fig. consistent with the observed accumulation of PIP3 at E-cadherin contacts (Fig. markers for tight junctions and desmosomes (Fig. We ﬁrst assessed overall cellular organization of epithelial junctions by immunoﬂuorescence analysis.398 J Mol Hist (2009) 40:395–405 Fig. as previously reported (Martin-Belmonte et al. 3a. Nor was the organization of E-cadherin affected in human mammary MCF7 cells (Fig. 2b. E-cadherin concentrated at cell–cell contacts in control MDCK cells and we found that its distribution was not materially affected by LY294002 (Fig. cell height appeared consistently reduced in cultures after inhibition of PI3-kinase. To conﬁrm this. we ﬁrst focused on E-cadherin function. Wortmannin reduced cell height to a similar degree (Fig. 1 Junctional localization of PIP3 in established MDCK epithelial monolayers. 2004). 2a). MDCK monolayers were treated with the PI3K inhibitors LY294002 (50 lM) or wortmannin (100 nM). Similarly. Both these drugs readily depleted PIP3 from cell contacts in our cells (data not shown). 2007). 1c). Strikingly. b) or with GFP alone (c). 2b). GFP expressed alone as a control distributed diffusely in the cytoplasm and did not co-localise with E-cadherin at the plasma membrane (Fig. Measuring maximal cell height at these apices. PIP2 was found in all plasma membrane domains (Fig. The effect of LY294002 was reversed upon wash-out of the drug (Fig. 1b).5-P2 (PH-PLCd. 2006) and raised the question of what role PIP3 might play in epithelial cell–cell interactions. Moreover. 1a. we found that LY294002-treated cells were consistently *20% shorter than control cells treated with DMSO alone (Fig. As shown in Fig. including at the apical surface. PIP3 was present at E-cadherinbased cell–cell contacts as well as on the basal plasma membrane. 2002a) and assembly of adhesive junctions during epithelial biogenesis (Laprise et al. were unchanged in MDCK cells after inhibition of PI3-kinase. E-cadherin is important for epithelial biogenesis and differentiation and cadherins can activate PI3-kinase signalling (Pece et al. This suggested that. 1a). 2d). respectively. Kovacs et al. 2a). The confocal optical planes shown were taken from the apical region. 2002b. 2c). Impact of PI3K inhibition on E-cadherin adhesion In order to explore the cellular mechanisms that might allow PI3-kinase to regulate cell height. PI-4. Gassama-Diagne et al. MDCK cells were transiently transfected with GFPtagged biosensors that identify PI-3. conﬁrming that its effect on cell height was not due to irreversible cellular toxicity.
ZO-1 and desmoplakin staining is shown for MDCK cells. these ﬁndings suggested that alterations in 123 . Overall. Apical views of E-cadherin staining are shown for both MDCK and MCF7 cells (a). Surface expression was assessed by testing the proportion of total cellular cadherin that was susceptible to surface trypsinization in the absence of calcium (Fig. They further suggest that the impact of PI3-kinase on cadherin function may be critically inﬂuenced by cell type and context.J Mol Hist (2009) 40:395–405 Fig. 1997). 8 h) or DMSO carrier as a control. 4a. than in already-established monolayers (Laprise et al. we used biochemical approaches to assess the total cellular and surface expression of E-cadherin (Fig. a–c Cells were ﬁxed and immuno-stained for E-cadherin (a). This is consistent with the observation that PI3-kinase inhibition had a more pronounced effect on junctional integrity and epithelial differentiation as Caco-2 cells grew to form monolayers.or wortmannintreated cells (Fig. To test whether this also occurred in epithelial cells we measured the adhesion of MCF7 cells to substrata coated with hE/Fc. both in control as well as in LY294002. bar is 2 lm 399 remained intact (Fig. 2). Earlier we reported that cadherin adhesion in CHO cells expressing E-cadherin (hE-CHO cells) was supported by PI3-kinase signalling (Kovacs et al. 2 Impact of PI3-kinase inhibition on junctional organization in MDCK and MCF7 epithelial cells. 4b) (Yap et al. This indicated that the vast majority of cellular cadherin was found on the cell surface. However. marking tight junctions (b). all the cellular E-cadherin was degraded by trypsinization in the absence of calcium. these ﬁndings indicate that the impact of PI3-kinase on cell height in epithelial cells was not due to a demonstrable change in cadherin function. PI3-kinase inhibition might affect more subtle aspects of cadherin function. 4a). Fulllength E-cadherin is protected from trypsin digestion in the presence of extracellular calcium. d Transmission electron micrographs of control or LY294002-treated MDCK cells. Tight junctions (arrows) and desmosomes (arrowheads) are identiﬁed in both specimens. 4d). 2002a). b). Conﬂuent MDCK or MCF7 cells were treated with LY294002 (50 lM. Furthermore. MCF7 cells were chosen for these experiments because hE/Fc derives from human E-cadherin. this differential sensitivity of cadherin thus gives a measure of the amount of cadherin that is present on the cell surface. 4c). Consistent with our earlier experience. Overall. ZO-1. the adhesion of MCF7 cells was unchanged upon treatment with LY294002 (Fig. with representative views at the apical plane and at mid-height through the cells. and surface expression was not perturbed by blocking PI3kinase. 4b). To probe cadherin biology further. a recombinant ligand that bears the complete ectodomain of E-cadherin. We found that total cellular levels of E-cadherin were unaffected by LY294002 when characterized by western blotting of cell lysates (Fig. adhesion of hE-CHO cells to hE/Fc was signiﬁcantly reduced by LY294002 (Fig. or desmoplakin marking desmosomes (c). but degraded when Ca2? is chelated (Takeichi 1977). Images of identical magniﬁcation are shown. 2002). Bars are 10 lm.
including apico-basal polarization in simple epithelia (Gassama-Diagne et al. increased epithelial height has often been interpreted to reﬂect apico-basal cell polarization (Gassama-Diagne et al.400 Fig. 3 Impact of PI3-kinase inhibition on epithelial cell height. Moreover. Martin-Belmonte et al. the Myosin regulatory light chain can be phosphorylated by PI3-kinase signalling pathways (Huang et al. then. Student’s t-test J Mol Hist (2009) 40:395–405 E-cadherin function were unlikely to account for the impact of PI3-kinase on epithelial cell height. 2006). PIP3. c Impact of LY294002 on cell height is reversible. Conﬂuent MDCK monolayers were treated with LY294002 (50 lM). 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. Chung et al. PI3-kinase and cell polarity PI3-kinase and its principal lipid product. green) or with DAPI (blue). Moreover. Myosin II activity and cell height We then turned to assess cytoskeletal molecules that can inﬂuence cell height. the basolateral determinants Lgl1. b Quantiﬁcation of cell height in control and LY294002. have been implicated in various forms of epithelial polarization.001. Notably. However.or wortmannin-treated cells. as previously described. a Representative X–Z images are shown. the actin-based motor. Vicente-Manzanares et al. ** P \ 0. The apical determinants Par3 and atypical PKC predominantly stained in the region of the apical junctional complex. cells treated with LY294002 (50 lM. Horizontal and vertical bars are 5 lm. potentially leading to activation of the motor. then ﬁxed and stained for Factin (phalloidin. 2009).01. 2005). wortmannin (100 nM) or DMSO carrier alone for 8 h. This prompted us to examine whether changes in epithelial polarity accompanied the loss of cell height in PI3-kinase-inhibited MDCK cells. 2006). 2007) and anterior-posterior polarization in a variety of migrating cells (Weiner et al. 2006. 8 h). neither the distribution of apical nor basolateral determinants was altered in LY-treated cells. These data made Myosin II an 123 . nonmuscle Myosin II. Overall. Conversely. Myosin II activity also supported cell height as keratinocytes differentiated in culture: lateral cell surfaces failed to grow when Myosin II was blocked with blebbistatin (Zhang et al. Dlg and Scribble localized to the lateral membrane at cell– cell contacts. 2001). division and adhesion (Conti and Adelstein 2008. facilitates changes in cell shape that accompany cell movement. 5). 1999. Cell heights in control cells. We assessed epithelial polarity by examining the subcellular distribution of a range of polarity determinants by immunoﬂuorescence analysis (Fig. or 2 h after removal of LY294002 (LY?WO). *** P \ 0. Arrows indicate the maximal apical dimensions used to calculate cell height.
2006). Adhesion was measured in CHO cells stably expressing E-cadherin (hE-CHO. This suggests that. *P \ 0. the active pool of Myosin II was not signiﬁcantly altered by inhibiting PI3-kinase.s. Conﬂuent MDCK cultures were treated with LY294002 (50 lM. 6a). total cellular levels of active phosphorylated MLC were unchanged by LY294002 (Fig. 8 h) or DMSO carrier. ppMLC stains in an apical junctional ring in control cells (Shewan et al. a direct inhibitor of Myosin II (Fig. as well as blebbistatin. E-cadherin levels were assessed by Western analysis. Parallel samples were lysed directly (WCE). We used both Y27632. b-tubulin was used as a loading control. As reported previously. 6a) as well as in actin-rich pools at the basal poles of our cells (not shown). after surface trypsinization in the presence of extracellular calcium (?Ca). 6b).: non-signiﬁcant attractive candidate to test as a possible downstream effector of PI3-kinase activity for the maintenance of cell height (Fig. identiﬁed using an antibody directed against the active phosphorylated state of the regulatory Myosin light chain (ppMLC). we directly assessed the potential relevance of Myosin II by testing the impact of its inhibition on cell height in our MDCK cell system.05. 6c). Myosin II was unlikely to substantively regulate cell height 123 . d Effect on E-cadherin homophilic adhesion. both Myosin IIA and Myosin IIB were found to concentrate in perijunctional apical rings (Fig. Cells were treated with LY294002 (50 lM) or DMSO carrier. LY294002 did not affect the apical localization of Myosin IIB. Then. or after surface trypsinization in the presence of EGTA to chelate extracellular calcium. 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. but this was not substantively affected in LY-treated cells (Fig. Instead of the intense band found in control cells. LY-treated cells showed a more loosely-organized perijunctional ring of Myosin IIA. b-tubulin was used as a loading control. but did appear to alter the precise perijunctional distribution of Myosin IIA. Of the three Myosin II isoforms found in mammalian cells (VicenteManzanares et al.J Mol Hist (2009) 40:395–405 401 Fig. Cadherin-deﬁcient parental CHO cells (pCHO) were used as negative controls. wortmannin (100 nM. we examined the localization of active Myosin II. 2005. c. 2009). 4 Impact of PI3-kinase inhibition on E-cadherin expression and function. n. Cell adhesion to hE/ Fc-coated substrata was measured as described in Methods. b Effect of PI3-kinase inhibition on surface expression of E-cadherin. c) or MCF7 cells (d). 8 h) or DMSO control. 8 h). This suggested that. despite some redistribution of perijunctional Myosin IIA organization. which blocks Myosin II activation by upstream ROCK signaling. Stehbens et al. in contrast to the keratinocyte system. 6). Similarly. In contrast to inhibiting PI3K. To characterize this further. whereas they were clearly reduced when ROCK was blocked with Y27632. neither Y27632 nor blebbistatin affected MDCK cell height. We ﬁrst examined the impact of PI3-kinase on the subcellular localization of Myosin II in MDCK cells by indirect immunoﬂuorescence microscopy.
Finally. and is well-known to affect the organisation of the actin cytoskeleton (Hall 1998. and complement the results of. 2005). rather than for the process of maintaining height in established monolayers that we studied. Lgl1. If Rac is a downstream mediator of PI3-kinase signalling in the regulation of MDCK cell height. Laprise et al. rapamycin had no statisticallysigniﬁcant effect on MDCK cell height during this period (Fig. 1996). It is possible that the impact of Myosin II on cell height is greatest during epithelial biogenesis. 2004). This is consistent with earlier evidence that dominant negative Rac1N17 decreased MDCK cell height (Bruewer et al. 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. our ﬁndings identify a role for PI3-kinase in supporting epithelial cell height in established polarized monolayers. Our current data are consistent with. As shown in Fig. the small GTPase. Rac signalling is activated by E-cadherin homophilic ligation through a pathway that is partially sensitive to PI3-kinase. Representative apical (Par3. Dlg1 and Scribble (Scrib). In contrast. using a range of concentrations incubated for periods where LY294002 clearly reduced cell height. NCS23766 caused a statistically-signiﬁcant reduction in cell height. placing it potentially downstream of PI3-kinase in cadherin signalling (Kovacs et al. To test this. we predicted that blocking Rac signalling should phenocopy the effects of PI3-kinase inhibition. with a trend towards a dose-dependent effect at higher concentrations.402 J Mol Hist (2009) 40:395–405 height. This suggested that our observed reductions in cell height might reﬂect an overall decrease in cell size. Moreover. in a range of concentrations over the same period where PI3kinase inhibitors clearly reduced cell height. Fig. 7b. 2004) and therefore identiﬁes Rac as a potential downstream target of PI3-kinase in the regulation of epithelial cell height. aPKC. In contrast to the impact of LY294002. 7a). Of note. blebbistatin prevented growth in keratinocyte height when the drug was added to cells in the process of differentiation (Zhang et al. rapamycin. Insall and Machesky 2009). This potentially involves the well-documented capacity for PI3-kinase to signal through Rac. Rac1. is commonly identiﬁed as a downstream mediator of PI3 Kinase signalling (Reif et al. two earlier reports. with downstream effects on cytoskeletal organization and membrane trafﬁcking. 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 123 . Conﬂuent MDCK cell monolayers were immunostained for Par3. (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. 5 PI3-kinase inhibition and epithelial cell polarity. Dlg1. MDCK cell polarity was not overtly affected by the shorter exposure to LY2094002 in established MDCK cell monolayers. LY-treated Caco-2 cells were ﬂatter and less-polarized than control cells (Laprise et al. Consequently. 2002). 2002a). Ramalingam and Khuri 2009). Machesky and Insall 1999. we incubated cells with the mTOR inhibitor. aPKC) or basolateral (Lgl1. Scrib) confocal sections are shown Conclusion Overall. We pursued this by directly inhibiting Rac with the small molecule inhibitor NSC23766 (Gao et al.
05. phosphorylated Myosin regulatory light chain (ppMLC). blebbistatin (100 lM) or Y27632 (50 lM) for 8 h. c Effect of Myosin II inhibition on cell height. a Impact on junctional localization of Myosin II isoforms. Cell height was measured by confocal microscopy as described in Fig. * P \ 0.s. Myosin IIB (MIIB) or active. Conﬂuent MDCK monolayers were treated with LY294002 (50 lM). b Lysates from MDCK cultures treated with LY294002. Bar is 10 lm. Conﬂuent MDCK monolayers were treated with LY294002 (50 lM. Y27632 Fig. 3. n. then ﬁxed and immunostained for Myosin IIA (MIIA). 7 Impact of mTOR and Rac on epithelial cell height. Cell height in conﬂuent MDCK monolayers was measured from x–z images as described in Fig. b-tubulin was used as a loading control.: non-signiﬁcant 123 . Confocal sections from apical junctional planes are shown. a Effect of mTOR was compared by treatment with either LY294002 (50 lM) or rapamycin (1–100 nM) for 8 h. 6 Impact of PI3-kinase on non-muscle Myosin II in established epithelial monolayers. 8 h) or DMSO alone. b Effect of Rac was tested by treatment with LY294002 (50 lM) or NSC 23766 (20– 200 lM) for 8 h (50 lM) or DMSO controls were immunoprobed for active phosphorylated Myosin regulatory light chain (pMLC) by Western analysis.J Mol Hist (2009) 40:395–405 403 Fig. 3.
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