RESEARCH ARTICLE

Phosphorylation hotspot in the C-terminal domain of occludinregulates the dynamics of epithelial junctional complexesBhargavi Manda, Hina Mir, Ruchika Gangwar, Avtar S. Meena, Shrunali Amin, Pradeep K. Shukla, Kesha Dalal,Takuya Suzuki and RadhaKrishna Rao*

ABSTRACTThe apical junctional complex (AJC), which includes tight junctions(TJs) and adherens junctions (AJs), determines the epithelial polarity,cell-cell adhesion and permeability barrier. An intriguing characteristicof a TJ is the dynamic nature of itsmultiprotein complex.Occludin is themost mobile TJ protein, but its significance in TJ dynamics is poorlyunderstood. On the basis of phosphorylation sites, we distinguished asequence in the C-terminal domain of occludin as a regulatory motif(ORM). Deletion of ORM and expression of a deletion mutant ofoccludin in renal and intestinal epithelia reduced the mobility ofoccludin at the TJs. ORM deletion attenuated Ca2+ depletion, osmoticstress and hydrogen peroxide-induced disruption of TJs, AJs and thecytoskeleton. The double point mutations T403A/T404A, but notT403D/T404D, in occludin mimicked the effects of ORM deletion onoccludin mobility and AJC disruption by Ca2+ depletion. Both Y398A/Y402A and Y398D/Y402D double point mutations partially blockedAJC disruption. Expression of a deletion mutant of occludin attenuatedcollective cell migration in the renal and intestinal epithelia. Overall, thisstudy reveals the role of ORM and its phosphorylation in occludinmobility, AJC dynamics and epithelial cell migration.

KEY WORDS: Tight junction, Occludin, Cell migration,Phosphorylation, Adherens junction, Cytoskeleton

INTRODUCTIONThe epithelial apical junctional complex (AJC) consists of two well-organized junctions, zonula occludens (also known as tight junctions,TJs) and zonula adherens (also known as adherens junctions, AJs)(Vogelmann and Nelson, 2005). TJs are localized to the apical end ofthe lateral membrane of polarized epithelial cells and the AJs arelocalized just beneath the TJs. The close proximity of TJs and AJs isindicative of crosstalk between these junctional complexes. Cell-celladhesion, maintenance of cell polarity and development of epithelialbarriers are important functions of the AJC during embryogenesis(Sheth et al., 2000) and postnatal life (Vogelmann et al., 2004). TheAJC forms a docking site for signaling elements that regulatecell proliferation and differentiation (Matter and Balda, 1999). Adysfunctional AJC leads to developmental defects as well as a varietyof diseases of the different organ systems (Bruewer et al., 2006; Jang,2014; Schlüter and Margolis, 2012).

TJs modulate the regulation of the paracellular permeabilitybarrier and prevent apical-to-basolateral diffusion of membraneproteins (Anderson and Van Itallie, 1995; Shin et al., 2006).Structural organization of TJs involves interactions betweentransmembrane proteins such as occludin, tricellulin, marvel D3,the claudin subfamily, cytoplasmic scaffold proteins such as zonulaoccludens 1, 2 and 3 (ZO-1, ZO-2 and ZO-3, respectively) and theactin cytoskeleton (Anderson and Van Itallie, 2009; Shen et al.,2011). The intracellular TJ plaque recruits protein kinases, such asPKC, MAPK, c-Src and c-Yes, and protein phosphatases, such asPP2A and PP1, that mediate the fine-tuning of signaling cascadesinvolved in the regulation of barrier function (Dörfel and Huber,2012; Rao, 2008). Recent observations have demonstrated that TJcomponents are highly dynamic, with occludin being the mostmobile among them (Shen et al., 2008). The structure of AJsinvolves interactions between E-cadherin, catenins and the actincytoskeleton (Vogelmann and Nelson, 2005).

Occludin was the first transmembrane protein of TJ to bediscovered (Furuse et al., 1993); however, its precise function in TJassembly and regulation is unclear. Information from occludinknockout mice ruled out its requirement in TJ assembly, but thecomplex phenotypes of these mice are not completely understood(Saitou et al., 2000). Evidence suggests that occludin is associatedwith many cellular functions such as adhesion, differentiation, cellmigration, apoptosis and Ca2+ homeostasis (Beeman et al., 2012;Rachow et al., 2013; Van Itallie and Anderson, 1997). Occludin is atetraspanin with two extracellular loops, one intracellular loop, alarge cytoplasmic C-terminal domain and a short N-terminaldomain. The coiled-coil region of the C-terminal domain interactswith the guanylate kinase domain of ZO-1 (Cummins, 2012). Ourprevious studies have shown that occludin undergoes tyrosine (Tyr)phosphorylation in Caco-2 cell monolayers during TJ disruption byhydrogen peroxide, osmotic stress or other insults (Elias et al., 2009;Kale et al., 2003; Rao et al., 1997, 2002; Samak et al., 2011). Tyrphosphorylation of occludin on Y398 and Y402 regulates itsinteraction with ZO-1 and ZO-3, respectively (Elias et al., 2009).We further showed that PKCη and PKCζ-mediated phosphorylationof occludin on T403 and T404 facilitated its assembly into epithelialTJs (Jain et al., 2011; Suzuki et al., 2009). Other studies havedemonstrated that S408 phosphorylation in occludin regulatesClaudin-2 (Cldn-2) localization at TJs (Raleigh et al., 2011),reduces association of occludin with ZO-1 and ZO-2, and delaysassembly of TJs (Dörfel et al., 2013). These studies highlight thepresence of a highly conserved sequence with a cluster ofphosphorylation sites (Y398-S408) in the occludin C-terminaldomain, and we refer to this region as the ‘occludin regulatorymotif’ (ORM).

In this study, we investigated the potential role of ORM in TJdynamics by using renal and intestinal epithelial models andmultiple models of TJ assembly and disassembly. We alsoReceived 30 May 2017; Accepted 21 February 2018

Departments of Physiology, University of Tennessee Health Science Center, 3North Dunlap, Memphis, TN, 38103 USA.

*Author for correspondence (rrao2@uthsc.edu)

B.M., 0000-0001-5618-3967; H.M., 0000-0003-1973-199X; R.G., 0000-0002-2907-0411; A.S.M., 0000-0003-2213-2488; P.K.S., 0000-0003-2515-9766; T.S.,0000-0003-2502-6496; R.K.R., 0000-0002-5906-8371

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investigated the potential role of ORM in directional cell migration.This study provides key insights into the role of occludin in theregulation of TJ dynamics, the crosstalk between TJs and AJs, andepithelial cell migration.

RESULTSDeletion of ORM enhances assembly of occludin into TJs inMDCK cell monolayersFirst, occludin deficient MDCK cell line (OD-MDCK) wasgenerated by transfection of MDCK cells with shRNA for canineoccludin and dilution cloning. The ORM sequence in humanoccludin (OCLNWT) was deleted to generate deletion mutant (DM)occludin (OCLNDM) (Fig. 1A). The pEGFP vector containing theOCLNWT or OCLNDM gene or the empty vector (Vec) wastransfected into OD-MDCK cells. Stable clones were isolated andcharacterized. Immunoblot analysis showed the expression ofEGFP-tagged OCLNWT and OCLNDM and EGFP in thecorresponding clones (Fig. 1B). The interaction of C-terminalsequence (358-504) with ZO-1 has been shown to be required forassembly of chicken occludin at the TJ (Furuse et al., 1994). Todetermine the interaction of OCLNDM with ZO-1, EGFP wasimmunoprecipitated and immunoblotted for ZO-1 or EGFP. Resultsshow that ZO-1 co-immunoprecipitates with OCLNDM, at a levelsimilar to that of OCLNWT (Fig. 1C), indicating that ORM deletiondoes not prevent the interaction of occludin with ZO-1. Live-cellfluorescence imaging showed that EGFP-OCLNDM localized to theintercellular junctions (Fig. 1D). Densitometric fluorescenceanalysis indicated that the junctional localization of EGFP-OCLNDM was significantly greater than that of EGFP-OCLNWT

(Fig. 1E). Z-section imaging (Fig. 1F) and z-profiling (Fig. 1G) ofGFP fluorescence in these monolayers indicated that EGFP-OCLNDM is localized predominantly to the TJs, with a slightlybroader distribution compared with that of EGFP-OCLNWT.Barrier development was evaluated by measuring transepithelial

electrical resistance (TER) and the unidirectional flux offluoresceinyl isothiocyanate-inulin (FITC-inulin). On post-seedingdays 3 and 4, TER in OCLNDM and Vec cell monolayers wassignificantly lower compared with those in MDCK and OCLNWT

cell monolayers (Fig. 1H); however, the inulin flux was similar in allcell monolayers (Fig. 1I). To determine whether this was due toaltered TJ pores, we examined the expression and distribution ofCldn-2, a major cationic pore-forming TJ protein. Immunoblotanalysis and confocal microscopy showed that Cldn-2 levels(Fig. 1J,K) and junctional distributions (Fig. 1L,M) were greaterin OCLNDM and Vec-cell monolayers compared with that inOCLNWT cell monolayers. These data indicate that the elevatedexpression and junctional localization of Cldn-2 might havecontributed to low TER in OCLNDM and Vec cell monolayers.

ORM deletion augments association of occludin with theactin-rich cell fraction and diminishes its mobility in TJsAs fluorescence imaging showed a relatively high localization ofEGFP-OCLNDM at the junctions, we examined the association ofoccludin with the actin-rich detergent-insoluble fraction of the cell.OCLNDM and Vec cell monolayers appear to have greater levels ofF-actin at the perijunctional region compared with that in OCLNWT

cell monolayers (Fig. 2A). Immunoblot analysis indicated thatEGFP-OCLNWT and EGFP-OCLNDM were distributed in bothTriton-insoluble (TI) and Triton-soluble (TS) fractions (Fig. 2B).Densitometric analysis showed that the TI fraction of EGFP-OCLNDM was significantly greater than that of EGFP-OCLNWT

(Fig. 2C), suggesting that lack of ORM enhances the association of

occludin with the actin cytoskeleton. The F-actin to G-actin ratiowas calculated by measuring the amount of actin in TI and TSfractions by densitometric analysis of immunoblots. The TI/TSratios for actin were 0.63±0.011 and 0.61±0.002 for OCLNWT andOCLNDM cell monolayers, suggesting that a difference in the ratesof actin polymerization in these two monolayers is unlikely. Todetermine the rate of F-actin disassembly, we evaluated the effect oflatrunculin A on the barrier function of monolayers prepared fromdifferent cell lines. Our data show that latrunculin induced arapid disruption of barrier function in MDCK and OCLNWT

cell monolayers, and the rates of disruption were significantly low inOCLNDM andVec cell monolayers (Fig. S1A,B). Immunofluorescencestaining for phospho-myosin light chain (pMLC) indicated that itwas distributed mainly in the intracellular compartment in all celllines, with no detectable distribution at the junctions (Fig. S1C,D).

Fluorescence recovery after photobleaching (FRAP) analysisshowed that EGFP-OCLNDM fluorescence was reduced comparedwith that of EGFP-OCLNWT (Fig. 2D,E). The mobile fraction ofEGFP-OCLNDM (∼29.2%) was estimated to be significantly lowerthan that of EGFP-OCLNWT (∼53.3%) (Fig. 2F). A previous studyshowed that TJ proteins including occludin are highly dynamic andare in constant exchange with extra-junction pools (Shen et al.,2008). Our data suggest that ORM regulates the proportion of themobile fraction of occludin in TJs.

ORM deletion attenuates Ca2+-depletion-mediateddisruption of the AJC and barrier dysfunction in MDCK cellmonolayersCa2+-switch assay was performed in MDCK, OCLNWT, OCLNDM

and Vec cell monolayers. Live-cell fluorescence imaging indicatedthat incubation with a low-Ca2+ medium (LCM) for 16 h resulted ina redistribution of EGFP-OCLNWT from the intercellular junctionsinto the intracellular compartment. Interestingly, LCM failed to alterthe junctional distribution of EGFP-OCLNDM (Fig. 3A). Theseresults were confirmed in multiple clones of OCLNWT andOCLNDM cells (Fig. S2). Immunofluorescence staining of fixedcell monolayers showed that LCM failed to induce redistribution ofEGFP-OCLN, ZO-1, E-cadherin and β-catenin in OCLNDM cellmonolayers (Fig. 3B). Images for individual proteins are providedin the supplemental information (Fig. S3A,B). Reorganization ofF-actin and β-tubulin was absent in LCM-treated OCLNDM cellmonolayers (Fig. 3C). These observations suggest that ORM isrequired for Ca2+-depletion-mediated redistribution of occludin aswell as other TJ and AJ proteins. LCM dramatically reduced TER(Fig. 3D) and increased inulin permeability (Fig. 3E) in MDCK andOCLNWT cell monolayers, but replacement of LCM with a normal-Ca2+ medium (NCM) gradually restored basal TER and inulin flux.These changes in TER and inulin flux indicate that barrier functionis disrupted by Ca2+ depletion and restored by Ca2+ replacement inOCLNWT cell monolayers. However, LCM showed only minoreffects on TER and inulin flux in OCLNDM and Vec cellmonolayers, suggesting that ORM is required for Ca2+-depletion-mediated barrier dysfunction.

The effect of ORMdeletion was also tested in an EGTA-mediatedCa2+ depletion model. In OCLNWT cell monolayers, EGTA rapidlyreduced TER (Fig. 3F) and increased inulin flux (Fig. 3G), but itseffect on TER and inulin flux was significantly low in OCLNDM cellmonolayers. Confocal microscopy for EGFP-OCLN and ZO-1showed that EGTA induces redistribution of these proteins from thejunctions in MDCK and OCLNWT cell monolayers (Fig. 3H), butnot in Vec andOCLNDM cell monolayers. Similarly, EGTA inducedrapid redistribution of E-cadherin and β-catenin from the junctions

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in MDCK and OCLNWT cell monolayers, but had only minimaleffect in Vec and OCLNDM cell monolayers (Fig. 3I); individualimages for occludin, ZO-1, E-cadherin and β-catenin are presentedin the supplemental information (Fig. S3C–F). These data togetherconfirm that ORM deletion attenuates Ca2+-depletion-induced TJand AJ disruption. EGTA treatment also induced remodeling of theactin cytoskeleton in MDCK and OCLNWT cell monolayers

(Fig. 3J), whereas EGTA had only minimal effect in Vec cellmonolayers and failed to modulate F-actin organization inOCLNDM cell monolayers.

Our previous studies have shown that dephosphorylation of TJand AJ proteins occurs during Ca2+-depletion-mediated disruptionof TJ and AJ (Seth et al., 2007). Therefore, we conducted a study todetermine whether Ca2+-depletion-mediated dephosphorylation ofE-cadherin, β-catenin and ZO-1 on threonine (Thr) residues occursin OCLNWT and OCLNDM cell monolayers. Immunoprecipitationof phospho-Thr (p-Thr) followed by immunoblot analysis showedthat LCM-induced Ca2+ depletion reduced Thr phosphorylation ofE-cadherin, β-catenin and ZO-1 in MDCK and OCLNWT cellmonolayers (Fig. 3K–M), whereas Thr dephosphorylation ofE-cadherin, β-catenin and ZO-1 was low in LCM-treated Vec cellmonolayers and absent in OCLNDN cell monolayers.

Deletion of ORM attenuates osmotic stress and hydrogenperoxide-mediated disruption of the AJCThe effect of ORM deletion on barrier function was examined inother models of TJ disruption. Osmotic stress induced an increase ininulin permeability in OCLNWT cell monolayers (Fig. 4A). OCLNDM

andVec cell monolayers were significantly resistant to osmotic stress-induced inulin permeability. Consistent with this, osmotic stresscaused a redistribution of occludin and ZO-1 from the junctions inOCLNWT cell monolayers, but less so in OCLNDM cell monolayers(Fig. 4B). Inulin permeability in control cell monolayers (Fig. S4A)and images for individual proteins (Fig. S4B,C) are presented in thesupplemental information. Osmotic stress-induced redistribution ofE-cadherin and β-catenin was also minimal in OCLNDM cellmonolayers (Fig. S5). Live-cell imaging revealed that exposure to

Fig. 1. Deletion of ORM enhances occludin association with the TJ.(A) A part of the sequence (G388-D415) in the C-terminal domain of occludinincluding ORM (Y398-S408) was deleted from the wild-type human occludin(OCLNWT) to generate a deletion mutant of occludin (OCLNDM). (B) Stableclones of OD-MDCK cells expressing EGFP-OCLNWT, EGFP-OCLNDM andEGFP vector (Vec) were generated. Total protein extracts were immunoblottedfor EGFP, occludin (OCLN) and β-actin (β-Act). (C) GFP wasimmunoprecipitated from the native extracts of OCLNWT, OCLNDM and Veccells and immunoblotted for ZO-1. Density of ZO-1 was measured andnormalized to the corresponding EGFP band density. Values presented in thegraph are means±s.e.m. (n=3). (D,E) OCLNWT, OCLNDM and Vec cellmonolayers were imaged live for EGFP (D). Junctional fluorescence wasevaluated by densitometric analysis (E). Values, in arbitrary units offluorescence intensity, are means±s.e.m. (n=3). Asterisks indicate that valuesare significantly (P<0.05) different from the OCLNWT value. (F,G) Z-sectionimages of GFP fluorescence in OLCNWT (WT) and OCLNDM (DM) cellmonolayers were captured (F), and Z-profiling of GFP fluorescence in thesecell monolayers were analyzed (G). Values are means±s.e.m. (n=4). Asterisksindicate that values are significantly (P<0.05) different from correspondingvalues for WT cell monolayers. (H,I) Equal numbers of MDCK (blue), OCLNWT

(OKD-WT; green), OCLNDM (OKD-DM; brown) and Vec (OKD-VEC; red) cellswere seeded onto transwell inserts. TER (H) and FITC-inulin flux (I) weremeasured at various time points post seeding. Values presented in the graphare means±s.e.m. (n=3; three different clones for each group and the value foreach clone is the average of six for 1 h, five for 2 h, four for 3 h and three for4 h). The experiment was performed twice. Asterisks indicate MDCK andOCLNWT values that are significantly (P<0.05) different from correspondingvalues for Vec and OCLNDM groups. (K,L) Total protein extracts fromOCLNWT,OCLNDM and Vec cells were immunoblotted for claudin-2 (Cldn-2) (K).Immunoblot bands were quantified by densitometric analysis (L). Values aremeans±s.e.m. (n=3). Asterisks indicate that values are significantly (P<0.05)different from the corresponding OCLNWT value. (J,M) Fixed cell monolayerswere stained for Cldn-2 by immunofluorescence method (J). Fluorescence atthe intercellular junctions wasmeasured by densitometric analysis (M). Valuesare means±s.e.m. (n=3). Asterisks indicate that values are significantly(P<0.05) different from the corresponding OCLNWT value. Scale bars: 50 μm.

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hydrogen peroxide caused redistribution of EGFP-OCLNWT from thejunctions, but it had minimal effect on EGFP-OCLNDM (Fig. 4C).Hydrogen peroxide caused a time-dependent increase in inulinflux (Fig. 4D), and redistribution of occludin/ZO-1 (Fig. 4E) andE-cadherin/β-catenin (Fig. 4F) in OCLNWT cell monolayers, butthese effects were significantly low in OCLNDM and Vec cellmonolayers. Inulin permeability in control cell monolayers(Fig. S6A) and images for individual proteins (Fig. S6B,C) arepresented in the supplemental information. These data indicate thatORM is required for an effective disruption of AJC by osmotic stressand hydrogen peroxide.

Phosphorylation of ORM on residues T403/T404 and Y398/Y402 determines the dynamic property of TJsTo determine the role of ORM phosphorylation in TJ dynamics,point mutants of full-length occludin were generated and expressedin OD-MDCK cells. Live-cell fluorescence imaging (Fig. 5A)showed that LCM caused redistribution of EGFP-OCLNT403/404D

from the intercellular junctions, similar to that of EGFP-OCLNWT, whereas the distribution of EGFP-OCLNT403/404A wasnot altered, similar to that of EGFP-OCLNDM. However,both EGFP-OCLNY398/402D and EGFP-OCLNY398/402A mutantsshowed significant resistance to LCM-mediated redistributionfrom the junctions (Fig. 5A). Confocal imaging of fixed cellmonolayers showed that LCM induced redistribution of bothEGFP-OCLN and ZO-1 from the intercellular junctions inOCLNWT and OCLNT403/404D cell monolayers, but not inOCLNDM, OCLNT403/404A, OCLNY398/402D and OCLNY398/402A

cell monolayers (Fig. 5B). Images for the distribution of individualproteins are provided in the supplemental information (Fig. S7).Similarly, LCM caused redistribution of E-cadherin and β-cateninfrom the intercellular junctions in OCLNWT andOCLNT403/404D cellmonolayers, but not in OCLNDM, OCLNT403/404A, OCLNY398/402D

and OCLNY398/402A cell monolayers (Fig. S8). LCM also decreasedTER (Fig. 5C) and increased inulin permeability (Fig. 5D) inOCLNWT

and OCLNT403/404D cell monolayers; these effects of LCMwere significantly attenuated in OCLNDM, OCLNT403/404A,OCLNY398/402D and OCLNY398/402A cell monolayers. FRAP analysis(Fig. 5E) demonstrated that the percentage mobile fraction of EGFP-OCLNT403/404D at the junctions was similar to that of EGFP-OCLNWT,whereas mobile fractions of EGFP-OCLNDM, EGFP-OCLNT403/404A,EGFP-OCLNY398/402D and EGFP-OCLNY398/402A were relativelylow (Fig. 5F). The FRAP half-life (FRAP t1/2) values for differentcell lines were not significantly different from that for EGFP-OCLNWT cell monolayers, except that FRAP t1/2 was significantlylower for EGFP-OCLNT403/404D cell monolayers (Fig. 5G).

The effect of Y398A/Y402A and Y398D/Y402D mutations onLCM-mediated TJ disruption indicated a potential role of tyrosinekinases and protein Tyr phosphorylation of proteins during Ca2+-depletion-induced TJ disruption. To determine the role of Tyrphosphorylation in Ca2+-depletion-mediated TJ disruption, TERand inulin flux were measured in MDCK cell monolayers treatedwith genistein, a tyrosine kinase inhibitor, prior to LCM. Decreasein TER (Fig. 6A) and increase in inulin flux (Fig. 6B) in cellscultured in LCM was significantly blocked by genistein. Confocalimaging showed that genistein also blocked LCM-inducedredistribution of occludin and ZO-1 (Fig. 6C–E) from thejunctions into the intracellular compartment.

Deletion of ORM reduces the mobile fraction of occludin andattenuates TJ dynamics in the intestinal epitheliumTo investigate whether the phenotype rendered by ORM deletion isa general epithelial property, we evaluated the role of ORM in TJdynamics in the intestinal epithelium. The OCLNWT or OCLNDM

gene or Vec was expressed in the rat intestinal epithelial cell line,IEC-6. Immunoblot analysis showed a comparable expressionof EGFP-OCLNWT and EGFP-OCLNDM, but there was noendogenous occludin detected in these cells (Fig. 7A). Live-cellimaging revealed that deletion of ORM did not prevent occludinlocalization at the junctions, confirming that ORM is not requiredfor occludin assembly into TJs (Fig. 7B); however, junctionaldistribution of EGFP-OCLNWT was much greater than that ofEGFP-OCLNDM. Densitometric analysis of fluorescence at thejunctions showed a higher fluorescence intensity in EGFP-

Fig. 2. Deletion of ORM enhances the association of occludin with theactin-rich fraction of cells and decreases its mobility at TJs. (A) Two daysafter seeding, OCLNWT, OCLNDM and Vec cells were fixed and stained forF-actin (F-Act), β-tubulin (β-Tub) and nucleus (Nuc). Scale bar: 50 µm.(B,C) Triton-soluble and insoluble fractions prepared from OCLNWT, OCLNDM

and Vec cells were immunoblotted for GFP and β-actin (β-Act) (B). GFP banddensities were measured and normalized to corresponding β-actin banddensities (C). Values are means±s.e.m. (n=3). Asterisks indicate the valuesthat are significantly (P<0.05) different from corresponding value for OCLNWT

cells. (D–F) FRAP analysis of EGFP was performed in OCLNWT and OCLNDM

cell monolayers. Time-lapse images of several ROIs (regions of interest) atintercellular junctions were collected before and after photobleaching (D).Fluorescence intensity in the bleached areawasmeasured (E) and percentagemobile fractions of OCLNWT and OCLNDM were calculated (F). Values inpanels E and F are means±s.e.m. (n=8). Asterisks indicate the values that aresignificantly (P<0.05) different from the corresponding values for OCLNWT

cells.

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OCLNDM cell monolayers (41.57±5.69, n=3, each an average ofthree regions; arbitrary units) than that in EGFP-OCLNWT cellmonolayers (31.20±2.02). FRAP analysis indicated that the

fluorescence (Fig. 7C,D) and mobile fraction of EGFP-OCLNDM

were significantly lower than those of EGFP-OCLNWT (Fig. 7E).Barrier function analysis showed the LCM-induced drop in TER

Fig. 3. See next page for legend.

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(Fig. 7F) and rise in inulin-permeability (Fig. 7G) in OCLNWT cellmonolayers, but the LCM effect was minimal in OCLNDM cellmonolayers. Consistently, immunofluorescence microscopyshowed that LCM induced redistribution of occludin and ZO-1(Fig. 7H) as well as E-cadherin and β-catenin (Fig. 7I) from thejunctions in OCLNWT-IEC-6 cell monolayers, but redistributionwas minimal in EGFP-OCLNDM-IEC-6 cell monolayers,

demonstrating that ORM is required for Ca2+-depletion-mediateddisruption of TJs in the intestinal epithelium.

To confirm the role of occludin in TJ dynamics in the intestinaltissue, we applied an ex vivo model of the intestinal epithelium byusing the intestinal loops prepared from (wild-type) WT andoccludin-deficient (OCLN−/−) mice and evaluated the effect ofEGTA-mediated Ca2+ depletion. Mucosal barrier function in theintestinal loops was evaluated by measuring the uptake of FITC-inulin from the lumen. Inulin uptake from the lumen of OCLN−/−

mouse intestine was significantly lower than that from WT mouseintestine (Fig. 7J). Confocal microscopy showed that EGTAinduced redistribution of ZO-1 (Fig. 7K) and E-cadherin/β-catenin(Fig. 7L) from the junctions in WT mouse intestines. EGTA causedonly a minimal effect on the junctional distributions of ZO-1,E-cadherin and β-catenin in OCLN−/− mouse intestines. These datasuggest that lack of occludin confers resistance to AJC disruption inthe intestinal tissue by depletion of Ca2+.

Deletion of ORM impairs collective cell migration in MDCKand IEC-6 cell monolayersTo determine the functional consequence of altered TJ dynamicscaused by lack of ORM, we investigated the role of ORM in cellmigration using OD-MDCK and IEC-6 cells that express EGFP-OCLNWT or EGFP-OCLNDM. Rates of cell migration followingscrape wounding were significantly lower in Vec and EGFP-OCLNDM MDCK cell monolayers than in EGFP-OCLNWT cellmonolayers (Fig. 8A,B). Similarly, Vec and EGFP-OCLNDM-IEC-6 cell monolayers showed lower rates of cell migrationfollowing scratch wounding than EGFP-OCLNWT-IEC-6 cellmonolayers (Fig. 8C,D). Taken together, these data indicate thatthe absence of ORM significantly attenuates collective cell

Fig. 3. ORM deletion attenuates Ca2+-depletion-mediated disruption ofthe AJC and barrier dysfunction. (A–C) OCLNWT and OCLNDM cellmonolayers were incubated with low-Ca2+ medium (LCM) or normal-Ca2+

medium (NCM) for 16 h. Live-cell images for EGFP fluorescence werecaptured before and after incubation (A). Fixed cell monolayers were stainedfor EGFP-occludin (GFP-OCLN), ZO-1, E-cadherin (E-Cad) and β-catenin(β-Cat) (B) or cytoskeletal proteins F-actin and β-tubulin (C). (D,E) MDCK(blue), OCLNWT (OKD-WT; green), OCLNDM (OKD-DM; brown) and Vec(OKD-VEC; red) cell monolayers on transwell inserts were incubated with LCMfor 16 h followed by incubation with NCM for up to 3 h. TER (D) and FITC-inulinflux (E) were measured at various time points. Values are mean±s.e.m. (n=6).Asterisks indicate the values for MDCK and OCLNWT cell monolayers that aresignificantly (P<0.05) different from corresponding values for OCLNDM andVec-cell monolayers. (F,G) MDCK (blue), Vec (red), OCLNWT (green) andOCLNDM (brown) cell monolayers on transwell inserts were incubated with(circles) or without (squares) 4 mM EGTA. TER (F) and FITC-inulin flux(G) were measured at various time points. Values are means±s.e.m. (n=6).Asterisks indicate the values for MDCK and OCLNDM monolayers that aresignificantly (P<0.05) different from corresponding values for Vec andOCLNWT monolayers. (H–J) Cell monolayers after EGTA treatment were co-stained for EGFP-occludin and ZO-1 (H), E-cadherin and β-catenin (I) orF-actin and β-tubulin (J). (K–M) Cell monolayers were incubated with LCM (L)or NCM (N) for 6 h. Phospho-threonine (p-Thr) was immunoprecipitated fromdenatured protein extracts. Immunoprecipitates and original extracts (Load)were immunoblotted for ZO-1 (K), E-cadherin (L), β-catenin and β-actin (M).Scale bars: 50 µm.

Fig. 4. ORM deletion attenuates disruptionof the AJC and barrier dysfunction causedby osmotic stress and hydrogen peroxide.(A) MDCK (blue), OCLNWT (green), OCLNDM

(brown) and Vec (red) cell monolayers ontranswell inserts were incubated in DMEM(Control; data in Fig. S4A) or DMEMcontaining 0.3 M mannitol to induce osmoticstress (OS). FITC-inulin flux (A) wasmeasured at various time points. Values aremeans±s.e.m. (n=6). Asterisks indicate thevalues for OCLNWT cell monolayers that aresignificantly (P<0.05) different fromcorresponding values for OCLNDM and Veccell monolayers. (B) Fixed cell monolayerswere stained for EGFP-occludin (GFP-OCLN) and ZO-1. (C) OCLNWT and OCLNDM

cell monolayers were treated with hydrogenperoxide (100 µM) and live-cell images forEGFP fluorescence were collected at varioustime points. (D) MDCK (blue), OCLNWT

(green), OCLNDM (brown) and Vec (red) cellmonolayers on transwell inserts were treatedwith hydrogen peroxide (100 µM) in DMEM.FITC-inulin flux wasmeasured at various timepoints. Values are means±s.e.m. (n=6).Asterisks indicate the values for OCLNWT cellmonolayers that are significantly (P<0.05)different from corresponding values forOCLNDM and/or Vec cell monolayers. Controlvalues are in Fig. S6A. (E,F) Fixed cellmonolayers at different time points werestained for EGFP-occludin and ZO-1 (E) orE-cadherin and β-catenin (F).

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migration in both renal and intestinal epithelia. To determinewhether lack of ORM affects single-cell migration, we evaluatedtransmigration of different lines of MDCK and IEC-6 cells.Transmigration of OD-MDCK cells expressing Vec or OCLNDM

was significantly greater than migration of MDCK cells andOD-MDCKcells expressingOCLNWT (Fig. 8E). Similarly,migrationof IEC-6 cells expressing Vec or OCLNDM was significantly greaterthan that of IEC-6 cells expressing OCLNWT (Fig. 8F).

DISCUSSIONBesides forming a seal that maintains tissue compartmentation, TJsserve to regulate the paracellular transport across the epithelium undervarious physiological and pathophysiological conditions. The TJ

protein complex is dynamic in nature (Shen, 2012), although theprecise function of this property is poorly understood. The importanceof phosphorylation in the assembly/disassembly of TJs (Rao, 2009)and the identification of phosphorylation sites in the C-terminaldomain of occludin (Elias et al., 2009; Kale et al., 2003; Suzuki et al.,2009) led to the recognition of a highly conserved phosphorylationhotspot in occludin – the ORM. We hypothesized that ORM plays aregulatory role in TJ dynamics. Although deletion of occludin preventsneither in vitro nor in vivo TJ assembly (Saitou et al., 1998, 2000), theresults of our current study provide evidence for a role of occludin andORM in the regulation of the dynamic property of TJs and AJs.

Interaction with ZO-1 is crucial for its assembly into the TJ. Ourresults indicate that ORM is not required for ZO-1 binding and,

Fig. 5. Phosphorylation of ORM on T403/T404 and Y398/402 determines the dynamic properties of TJs. (A,B) OCLNWT (WT), OCLNDM (DM), OCLNT403/404A

(T403/404A), OCLNT403/404D (T403/404D), OCLNY398/402A (Y398/402A) and OCLNY398/402D (Y398/402D) cell monolayers were incubated with low-Ca2+ medium(LCM) or normal-Ca2+ medium (NCM) for 1–24 h. Live-cell images for EGFP fluorescence were collected before and after incubation (A). Cell monolayers fixedat 1 h and 16 h were stained for EGFP-Occludin and ZO-1 (B). Scale bars: 50 µm. (C,D) OCLNWT, OCLNDM, OCLNT403/404A, OCLNT403/404D, OCLNY398/402A andOCLNY398/402D cell monolayers on transwell inserts were incubated with LCM for 16 h and TER (C) and FITC-inulin flux (D) were measured. Values are means±s.e.m. (n=6). Asterisks indicate the values that are significantly (P<0.05) different from corresponding values for OCLNWT cell monolayers. (E–G) FRAP analysis ofEGFP was performed in OCLNWT (WT), OCLNDM (DM), OCLNT403/404A (T2A), OCLNT403/404D (T2D), OCLNY398/402A (Y2A) and OCLNY398/402D (Y2D) cellmonolayers. Time-lapse images were collected before and after photobleaching of several ROIs at intercellular junctions. Fluorescence intensity was measured(E) and%mobile fraction (F) and t1/2 values (G) were calculated. Values aremeans±s.e.m. (n=4). Asterisks indicate the values that are significantly (P<0.05) differentfrom the value for OCLNWT cell monolayers.

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therefore, ORM deletion does not prevent TJ assembly or barrierfunction. On the contrary, assembly of OCLNDM at the junctions issignificantly greater than that of OCLNWT. On days 3–4 afterseeding, OCLNDM and Vec cell monolayers maintained low TERcompared with OCLNWT and MDCK cell monolayers, but theinulin permeability in OCLNDM and Vec cell monolayers was aslow as that in OCLNWT and MDCK monolayers. This raised thequestion whether low resistance on days 3–4 after seeding is causedby higher expression of pore-forming claudins. A previous studyshowed that occludin regulates the localization of Cldn-2, a cation-selective pore-forming claudin, in Caco-2 cell monolayers through amechanism that depends on phosphorylation of S408 (Raleigh et al.,2011). The present study shows that the levels and junctionaldistribution of Cldn-2 were significantly higher in Vec and OCLNDM

cell monolayers. Occludin and ORM may indirectly regulate theexpression and distribution of Cldn-2. However, on post seeding days1–2, OCLNDM and Vec cell monolayers maintained low TER andhigh inulin permeability compared with those in OCLNWT andMDCK cell monolayers. Therefore, in addition to the difference inCldn-2 levels, the TJ barrier properties or TJ assembly kinetics mayalso be altered in OCLNDM and Vec cell monolayers. To draw ameaningful conclusion regarding the interaction between occludinand Cldn-2, detailed future studies in cell lines and mouse tissuesare necessary. A previous study has shown that TER of OD-MDCKcells is significantly lower than that in control cell monolayersduring post-seeding assembly or Ca2+-mediated reassembly of TJs(Yu et al., 2005). This study has also shown that the FITC-dextranpermeabilities of 6-day-old MDCK and OD-MDCK cell monolayerswere not different, although the permeability characteristics weredifferent for small molecular weight solutes.

The cytoplasmic TJ plaque is closely associated with theperijunctional actomyosin ring through its interaction with ZO-1,and, therefore, indirectly with occludin; this interaction is essentialfor the assembly and maintenance of TJs (Fanning et al., 1998). Ourdata show that lack of ORM enhances localization of occludin in theactin-rich detergent-insoluble fraction of the cell, suggesting anenhanced interaction of occludin with the actin cytoskeleton in theabsence of ORM. However, it should be noted that the detergent-insoluble fraction may also contain other components, such as lipidrafts. FRAP analysis demonstrated that TJ proteins are highlydynamic at steady state and each TJ protein has a distinct dynamicbehavior, with occludin having the highest mobile fraction (Shenet al., 2008). Our data show that ORM deletion reduces the mobilefraction of occludin, indicating that it is more static at theintercellular junctions in the absence of ORM. We speculate thatthe increased association of OCLNDM with the actin cytoskeletondetermines the reduced mobile fraction or vice versa.

Many previous studies have associated hyperphosphorylatedoccludin with TJs, mostly assessed by the association ofhyperphosphorylated occludin with the detergent-insoluble fraction.The results of the present study show that non-phosphorylatedoccludin also associates with the detergent-insoluble fraction and TJs.Although hyperphosphorylated occludin has been shown to beassociated with TJs and the detergent-insoluble fraction in severalstudies, it is unclear whether occludin is phosphorylated before orafter its assembly into TJs. A previous study has indicated that lack ofoccludin phosphorylation delays, but does not prevent, assembly ofoccludin into TJs (Suzuki et al., 2009). Our present studydemonstrates that phosphorylation of occludin is not a prerequisitefor its assembly into TJs, and that absence of phosphorylation confersresistance to disassembly of occludin from TJs. Therefore,irrespective of whether the phosphorylation occurs prior to or after

Fig. 6. Inhibition of tyrosine kinase activity blocks Ca2+-depletion-mediated disruption of TJs and barrier function. (A,B) MDCK cellmonolayers on transwell inserts were treated with 100 µM genistein (Gen)30 min prior to LCM treatment. TER (A) and FITC-inulin flux (B) weremeasuredafter 1 h. Values aremeans±s.e.m. (n=6). Asterisks indicate the values that aresignificantly (P<0.05) different from corresponding value for OCLNWT cellmonolayers and hash signs indicate the values that are significantly (P<0.05)different from that of OCLNDM cell monolayers. (C,D) Cell monolayers were co-stained for GFP-occludin (C) and ZO-1 (D). Merged images are presented inpanel E. Scale bar: 50 µm.

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assembly, it is required for optimal disassembly of occludin from theTJ. Interestingly, our study also shows that non-phosphorylatedoccludin confers resistance to disassembly of an entire TJ. Therefore,

occludinmay not be needed for TJ assembly but, once it is assembledinto a TJ, it regulates the TJ dynamics by a phosphorylation-dependent mechanism.

Fig. 7. Deletion of ORM attenuates occludin mobility and Ca2+-depletion-mediated disruption of the AJC in the intestinal epithelium. (A,B) Total proteinextracts from IEC-6 cells expressing EGFP-OCLNWT, EGFP-OCLNDM and EGFP vector (Vec) were immunoblotted for EGFP, occludin and β-actin (β-Act) (A).OCLNWT, OCLNDM and Vec cell monolayers were imaged live for EGFP (B). (C–E) FRAPanalysis of EGFPwas performed inOCLNWT (WT) andOCLNDM (DM) cellmonolayers. Time-lapse images of several ROIs at intercellular junctions were collected before and after photobleaching (C). Fluorescence intensity in the bleachedarea was measured (D) and percentage mobile fractions of OCLNWT and OCLNDM were calculated (E). Values are means±s.e.m. (n=8). Asterisks indicatevalues that are significantly (P<0.05) different from the value for OCLNWT cells. (F,G) OCLNWT and OCLNDM cell monolayers on transwell inserts were incubated inLCM, and TER (F) and FITC-inulin flux (G) were measured at various time points. Values presented in the graph are means±s.e.m. (n=6). Asterisks indicate thevalues that are significantly (P<0.05) different from corresponding values for the OCLNWT group. (H,I) Fixed cell monolayers were stained for TJ proteins EGFP-occludin and ZO-1 (H) or AJ proteins E-cadherin and β-catenin (I). (J-L) Intestinal loops (∼2 cm long) from the mid small intestine of wild-type (WT) and occludin-deficient (OCLN−/−) mice were prepared and filled with saline containing FITC-inulin with or without 1 mM EGTA. Loss of inulin from the loops was measured after30 min incubation in DMEM (J). Values are means±s.e.m. (n=3). The asterisk indicates the value for the OCLN−/− group that is significantly (P<0.05) different fromthe value for the WT group. Cryosections of the loops were immunostained for ZO-1, F-actin and nucleus (K) or E-cadherin and β-catenin (L). Scale bars: 50 µm.

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The Ca2+-switch assay is a widely used technique to study the denovo assembly of TJs. We used this method to investigate the role ofORM in TJ disruption and assembly. Live cell imaging showed thatincubation with LCM induced redistribution of EGFP-OCLNWT

from the intercellular junctions into the intracellular compartment.Intriguingly, LCM failed to induce redistribution of EGFP-OCLNDM

from the intercellular junctions even after overnight incubation,indicating that ORM is required for Ca2+-depletion-mediatedredistribution of junctional occludin. Immunofluorescence stainingof fixed cell monolayers indicated that the lack of occludin or ORMblocks the redistribution of both occludin and ZO-1. Furthermore,absence of occludin or ORM prevents Ca2+-depletion-mediateddecrease in electrical resistance and inulin permeability. Theseobservations demonstrate that ORM in occludin is required for Ca2+-depletion-mediated disruption of TJs and barrier dysfunction.Absence of occludin or ORM also blocked the Ca2+-depletion-

mediated redistribution of E-cadherin and β-catenin from thejunctions, indicating that the absence of ORM interferes with theCa2+-depletion-mediated disruption of AJs. The mechanisminvolved in Ca2+-depletion-mediated TJ disassembly remains tobe elucidated. It could be assumed that the extracellular Ca2+

depletion leads to AJ disruption – as E-cadherin requires Ca2+ for itstrans homophilic interactions – in turn, disrupting the TJs. Another

mechanism that is likely to be involved is that the depletion ofextracellular Ca2+ leads to a decrease in intracellular Ca2+, whichactivates a signaling cascade that mediates the disassembly of AJCs.Furthermore, the absence of ORM blocks Ca2+-depletion-mediateddisruption of actin cytoskeleton and microtubules. Therefore, it islikely that Ca2+ depletion triggers a common signaling pathway thatleads to disruption of TJs, AJs and the cytoskeleton, and that ORMis required for activation of this signaling cascade. The lack ofdifferences in detergent insoluble to soluble ratios of actindistribution suggests that actin polymerization rates in OCLNWT

and OCLNDN cell monolayers are the same. However, a resistanceto latrunculin-mediated barrier disruption in EGFP-OCLNDM andVec cell monolayers suggested that the rate for F-actin disassemblyis reduced by ORM deletion.

To determine whether ORM is involved in TJ disruption by otherstressors, we evaluated its influence on TJ disruption in response toosmotic stress or hydrogen peroxide. Osmotic stress and oxidativestress are known to induce TJ disruption by Src-mediated Tyrphosphorylation and PP2A-induced Thr dephosphorylation ofoccludin (Basuroy et al., 2003; Rao, 2008; Samak et al., 2015,2011; Sheth et al., 2003). Our current data show that the absence ofORM attenuates both osmotic stress-mediated and hydrogenperoxide-mediated disruption of TJs and AJs. These observations

Fig. 8. Absence of ORM impairs directional cell migration in renal and intestinal epithelia. (A,B) OD-MDCK cells expressing EGFP-OCLNWT (WT), EGFP-OCLNDM (DM) and EGFP vector (Vec) were grown to confluence, and cell migration assay was performed by scrape wounding. Phase-contrast images werecaptured at various time points (A); the purple lines indicate the origin of migration. Area of migration wasmeasured using ImageJ and presented in arbitrary units(B). Values are means±s.e.m. (n=5; each value is an average of five images from the same monolayer). Asterisks indicate the values that are significantly(P<0.05) different from corresponding values for Vec cells, and the hash signs indicate the values that are different from corresponding values for EGFP-OCLNWT

cells. (C,D) IEC-6 cells expressing EGFP-OCLNWT (WT), EGFP-OCLNDM (DM) and EGFP vector (Vec) were grown to confluence, and cell migration assay wasperformed by scratch wounding. Phase-contrast images were captured at 12 h or 24 h (C). The wound area was measured using ImageJ and presented inarbitrary units (D). Values are means±s.e.m. (n=5; each value is an average of five images from the same monolayer). Asterisks indicate the values that aresignificantly (P<0.05) different from corresponding values for Vec cells, and the hash signs indicate the values that are different from corresponding values forEGFP-OCLNWT cells. (E,F) OD-MDCK cells (E) and IEC-6 cells (F) expressing EGFP vector (Vec), EGFP-OCLNWT (WT) or EGFP-OCLNDM (DM) (105 cells/well), and MDCK cells (E) were seeded at low density on to transwells. Cells that migrated to the bottom surface of transwells were counted (E). Similar to MDCKcells, IEC-6 cells transfected with EGFP vector (Vec), EGFP-OCLNWT (WT) or EGFP-OCLNDM (DM) were seeded on to transwells to measure transmigration (F).Values are means±s.e.m., and the values within the bars are the number of samples per group. Asterisks indicate significant differences (P<0.05) between thegroups.

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confirm that the role of ORM is not restricted to Ca2+-depletion-mediated TJ disruption. Rather, it is a general phenomenonaffecting TJ and AJ disruption by several factors. This view isfurther supported by the previous observation that OD-MDCK cellmonolayers are resistant to methyl-β-cyclodextrin-induced barrierdisruption, whereas native MDCK cell monolayers are not (Yuet al., 2005).Resistance to AJC disruption due to multiple stressors, such as

Ca2+ depletion, osmotic stress, hydrogen peroxide and latrunculinA, in occludin-deficient Vec cell monolayers suggests that occludinhas a role in promoting AJC disruption. This is an intriguingobservation. Although it appears that occludin negatively affects theAJC, the ability to disassemble is likely an important characteristicfeature of the epithelial TJs, AJs and the cytoskeleton. It is alreadyestablished that the ability of the actin cytoskeleton to disassemble isan essential feature of actin dynamics as actin depolymerization isneeded for cell migration and other cellular functions. Our data raisethe question whether the presence of occludin in TJs enhancesthe flexibility of the actin cytoskeleton to dissociate when neededfor certain cellular functions. Our data demonstrate that thephosphorylation hotspot, i.e. ORM, in the C-terminal domain ofoccludin is necessary for this function of occludin.Our previous studies have demonstrated that occludin is

phosphorylated during the assembly and disassembly of TJs (Rao,2009; Rao et al., 2002; Seth et al., 2007). Phosphorylation ofoccludin on T403 and T404 promotes TJ assembly (Suzuki et al.,2009), whereas phosphorylation of Y398 and Y402 prevents TJinteraction with ZO-1 (Kale et al., 2003) and is involved in thedisruption of TJs (Elias et al., 2009). As these phosphorylation sitesare located in the ORM sequence, we conducted studies todetermine whether the response to ORM deletion could bemimicked by point mutations to these residues. Attenuation ofoccludin mobility, Ca2+-depletion-mediated disruption of the AJCand barrier dysfunction by T403A/T404A double mutations aresimilar to the effects of ORM deletion, demonstrating that thephosphorylation of ORM determines the dynamic property ofoccludin and, therefore, AJC dynamics. This observation isin contrast to our previous observation that T403/T404phosphorylation promotes TJ assembly. The likely explanation forthis discrepancy is that, in our previous study, we expressed T403/404 mutants in native MDCK cells with unaltered endogenous WToccludin. Therefore, T403/404 mutants were likely to oligomerizewith the endogenous WT occludin. Although the absence of T403/404 phosphorylation delayed the assembly of occludin into TJs,eventually it did assemble. In the present study, we expressedoccludin mutants in occludin-deficient cells. Occludinphosphorylation dynamics can promote TJ assembly and, oncethe TJ is formed, the phosphorylation dynamics are required torecognize the signaling elements needed to disrupt the AJC.Interestingly, LCM-mediated barrier dysfunction and occludin/

ZO-1 redistribution was partially blocked by Y398A/Y402A orY398D/Y402D mutations. The likely explanation is that thesetyrosine residues are transiently phosphorylated during LCM-induced TJ disruption and, therefore, both phosphorylation anddephosphorylation of these residues are necessary for TJ disruption.The importance of Tyr phosphorylation in LCM-mediated TJdisruption and barrier dysfunction was confirmed by theobservation that genistein, a tyrosine kinase inhibitor, prevents theCa2+-depletion-mediated loss of TJ integrity. FRAP analysis showedthat mutation of T403/404 and Y398/402 alters the mobile fraction ofoccludin in parallel to its effect on Ca2+-depletion-mediated TJdisruption. We speculate that the interaction of signaling elements

with ORM by a phosphorylation-dependent mechanism may beinvolved in the regulation of the dynamic property of the AJC indifferent epithelial tissues.

It is unclear whether the reduction in mobile fraction of occludinis related to the resistance of EGFP-OCLNDM cell monolayers to TJdisruption. The effect of ORM deletion on the resistance to AJCdisruption upon Ca2+ depletion is greater than that observed on themobile fraction of occludin. It is possible that these two responsesare independent of each other. However, FRAP is likely to be aresult of multiple pools of mobile fraction. The lack of ORM mightaffect only one pool of mobile fraction that is relevant to TJdisruption. The logical explanation for AJC disruption by Ca2+

depletion is loss of cadherin-dependent adhesion that requiresextracellular Ca2+ for homophilic interactions between extracellulardomains of E-cadherin. The results of the present studyunexpectedly showed that the non-phosphorylated occludinmutant confers cell monolayers with the resistance to Ca2+-mediated disruption of AJs and the actin cytoskeleton. Thisobservation suggests that the process of Ca2+-depletion-mediateddisruption of TJs, AJs and the actin cytoskeleton involvesmodification of a common intracellular mechanism. Previousstudies have demonstrated the role of Rho-associated kinase II(ROCKII) (Samarin et al., 2007) and c-Jun N-terminal kinase (JNK)(Naydenov et al., 2009) in AJC disruption by Ca2+ depletion. Ourpresent observation confirms that AJ disruption is not the primarymechanism involved in Ca2+-depletion-mediated TJ disruption. Ourprevious studies have shown that dephosphorylation of TJ and AJproteins occurs during Ca2+-depletion-mediated disruption ofTJs and AJs (Seth et al., 2007). Data presented in this article(Fig. 3K-M) show that Ca2+ depletion mediates dephosphorylationof E-cadherin, β-catenin and ZO-1 on Thr residues in MDCK andOCLNWT cell monolayers, whereas the Ca2+-depletion-mediatedThr dephosphorylation of E-cadherin, β-catenin and ZO-1 is absentin OCLNDM cell monolayers. This suggests that ORM forms aplatform to recruit signaling molecules such as protein kinases andphosphatases involved in the regulation of the phosphorylationstatus of TJ and AJ proteins.

The studies described here were conducted in cell lines derivedfrom MDCK cells, a renal tubular epithelial cell line. To determinewhether the ORM-mediated regulation of AJC dynamics isobserved in another epithelium, we performed similar studies inthe intestinal epithelium using IEC-6 cells that lack occludin andOCLN−/− mice. Similar to the observation made in MDCK cells,FRAP analysis in IEC-6 cells expressing OCLNWT or OCLNDM

revealed that the mobile fraction of occludin is reduced in theabsence of ORM. ORM deletion also attenuated Ca2+-depletion-mediated barrier dysfunction and disruption of TJs and AJs,suggesting that ORM is also required for occludin and TJ dynamicsin the intestinal epithelium as well. Resistance to EGTA-mediatedinulin absorption and redistribution of AJC proteins from theintestinal epithelial junctions of OCLN−/−mice compared with WTmouse intestines further supports the role of occludin in theregulation of TJ and AJ dynamics. Therefore, the role of occludinand ORM appears to be not only universal to different types ofstress, but also a common property of different epithelia.

Although the precise function of occludin is unclear at this time,there are pieces of evidence that indicate its potential role in severalcellular functions, one of which is regulation of cell migration. Aprevious study has shown that occludin is involved in directionalcell migration (Du et al., 2010). During directional cell migration,occludin appears to be localized to the leading edge, andknockdown of occludin attenuates cell migration. Phosphorylation

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of occludin on Y473 allowed recruitment of p85α to the leadingedge. Knockdown of occludin blocked activation of PI 3-kinase,leading to disorganization of the actin cytoskeleton and preventionof lamellipodia extension. Our data show that occludin deficiencyor the absence of ORM attenuates cell migration in MDCK andIEC-6 cells; cell migration was rescued by OCLNWT but not byEGFP-OCLNDM. In addition to its suggested role at the leadingedge, the reduced mobile fraction of occludin and, in turn, reducedTJ fluidity in the absence of ORM might be responsible for theobstruction of cell migration. These data suggest that interaction ofsignaling elements with ORM is involved in the mechanism of cellmigration. ORM and TJ dynamics may be important in collectiveepithelial migration rather than migration of an individual cell.Collective cell migration is implicated in physiological conditions,such as crypt-to-villus migration of intestinal epithelial cells, andpathophysiological conditions, such as wound healing (Friedl andGilmour, 2009).To determine whether the effect of ORM is specific for collective

cell migration, we evaluated single-cell migration by transmigrationassay. Surprisingly, our preliminary data show that occludindeficiency and ORM deletion increases single-cell migration inboth MDCK and IEC-6 cells, which is in contrast to their effect oncollective cell migration. Single-cell migration may be relevant totumor metastasis, and our data open a new avenue for futureinvestigations to understand the differences in the mechanismsinvolved in collective cell migration and single-cell migration inreference to their regulation by occludin.In summary, this study demonstrates that ORM, a highly

conserved phosphorylation hotspot in the C-terminal domain ofoccludin, determines the dynamic property of TJs in renal andintestinal epithelial monolayers. ORM confers TJs with a dynamicproperty under multiple conditions of stress in renal and intestinalepithelial tissues. The results of this study also indicate that ORMmay directly or indirectly influence the integrity of AJs and thecytoskeleton. Therefore, the TJ dynamics regulated by ORM mightplay an important role in collective cell migration in epithelialtissues.

MATERIALS AND METHODSPlasmids, gene knockdown and mutationsFor occludin gene silencing in canine MDCK cells, vector-based shorthairpin RNAs (shRNAs) were designed using the Dharmacon website(siDesign® Center, http://www.dharmacon.com/DesignCenter/) and clonedinto pRNAtin-U6 vector (GenScript), as described previously (Suzuki et al.,2008). Two targeting sequences were chosen against the nucleotidesequence of the canine occludin gene (Target 1: 5′-TATGTCAGACCTT-ATAACG-3′; Target 2: 5′-TATGCTACCACCCATTAAG-3′). Thesequences were further verified by a BLAST search against the caninegenome database to confirm the uniqueness of these sequences.To construct the shRNA vectors, two pairs of oligonucleotides containingthe antisense sequence, a hairpin loop region (TTGATATCCG) andthe sense sequence with cohesive BamHI and HindIII sites weresynthesized (Sigma Genosys, St Louis, MO). Primer sequences areprovided in Table S1. For a control, a mutant form of shRNA wasdesigned by replacing adenine and guanine nucleotides with cytosine andadenine, respectively.

The construct pEGFP-occludin (human) was used for ORM deletion byinverse PCR using primers flanking the ORM region. The primers were asfollows: sense primer 5′-TGGATCAGGGAATATCCACCTATC-3′ andantisense primer 5′-TGCTCTTCCCTTTGCAGGTGCTCT-3′. The DPn1-treated PCR product was ligated by incubation with T4 polynucleotidekinase and T4 DNA ligase. The mutation was confirmed by restrictiondigestion, PCR amplification and sequencing. This resulted in an ORMdeletion mutant that is missing 28 amino acids in its C-terminal domain

(Δ388-415: GRSKRTEQDHYETDYTTGGESCDELEED). Double pointmutations of T403A/T404A, T403D/T404D, Y398A/Y402A and Y398D/Y402D were introduced in wild-type pEGFP-occludin, as describedpreviously (Elias et al., 2009; Suzuki et al., 2009), by using QuikChangesite-directed mutagenesis kit (Stratagene).

Cell culture and transfectionStable clones of OD-MDCK cells were generated by shRNA transfection ofMDCK.2 cells (ATCC, CRL-2936), and stable knockdown clones wereisolated by dilution cloning. The pEGFP-OCLNWT or pEGFP-OCLNDM

constructs or empty vector was transfected into stable OD-MDCK cells. Cellsgrown in six-well cluster plates to 70% confluence were transfected in opti-MEM (Invitrogen) containing 1 µg plasmid DNA and 3 µl lipofectamine2000 (Invitrogen) and incubated at 37°C for 6 h. Fetal bovine serum (FBS)was then added to the medium to make a final concentration of 10% serumand the cells were incubated at 37°C. After 24 h, the cell monolayers weretrypsinized and seeded onto 100 mm Petri dishes for selection. Stable clonesexpressing EGFP (Vector), EGFP-OCLNWT or EGFP-OCLNDM wereisolated by geneticin (G418) selection and dilution cloning. At least 12clones from each group were screened for EGFP fluorescence by live-cellimaging and western blot analysis. Point mutant occludin construct pEGFP-OCLNT403/404A or pEGFP-OCLNT403/404D or pEGFP-OCLNY398/402A orpEGFP-OCLNY398/402D was transfected into OD-MDCK clones as describedabove. IEC-6 cells expressing EGFP-OCLNWT, EGFP-OCLNDM or EGFPwere generated by transfection with FuGENE HD (Promega).

Detergent-insoluble fractionCell monolayers in 60 mm culture plates were incubated with 500 µl lysisbuffer-CS [Tris buffer containing 10 µl/ml protease inhibitor cocktail, 1 mMvanadate and 1 mM phenylmethane sulfonyl fluoride (PMSF)] on ice for15 min. Cell lysates were centrifuged at 15,600 g for 4 min at 4°C tosediment the high-density actin cytoskeleton. The supernatant was used asthe triton-soluble fraction. The pellet, which is known to contain membrane-associated TJ components, was suspended in 200 μl of lysis buffer-CS andsonicated to homogenize the actin cytoskeleton. This was used as the triton-insoluble fraction. Protein contents in both fractions were measured by theBCA assay (Pierce Biotechnology, Rockford, IL). The protein fractionswere mixed with an equal volume of 2× Laemmli’s sample buffer andheated at 100°C for 10 min.

ImmunoprecipitationEGFP was immunoprecipitated from total protein extracts under nativeconditions as previously described (Suzuki et al., 2009). Confluentmonolayers (60 mm culture plates) were washed with ice-cold PBS, andproteins were extracted in 500 µl ice-cold lysis buffer (10 mM Tris-ClpH 7.5 containing 150 mM NaCl, 0.5 mM EDTA, 0.5% NP40, 1 mMPMSF, 1× Protease inhibitor cocktail). The lysates were centrifuged at15,600 g for 4 min at 4°C. The supernatant was separated, and the pellet wassuspended in 200 µl lysis buffer and sonicated for 10 s to inducefragmentation of F-actin filaments and release actin-bound proteincomplexes in the supernatant fraction. This was centrifuged at 17,135 gfor 10 min and the supernatants were pooled. The protein concentration wasquantified by the BCA method. The total lysates (1.0 mg protein/ml) wereincubated with prewashed 25 µl GFP-Trap_A beads at 4°C for 1 h. Immunecomplexes were isolated by spinning at 4500 g for 2 min at 4°C, after whichthey were washed with Tris buffer, mixed with an equal volume ofLaemmli’s sample buffer (2× concentrated), heated at 100°C for 10 min andcentrifuged at 17,135 g for 10 min.

For p-Thr analysis of TJ and AJ proteins, proteins from MDCK, Vec,OCLNWT and OCLNDM cell monolayers were placed under denaturingconditions using lysis buffer-D (0.3% SDS w/v, 10 mM Tris-HCl, pH 7.4,containing 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 10 µg/ml aprotinin,10 µg/ml bestatin and 0.1 mM PMSF) and pre-heated to 100°C. Afterrepeated pipetting to homogenize samples, they were heated at 100°C for10 min. Protein extracts (400 μg) were incubated with anti-p-Thr antibodiesovernight as described above. Protein complexes were immunoprecipitatedwith Protein A/G-Sepharose beads and extracted in Laemmli sample bufferfor immunoblot analysis for ZO-1, E-cadherin and β-catenin.

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Immunoblot analysisEGFP immunoprecipitates and protein extracts were separated by SDS-polyacrylamide gel (7%) electrophoresis and transferred to PVDFmembranes. Proteins on the membrane were probed with specific primaryantibodies in combination with HRP-conjugated anti-mouse IgG or HRP-conjugated anti-rabbit IgG antibodies. The blots were developed by anenhanced chemiluminescence (ECL) method (Amersham, ArlingtonHeights, IL). The bands were quantified by densitometric analysis usingImageJ software (National Institutes of Health, Bethesda, MD).

Analysis of barrier function and junction integrityBarrier function of epithelial monolayers was evaluated by measuring TERand the flux of FITC-inulin as described before (Suzuki et al., 2008). TJ andAJ integrity was assessed by immunofluorescence staining of fixed cellmonolayers for GFP-occludin, ZO-1, E-cadherin and β-catenin as describedbefore (Suzuki et al., 2009).

FRAP analysisBy using a FRAP module in a Zeiss 710 confocal microscope and Zensoftware, FRAP analyses were performed in cell lines expressing EGFP-OCLNWT and its mutants. Three to four regions of interest (ROIs), providedin the module, were defined at the intercellular junctions of the monolayer.A high-intensity laser was used at 488 nm to bleach the ROIs, with a specificnumber of iterations. The module was set to capture at least three imagesbefore the bleach and time-lapse images up to ∼25 min after the bleach. Allthe time-lapse images were collected at the same focal plane. The time-series images were processed by using ImageJ software and confirmed bymanual calculation. Fluorescence intensity was measured at different timesafter bleaching to calculate percentage recovery. Percentage recovery at25 min was considered to be the percentage mobile fraction.

Ca2+-switch assayOD-MDCK and IEC-6 cells expressing EGFP-OCLNWT or its mutants weregrown to confluence in transwells (12 or 6.5 mm) or on 60 mm culture platesand incubated in LCM (low-Ca2+ medium; DMEM containing only 10 µMCaCl2). After 16 h in LCM, the medium was switched with NCM (normal-Ca2+ medium). Barrier function was monitored at different time points bymeasuring TER and inulin flux. TJ integrity was monitored by live-cellimaging, in the case of culture plates, or by fixing cell monolayers, in thecase of transwells. In another model, cell monolayers were treated with4 mM EGTA to induce Ca2+ depletion.

Cell migration assayFor collective cell migration, OD-MDCK and IEC-6 cells expressingEGFP-OCLNWT, EGFP-OCLNDM and EGFP were grown to confluence insix-well cluster plates. The cell monolayers were scraped with a razorblade in MDCK cells, whereas a pipette tip was used for scratch woundingin IEC-6 cells. Phase-contrast images of the wounded area were captured atdifferent time points and processed using ImageJ software. The averagerate of cell migration in each monolayer was calculated from at least fivespots.

For single-cell migration, EGFP-OCLNWT, EGFP-OCLNDM, Vector andMDCK cells (1×105 cells/100 µl) in DMEM containing 0.1% BSAwithoutserum were seeded on top of an 8 µm filter membrane in a 12-well transwellinsert. DMEM containing 10% FBS was added to the bottom chamber oftranswells. Cells were allowed to incubate for 6 h at 37°C and 5% CO2.Migrated cells were fixed with 90% ethanol for 10 min and air dried. Cellswere stained with 2% Crystal Violet at room temperature for 10 min.Cells were photographed, photographs converted to 16-bit images, and cellscounted using ImageJ software.

Osmotic stress and oxidative stressCell monolayers in transwell inserts were treated with DMEM containingmannitol (0.3 M) to induce osmotic stress or incubated with hydrogenperoxide (100 µM) to induce oxidative stress, as previously described (Eliaset al., 2009; Samak et al., 2011). Barrier function and TJ integrity weremonitored at various time points.

Intestinal loops ex vivoAdult WT and OCLN−/− mice that had ad libitum access to regularlaboratory chow and water were used in this study. OCLN−/− micebackcrossed to C57BL/6 mice were provided by Dr Jerrold Turner (HarvardUniversity, Boston, MA). All experiments were conducted according to anIACUC approved protocol. Mid small intestine was harvested and intestinalsacs (∼2 cm long) were prepared. One end of the segment was ligated with anylon thread. Saline-containing FITC-inulin (0.5 mg/ml) with or without1 mM EGTA was injected from the other end immediately before ligatingthe loop to prepare sacs. The sacs were incubated in DMEM at 37°C for30 min. After the incubation, contents of the sac were collected, andfluorescence in the luminal flushing was measured. Cryosections of loopswere stained for ZO-1, F-actin, E-cadherin, β-catenin and nucleus forimmunofluorescence analysis.

ReagentsDulbecco’s modified Eagle’s medium (DMEM), FBS and antibiotics werepurchased from Cellgro® (Manassas, VA) whereas Ca2+ free DMEM wasfrom Thermo Fisher Scientific (Tustin, CA). Rabbit polyclonal anti-ZO-1,rabbit polyclonal anti-p-Thr, mouse monoclonal anti-occludin, anti-Cldn-2,Alexa Fluor 488-conjugated anti-mouse IgG and phalloidin were purchasedfrom Invitrogen (Carlsbad, CA). Mouse monoclonal anti-EGFP antibodywas purchased from Clontech (Mountain View, CA). Hoechst 33342 wasobtained from Life Technologies (Carlsbad, CA). Mouse monoclonal anti-E-cadherin and rabbit polyclonal anti-β-catenin were purchased from BDBiosciences (San Jose, CA). Latrunculin-A, genistein, Cy3-conjugated anti-rabbit IgG, HRP-conjugated anti-mouse IgG, HRP-conjugated anti-rabbitIgG and mouse monoclonal anti-β-actin antibodies were obtained fromSigma (St Louis, MO). For immunofluorescence staining, all antibodieswere used at 1:100 dilution and Hoechst dyewas used at 1:1000 dilution. Forimmunoblot analysis, all primary antibodies were used at 1:1000 dilutionand secondary antibodies were used at 1:10,000 dilution. GFP-Trap A waspurchased from Chromotek (Hauppauge, NY). Restriction enzymes T4DNA ligase, polynucleotide kinase and DPn1 were obtained from NewEngland Biolabs (Beverly, MA). Full details of all antibodies used areprovided in Table S2.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: B.M., R.K.R.; Methodology: B.M., H.M., R.G., A.S.M., S.A.,P.K.S., K.D., T.S.; Software: B.M.; Validation: B.M.; Formal analysis: B.M.;Investigation: B.M., A.S.M., S.A., K.D.; Resources: H.M.; Data curation: B.M.,A.S.M.; Writing - original draft: B.M.; Writing - review & editing: R.K.R.; Supervision:R.K.R.; Project administration: R.K.R.; Funding acquisition: R.K.R.

FundingBhargavi Manda is a recipient of J. Paul Quigley memorial scholarship and DorothyK. and Daniel L. Gerwin graduate scholarship award from the University ofTennessee Health Science Center. This study was funded by National Institutes ofHealth grants DK55532 and AA12307. Deposited in PMC for release after 12months.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.206789.supplemental

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