The Tight Junction Protein, Occludin, Regulates the Directional Migration of Epithelial Cells

Developmental Cell

Article The Tight Junction Protein, Occludin, Regulates the Directional Migration of Epithelial Cells Dan Du,1 Feilai Xu,1 Lihou Yu,1 Chenyi Zhang,1 Xuefeng Lu,1 Haixin Yuan,1 Qin Huang,1 Fan Zhang,1 Hongyan Bao,1 Lianghui Jia,1 Xunwei Wu,3 Xueliang Zhu,2 Xiaohui Zhang,1 Zhe Zhang,1 and Zhengjun Chen1,* 1State

Key Laboratory of Molecular Biology Laboratory of Molecular Cell Biology Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China 3Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA *Correspondence: [email protected] DOI 10.1016/j.devcel.2009.12.008 2Key

SUMMARY

Cell polarity proteins regulate tight junction formation and directional migration in epithelial cells. To date, the mechanism by which these polarity proteins assemble at the leading edge of migrating epithelial cells remains unclear. We report that occludin, a transmembrane protein, is localized at the leading edge of migrating cells and regulates directional cell migration. During migration, occludin knockdown disrupted accumulation of aPKC-Par3 and PATJ at the leading edge, and led to a disorganized microtubule network and defective reorientation of the microtubule organization center (MTOC). Phosphorylation of occludin at tyrosine 473 residue allowed recruitment of p85a to the leading edge via association with its C-terminal SH2 domain. Loss of occludin attenuated activation of PI3K, leading to disorganization of the actin cytoskeleton and reduced cell protrusions. Our data indicate that occludin is required for the leading-edge localization of polarity proteins aPKC-Par3 and PATJ and promotes cell protrusion by regulating membrane-localized activation of PI3K.

INTRODUCTION Directed cell migration is an integrated process that is essential for embryonic development and plays a key role in wound healing and immune system function (Gupta et al., 2002; Ridley et al., 2003). Migration is a cyclical process, beginning with cellular responses to an external signal such as growth factors or the extracellular matrix (ECM) that elicits polarized protrusion extension in the direction of movement (Raftopoulou and Hall, 2004). During migration, the MTOC and Golgi apparatus reorient between the leading edge and the nucleus to direct and maintain migration (Etienne-Manneville, 2004; Gomes et al., 2005). The adhesions attaching the protrusion to the substratum serve, in part, as traction points for migration. Contraction then moves the cell body forward, and release of the

attachments at the rear as the cell retracts completes the cycle (Ridley et al., 2003). Epithelial sheets consist of fully polarized epithelial cells with an apical domain and basal domain separated by tight junctions (TJ), which are crucial for barrier function (Gumbiner, 1993; Tsukita et al., 2001). The conserved polarity complexes, Cdc42/Rac1Par3-Par6-aPKC, PATJ-PLAS-CRB, and Scrib-Dlg-Lgl, are crucial for the polarization of epithelial cells via recruitment to cell junctions by junction proteins (Goldstein and Macara, 2007; Shin et al., 2006). During wound healing, the apical-basal polarity of epithelial cells is disrupted and polarity protein complexes are redistributed. After wounding a monolayer of MCF10A cells, Scrib is recruited to the leading edge, causing bPIX, a Cdc42/Rac1 activator, to localize at the leading edge and activate Cdc42 and Rac1 there (Dow et al., 2007; Osmani et al., 2006). In astrocytes, integrin-stimulated active Cdc42 binds Par6 and causes aPKC activation at the leading edge (Etienne-Manneville and Hall, 2001). Active aPKC regulates the cortical recruitment of Dlg1 and inactivates GSK3b through serine 9 phosphorylation, leading to the association of Adenomatous Polyposis Coli (APC) with microtubule plus ends at the leading edge and subsequent centrosome polarization and nuclear translocation (EtienneManneville and Hall, 2001, 2003; Etienne-Manneville et al., 2005; Gomes et al., 2005). However, the later study showed that the GSK3aS21A and GSK3bS9A double knockin fibroblast had no defect in reorientation of the centrosome or the Golgi after wounding (Schlessinger et al., 2007). In keratinocytes, the ParTiam1 complex stabilizes the front-rear polarization of migratory cells and stimulates both persistent and chemotactic migration (Pegtel et al., 2007); a Par3-aPKC complex is also recruited to the leading edge of migrating MDCK II cells via association with PATJ, regulating the polarization of MTOC during wound healing (Shin et al., 2007). However, Par3 was not detected at the leading edge of astrocytes during migration, and junction proteins associate with polarity proteins in epithelial cells, suggesting that different signal pathways are involved in epithelial cells and astrocyte migration. Class IA PI3Ks are heterodimers composed of a regulatory (p85) and a catalytic (p110) subunit, and phospho-peptide binding to the SH2 domain of p85 induces a conformational change that mediates the activation of p110, leading to the phosphorylation of phosphoinositides at position 3 of the inositol ring (Yu et al., 1998). In leukocyte migration, the localized

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Developmental Cell Occludin Regulates Epithelial Migration

accumulation of PIP3 defines the migration direction and triggers the activation of the Rho-family GTPase at the leading edge (Barber and Welch, 2006; Funamoto et al., 2002; Jimenez et al., 2000; Rickert et al., 2000; Srinivasan et al., 2003). Rhofamily GTPases are critical molecular switches that modulate the actin cytoskeleton. For example, RhoA triggers actin stress fiber formation; Rac1 induces lamellipodia and membrane ruffling; and Cdc42 elicits the formation of filopodia, i.e., protrusive actin (Etienne-Manneville and Hall, 2002). Occludin, the first identified integral TJ protein, is a 60 kDa tetraspan membrane protein that forms two extracellular loops flanked by cytoplasmic N and C termini. Occludin has a role in the TJ barrier and defense functions (Balda et al., 1996; Chen et al., 1997; Furuse et al., 1996; Hirase et al., 1997; McCarthy et al., 1996; Tsukita et al., 2001; Yu et al., 2005), but occludin disruption in knockout mice or cell culture does not alter TJ structures (Saitou et al., 2000; Yu et al., 2005). Occludin associates with many signal transduction molecules, suggesting a more broad spectrum of biological roles (Barrios-Rodiles et al., 2005; Feldman et al., 2005; Nusrat et al., 2000; Seth et al., 2007). Expression of occludin is upregulated in MDCK cells upon TGFb stimulation, and occludin allows the TGFb type I receptor to be anchored at TJs for efficient TGFb-dependent dissolution of TJs during epithelial-to-mesenchymal transitions (Barrios-Rodiles et al., 2005; Kojima et al., 2007). Occludin also regulates the organization of actin and is required for translocation of p85a to TJs to modulate actin organization after oxidative stress (Atsumi Si et al., 1999; Sheth et al., 2003). Furthermore, overexpression of occludin in AC2M2 cells induces cell spreading (Osanai et al., 2007). These studies suggest occludin may be involved in cell migration. Here, we analyzed the role of occludin in the directional migration of Madin-Darby canine kidney (MDCK) epithelial cells. RESULTS Occludin Localizes at the Leading Edge of Migrating MDCK Cells and Is Required for Wound Healing To investigate the role of occludin in cell migration, an occludin RNAi (occRi) plasmid was generated to suppress the exogenous as well as endogenous occludin in HEK293 and MDCK cells, respectively, whereas a scramble RNAi control (occRiC) plasmid had no effect on expression of occludin. Expression of an RNAiresistant mutant of occludin (occRiR) was not affected by occRi plasmid (see Figures S1A–S1C available online). The control MDCK cells showed approximately 90% wound closure 17 hr after wounding, but the occRi cells covered only 28.6% ± 6.0% of the control area (Figure 1A). This migration defect could be rescued by expressing EGFP-tagged wild-type occludin (EGFP-Occ/wt) in the occRi cells, but not by EGFP alone (see below). To determine cellular redistribution of occludin, we performed immunofluorescence analysis of occludin in migrating epithelial cells. Occludin is not only localized at cell junctions in most of the cells (Figures 1B and 1G; Figure S1D), but also, strikingly, localized to the leading edge along the actin cortex at the front lines of both directional (Figure 1B) and random (Figure S1D) migrating epithelia. Overexpressed EGFP-Occ/wt localized similarly to the leading edge of migrating MDCK cells during

wound healing (Figure 1G; Movie S1). Knocking down occludin led to the loss of occludin signal at the leading edge (Figure S1C). To exclude the possibility that leading edge accumulation of occludin was due to the thickening of the cell/membrane, we used EGFP and wheat germ agglutinin (WGA), which has been used as a membrane marker, as controls (Chemin et al., 2005; Shen et al., 2008). When the cytoplasmic fluorescence of EGFP/WGA-TRITC and occludin were adjusted to the same level, ratio of the occludin staining at the edge versus within the average cytoplasm signal was higher than that of EGFP/ WGA-TRITC fluorescence, suggesting that the thickening of the cell at the leading edge contributed little to the enrichment of occludin signal (Figures 1C–1F). Atomic force microscopy (AFM) was also employed to explore the structure of cell surface and the localization of occludin at the leading edge. As shown in Figures S1E–S1G, within the measured area, the peak of occludin distribution was at the cortical membrane and did not overlap with that of cell membrane height. It suggested that the leading edge accumulation of occludin detected by AFM was not a result of the cell membrane folding. In conclusion, above-mentioned data from different experiments strongly suggest that occludin preferentially accumulates on the leading edge membrane of migrating cells. Occludin Knockdown Impairs the Leading Edge Localization of aPKC-Par3/PATJ and Reorientation of MTOC during Wound Healing aPKC, which regulates polarized migration with Par6 or Par3PATJ at the leading edge of astrocytes and MDCK II cells (Etienne-Manneville and Hall, 2001; Shin et al., 2007), can be pulled down by a peptide of the occludin coiled-coil domain (Nusrat et al., 2000). Interestingly, in our control MDCK cells, aPKCPar3 and PATJ localized to the leading edge of migrating cells, but not when occludin expression was knocked down (Figure 2A). Consistent with this, overexpressed EGFP-Par3, EGFPaPKC, and EGFP-PATJ accumulated at the leading edge of control cells but not at that of the occRi cells during migration (Figure S2A), suggesting that occludin retains aPKC-Par3 and PATJ at the leading edge during migration. Re-expression of EGFP-Occ/wt, but not EGFP alone, in occRi cells could rescue the mislocalization of this polarity complex (Figures 2B and 2C). Defective enrichment of Par3-aPKC at the leading edge is known to disrupt polarized organization of the microtubule network (Shin et al., 2007). During wound healing, occludin silencing blocked the normal elongation of microtubules perpendicular to the wound, leading to microtubules being parallel to the wound (Figure 2D). Re-expression of EGFP-Occ/wt restored microtubule elongation toward the leading edge, indicating that occludin regulates the microtubule network during wound healing (Figure 2E). The disorganized microtubule network indicated defective MTOC reorientation during wound healing. In control cells, 68.3% ± 3.8% of front row cells had MTOC in the direction of migration 6 hr after wounding, but only 47.0% ± 5.3% of occRi cells showed the correct MTOC reorientation (Figures 2F and 2G). This defective MTOC reorientation could be rescued by EGFP-tagged occludin, with 62.3% ± 4.0% of cells showing the correct orientation (Figures 2F and 2G). Thus, we believe loss of occludin disrupted accumulation of aPKC-Par3/PATJ at the leading edge and led to subsequent disorganization of the

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Developmental Cell Occludin Regulates Epithelial Migration

Figure 1. Occludin Localizes at the Leading Edge of Migrating Epithelial Cells and Is Required for Wound Healing (A) Wound healing assays were performed with control (con) and occludin RNAi (occRi) MDCK cells. Scale bar, 100 mm. Wound areas were measured with ImageJ software. Relative areas, which are defined as (recovered area of occRi cells)/(recovered area of con cells), of the wound were measured in three independent experiments. (B) MDCK cells were grown until confluence and fixed 6 hr after scratching, followed by immunostaining for occludin. (C) EGFP-transfected MDCK cells were immunostained with a-occludin antibody at 6 hr postwounding. EGFP and occludin are shown in green and red, respectively, and the nucleus is in blue. (D) The y axis denotes the relative average fluorescence intensity, which is defined as (fluorescence intensities)/(mean cytoplasmic fluorescence intensities), of occludin and EGFP at the three indicated lines. The x axis denotes the distance along the line from outside to inside of the cell. (E) MDCK cells were stained with WGA-TRITC and a-occludin antibody after wounding. WGA-TRITC and occludin are shown in red and green, respectively, and the nucleus is in blue. (F) Relative average fluorescence intensities (FI; y axis) of occludin and WGA-TRITC at the indicated lines (x axis, distance from outside to inside of the cell). The red arrowhead indicates the start point of cytoplasm. OC: outside cell region, M: membrane region, IC: inside cell region. (G) Localization of overexpressed EGFP-Occ/wt in MDCK cells during wound healing. Arrows indicate the leading edge localization of occludin, and arrowheads indicate the cell junction localization of occludin. Scale bar: 20 mm.

microtubule network and defective MTOC reorientation during wound healing. Occludin Recruits aPKC-Par3/PATJ to the Leading Edge and Regulates Polarized Migration Occludin and aPKC-Par3/PATJ could coimmunoprecipitate each other in MDCK cells (Figure 3A; Figure S3A), though when cotransfected, aPKC, but neither Par3 nor PATJ, could be coimmunoprecipitated with occludin (Figure S3B), suggesting that occludin might be associated with aPKC-Par3 via aPKC. Using an in vitro binding assay, we could confirm that purified His-aPKC interacted with the GST-fused C-terminal of occludin (GST-Occ/CT) (Figure 3B). Furthermore, aPKC, Par3,

and PATJ colocalized with occludin at the leading edge of migrating MDCK cells during wound healing (Figure 3C Figures S3C–S3E). Knockdown of aPKC (Figure S2B) did not disrupt occludin localization (Figure S2C). Overexpression of EGFP-Occ/ CT (EGFP-tagged occludin cytoplasmic domain, which mainly localized in the nucleus and cytoplasm; Figure S2D) in MDCK cells interfered with the leading edge localization of aPKC (Figure S2D) and MTOC reorientation (Figures 3D and 3E): 68.0 ± 3.0% MTOC had the right position in EGFP-expressing cells; only 45.0 ± 8.0% MTOC correctly reoriented in EGFPOcc/CT expressing cells. These data were consistent with Figure 2A, indicating that occludin mediates recruitment of aPKCPar3/PATJ to the leading edge during wound healing.

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Developmental Cell Occludin Regulates Epithelial Migration

Figure 2. Occludin Knockdown Impairs the Leading Edge Localization of PATJ/Par3-aPKC and Reorientation of MTOC during Wound Healing (A) Control (con) and occludin RNAi (occRi) MDCK cells were fixed 6 hr after scratching. Localization of aPKC (red), Par3 (green), and PATJ (red) was visualized by immunostaining. (B and C) Localization of aPKC (red), PATJ (red), and Par3 (red) in EGFP-Occ/wt (B) or EGFP (C)-rescued occRi cells. Arrows indicate the leading edge localization of the indicated protein, and gray lines indicate the leading edge of occRi cells. (D) Microtubule distribution was visualized by immunostaining using a-tubulin. (E) Microtubule distribution in EGFP-Occ/wt or EGFP-rescued occRi cells. Nuclei are shown in blue. (F) Con, occRi, EGFP-Occ/wt-rescued occRi, and EGFP-rescued occRi cells were wounded and MTOCs were visualized by staining g-tubulin (red). Nuclei are shown in blue. Arrows indicate the direction of cell migration, and arrowheads indicate MTOC. Scale bars, 20 mm. (G) Quantification of reorientated MTOC in (F). **p < 0.01, Student’s t test. Standard deviations are shown as error bars (n = 3).

Occludin Knockdown Impairs Lamellipodia Formation and Inhibits PI3K Activation Loss of occludin decreased lamellipodia formation, enhanced actin bundle near the leading edge, and decreased cortactin levels at the leading edge (Figure 4A). Lamellipodia formation requires localized activation of the small GTPase, Rac1 (Nobes and Hall, 1999). Serum stimulation activated Rac1, and occludin RNAi attenuated this activation (Figure 4B) and disrupted Rac1

localization to the leading edge (Figure 4C); overexpressed EGFP-Rac1 also accumulated at the leading edge, but this enrichment was significantly reduced in occRi cells (Figure S4A). To exclude the possibility that the loss of the polarity proteins (aPKC-Par3/PATJ) from the leading edges might be a result of the failure of lamellipodia extension, we overexpressed EGFP-Rac1L61 to rescue the lamellipodial extension, and found it could not rescue the leading edge localization of

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Developmental Cell Occludin Regulates Epithelial Migration

Figure 3. Occludin Is Associated with aPKC-Par3/PATJ during Polarized Migration (A) Total cell lysates of MDCK cells were immunoprecipitated with nonimmune serum (NS), a-occludin, or a-PKC antibodies, and precipitated proteins were detected with indicated antibodies. (B) Purified His-aPKC was incubated with GST, GST-Occ/CT, or GST-Par6 and immunoblotted with a-aPKC or a-GST antibodies. (C) Localization of occludin (green) and aPKC (red), Par3 (red), or PATJ (red) in migrating MDCK cells during wound healing. (D) EGFP and EGFP-Occ/CT expressing cells were wounded, and MTOCs (red) were visualized by staining g-tubulin. Nuclei are shown in blue. Arrows indicate the direction of cell migration, and arrowheads indicate MTOC. Scale bars, 20 mm. (E) Quantification of MTOC reorientation. **p < 0.01, Student’s t test. Standard deviations are shown as error bars (n = 3).

aPKC in occludin-silenced MDCK cells in our case (Figures S2E– S2H). It suggests that reduced lamellipodia extension was not the main reason for the mislocalization of polarity proteins at the leading edge. PI3K regulates Rac1 activation at the leading edge and mediates actin dynamics and protrusion formation in migrating cells (Barber and Welch, 2006; Rickert et al., 2000; Srinivasan et al., 2003). Occludin knockdown reduced serum-stimulated Akt phosphorylation, a marker of PI3K activity (Figure 4D, left panels), but had no effect on Erk1/2 activation (Figure 4D, right panels). A PH-Akt-EGFP construct, indicating the presence of active PI3K, localized at cell junctions and the leading edge during migration (Figure 4E; Movies S2 and S3). This leading edge localization was disrupted by occludin RNAi, suggesting that occludin regulates the spatial activation of PI3K, which is crucial for lamellipodia formation. Inhibition of PI3K by LY294002 dramatically hindered wound closure and reduced protrusions in MDCK cells (Figures S4B–S4D), but did not affect MTOC reorientation (Figure S4E), although PI3K can define the direction of motion (Funamoto et al., 2002; Rickert et al., 2000; Srinivasan et al., 2003; Wu et al., 2007). Occludin Interacts with the cSH2 Domain of p85a via Src-Mediated Phosphorylation of Y473 Tyrosine phosphorylation of occludin correlates its distribution and function (Rao et al., 2002; Sakakibara et al., 1997; Tsukamoto

and Nigam, 1999; Wong, 1997). Occludin was significantly tyrosine phosphorylated after serum stimulation or POV treatment in MDCK cells (Figure 5A). Both the POV-induced and basal phosphorylation of occludin were significantly attenuated by PP2, a Src family kinase (SFK) inhibitor (Figures S5A and S5B). The phosphorylation sites might exist in the C terminus, because the occludin mutant without its cytoplasmic terminus (Occ/DC) was not tyrosine phosphorylated (Figure S5C). An in vitro kinase assay confirmed that GST-Occ/CT was a substrate of Src kinase (Figure S5D). POV treatment and serum stimulation also promoted the association of occludin and p85a, the regulatory subunit of PI3K (Figure 5A), while PP2 treatment inhibited the basal association of occludin and p85a (Figure S5B), suggesting that this association may require tyrosine phosphorylation of occludin cytoplasmic terminus. Via pull-down assay, the cSH2 domain of p85a was revealed to mediate the POV-induced association (Figure S5E). Further study found that an R649A mutation in p85a inhibited the association of GST-cSH2 with tyrosine-phosphorylated occludin (Figure S5F). As predicted in the Scansite program (http://scansite.mit.edu/), Y473 of occludin is a potential binding site for p85a. Indeed, a mutant of occludin, EGFP-Occ/ Y473F, in which Y473 was substituted by phenylalanine (F), abolished the association of occludin and p85a, indicating that the cSH2 domain of p85a bound to phosphorylated Y473 (Figure 5B). To detect phosphorylation of occludin at residue Y473 (pY473-occludin) at the leading edge, a phospho-specific

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Developmental Cell Occludin Regulates Epithelial Migration

Figure 4. Occludin Knockdown Impairs Lamellipodia Formation and PI3K Activation (A) Control (con) and occludin RNAi (occRi) cells were fixed 6 hr after wounding. Cortactin is in green and F-actin is in red. (B) Starved con and occRi MDCK cells were stimulated with or without 10% FCS for 10 min. Active Rac1 was pulled down by GST-PAK-CRIB. (C) Localization of Rac1 (red) and F-actin (cyan) in con and occRi cells during wound healing. Scale bar, 20 mm. (D) The activation of Akt (left panels) and Erk (right panels) in serum-stimulated con and occRi cells. (E) Con and occRi cells were transfected with PH-AktEGFP, 48 hr later the cells were wounded and fixed, and the localization of PH-Akt-EGFP was imaged by confocal microscopy. Scale bar, 20 mm. Arrows indicate the leading edge localization of the proteins. Arrowheads indicate the leading edge of migrating occRi cells.

localize to the leading edge, indicating that this localization is independent of Y473 phosphorylation. However, expression of EGFPOcc/Y473D, but not EGFP-Occ/Y473F, could recruit p85a (Figure 6D) and Rac1 (Figure S6) to the leading edge, demonstrating that phosphorylation of occludin at 473 tyrosine residue was critical for the leading edge localization of p85a and Rac1.

antibody against phosphorylated Y473 (pY473)-peptide derived from occludin was generated and purified. The specificity of anti-pY473 antibody was proved by antigen block and western blot assays (Figure 5C). The most important experimental result was that the antibody could recognize tyrosine phosphorylated occludin/wt, but not its mutant occludin/Y473F, though this mutated protein was still tyrosine phosphorylated (at other tyrosine residues of occludin) (Figure 5D). Using anti-pY437 antibody, we were able to show that serum stimulation obviously increased pY473 of occludin (Figure 5D, upper panel). Furthermore, immunostaining analysis with anti-pY473 antibody clearly revealed that pY473-occludin specifically localized at the leading edge of migrating MDCK cells (Figures 5E and 5F), and this accumulation could be inhibited by PP2 treatment (Figure S5G). Phosphorylation of Y473-Occludin Regulates the Leading Edge Localization of p85a Occludin and p85a colocalized at the leading edge of migrating MDCK cells during wound healing (Figure 6A). Localization of p85a at the leading edge was disrupted by occludin knockdown, yet rescued by EGFP-Occ/wt (Figures 6A and 6B). EGFP-Occ/ Y473F and EGFP-Occ/Y473D, two mutants, were generated to mimic the nonphosphorylation and phosphorylation status of occludin, respectively. As shown in Figure 6C, both could

Phospho-Y473 Is Required for Localization of Active PI3K to the Leading Edge and Cell Migration during Wound Healing EGFP-Occ/Y473D, but not EGFP, EGFP-Occ/ wt, or EGFP-Occ/Y473F, could enhance phosphorylated Akt (pAkt) (Figure 7A), suggesting that Y473 phosphorylation was important for PI3K activation. To study the role of pY473-p85a interaction in cell migration, effects of different occludin constructs were compared. EGFPOcc/wt expression increased wound area coverage 1.27 ± 0.11 times (Figures S7A–S7C). EGFP-Occ/Y473D increased wound coverage 1.47 ± 0.15 times versus EGFP alone and 1.15 ± 0.11 times versus EGFP-Occ/wt. EGFP-Occ/Y473F, which cannot bind to p85a, weakly promoted cell migration (1.10 ± 0.17 times versus EGFP alone and 0.86 ± 0.12 times versus EGFP-Occ/wt). EGFP-Occ/wt and EGFP-Occ/Y473D could rescue wound healing in occludin knockdown cells by 0.85 ± 0.08 and 0.89 ± 0.12 times, while EGFP-Occ/Y473F only weakly rescued this defective migration by 0.73 ± 0.12 times (Figures 7B and 7C). DISCUSSION Tissue disruption in higher vertebrates produces a rapid repair process necessary for normal biological function. Wound healing, whether initiated by trauma, microbes, or foreign materials, proceeds via an overlapping series of events, including cell migration across the wound bed to cover it. During migration, cells have to determine the correct migration direction to the wound area and form protrusions to move. A conserved polarity

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Developmental Cell Occludin Regulates Epithelial Migration

Figure 5. Occludin Interacts with the cSH2 Domain of p85a via Src-Mediated Phosphorylation of Y473 (A) Starved MDCK cells were treated with POV or stimulated by 10% FCS for 10 min. Cell lysates were immunoprecipitated by a-occludin antibody and immunoblotted with 4G10 (a-phospho-tyrosine), a-p85a, or a-occludin antibodies. (B) EGFP, EGFP-Occ/wt, or EGFP-Occ/Y473F-overexpressed MDCK cells were treated by POV. Cell lysates were incubated with purified GST-cSH2, followed by immunoblotting using a-GFP (upper panel) or a-GST (bottom panel) antibody. (C) Upper panel: Phospho-peptides (pPT) used to generate the antibody and the control non-phospho-peptides (PT) were dotted on the nitrocellulose and immunoblotted by a-pY473 antibody. Bottom panel: MDCK cells were serum starved and treated with or without POV for 10 min. Cell lysates were immunoprecipitated with a-occludin antibody and immunoblotted by a-pY473 antibody in the presence or absence of antigen peptide (pPT). (D) Upper panel: Starved MDCK cells were stimulated by 10% FCS for 10 min. Cell lysates were immunoprecipitated by a-pY473 antibody and immunoblotted with a-occludin antibody. Bottom panel: MDCK cells were cotransfected with src kinase and EGFP, EGFP-Occ/wt, or EGFP-Occ/Y473F as indicated. The cell lysates were immunoblotted by a-pY473, 4G10, or a-occludin antibody. v: empty vector. (E) MDCK cells were fixed at 6 hr postwounding, then stained with WGA-TRITC and a-pY473 antibody with or without blocking antigen. WGA-TRITC and pY473 occludin are shown in red and green, respectively, and the nucleus is in blue. Scale bar, 20 mm. Arrows indicate the leading edge localization of the proteins. (F) Quantification of the average fluorescence intensities (y axis) of WGA-TRITC and pY473 occludin at the indicated lines. The x axis denotes the distance along the line from outside toward the inside of the cell. The red arrowhead indicates the start point of cytoplasm. OC: outside cell region, M: membrane region, IC: inside cell region.

complex, Par3-aPKC-Par6, is critical for directional migration, but the mechanism of its leading edge localization is unknown. Here we showed that the tight junction protein, occludin, recruits aPKC-Par3 and PATJ to the leading edge to stabilize polarized migration in response to external stimuli (Figure 7D). In addition, occludin recruits p85a at the leading edge via phosphorylation of occludin at Y473 residue to promote PI3K activation, leading to Rac1 activation and the formation of lamellipodia (Figure 7D). Occludin Is Essential for Polarized Epithelial Migration Occludin is a TJ transmembrane protein that modulates the barrier function of TJs, but disruption of occludin expression

does not affect TJ structure (Saitou et al., 2000; Yu et al., 2005). Occludin can recruit signal transduction molecules to TJs. Here, we identified occludin as a crucial regulator in epithelial migration. During wound healing in MDCK cells, occludin was localized at the leading edge, where numerous signaling processes occur to regulate cell migration (Ridley et al., 2003). Knockdown of occludin blocked wound closure, MTOC reorientation, and cell protrusion. Par3-aPKC-Par6 is recruited to tight junctions by transmembrane junction proteins and promotes tight junction formation to separate apical and basal domains (Joberty et al., 2000; Lin et al., 2000). Thus, the polarity complex could regulate polarized

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Developmental Cell Occludin Regulates Epithelial Migration

Figure 6. Occludin Controls the Leading Edge Localization of p85a via Phosphorylation of Y473 (A) Control (con) and occludin RNAi (occRi) cells were fixed 6 hr postwounding and immunostained with occludin, p85a, or F-actin as indicated. (B) EGFP-Occ/wt or EGFP-rescued occRi cells were fixed 6 hr after wounding; p85a is shown in red, and EGFP-tagged proteins are in green. (C) EGFP-Occ/Y473F or EGFP-Occ/Y473D was transfected in MDCK cells. EGFP-tagged proteins are shown in green and F-actin in red. (D) EGFP-Occ/Y473F or EGFP-Occ/Y473D was transfected in occRi cells. 6 hr after wounding, the cells were stained with a-p85a antibody. EGFP-tagged proteins are visualized in green and p85a in red. Nuclei are shown in blue. Arrows indicate the leading edge localization of the proteins. Scale bar, 20 mm.

epithelial migration via recruitment to the leading edge by transmembrane polarity proteins such as occludin. Because occludin has been reported to regulate actin organization, and overexpression of occludin in AC2M2 cells induces cell spreading (Atsumi Si et al., 1999; Osanai et al., 2007), it is not surprising that depletion of occludin resulted in reduced cell protrusion, which was crucial for cell migration. However, occludin null mice survived and were grossly normal. One of the explanations might be that other junction protein(s), such as claudins/JAMs, could compensate for the lack of occludin in critical processes, so that the mice can survive. For example, claudin1, which could bind to PATJ directly (Roh et al., 2002), is known to promote cell migration during wound healing (Leotlela et al., 2007). It is also possible that other proteins, including RTKs/GPCRs, might activate PI3K in the absence of occludin. Consistent with our data, knockout of many proteins, which are critical for directional migration, does not affect the early development. For example, aPKCz is critical for polarized migration in fibroblast cells and astrocytes (Etienne-Manneville and Hall, 2001; Gomes et al., 2005), but early development is not impaired in aPKCz null mice (Leitges et al., 2001).

Occludin Is Required for MTOC Reorientation aPKC-Par3 and PATJ have been reported to be crucial for epithelial cell polarization during migration (Shin et al., 2007). Yet, the mechanism of its leading edge localization is not clear. A recent study showed that a peptide in the coiled-coil domain of occludin could pull down aPKC from T84 cells (Nusrat et al., 2000). Here, we identified a direct interaction between occludin and aPKC in vitro, and found they were colocalized at both tight junctions and the leading edge of migrating cells. This provides a mechanism for how occludin can recruit aPKC to the leading edge to regulate polarized migration. Par3-PATJ could then be recruited to the leading edge via association with aPKC as a complex. Another possibility for PATJ to be recruited to the leading edge would be via ZO-3-occludin because ZO-3 was reported to interact with both of them (Roh et al., 2002). Therefore, we propose that occludin could recruit aPKC-Par3/PATJ to the leading edge in response to external stimuli. The interaction might promote or sustain the polarity complex at the leading edge, and regulate the correct microtubule elongation and MTOC reorientation (Figure 7D). In the absence of occludin, the enrichment of aPKC-Par3/PATJ at the leading edge is disrupted and results in an impaired microtubule network and defective MTOC reorientation. At the moment, the precise mechanism of how occludin and its associated proteins including aPKC-Par3/ PATJ regulate MTOC reorientation is unclear. Several previous studies have indicated that aPKC might be involved in MTOC orientation and nuclear translocation (Etienne-Manneville and Hall, 2001, 2003; Etienne-Manneville et al., 2005; Gomes et al., 2005). We thought that Dlg1 and GSK3b are two good candidates for aPKC downstream effectors to be studied, though conflicting conclusions on the involvement of phosphorylated GSK3b by aPKC in MTOC reorientation were drawn by different studies from Hall’s group (Etienne-Manneville and Hall, 2003; EtienneManneville et al., 2005; Schlessinger et al., 2007). Pawson’s group reported that the mammalian ortholog of the Drosophila melanogaster tumor suppressor Lethal (2) giant larvae (Mlgl), as a functional target of the aPKC–Par6 complex, accumulates at the leading edge of migrating embryonic fibroblasts, regulating cell migration (Plant et al., 2003). Loss and mutation of Mlgl have been reported to be associated with tumor cell migration

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Figure 7. Phosphorylation of Y473 Contributes to PI3K Activation and Cell Migration (A) EGFP, EGFP-Occ/wt, EGFP-Occ/Y473F, or EGFP-Occ/Y473D was transfected in MDCK cells, and cell lysates were immunoblotted with a-GFP, a-Akt, a-pAkt, a-pErk, or a-Erk antibodies. GAPDH was used as loading control. (B) The wound healing assay was performed in occRi cells overexpressing EGFP, EGFP-Occ/wt, EGFP-Occ/Y473F, or EGFP-Occ/Y473D, and photographed at the indicated time. (C) Quantification of wound closure analyzed in (B). *p < 0.05, **p < 0.01, Student’s t test. Standard deviations are shown as error bars (n = 3). (D) Hypothetical model of occludin-regulated epithelial cell migration.

and invasion (Lu et al., 2009; Pagliarini and Xu, 2003). It would be very interesting to test whether Mlgl and its phosphorylation participate in regulation of occludin in MTOC reorientation. Nevertheless, the identity of the downstream target(s) of the occludin-aPKC complex at the leading edge regulating MTOC reorientation remains to be discussed and further clarified. Occludin Is Critical for the Leading Edge Activation of PI3K and Lamellipodia Formation Occludin knockdown also disrupted lamellipodia formation at the leading edge. Lamellipodia formation is largely dependent on activation of Rac1 at the membrane, which promotes actin polymerization and membrane extension (Nobes and Hall, 1999). Occludin depletion impaired Rac1 activation and translocation to the leading edge upon serum stimulation. In the singlecell polarization and migration model, binding of a chemoattractant stimulates activation of PI3K, and active PI3K synthesizes the lipid second messenger PI(3,4,5)P3 (or PIP3) at the inner surface of plasma membrane by phosphorylating the integral membrane lipid PI(4,5)P2 (Chen et al., 2003; Funamoto et al., 2002). PIP3 can then recruit guanine-nucleotide exchange factors (GEFs) via PH/DHR-1 domains to the membrane and modulate the activation of Rho GTPase such as Rac1 (Barber and Welch, 2006; Srinivasan et al., 2003; Wu et al., 2007). Occludin depletion also impaired PI3K localization and activation upon serum stimulation. Loss of PI3K activation at the leading edge could explain why Rac1 was not enriched at the leading

edge and lamellipodia formation was attenuated. Thus, occludin controls the leading edge activation of PI3K and Rac1 to direct lamellipodia formation. PI3K has an important role in polarized migration during chemotaxis (Chen et al., 2003; Funamoto et al., 2002; Srinivasan et al., 2003). We found that inhibition of PI3K by LY294002 significantly attenuated cell migration, confirming the requirement of PI3K activity in epithelial migration. In single-cell migration, PI3K produces PIP3 to stimulate cell protrusion and define the direction of motion. PI3K inhibition also reduced lamellipodia formation. PI3K also regulates Cdc42, which can influence migration and polarization (Chen et al., 2003; Funamoto et al., 2002; Jimenez et al., 2000; Srinivasan et al., 2003). However, although PI3K inhibition reduced cell migration, it did not disrupt MTOC polarization in MDCK cells during wound healing. This result suggests that occludin regulates the two downstream pathways (aPKC-Par3/PATJ and PI3K) independently. pY473 Links Occludin to p85a and Activation of PI3K Occludin interacts with p85a upon oxidative stress, but the association mechanism is not clear (Sheth et al., 2003). Here we found that the occludin Y473 residue is tyrosine phosphorylated upon serum stimulation or POV treatment and binds to the cSH2 domain of p85a, enhancing the association of occludin and p85a. Overexpression of EGFP-Occ/Y473D, but not EGFP-Occ/ Y473F, could promote PI3K activation, which was in agreement with reduced PI3K activation upon serum stimulation in occRi

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MDCK cells. Moreover, EGFP-Occ/wt could promote cell migration but EGFP-Occ/Y473F lost the ability. At the same time, EGFP-Occ/Y473D augmented cell migration more than EGFPOcc/wt, indicating that occludin enhances cell migration via PI3K activation. In addition, colocalization of occludin and p85a at the leading edge confirms their regional association during migration. Staining by phospho-Y473 antibody reveals that phospho-Y473 occludin localizes at the leading edge, and this localization seems to be independent of Y473 phosphorylation, as both EGFP-Occ/Y473D and EGFP-Occ/Y473F are localized to the leading edge. But this phosphorylation of occludin is required for the leading edge localization of p85a, as expression of EGFP-Occ/Y473D, but not EGFP-Occ/Y473F, could rescue the defective leading edge localization of p85a in occRi cells. This is in agreement with the impaired regional activation of PI3K, indicated by PH-Akt-GFP in occRi cells, which might be the reason for the impaired leading edge localization and activation of Rac1. We therefore propose that occludin is located at the leading edge after wounding and phosphorylated at the Y473 residue in response to external stimuli. The phosphorylated Y473 recruits p85a to the leading edge and promotes PI3K activation, leading to activation of downstream effectors, such as Rac1, and lamellipodia formation during cell migration (Figure 7D). In addition to occludin-dependent activation of PI3K, GPCRs and RTKs can also regulate the activation of PI3K during wound healing (Graupera et al., 2008; Jimenez et al., 2000; Jones et al., 2003; Meng and Lowell, 1998; Vanhaesebroeck et al., 1999; Yip et al., 2007). Therefore, occludin-mediated regulation of PI3K presumably cooperates with other mechanisms to control the precise tuning of temporal and spatial PI3K activity at the leading edge of migrating cells. Taken together, the recruitment and colocalization of occludin with PI3K and aPKC-Par3/PATJ at the leading edge, combined with disrupted recruitment following occludin depletion, point to an important role for these polarity proteins in the regulation of occludin-dependent directional migration. EXPERIMENTAL PROCEDURES Cell Culture and Transfection MDCK, A431, and HEK293 were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen). The transfection was performed using Fugene6 (Roche Diagnostics) or Lipofectamin 2000 (Invitrogen), according to the manufacturer’s instructions. MDCK occRi stable clones were selected with 500 mg/ml G418. Wound Healing Assay Wound-healing assays were performed as described (Fenteany et al., 2000), with minor modification. Briefly, the cells were grown in 6-well plates until confluence, and the monolayer was scratched with a 1 ml Pipetman tip. Cells were then photographed with an Olympus IX81 microscope at different time points. For wound-closure experiments, relative wound areas were measured using ImageJ software (NIH) 17 hr after wounding. Western Blotting The cells were lysed in RIPA lysis buffer (1% Triton X-100; 0.5% sodium deoxycholate; 0.2% SDS; 150 mM NaCl; 10 mM HEPES [pH 7.3]; 2 mM EDTA; 10 mg/ml each of chymostatin, leupeptin, and pepstatin A; 20 mM phenylmethylsulfonyl fluoride; 2 mM sodium orthovanadate; 10 mM sodium pyrophosphate; and 20 mM sodium fluoride). Total protein amount was quantified using the Bradford protein assay. The proteins were separated by one-dimensional SDS-PAGE, transferred to a nitrocellulose membrane, and analyzed by

immunoblotting with the indicated antibodies. The densities of western bands were measured with Quantity One software (Bio-Rad). Immunoprecipitation For immunoprecipitation, the cells were lysed in NP-40 lysis buffer (0.2% NP-40; 0.1% sodium deoxycholate; 20 mM Tris-HCl [pH 7.4]; 10 mg/ml each of chymostatin, leupeptin, and pepstatin A; 20 mM phenylmethylsulfonyl fluoride; 2 mM sodium orthovanadate; 10 mM sodium pyrophosphate; and 20 mM sodium fluoride). Total protein amount was quantified using the Bradford protein assay. After preclearing with protein A- or protein G-Sepharose beads, the supernatants were incubated with polyclonal a-occludin and protein A-Sepharose at 4 C overnight. The beads were washed three times with lysis buffer. Bound proteins were eluted from the beads in SDS sample buffer and boiled for 10 min. For POV treatment or serum stimulation experiments, the cells were grown until subconfluence, starved overnight, and incubated with 10 mM pervanadate or 10% FCS for 10 min, 37 C. The cells were lysed with NP-40 buffer on ice followed by immunoprecipitation. Immunostaining Immunostaining was performed as described previously (Fang et al., 2007) with modest modification. Cells were grown on glass coverslips (Fisher) until confluence, the wound healing assay was then performed, and cells were fixed with 4% paraformaldehyde for 20 min at room temperature 6 hr after scratching and then permeabilized with 0.1% Triton X-100 in PBS for 5 min. Alternatively, they were fixed with 100% methanol for 5 min at 20 C. After blocking with 3% BSA in PBS for 30 min at room temperature, the cells were incubated with primary antibodies diluted in 1% BSA in PBS overnight at 4 C. Cells were then washed and incubated with secondary antibodies for 1 hr at room temperature. Coverslips were mounted using PERMAFLUOR aqueous mounting medium (Immunotech, France) and analyzed with a laser-scanning confocal microscope (Leica SP2 or SP5, Germany). Normalization of Occludin Staining at the Leading Edge MDCK cells were transfected with EGFP, and 48 hr later the cells were wounded. After 6 hr, the cells were fixed and stained with a-occludin antibody. Three individual lines were drawn from the outside to the inside of the cells across the cell membrane. The fluorescent intensities (FI) of interested molecules at the lines were measured by ImageJ software, and the average was calculated. We hypothesized that the first pixel after the major peak of occludin was the starting point of cytoplasm. Then we calculated the average fluorescent intensity of all the pixels in the measured cytoplasm area. The absolute fluorescent intensity of proteins at every pixel was divided by their average cytoplasmic fluorescent intensity. Then the relative fluorescent intensities of different proteins were compared. In Vitro Kinase Assay The C terminus (264–521 aa) of mouse occludin was cloned into pGEX-2T. The GST fusion protein (2 mg) or control GST (2 mg) were purified and incubated in the presence or absence of 1 U Src kinase (Oncogene) with 350 mM ATP, 0.05 mg/ml BSA, and 0.1%b-mercaptoethanol in kinase buffer (20 mM HEPES [pH 7.5], 10 mM MgCl2, 1 mM dithiothreitol, and 200 mM sodium orthovanadate) for 15 min. After centrifugation, the supernatant was discarded, and the pellets were resuspended in SDS sample buffer, boiled, and separated on SDS-PAGE. In Vitro Binding Assay Proteins fused to GST were synthesized in Escherichia coli and absorbed on GSH-Sepharose (Amersham Biosciences). The cells were lysed in NP-40 lysis buffer and mixed with 5 mg of recombinant GST fusion protein bound to glutathione-Sepharose beads for 4 hr, then washed extensively with binding buffer. Proteins adhering to the beads were analyzed by SDS-PAGE followed by immunoblotting analysis. Statistical Analysis For wound-closure assays, wound areas were measured in triplicate. For MTOC reorientation assays, a minimum of 100 cells from the front row of migrating epithelial sheets were counted, and Centrosomes (arrow heads) located in the forward-facing 120 sector with its vertex at the nuclear center

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were considered as polarized (Palazzo et al., 2001; Shen et al., 2008). Experiments were repeated three times. Student’s t test was used to analyze the significance of difference between groups. A p value <0.05 was considered to be statistically significant. Data were presented as means ± SD. Time-Lapse Microscopy for Cell Migration Cells were cultured in L-15 medium (Invitrogen) supplemented with 10% (v/v) fetal calf serum, 100 mg/ml streptomycin, and 100 U/ml penicillin. Image sequences for cell migration were collected with a laser-scanning confocal microscope (Leica SP5, Germany). ImageJ software was used for measurement. GTPase Activation Assays Cells were lysed in buffer containing 2% NP-40, 50 mM Tris–HCl (pH 7.5), 10 mM MgCl2, and 0.3 M NaCl. To detect the active GTP-bound form of GTPases in the cell lysates, the supernatants were mixed with 5 mg of recombinant GST-PAK-CRIB, bound to glutathione-Sepharose beads for 4 hr and washed twice with wash buffer containing 25 mM Tris-HCl (pH 7.5), 30 mM MgCl2, and 40 mM NaCl, and then the bound proteins were analyzed by SDS–PAGE followed by immunoblotting analysis. SUPPLEMENTAL INFORMATION Supplemental Information includes three movies and seven figures and Supplemental Experimental Procedures and can be found with this article online at doi:10.1016/j.devcel.2009.12.008. ACKNOWLEDGMENTS We thank Dangsheng Li for critical comments on the manuscript. We also thank Cord Brakebusch and Shaohua Li for providing Rac1/ ES cells. This research was supported by grants from National Natural Science Foundation of China (30730055, 30871270, and 30623002), from the National Key Scientific Program of China (2007CB914504), from the National High Technology and Development Program of China (2006AA021308), from the Chinese Academy of Sciences (KSCX2-YW-R-108 and KSCX1-YW-R-67), and also from the Program of Shanghai Subject Chief Scientist (08XD14051). Received: December 1, 2008 Revised: October 16, 2009 Accepted: December 16, 2009 Published: January 19, 2010 REFERENCES Atsumi Si, S., Kokai, Y., Tobioka, H., Kuwahara, K., Kuwabara, H., Takakuwa, Y., Sasaki Ki, K., Sawada, N., Mitaka, T., Mochizuki, Y., et al. (1999). Occludin modulates organization of perijunctional circumferential actin in rat endothelial cells. Med. Electron Microsc. 32, 11–19. Balda, M.S., Whitney, J.A., Flores, C., Gonzalez, S., Cereijido, M., and Matter, K. (1996). Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein. J. Cell Biol. 134, 1031–1049.

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