USP9x-mediated deubiquitination of EFA6 regulates de novo tight junction assembly
2010; Springer Nature; Volume: 29; Issue: 9 Linguagem: Inglês
10.1038/emboj.2010.46
ISSN1460-2075
AutoresDelphine Théard, F. Labarrade, Mariagrazia Partisani, Julie Milanini, Hiroyuki Sakagami, Edward A. Fon, Stephen A. Wood, Michel Franco, Frédéric Luton,
Tópico(s)Neuroinflammation and Neurodegeneration Mechanisms
ResumoArticle25 March 2010free access USP9x-mediated deubiquitination of EFA6 regulates de novo tight junction assembly Delphine Théard Delphine Théard CNRS UMR6097, Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia-Antipolis, Valbonne, France Search for more papers by this author Florian Labarrade Florian Labarrade CNRS UMR6097, Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia-Antipolis, Valbonne, France Search for more papers by this author Mariagrazia Partisani Mariagrazia Partisani CNRS UMR6097, Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia-Antipolis, Valbonne, France Search for more papers by this author Julie Milanini Julie Milanini CNRS UMR6097, Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia-Antipolis, Valbonne, France Search for more papers by this author Hiroyuki Sakagami Hiroyuki Sakagami Department of Anatomy, Kitasato University School of Medicine, Kitasato, Japan Search for more papers by this author Edward A Fon Edward A Fon Montreal Neurological Institute, McGill University, Montréal, Québec, Canada Search for more papers by this author Stephen A Wood Stephen A Wood National Centre for Adult Stem Cell Research, Griffith University, Nathan, Queensland, Australia Search for more papers by this author Michel Franco Michel Franco CNRS UMR6097, Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia-Antipolis, Valbonne, France Search for more papers by this author Frédéric Luton Corresponding Author Frédéric Luton CNRS UMR6097, Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia-Antipolis, Valbonne, France Search for more papers by this author Delphine Théard Delphine Théard CNRS UMR6097, Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia-Antipolis, Valbonne, France Search for more papers by this author Florian Labarrade Florian Labarrade CNRS UMR6097, Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia-Antipolis, Valbonne, France Search for more papers by this author Mariagrazia Partisani Mariagrazia Partisani CNRS UMR6097, Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia-Antipolis, Valbonne, France Search for more papers by this author Julie Milanini Julie Milanini CNRS UMR6097, Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia-Antipolis, Valbonne, France Search for more papers by this author Hiroyuki Sakagami Hiroyuki Sakagami Department of Anatomy, Kitasato University School of Medicine, Kitasato, Japan Search for more papers by this author Edward A Fon Edward A Fon Montreal Neurological Institute, McGill University, Montréal, Québec, Canada Search for more papers by this author Stephen A Wood Stephen A Wood National Centre for Adult Stem Cell Research, Griffith University, Nathan, Queensland, Australia Search for more papers by this author Michel Franco Michel Franco CNRS UMR6097, Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia-Antipolis, Valbonne, France Search for more papers by this author Frédéric Luton Corresponding Author Frédéric Luton CNRS UMR6097, Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia-Antipolis, Valbonne, France Search for more papers by this author Author Information Delphine Théard1, Florian Labarrade1, Mariagrazia Partisani1, Julie Milanini1, Hiroyuki Sakagami2, Edward A Fon3, Stephen A Wood4, Michel Franco1 and Frédéric Luton 1 1CNRS UMR6097, Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia-Antipolis, Valbonne, France 2Department of Anatomy, Kitasato University School of Medicine, Kitasato, Japan 3Montreal Neurological Institute, McGill University, Montréal, Québec, Canada 4National Centre for Adult Stem Cell Research, Griffith University, Nathan, Queensland, Australia *Corresponding author. CNRS UNSA UMR6097, Institut de Pharmacologie Moléculaire et Cellulaire, 660 Route des Lucioles, Valbonne 6560, France. Tel.: +33 04 93 95 77 70; Fax: +33 04 93 95 77 08; E-mail: [email protected] The EMBO Journal (2010)29:1499-1509https://doi.org/10.1038/emboj.2010.46 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In epithelial cells, the tight junction (TJ) functions as a permeability barrier and is involved in cellular differentiation and proliferation. Although many TJ proteins have been characterized, little is known about the sequence of events and temporal regulation of TJ assembly in response to adhesion cues. We report here that the deubiquitinating enzyme USP9x has a critical function in TJ biogenesis by controlling the levels of the exchange factor for Arf6 (EFA6), a protein shown to facilitate TJ formation, during a narrow temporal window preceding the establishment of cell polarity. At steady state, EFA6 is constitutively ubiquitinated and turned over by the proteasome. However, at newly forming contacts, USP9x-mediated deubiquitination protects EFA6 from proteasomal degradation, leading to a transient increase in EFA6 levels. Consistent with this model, USP9x and EFA6 transiently co-localize at primordial epithelial junctions. Furthermore, knockdown of either EFA6 or USP9x impairs TJ biogenesis and EFA6 overexpression rescues TJ biogenesis in USP9x-knockdown cells. As the loss of cell polarity is a critical event in the metastatic spread of cancer, these findings may help to understand the pathology of human carcinomas. Introduction Polarized epithelial cells are adjoined through intercellular junctions arranged along their lateral membrane domains. In general, adhesion is provided by the adherens junction (AJ), mediated by homotypic calcium-dependent interactions between transmembrane E-cadherin molecules. The tight junction (TJ) is located at the most apical point of the lateral membrane, where it functions as a permeability barrier and is involved in various functions including cellular differentiation and proliferation (Tsukita et al, 2001, 2008). Both, AJ and TJ are associated with the actin cytoskeleton through cytosolic proteins that bridge the cytoplasmic tail of the junctional transmembrane proteins with the actin filaments. A model of TJ biogenesis during epithelial polarization is emerging whereby cell–cell contacts are initially engaged by nectin and E-cadherin. At these newly formed contact zones, the underlying actin is rearranged to stabilize and strengthen the cell–cell adhesion. TJ proteins are then recruited to the sites of cadherin-mediated cell–cell contacts and eventually segregate away from the contact zone to form a distinct junctional complex bound to a dense circumferential ring of acto-myosin (Drubin and Nelson, 1996; Miyoshi and Takai, 2005). During this process, the polarity complexes Crumbs/Pals/PATJ and Scribble/Lgl function to determine the apico-basal polarity, whereas the Par3/Par6/aPKC complex is believed to define the landmark where the TJ will form (Shin et al, 2006; Goldstein and Macara, 2007). Despite the intense effort to characterize the composition and organization of TJs at the molecular level, the temporal regulation of TJ biogenesis in response to de novo E-cadherin-mediated cell–cell adhesion remains poorly understood. The EFA6 family is composed of four members EFA6A, B, C and D, each encoded for by a different gene. Additional isoforms generated by alternative splicing have been described for EFA6A (Sironi et al, 2009), and possibly EFA6B (Derrien et al, 2002; Luton et al, 2004; Supplementary Figure S1A and B) and EFA6C (Matsuya et al, 2005). The tissue distribution has been mostly studied at the mRNA level in the mouse brain. Three EFA6 members (EFA6A, EFA6C and EFA6D) are abundantly expressed in the brain where they display overlapping but distinct expression patterns (Sakagami, 2008). EFA6C seems to be restricted to neuronal tissues (Derrien et al, 2002; Matsuya et al, 2005), whereas a small EFA6A RNA messenger was also found in the small intestin and colon (Derrien et al, 2002; Sironi et al, 2009). EFA6D mRNA is ubiquitously expressed with the highest levels in liver, lung and thymus (Sakagami et al, 2006). EFA6B mRNA was found widely expressed in epithelial tissues (Derrien et al, 2002). In addition, anti-EFA6B immunoblots reveal the presence of a 66 kDa protein in most tissues specially in the kidney, and a protein of 175 kDa with high expression in the thymus, spleen and lungs (Derrien et al, 2002; Supplementary Figure S1A and B). All EFA6 proteins follow a general structure that is comprised of a divergent N-terminal domain, the conserved catalytic Sec7 domain, a PH domain responsible for membrane localization and a C-terminal region involved in the rearrangement of the actin cytoskeleton (Franco et al, 1999; Derrien et al, 2002; Luton et al, 2004; Klein et al, 2008). The best-characterized isoforms, EFA6A and EFA6B, have been found to function similarly regarding their ability to catalyse Arf6 nucleotide exchange activity, their subcellular distribution and their effects on the actin cytoskeleton organization. Further, the PH-Cter constructs of EFA6A and EFA6B both localize and affect the actin cytoskeleton organization similarly (Derrien et al, 2002; Luton et al, 2004; Klein et al, 2008; unpublished data). Previously, we have shown that overexpression of EFA6A accelerated the formation of the TJ by contributing to the reorganization of the apical actin cytoskeleton in Madin-Darby canine kidney (MDCK) cells (Luton et al, 2004). Our current results show that endogenous EFA6B is required for efficient TJ biogenesis. Protein levels of EFA6B increase both temporally and spacially just at the point of cell–cell contact. In addition, we report here that EFA6B is constitutively degraded by the proteosome machinary, and that USP9x-mediated deubiquitination is responsible for the build-up of EFA6B levels during the establishment of cell polarity. USP9x has been found to translocate transiently to primordial cell–cell contacts, thereby ensuring the proper spatiotemporal activity of EFA6B, and thus, coordinating the timing of TJ assembly. Results To further investigate the function of EFA6 during TJ biogenesis, we used small interfering RNA (siRNA) to reduce endogenous EFA6B levels in MDCK cells (Supplementary Figure S1C). As a control, the human vsvg-EFA6A, which is insensitive to the canine EFA6B siRNA, was expressed in EFA6B-knockdown cells (Figure 1A). EFA6B-knockdown cells were much slower at forming TJ with only a few cells displaying a partial ZO-1 staining at their periphery (Figure 1B). The delay in TJ assembly was completely rescued by vsvg-EFA6A overexpression. EFA6B knockdown did not affect the expression of Arf6 or of the constitutive TJ or AJ proteins (Supplementary Figure S1C). Cells overexpressing vsvg-EFA6A assembled their TJ at a faster rate than control cells with a majority (≈60% of the cells) being totally surrounded by ZO-1 staining compared with <20% for the control cells. At the level of TJ function, EFA6B knockdown increased the diffusion of TRITC-dextran and delayed the acquisition of transepithelial resistance (TER), whereas vsvg-EFA6A overexpression reduced TRITC-dextran diffusion and accelerated the gain of TER (Figure 1C). Importantly, the effects of EFA6B knockdown on TRITC-dextran diffusion and TER acquisition could be rescued by overexpressing wild type, but not by mutants of EFA6A that are either catalytically inactive (vsvg-EFA6A-E242K) or defective in actin remodelling (vsvg-EFA6A-ΔCter) (Supplementary Figure S2A). Thus, both the GEF activity in the Sec7 domain and a non-catalytic actin remodelling activity in the C-terminal domain are required for EFA6 to efficiently orchestrate TJ assembly. Next, we examined endogenous EFA6B levels in MDCK cells that were induced to polarize by a calcium switch procedure. We found that EFA6B levels increased 30 min after calcium repletion (Figure 1D). EFA6B protein levels reached a peak of 8.7±0.8 fold increase compared with cells grown in normal calcium (n=9) at 60–90 min, and returned to baseline after 4–6 h. In contrast, the levels of Arf6 did not vary significantly. As previously shown, occludin, claudin 1 and E-cadherin levels decreased rapidly after calcium withdrawal and slowly recovered upon calcium repletion. Importantly, EFA6B accumulation did not coincide with changes in other TJ proteins and occurred before the appearance of the high molecular weight phosphorylated forms of occludin, which is associated with its incorporation into TJs (Sakakibara et al, 1997; Wong, 1997). Figure 1.EFA6B is required for efficient TJ assembly. (A) Immunoblot showing EFA6B, vsvg-EFA6A and actin levels in tet-off regulated vsvg-EFA6A MDCK cells grown in the absence or presence of doxycyclin (−/+Dox) and transiently transfected with control or EFA6B-specific siRNA #637. (B) The indicated cells were subjected to a calcium switch, fixed 90 min after calcium repletion, and processed for immunofluorescence analysis. The expression and localization of EFA6B (green), ZO-1 (red) and vsvg-EFA6A (blue) were examined. Scale bars, 25 μm. (C) The gain of TJ barrier function was analysed in a calcium switch assay by measuring the TER over time and the paracellular diffusion of TRITC-dextran at 2 h after calcium repletion +/−siEFA6B #637. For TER measurement n=5 and error bars represent the s.e.m. For siControl cells +/−Dox P<0.001 at all times, for siEFA6B cells +/−Dox P<0.002 at all times, for +Dox cells +/−siEFA6B P<0.005 after 30 min, for –Dox cells +/−siEFA6B P<0.01 at all times. For the paracellular diffusion of the TRITC-dextran n=3 and error bars represent s.e.m. For siControl cells +/−Dox P=0.0017, for siEFA6B cells +/−Dox P=0.0011, for +Dox cells +/−siEFA6B P=0.0073, for –Dox cells +/−siEFA6B P=0.0066. (D) The expression of EFA6B, occludin, claudin 1, E-cadherin, Arf6 and actin was analysed by immunoblotting during a calcium switch assay at the indicated times (min) after calcium repletion. NC, cells kept in normal calcium medium; LC, cells incubated 4 h in low calcium medium and lysed before calcium repletion. Download figure Download PowerPoint Considering the dramatic variations in EFA6B levels during the induction of cell polarity, we asked whether EFA6B levels could be under the control of the ubiquitin-proteasome system, the main pathway for degradation of cytosolic proteins (Hershko and Ciechanover, 1998). In both non-polarized (Figure 2A) and fully polarized (data not shown) MDCK cells, EFA6B accumulation correlates with the concentration and duration of exposure to the proteasome inhibitors lactacystin and MG-132. These results suggest that, at steady state, EFA6B is rapidly turned over by the proteasome. Indeed, EFA6B displays a short half-life and a rapid rate of protein synthesis that can account for the rapid modulation of EFA6B levels during cell polarization (Supplementary Figure S2B). Next, we analysed whether EFA6B was ubiquitinated in MG-132 treated MDCK cells. The anti-ubiquitin antibody P4G7 revealed a major band at ≈97 kDa corresponding to EFA6B (66 kDa) attached with four molecules of ubiquitin (Figure 2B, left panel). When the same samples were analysed with the FK1 antibody that recognizes only poly-ubiquitin chains (Figure 2B, right panel), we observed the same major band at ≈97 kDa demonstrating that EFA6B is not multi-monoubiquitinated but rather poly-ubiquitinated. The poly-ubiquitination status was confirmed in pull-down experiments using the ubiquitin-binding domain of the proteasome subunit S5a that recognizes poly-ubiquitin compared with the very weak signal using the domain of Eps15 that preferentially bind mono-ubiquitin (Woelk et al, 2006) (Supplementary Figure S2C). Thus, EFA6B is constitutively poly-ubiquitinated and degraded by the proteasome. Next, we asked whether endogenous EFA6B was poly-ubiquitinated during epithelial cell polarization. In the absence of proteasome inhibitors, filter-grown MDCK cells were subjected to a calcium switch and the EFA6B immunoprecipitated at different times after calcium repletion. The anti-poly-ubiquitin antibody revealed a major band at ≈97 kDa and some much higher molecular weigh bands appearing at about 60 min after calcium repletion (Figure 2C, left panel). After stripping, the membrane was re-probed with the anti-EFA6B antibody to ascertain whether those bands were poly-ubiquitinated forms of EFA6B (Figure 2C, right panel). The unmodified EFA6B migrates at 66 kDa, indicating that the ≈97 kDa band corresponds to the addition of a chain of four ubiquitin molecules. An extra band at about 105 kDa corresponding to the addition of five ubiquitin molecules is also detected though barely visible in the anti-ubiquitin immunoblot. In our calcium switch experiments, we have always observed a strong and robust poly-ubiquitination of EFA6B with high molecular weight moieties. However, we have never detected poly-ubiquitinated forms smaller than ≈97 kDa, suggesting that a minimal chain of four ubiquitin molecules is added at once. Typically, the poly-ubiquitination of EFA6B was only seen during a very narrow time window, suggesting that it is subsequently degraded very rapidly. At the peak of ubiquitination, 53.3±17.5% (n=8) of the total EFA6B was poly-ubiquitinated, which is considerable considering that the cells were not transfected with ubiquitin nor treated with proteasome inhibitors. Together, our data identify a pool of EFA6B, which after accumulating during the initial stages of epithelial polarization is rapidly poly-ubiquitinated and degraded by the proteasome. Figure 2.EFA6B is a substrate of the ubiquitin-proteasome system. (A) MDCK cells were grown in the presence of lactacystin or MG-132 and lysed at the indicated times. The lysates were resolved by SDS–PAGE and immunoblotted to determine the amount of EFA6B and, as a loading control, actin. The left panels show a dose response to increasing amounts of the indicated drugs for 2 h and the right panels show a time course for the indicated concentration of the drugs. Cells toxicity to 50 μM MG-132 at 10 h explains the weak signal. (B) MDCK cells were treated or not with MG-132 (50 μM) for 4 h and endogenous EFA6B (66 kDa) immunoprecipitated. The two samples were deposited twice side-by-side on a poly-acrylamide gel and resolved by electrophoresis. After transfer, the immunoblots were probed separately either with the anti mono- and poly-ubiquitin antibody (P4G7) or the anti poly-ubiquitin antibody (FK1). (C) MDCK cells grown on filters in the absence of proteasome inhibitors were subjected to a calcium switch and incubated after calcium repletion for the indicated times. NC, normal calcium medium; LC, low calcium medium. After lysis, the samples were resolved on SDS–PAGE, immunoblotted and probed first with the anti poly-ubiquitin (FK1) antibody. After stripping, the membrane was then probed with the anti-EFA6B antibody. The arrows point to the non-ubiquitinated (66 kDa) and poly-ubiquitinated EFA6B. HC, heavy chain from the immunoprecipitating antibody. Download figure Download PowerPoint We reasoned that a straightforward mechanism to account for the rapid increase in EFA6B levels would be through protection from the proteasome by deubiquitination. Thus, we looked for a DUB that would act on EFA6B upon E-cadherin engagement. USP9x is a DUB that has been reported to affect two junctional proteins, β-catenin and afadin, and to associate with E-cadherin in subconfluent epithelial cells, raising the possibility that USP9x might be a key DUB in cell polarization (Taya et al, 1998, 1999; Murray et al, 2004). We set out to observe an interaction between endogenous USP9x and EFA6B through co-precipitation. To co-precipitate USP9x and EFA6B from MDCK cells, we performed a calcium switch and lysed the cells at various times after calcium repletion. USP9x could be co-precipitated together with EFA6B only at 45 min after calcium repletion, indicating that the interaction between endogenous EFA6B and USP9x is transient and occurs predominantly at early time points during cell–cell adhesion (Figure 3A). Next, we performed pull-down experiments using four fragments of USP9x, encompassing the entire molecule, fused to GST. Only the fragment N2 (674–1218) could bind directly to purified his-EFA6A (Figure 3B). We also performed pull-down experiments using several fragments of EFA6A fused to GST (Figure 3C). Endogenous USP9x from MDCK lysate was pulled down with GST-EFA6A full length, the GST-PHCter, and the GST-PH fragments, but not with GST-Sec7, GST-Cter or GST alone. Thus, the PH domain alone is efficient enough to bind USP9x, and can be used in the cell as a dominant negative for USP9x activity. When used as a dominant negative in MDCK cells, it dramatically slowed down the TJ formation compared with untransfected MDCK cells, whereas consistent with earlier findings, GFP-EFA6A stimulated the TJ formation (Figure 3D; Supplementary Figure S3A). This result suggests that EFA6 binding to USP9x has a function in TJ assembly. Thus, we tested whether USP9x could promote EFA6 deubiquitination in baby hamster kidney (BHK) cells. Expression of V5-USP9x, but not the catalytic point mutant V5-USP9x C1566S, decreased vsvg-EFA6A ubiquitination and increased its levels in BHK cells (Figure 3E). Figure 3.EFA6 binds directly to and is a substrate of the DUB USP9x (A) EFA6B co-immunoprecipitates with USP9x. MDCK cells grown on 24-mm filters and submitted to a calcium switch were solubilized in NP-40 lysis buffer at the indicated times after calcium repletion. The cleared lysates were subjected to immunoprecipitation (IP) with a whole IgG fraction as a negative control or with the anti-EFA6B antibody. After electrophoresis and transfer, the immunoblots (IB) were probed separately with an anti-USP9x antibody and an anti-EFA6B antibody. H, fraction (1/20) of the homogenate used in the IP. (B) The N2 (674–1218) domain of USP9x is required for EFA6A direct binding. Schematic representation of the different domains of USP9x fused to GST: N1 (1–674), N2 (674–1218), C1 (1216–2107), C2 (2102–2560). Purified his-EFA6A was incubated for 2 h at 4°C with the indicated GST fusion proteins followed by precipitation with glutathione beads. The precipitates were resolved by SDS–PAGE and the membrane probed with the anti-histidine antibody (upper panel). The middle and lower panels show anti-GST and anti-histidine immunoblots to control for the amount of GST fusion protein and his-EFA6A present in the assay, respectively. (C) The PH domain of EFA6A is required for USP9x binding. Schematic representation of the different domains of EFA6A fused to GST used in the pull-down assay. An MDCK cell lysate was incubated for 4 h at 4°C with the indicated GST fusion proteins and precipitated with glutathione beads. The precipitates were resolved by SDS–PAGE and the membrane probed with the anti-USP9x antibody (upper panel). The middle and lower panels show an anti-GST and anti-USP9x immunoblots to control for the amount of GST fusion protein and USP9x present in the assay, respectively. (D) The gain of TJ barrier function was analysed in a calcium switch assay by measuring the TER over time. Two MDCK cell lines expressing the PH domain of EFA6A fused to GFP were analysed (GFP-PH #1 and GFP-PH #2) and compared with control or MDCK cells expressing GFP-EFA6A full length. n=3 and error bars represent the s.e.m. For both GFP-PH cell lines and GFP-EFA6A versus MDCK control, P<0.05 at all times. (E) USP9x promotes EFA6A deubiquitination in cells. BHK cells were transfected with the indicated constructs. At 24 h post-transfection, the cells were lysed in SDS and vsvg-EFA6A immunoprecipitated with an anti-vsvg antibody. The immunoprecipitates were resolved by SDS–PAGE and the membrane probed with an anti-myc antibody to detect ubiquitinated vsvg-EFA6A. The corresponding lysates were analysed by immunoblotting for their ubiquitination pattern and the expression of vsvg-EFA6A, V5-USP9x and actin. In the immunoprecipitation gel, the non-ubiquitinated vsvg-EFA6A is not visible, but its known position is indicated with an arrow, and the IgG HC arrow points to the heavy chain from the anti-vsvg immunoprecipitating antibody. The asterisks point to the major high molecular weigh bands of the ubiquitinated vsvg-EFA6. Download figure Download PowerPoint These results indicate that USP9x deubiquitinates EFA6 in cells, thereby protecting it from degradation by the proteasome. To further explore the function of USP9x, we analysed the effects of siRNA-mediated knockdown of USP9x (Supplementary Figure S3B) on EFA6B, AJ and TJ protein levels. In MDCK USP9x-knockdown cells, afadin expression was dramatically decreased, indicating that it is a constitutive substrate of USP9x (Figure 4A). However, the protein levels of neither EFA6B nor any of the other AJ or TJ proteins were affected by the USP9x knockdown. Nevertheless, the kinetics of EFA6B accumulation suggested that USP9x might only affect EFA6B levels during the early stages of cell polarization. Indeed, over the course of a calcium switch experiment and in the absence of proteasome inhibitors, no accumulation or poly-ubiquitination of EFA6B could be detected in USP9x-knockdown cells (Figure 4B). In contrast, in USP7-knockdown cells, used as a control, EFA6B accumulation and poly-ubiquitination occurred similarly to control MDCK cells (Figures 2D and 4B). These results support our model whereby USP9x is responsible for the transient accumulation of EFA6 protein, and that the observed poly-ubiquitination is due to the release of a synchronized pool of EFA6B no longer under the protective effect of USP9x. It also excluded the possibility that EFA6B accumulation was solely due to an arrest of its poly-ubiquitination. Note that the levels of USP9x remained unchanged during the calcium switch-induced cell–cell contact formation in control USP7-knockdown cells. In addition, except for afadin levels, which were reduced throughout the polarization time course in USP9x-knockdown cells, we never observed changes in the levels of any of the other AJ or TJ components (Supplementary Figure S3C). These results indicate that subsequent to E-cadherin engagement USP9x activity results in EFA6B accumulation. Figure 4.USP9x controls EFA6B levels and poly-ubiquitination in polarizing MDCK cells. (A) Lysates from non-polarized MDCK cells transfected with siRNA directed against USP7 (siRNA #1118) or USP9x (siRNA #182) were resolved by SDS–PAGE and the levels of the indicated proteins analysed by immunoblotting. The same results were obtained with fully polarized cells (data not shown). (B) MDCK cells, depleted for USP7 (as a control) or USP9x by RNA interference, were subjected to a calcium switch to induce polarization in the absence of proteasome inhibitors. At the indicated times, endogenous EFA6B was immunoprecipitated. The immunoprecipitates were resolved on SDS–PAGE, and after transfer, the membrane was probed with an anti-EFA6B antibody. The arrows point to the non-ubiquitinated (66 kDa) and poly-ubiquitinated EFA6B of higher molecular weights. A fraction (1/40) of the corresponding lysates at each time point was analysed by immunoblot for the expression of USP7, USP9x and actin. The expression of afadin, β-catenin, E-cadherin, occludin and claudin 1 is shown in Supplementary Figure S3C. Download figure Download PowerPoint Next, we compared the subcellular localization of EFA6 and USP9x during the formation of cell–cell contacts. Adjacent cells contact through membrane protrusions such as lamellipodia or filopodia. At the initial stage of cell–cell contacts, which yield to the formation of AJ, primordial spot-like junctions first form at the periphery or tips of these cellular protrusions. E-cadherin and filamentous actin concentrate into these newly formed adhesion points that extend and fuse followed by the recruitment and accumulation of the components of the TJ (Adams et al, 1998; Vasioukhin and Fuchs, 2001; Miyoshi and Takai, 2005). Our results show that in non-contacting cells, vsvg-EFA6A was detected at the periphery of lamellipodia and at the tip of filopodia (Figure 5A–A"). When we followed in real-time GFP-EFA6A localization in comparison to E-cadherin-RFP, we detected E-cadherin-RFP at the primordial contacts followed rapidly by co-localization of GFP-EFA6A, typically within 5 min (Figure 5B; full movie Supplementary Figure S4M). As the contact matures, large E-cadherin plaques gradually emerge at the margins (Adams et al, 1998) where the GFP-EFA6A signal is increasing and remains tightly co-localized with E-cadherin-RFP (Figure 5C; full movie Supplementary Figure S4N). This increase in the GFP-EFA6A signal at primordial contact supports our biochemical observations where we describe the accumulation of EFA6 (Figure 1; Luton et al, 2004). It is interesting to note that the maturation of the contact is coordinated with and dependent on the reorganization of the underlying actin cytoskeleton, and that in several model systems, EFA6 has been shown to affect cytoskeletal organization, which includes the apical actin ring of the TJ (Franco et al, 1999; Derrien et al, 2002; Luton et al, 2004). In mature contacts, vsvg-EFA6A is no longer enriched (Figure 5A"). Subsequently, we investigated the localization of endogenous USP9x using a rabbit polyclonal antibody (
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