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First published online March 18, 2009
doi: 10.1242/10.1242/jcs.040055

Journal of Cell Science 122, 985-994 (2009)
Published by The Company of Biologists 2009
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The E3 ubiquitin ligase LNX1p80 promotes the removal of claudins from tight junctions in MDCK cells

Senye Takahashi1,*,{ddagger}, Noriko Iwamoto1,2,*, Hiroyuki Sasaki3, Masato Ohashi4, Yukako Oda2, Shoichiro Tsukita1,§ and Mikio Furuse2,¶

1 Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan
2 Division of Cell Biology, Department of Physiology and Cell Biology, Graduate School of Medicine, Kobe University, Kusunoki-cho 7-5-1, Chuo-ku, Kobe 650-0017, Japan
3 Department of Molecular Cell Biology, Institute of DNA Medicine, The Jikei University School of Medicine, Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan
4 Laboratory of Nano-Structure Physiology, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Higashiyama 5-1, Myodaiji-cho, Okazaki 444-8787, Japan


Figure 1
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  		Fig. 1. Identification of LNX1p80 as a claudin-1-binding protein. (A) Schematic representation of LNX1p80. LNX1p80 contains a RING-finger domain, followed by a NPAY motif for interaction with Numb, and four PDZ domains. A cDNA fragment encoding residues 256-729 of mouse LNX1p80 (mLNX1p80), which includes four PDZ domains, was obtained in yeast two-hybrid screening with the C-terminal cytoplasmic domain of claudin-1 as bait. (B) Interaction of mLNX1p80 with the cytoplasmic domain of claudin-1 in vitro. HA-tagged mLNX1p80 bound to the GST-fusion proteins with the C-terminal cytoplasmic domain of claudin-1 (GST-cld1C) or its mutant lacking C-terminal tyrosine and valine (GST-cld1C{Delta}YV) was detected in western blots with anti-HA mAb (IB: HA). The amount of eluted GST-fusion proteins is shown by Coomassie brilliant blue staining (CBB).

 

Figure 2
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  		Fig. 2. Localization of exogenous LNX1p80 in MDCK cells. (A) MDCK cells stably expressing EGFP-tagged mouse LNX1p80 (EGFP-mLNX1p80) were immunofluorescently stained with anti-ZO-1 mAb. The localization of EGFP-mLNX1p80 in the same field was detected with its fluorescent signal. EGFP-mLNX1p80 colocalized with ZO-1 at cell-cell junctions and was also distributed in the cytoplasm. Vertical sectional images obtained from confocal microscopy showed that EGFP-mLNX1p80 was colocalized with ZO-1 at the most apical portion of lateral membranes. Arrow indicates the thickness of the cellular sheet. (B) EGFP-mLNX1p80-expressing MDCK cells were co-cultured with parental MDCK cells and immunostained with anti-claudin-1 pAb. The concentration of claudin-1 in TJs was remarkably reduced in EGFP-mLNX1p80-expressing cells (asterisk). Scale bars: 10 µm.

 

Figure 3
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  		Fig. 3. Downregulation of claudins from TJs by inducible expression of LNX1p80 in MDCK cells. (A) Confirmation of the induced expression of EGFP-tagged canine LNX1p80. An MDCK clone that expressed EGFP-tagged canine LNX1p80 (EGFP-LNXp80) under the control of the Tet-Off inducible system (Tet-EGFP-LNX1p80 MDCK cells) was cultured in the presence (+) or absence (–) of doxycycline. Lysates from these cells were separated by SDS-PAGE, followed by immunoblotting with anti-GFP mAb (IB: GFP). (B) Localization of EGFP-LNX1p80 and claudin-1, claudin-2 and claudin-4 in Tet-EGFP-LNX1p80 MDCK cells. Cells were cultured in the presence [Dox (+)] or absence [Dox (–)] of doxycycline and immunofluorescently labeled with pAbs against claudin-1, claudin-2 and claudin-4 (right column). The expression of EGFP-LNX1p80 in the same field was detected by its fluorescent signal (left column). In the presence of doxycycline, there was no expression of EGFP-LNX1p80 and claudin-1 was localized at TJs in all cells (a). By contrast, the junctional concentration of claudin-1 was markedly reduced in EGFP-LNX1p80-induced cells in the absence of doxycycline (b). Even under the induced conditions, EGFP-LNX1p80 was not detected in some cells where claudin-1 was clearly localized at TJs (asterisk in c and d). The junctional concentration of claudin-2 (c) and claudin-4 (d) was also markedly reduced in these cells in EGFP-LNX1p80-induced conditions, except for uninduced cells (asterisks). (C) Localization of occludin, ZO-1, and E-cadherin in Tet-EGFP-LNX1p80 MDCK cells. Occludin was slightly downregulated in induced cells (a). By contrast, no significant change was observed in the localization of ZO-1 (b) and E-cadherin (c). Scale bar: 10 µm.

 

Figure 4
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  		Fig. 4. Freeze-fracture replica images of TJ strands in LNX1p80-expressing MDCK cells. (A) Confirmation of downregulation of claudin-1 from TJs in EGFP-LNX1p80-expressing MDCK cells obtained by retrovirus-mediated gene transfer followed by FACS. The signals of EGFP and immunolabeled claudin-1 in EGFP-expressing cells (left) and EGFP-LNX1p80-expressing cells (right) were fluorescently detected. Scale bar: 10 µm. (B) Freeze-fracture replica images of TJ strands. The cells were plated on 24-mm filters, cultured for 3 days, fixed with glutaraldehyde, and then processed for freeze-fracture replica electron microscopy. The number of TJ strands in EGFP-LNX1p80-expressing cells (bottom panels) was obviously lower than that in EGFP-expressing cells (top panels). Scale bar: 200 nm.

 

Figure 5
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  		Fig. 5. Barrier function of TJs in LNX1p80-expressing MDCK cells. (A) TER measurements. TER in EGFP-expressing MDCK cells transiently reached a maximum at day 2, and then settled to a lower value. This rapid raise of TER around day 2 was never observed in EGFP-LNX1p80-expressing MDCK cells (n=6). (B) FITC-dextran flux measurements. Paracellular flux of 4 kDa dextran was significantly increased in EGFP-LNX1p80-expressing MDCK cells compared with control cells (mean ± s.e.m.; n=5).

 

Figure 6
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  		Fig. 6. LNX1p80-mediated polyubiquitylation of claudin-1 in HEK293 cells. (A) The expression constructs for Flag-tagged canine LNX1p80, HA-tagged ubiquitin and Myc-tagged claudin-1 were transiently cotransfected into HEK293 cells. Claudin-1 was then immunoprecipitated with anti-claudin-1 pAb, followed by immunoblotting with anti-HA mAb. The combination of Flag-tagged LNX1p80, claudin-1 and HA-ubiquitin exhibited dense ubiquitylation as a smear (LNX1WT, lane 2) compared with the lysate without Flag-tagged LNX1p80 (Vector, lane 1) or that with its C48A mutant (LNX1C48A, lane 3). When claudin-1 was replaced with the C-terminal YV-deleted mutant (claudin-1{Delta}YV), only a background level of ubiquitylation was detected. (B) HEK293 cells were transfected as described in A. Flag-tagged LNX1p80 was immunoprecipitated with anti-Flag mAb, followed by immunoblotting with anti-Myc mAb. Myc-tagged claudin-1 was co-precipitated with Flag-tagged LNX1p80 (LNX1WT) and LNX1p80C48A (LNX1C48A), whereas claudin-1{Delta}YV was not. (C) Effects of ubiquitin mutants on LNX1p80-mediated ubiquitylation of claudin-1. Lanes 1-3 show a reproduction of lanes 1-3 in A. The K0 mutant of ubiquitin completely abolished the ubiquitylation of claudin-1 by LNX1p80 (lane 4), whereas the K48R mutant never affected it (lane 5).

 

Figure 7
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  		Fig. 7. Lysosomal degradation and LNX1p80-promoted ubiquitylation of claudin-1 in MDCK cells. (A) The effect of the lysosome inhibitor chloroquine and the proteasome inhibitor MG132 on the amount of claudin-1. MDCK cells were treated with 100 mM chloroquine or 10 mM MG132 for 18 hours and processed for SDS-PAGE followed by immunoblotting with anti-claudin-1 pAb, anti-β-actin mAb and anti-ZO-1 mAb. Chloroquine treatment increased the amount of claudin-1, indicating that claudin-1 is degraded in the lysosome. MG132 treatment did not affect the amount of claudin-1. With both treatments, the amounts of β-actin and ZO-1 did not significantly change. (B) Ubiquitylation of claudin-1 by LNX1p80 in MDCK cells. Tet-EGFP-LNX1p80 MDCK cells and EGFP-tagged canine LNX1p80C48A-expressing MDCK cells under the control of the Tet-Off system (Tet-EGFP-LNX1p80C48A MDCK cells) were cultured with or without doxycycline (Dox+ and Dox–). Claudin-1 was immunoprecipitated, followed by immunoblotting with anti-ubiquitin mAb. Ubiquitylation of claudin-1 was increased in Tet-EGFP-LNX1p80 MDCK cells treated with 100 mM of chloroquine for 18 hours. In Tet-EGFP-LNX1p80C48A MDCK cells, only the background level of ubiquitylation was detected.

 

Figure 8
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  		Fig. 8. Colocalization of claudin-2 with LNX1p80 and an endocytic marker in LNX1p80-expressing MDCK cells. Tet-EGFP-LNX1p80 MDCK cells (described in Fig. 3) were immunofluorescently stained with anti-claudin-2 mAb and anti-Rab7 pAb (A) or anti-cathepsin D pAb (B) under the induction of EGFP-LNX1p80 expression. Claudin-2, EGFP-LNX1p80, and Rab7 or cathepsin D were often colocalized in intracellular vesicular structures (arrowheads). Since different planes were analyzed using a x100 objective lens with a large numerical aperture, the concentration of EGFP-LNX1p80 at cell-cell contacts was not visible in these images. Scale bars: 10 µm.

 

Figure 9
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  		Fig. 9. Effects of LNX1p80 expression on the amount of endogenous claudin-1 protein in MDCK cells. (A) Reduction in the amount of claudin-1 by LNX1p80 expression in MDCK cells. Western blot of the whole lysate (whole), 1% NP-40-soluble (sup) and insoluble (pellet) fraction of MDCK cells constitutively expressing EGFP or EGFP-LNX1p80 with anti-claudin-1 pAb. The amount of NP-40-insoluble claudin-1 (pellet) was remarkably reduced in cells expressing EGFP-LNX1p80 compared with that in EGFP-expressing MDCK cells. (B) The effect of RING-domain mutant of LNX1p80 on claudin-1. Tet-EGFP-LNX1p80 MDCK cells and Tet-EGFP-LNX1p80C48A MDCK cells were cultured with (+) or without (–) doxycycline. The amounts of claudin-1, E-cadherin, EGFP-LNX1p80 and EGFP-LNX1p80C48A in total cell lysates were analyzed by immunoblotting with anti-claudin-1 pAb, anti-E-cadherin mAb and anti-GFP mAb. The total claudin-1 protein level was decreased in EGFP-LNX1p80-expressing cells, but not in EGFP-LNX1p80C48A-expressing cells. The amount of E-cadherin did not change in this experiment. (C) Immunofluorescence localization of claudin-1 in MDCK cells expressing EGFP-LNX1p80C48A. Tet-EGFP-LNX1p80C48A MDCK cells cultured without doxycycline were immunostained with anti-claudin-1 pAb. Vertical sections were obtained by confocal microscopy (lower panel). The concentration of claudin-1 was not limited to TJ regions but also extended to the lateral membrane (arrow). EGFP-LNX1p80C48A was colocalized with claudin-1. Scale bar: 10 µm.

 

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© The Company of Biologists Ltd 2009