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. 2019 Oct 7;218(10):3372-3396.
doi: 10.1083/jcb.201812157. Epub 2019 Aug 29.

Claudins and JAM-A coordinately regulate tight junction formation and epithelial polarity

Tetsuhisa Otani  1   2 , Thanh Phuong Nguyen  1   2 , Shinsaku Tokuda  3 , Kei Sugihara  4 , Taichi Sugawara  1   2 , Kyoko Furuse  1 , Takashi Miura  4 , Klaus Ebnet  5 , Mikio Furuse  6   2
Affiliations

Claudins and JAM-A coordinately regulate tight junction formation and epithelial polarity

Tetsuhisa Otani et al. J Cell Biol. .

Abstract

Tight junctions (TJs) establish the epithelial barrier and are thought to form a membrane fence to regulate epithelial polarity, although the roles of TJs in epithelial polarity remain controversial. Claudins constitute TJ strands in conjunction with the cytoplasmic scaffolds ZO-1 and ZO-2 and play pivotal roles in epithelial barrier formation. However, how claudins and other TJ membrane proteins cooperate to organize TJs remains unclear. Here, we systematically knocked out TJ components by genome editing and show that while ZO-1/ZO-2-deficient cells lacked TJ structures and epithelial barriers, claudin-deficient cells lacked TJ strands and an electrolyte permeability barrier but formed membrane appositions and a macromolecule permeability barrier. Moreover, epithelial polarity was disorganized in ZO-1/ZO-2-deficient cells, but not in claudin-deficient cells. Simultaneous deletion of claudins and a TJ membrane protein JAM-A resulted in a loss of membrane appositions and a macromolecule permeability barrier and in sporadic epithelial polarity defects. These results demonstrate that claudins and JAM-A coordinately regulate TJ formation and epithelial polarity.

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Figures

Figure 1.
Figure 1.
ZO-1/ZO-2 are required for apical junction localization of TJ proteins. (A–J) Immunofluorescence analyses of parental MDCK II cells (A–J) and ZO-1/ZO-2 dKO cells (A′–J′). (A and B) ZO-1 (A) and ZO-2 (B) expression was abolished in ZO-1/ZO-2 dKO cells. (C–J) TJ markers including ZO-3 (C), occludin (D), JAM-A (E), claudin-1 (F), claudin-2 (G), claudin-3 (H), claudin-4 (I), and claudin-7 (J) were not concentrated to the apical junctions in ZO-1/ZO-2 dKO cells, and JAM-A (E) and claudins (F–J) were diffusely localized along the lateral and apical plasma membrane. Occasional apical junction accumulation of occludin and claudins, but not JAM-A was observed (arrowheads), colocalizing with ZO-3 (z-sections in D and G). Graphs are quantitation of the fluorescence intensity and represent mean ± SD (n = 2–9). *, P < 0.05; ***, P < 0.0005, compared by t test. Scale bar: 20 µm. n.s., not significant.
Figure 2.
Figure 2.
Localization of TJ proteins in claudin quinKO cells. (A–J) Immunofluorescence analyses of parental MDCK II cells (A–J) and claudin quinKO cells (A′–J′). (A–E) Claudin-1 (A), claudin-2 (B), claudin-3 (C), claudin-4 (D), and claudin-7 (E) expression was abolished in claudin quinKO cells. (F) Occludin localization to apical junctions was reduced. (G–I) ZO-1 (G) and ZO-2 (H) were more concentrated at apical junctions, while ZO-3 (I) localization was not altered in claudin quinKO cells. (J) JAM-A was more concentrated at apical junctions in claudin quinKO cells. Graphs are quantitation of the fluorescence intensity and represent mean ± SD (n = 3 each). *, P < 0.05; **, P < 0.005; ***, P < 0.0005, compared by t test. Scale bar: 20 µm. n.s., not significant.
Figure 3.
Figure 3.
Claudins are required for TJ strand formation but not membrane appositions. (A–D) Freeze-fracture replica EM analyses. (A) TJ strands were observed beneath the apical microvilli in parental MDCK II cells. (B) TJ strands were not found in ZO-1/ZO-2 dKO cells. (C) Expression of ZO-1–GFP restored the TJ strands in ZO-1/ZO-2 dKO cells. (D) Claudin quinKO cells lacked TJ strands, but intramembrane particles were occasionally accumulated beneath the apical microvilli. (E–H) Transmission EM analyses of ultrathin sections. Black squares indicate the regions shown in high-magnification images. (E) TJs with membrane appositions were observed at the most apical cell junctions in MDCK II cells. (F) TJs were absent, and the intercellular space was widened in ZO-1/ZO-2 dKO cells. AJ-like structures associated with actin bundles were observed (asterisk). (G) Expression of ZO-1–GFP restored the formation of TJs in ZO-1/ZO-2 dKO cells. (H) TJ-like structures with membrane appositions were found in claudin quinKO cells. (I) Low-magnification view of a freeze-fracture replica from ZO-1/ZO-2 dKO cells. No TJ strands were found throughout the lateral plasma membrane. (J) An example of fragmented TJ strand-like structures in ZO-1/ZO-2 dKO cells. (K) Quantitation of TJ strand length normalized to the apical surface length of the corresponding fractured region. Graphs represent mean ± SD (n = 15 for MDCK II, n = 24 for ZO-1/ZO-2 dKO, n = 13 for ZO-1/ZO-2 dKO + ZO-1–GFP, n = 22 for claudin quinKO). ***, P < 0.0005, compared by t test. (L–O) TJ membrane kissing points were observed after ferrocyanide-reduced osmium/tannic acid/osmium postfixation. (L and M) TJ kissing points were observed in MDCK cells. (L′ and M′) Tracing of TJs. (N and O) Membranes were closely apposed to one another, but membrane kissing points were not observed in claudin quinKO cells. The most apical cell junctions with membrane appositions were observed. (N′ and O′) Tracing of TJ-like structures. Mv, microvilli. Scale bars: 200 nm (A–D); 100 nm (E–H); 500 nm (I and J); 100 nm (L–O).
Figure 4.
Figure 4.
Size-dependent permeability barrier defects in claudin quinKO cells. (A) TER measurements. Unit area resistance was markedly reduced in ZO-1/ZO-2 dKO and claudin quinKO cells. (B–E) Paracellular flux measurements. (B) Apical-to-basal permeability of fluorescein (332.31 D) was dramatically increased in ZO-1/ZO-2 dKO and claudin quinKO cells. (C) Apical-to-basal permeability of 4-kD FITC-dextran was markedly increased in ZO-1/ZO-2 dKO cells and significantly increased in claudin quinKO cells. (D) Apical-to-basal permeability of 40-kD FITC-dextran was dramatically increased in ZO-1/ZO-2 dKO cells, but only moderately increased in claudin quinKO cells. (E) Apical-to-basal permeability of 150-kD FITC-dextran was dramatically increased in ZO-1/ZO-2 dKO cells, but not in claudin quinKO cells. (F) Model illustrating the barrier defects in ZO-1/ZO-2 dKO cells and claudin quinKO cells. In MDCK II cells, TJs are formed, and paracellular passage of electrolytes and macromolecules is restricted. In ZO-1/ZO-2 dKO cells, the intercellular space is widened, and electrolytes and macromolecules can diffuse along the intercellular space. In claudin quinKO cells, membrane kissing points are lost, but neighboring cell membranes are closely apposed to one another, allowing paracellular diffusion of electrolytes, but not macromolecules. Graphs represent mean ± SD (n = 2 for A; n = 3 for B–E). *, P < 0.05; ***, P < 0.0005, compared by t test. n.s., not significant.
Figure 5.
Figure 5.
ZO-1/ZO-2 regulates epithelial polarity. (A–D) Immunofluorescence analyses of polarity markers. (A1–F1) In MDCK II cells, ezrin (A1), gp135 (B1), and Forssman antigen (C1) were selectively localized to the apical membrane, while Na-K ATPase α1 subunit (D1) and Scribble (E1) were restricted to the basolateral membrane. γ-Tubulin staining (F1) shows that centrosomes are aligned in the apical cytoplasm. (A2–F2) In ZO-1/ZO-2 dKO cells, epithelial polarity was disorganized, and ezrin (A2), Forssman antigen (C2), Na-K ATPase α1 subunit (D2), and Scribble (E2) were detected on both the apical and basolateral membranes, while gp135 (B2) or γ-tubulin (F2) localization was not severely perturbed. (A3–F3) Expression of ZO-1–GFP rescued the epithelial polarity phenotypes of ZO-1/ZO-2 dKO cells. (A4–F4) No epithelial polarity defects were observed in claudin quinKO cells. (G–J) Quantitation of polarization index of ezrin (G), gp135 (H), Na-K ATPase α1 subunit (I), and Scribble (J). Graphs represent mean ± SD (n = 3–9). (K) Forssman antigen localization in apical (K1–K4) and lateral (K′1–K′4) confocal sections. (L) Quantitation of polarization index of Forssman antigen. Graphs represent mean ± SD (n = 6–15). All cells were cultured on Transwell filters for 5–7 d. *, P < 0.05; **, P < 0.005; ***, P < 0.0005, compared by t test. Scale bars: 10 µm.
Figure 6.
Figure 6.
ZO-1/ZO-2 regulates polarized cyst formation. (A–D) Localization of apical marker gp135 (green) and basolateral marker Scribble (magenta) in cells embedded in collagen I gels and cultured for 5–7 d. (A) MDCK II cells formed polarized cysts. (B) ZO-1/ZO-2 dKO cells failed to expand their lumens and did not form polarized cysts. Occasional colocalization of gp135 and Scribble was observed. (C) ZO-1/ZO-2 dKO cells were able to form polarized cysts upon expression of ZO-1–GFP. (D) Claudin quinKO cells formed polarized cysts. (E) Quantitation of cyst phenotypes (n = 343 for MDCK II, n = 536 for ZO-1/ZO-2 dKO, n = 311 for ZO-1/ZO-2 dKO + ZO-1–GFP, n = 752 for claudin quinKO). (F–H) Transmission EM observation of cysts formed by MDCK II cells. MDCK II cells formed a polarized epithelium in the collagen gels, and TJs were formed at the most apical region of the intercellular junctions (H). (I–L) Transmission EM observation of ZO-1/ZO-2 dKO cells embedded in collagen I gels. The lumens failed to expand in ZO-1/ZO-2 dKO cells, and microvilli and microlumens were observed between the cells (K and L). The cells were pseudocolored in J′. White squares indicate the regions shown in high-magnification images. Scale bars: 10 µm (A–D); 2 µm (F and I); 1 µm (G and J); 500 nm (H, K, and L).
Figure 7.
Figure 7.
ZO-1/ZO-2 is required for zonula adherens formation. (A and B) AJ markers afadin (A) and E-cadherin (B) were apically localized but fragmented in ZO-1/ZO-2 dKO cells. (C and D) Afadin (C) and E-cadherin (D) localizations were not altered, but the junctions appeared to be straighter in claudin quinKO cells. Graphs are quantitation of the fluorescence intensity and represent mean ± SD (n = 3–5). (E–G) F-actin (E), Myosin IIA (F), and IIB (G) organization in ZO-1/ZO-2 dKO cells and claudin quinKO cells. Apical confocal sections are shown in E–G. Actin bundles (E′) and myosin II foci (F′ and G′) were observed in the apical cytoplasm of ZO-1/ZO-2 dKO cells, while F-actin (E″) and Myosin II (F″ and G″) were more enriched at cell junctions in claudin quinKO cells (also see line scan). Line scans represent the fluorescent intensity along the yellow arrows, and black arrows indicate the position of cell junctions. Bar graphs are quantitation of the fluorescence intensity of Myosin II in the apical confocal sections and represent mean ± SD (n = 3–5). (H and I) Tension applied to AJ was monitored by vinculin (H) and α18 (I) localization. Vinculin and α18 localization were increased in ZO-1/ZO-2 dKO and claudin quinKO cells, suggesting that AJs are subjected to increased tension. In ZO-1/ZO-2 dKO cells, vinculin and α18 accumulated at vertices (arrowheads) indicating anisotropy in tension, while vinculin and α18 were increased in an isotropic manner in claudin quinKO cells. Apical confocal sections are shown in H. White squares indicate the regions shown in high-magnification images. Graphs are quantitation of the fluorescence intensity of apical vinculin and total α18 signals and represent mean ± SD (n = 3 each). *, P < 0.05; **, P < 0.005, compared by t test. Scale bar: 20 µm (A–D and F–I); 10 µm (E). n.s., not significant.
Figure 8.
Figure 8.
The effect of AJs on epithelial polarity is distinct from that of ZO-1/ZO-2. (A) TER measurements showed that the epithelial barrier was not disorganized in afadin KO or E-cadherin KO cells. Graphs represent mean ± SD (n = 3). (B) No epithelial polarity defects were observed in afadin KO and E-cadherin KO cells cultured on Transwell filters. See Fig. 6 A for control images. (C–F) Quantitation of polarization index of ezrin (C), Forssman antigen (D), Na-K ATPase α1 subunit (E), and Scribble (F). Graphs represent mean ± SD (n = 2–9). Data for MDCK II cells are identical to Fig. 6. (G and H) Localization of apical marker gp135 (green) and basolateral marker Scribble (magenta) in afadin KO cells (G) and E-cadherin KO cells (H) embedded in collagen I gels and cultured for 5–7 d. (G) Afadin KO cells showed multilumen phenotypes. (H) E-cadherin KO cells were able to form polarized cysts. See Fig. 7 A for control images. (I) Quantitation of cyst phenotypes (n = 318 for MDCK II, n = 332 for Afadin KO, n = 457 for E-cadherin KO). (J) Phase-contrast images of MDCK II cells and αE-catenin KO cells. Strong cell–cell adhesion is lost in αE-catenin KO cells. (K) Epithelial polarity defects in αE-catenin KO cells. Ezrin and Na-K ATPase α1 subunit were detected on both the apical and basolateral membranes, and centrosome localization was randomized. Scale bars: 10 µm. n.s., not significant.
Figure 9.
Figure 9.
Polarity signaling molecules are mislocalized in ZO-1/ZO-2 dKO cells. (A–C) Par-3 (A), aPKC (B), and Pals1 (C) were localized to apical junctions in MDCK II cells. (D–F) Par-3 (D) and aPKC (E) were diffuse and fragmented in ZO-1/ZO-2 dKO cells. Pals1 (F) localization was fragmented to a smaller extent in ZO-1/ZO-2 dKO cells. (G–I) Apical junction localization of Par-3 (G), aPKC (H), and Pals1 (I) was restored by expression of ZO-1–GFP in ZO-1/ZO-2 dKO cells. (J–L) Par-3 (J), aPKC (K), and Pals1 (L) were able to localize to apical junctions in claudin quinKO cells. Apical confocal sections are shown for Pals1, and the intracellular signals are nonspecific staining. (M and N) Willin-GFP overexpression promoted apical junction localization of aPKC in ZO-1/ZO-2 dKO cells. (M) aPKC localization was diffuse and fragmented in ZO-1/ZO-2 dKO cells. (N) Willin-GFP overexpression promoted apical junction localization of aPKC. GFP (M and N); aPKC (M′ and N′). (O) Willin-GFP overexpression did not restore the polarized localization of ezrin and Na-K ATPase α1 subunit in ZO-1/ZO-2 dKO cells. (P and Q) Quantitation of polarization index of ezrin (P) and Na-K ATPase α1 subunit (Q). Graphs represent mean ± SD (n = 10 each). ***, P < 0.0005, compared by t test. (R–T) Localization of apical marker gp135 (green) and basolateral marker Scribble (magenta) in MDCK II cells (R), ZO-1/ZO-2 dKO cells (S), and ZO-1/ZO-2 dKO + Willin-GFP cells (T) embedded in collagen I gels and cultured for 5–7 d. Willin-GFP expression did not rescue the lumen phenotype. (U) Quantitation of cyst phenotypes (n = 368 for MDCK II, n = 536 for ZO-1/ZO-2 dKO, n = 1,061 for ZO-1/ZO-2 dKO + Willin-GFP). Data for ZO-1/ZO-2 dKO are identical to Fig. 7 E. Scale bars: 20 µm (A–L and O); 10 µm (M, N, and R–T).
Figure 10.
Figure 10.
JAM-A regulates membrane appositions and epithelial polarity in claudin quinKO cells. (A–M) Immunofluorescence analyses of cell junction proteins in claudin/JAM-A KO cells. (A) Discontinuity in ZO-1 staining is observed (arrowheads), and large gaps of ZO-1 were occasionally found (asterisk). (B–E) Discontinuity (arrowheads) and gaps (asterisk) were also observed for ZO-2 (B), ZO-3 (C), and Afadin (D) in claudin/JAM-A KO cells. E-cadherin (E) staining was continuous, suggesting that cell–cell contact is maintained. (F and G) F-actin staining shows thickening of circumferential actin bundles. Apical confocal sections are shown. Line scans represent the fluorescent intensity along the yellow arrow in F, and black arrows in G represent the position of cell junctions. (H) Linearity of cell junctions were quantified by measuring the length of deviation from a straight line drawn between the vertices of the corresponding cell junction. Cell junctions of claudin quinKO cells were straighter than MDCK II cells, and extremely straight in claudin/JAM-A KO cells. Graphs represent mean ± SD (n = 15 each). (I–L) Occludin immunostaining was reduced in claudin quinKO cells, and further reduced in claudin/JAM-A KO cells. (M) Quantitation of the fluorescence intensity of occludin. Graphs represent mean ± SD (n = 3 each). Data for MDCK II and claudin quinKO are identical to Fig. 2 F. (N–P) Transmission EM analyses of ultrathin sections of claudin/JAM-A KO cells. Black squares indicate the regions shown in high-magnification images. Intercellular spaces were open in claudin/JAM-A KO cells, and AJ-like structures were found (asterisk). Focal membrane appositions were found in some cases (arrow). Occasionally, apical cell junctions were not formed, and microvilli-like structures were observed along the lateral membranes (P). Mv, microvilli. (Q and R) Large gaps (asterisks) were occasionally observed in Par-3 (Q) and aPKC (R) staining. (S) Sporadic epithelial polarity defects were observed in claudin/JAM-A KO cells (yellow brackets), where ezrin was mislocalized to the lateral cell junctions, and Na-K ATPase α1 subunit was found on both apical and basolateral membranes. (T and U) Localization of apical marker gp135 (green) and basolateral marker Scribble (magenta) in JAM-A KO cells (T) and claudin/JAM-A KO cells (U) embedded in collagen I gels and cultured for 5–7 d. Multilumen phenotypes were observed in ∼20% of the cysts formed by JAM-A KO cells (T′). Majority of the cysts formed by claudin/JAM-A KO cells showed closed lumen phenotypes (U), while ∼20% of them had single lumens, although the lumen morphology was abnormal (U′). (V) Quantitation of cyst phenotypes (n = 343 for MDCK II, n = 398 for JAM-A KO-1, n = 502 for JAM-A KO-2, n = 752 for claudin quinKO, n = 970 for claudin/JAM-A KO-1, n = 1186 for claudin/JAM-A KO-2). Data for MDCK II and claudin quinKO are identical to Fig. 7 E. *, P < 0.05; ***, P < 0.0005. See Figs. 1, 2, 3, 6, 7, and 9 for control images. Scale bars: 20 µm (A–L and Q–S); 100 nm (N and O); 1 µm (P); 10 µm (T and U).
Figure 11.
Figure 11.
JAM-A is required for the claudin-independent macromolecule permeability barrier formation. (A) TER measurements. Unit area resistance was markedly reduced in claudin quinKO and claudin/JAM-A KO cells. (B–E) Paracellular flux measurements. (B) Apical-to-basal permeability of fluorescein (332.31 D) was dramatically increased in claudin quinKO and claudin/JAM-A KO cells. (C) Apical-to-basal permeability of 4-kD FITC-dextran was markedly increased in claudin/JAM-A KO cells, and significantly increased in claudin quinKO cells. (D) Apical-to-basal permeability of 40-kD FITC-dextran was dramatically increased in claudin/JAM-A KO cells, but only moderately increased in claudin quinKO cells. (E) Apical-to-basal permeability of 150-kD FITC-dextran was dramatically increased in claudin/JAM-A KO cells, but only modestly in claudin quinKO cells. (F) Summary of the phenotypes of MDCK II, ZO-1/ZO-2 dKO, claudin quinKO, JAM-A KO, and claudin/JAM-A KO cells. In MDCK II cells, claudins and JAM-A are concentrated at the TJs with ZO-1/ZO-2. Membranes closely appose to each other and kissing points are formed, and paracellular diffusion of electrolytes and macromolecules are prohibited. Epithelial polarity is maintained. In ZO-1/ZO-2 dKO cells, claudins and JAM-A are diffusely localized, and membrane appositions and kissing points are lost. Intercellular space is widened, and electrolytes and macromolecules diffuse across the paracellular space. Epithelial polarity is disorganized. In claudin quinKO cells, JAM-A and ZO-1/2 are concentrated at the apical junctions, and membrane appositions are formed despite the lack of kissing points. Although electrolytes can diffuse across the paracellular space, the paracellular diffusion of macromolecules is prohibited. No epithelial polarity defects are observed. In JAM-A KO cells, claudins and ZO-1/2 localize to TJs, and kissing points are formed. Epithelial barrier and polarity are not perturbed. In claudin/JAM-A KO cells, ZO-1 can localize to apical junctions, but discontinuity is observed. Intercellular space is widened although focal membrane appositions are observed in some cases. Electrolytes and macromolecules can diffuse across the paracellular space, and epithelial polarity is disorganized in some regions. Graphs represent mean ± SD (n = 3 each). **, P < 0.005; ***, P < 0.0005, compared with MDCK II cells (or with claudin quinKO cells when notified) by t test. n.s., not significant.
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