Limits...
Exogenous expression of the amino-terminal half of the tight junction protein ZO-3 perturbs junctional complex assembly.

Wittchen ES, Haskins J, Stevenson BR - J. Cell Biol. (2000)

Bottom Line: Similarly, the adherens junction proteins E-cadherin and beta-catenin were also delayed in their recruitment to the cell membrane, and NZO-3 expression had striking effects on actin cytoskeleton dynamics.NZO-3 expression did not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100-soluble, signaling-active beta-catenin was increased in NZO-3-expressing cells during junction assembly.We hypothesize that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or beta-catenin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

ABSTRACT
The functional characteristics of the tight junction protein ZO-3 were explored through exogenous expression of mutant protein constructs in MDCK cells. Expression of the amino-terminal, PSD95/dlg/ZO-1 domain-containing half of the molecule (NZO-3) delayed the assembly of both tight and adherens junctions induced by calcium switch treatment or brief exposure to the actin-disrupting drug cytochalasin D. Junction formation was monitored by transepithelial resistance measurements and localization of junction-specific proteins by immunofluorescence. The tight junction components ZO-1, ZO-2, endogenous ZO-3, and occludin were mislocalized during the early stages of tight junction assembly. Similarly, the adherens junction proteins E-cadherin and beta-catenin were also delayed in their recruitment to the cell membrane, and NZO-3 expression had striking effects on actin cytoskeleton dynamics. NZO-3 expression did not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100-soluble, signaling-active beta-catenin was increased in NZO-3-expressing cells during junction assembly. In vitro binding experiments showed that ZO-1 and actin preferentially bind to NZO-3, whereas both NZO-3 and the carboxy-terminal half of the molecule (CZO-3) contain binding sites for occludin and cingulin. We hypothesize that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or beta-catenin.

Show MeSH

Related in: MedlinePlus

NZO-3 expression alters the distribution of ZO-1 during the early stages of TJ assembly. (A) Parental MDCK (P) and MDCK/NZO-3 (NZO-3) cells were subjected to a calcium switch, and the localization of ZO-1 at 6, 12, 24, and 48 h after the re-addition of calcium was determined by immunofluorescence with anti–ZO-1 antibody. In parental cells, ZO-1 is completely localized to cell borders after 6 h in calcium-containing media. Over 48 h the cells become more tightly packed and uniform in size. In NZO-3 expressing cells, only scattered islands of cells showed ZO-1 localization at cell borders by 6 h. The number of cells with ZO-1 localized to cell–cell contact sites increases over time and is complete by 48 h after re-addition of calcium. (B) No MDCK/NZO-3 cells show ZO-1 localization around their peripheries at t = 0 in the calcium switch. Phase-contrast image shows that cells form a confluent monolayer at t = 0 of calcium switch. Anti–ZO-1 immunofluorescence shows ZO-1 staining in large intracellular aggregates and in thick bars at sites where two cells make contact. Bars: (A and B) 30 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169439&req=5

Figure 4: NZO-3 expression alters the distribution of ZO-1 during the early stages of TJ assembly. (A) Parental MDCK (P) and MDCK/NZO-3 (NZO-3) cells were subjected to a calcium switch, and the localization of ZO-1 at 6, 12, 24, and 48 h after the re-addition of calcium was determined by immunofluorescence with anti–ZO-1 antibody. In parental cells, ZO-1 is completely localized to cell borders after 6 h in calcium-containing media. Over 48 h the cells become more tightly packed and uniform in size. In NZO-3 expressing cells, only scattered islands of cells showed ZO-1 localization at cell borders by 6 h. The number of cells with ZO-1 localized to cell–cell contact sites increases over time and is complete by 48 h after re-addition of calcium. (B) No MDCK/NZO-3 cells show ZO-1 localization around their peripheries at t = 0 in the calcium switch. Phase-contrast image shows that cells form a confluent monolayer at t = 0 of calcium switch. Anti–ZO-1 immunofluorescence shows ZO-1 staining in large intracellular aggregates and in thick bars at sites where two cells make contact. Bars: (A and B) 30 μm.

Mentions: The physiological data suggesting impaired TJ-forming ability in NZO-3–expressing cells prompted us to assess the localization of proteins associated with both TJs and AJs in the calcium switch experimental system. Parental and MDCK/NZO-3 cells grown on filters were subjected to a calcium switch and protein localization was determined 6, 12, 24, and 48 h after re-addition of calcium. As shown in Fig. 4 A, ZO-1 is localized at cell–cell contacts within 6 h after re-addition of calcium. In the 12–48-h time period, the parental cells develop the cobblestone uniformity of shape and size that is typical of an MDCK epithelial cell monolayer at steady-state. The appearance of ZO-1 at cellular contacts 6 h after re-addition of calcium correlates well with the rapid increase in TER observed in the parental cells (Fig. 3 A). In contrast, cells expressing NZO-3 showed a significant delay in ZO-1 recruitment to cell borders after re-addition of calcium (Fig. 4 A). At 6 h, only small, isolated clusters of cells showed ZO-1 staining at cell borders. Over time the areas of cell monolayer that show ZO-1 localization at cell–cell contacts increase in size. At 6, 12, and 24 h when ZO-1 is not yet fully localized to cell borders, ZO-1 also localizes to large intracellular aggregates and linear planes of contact between neighboring cells. Complete localization of ZO-1 at the junctional membrane in NZO-3–expressing cells did not occur until 48 h after re-addition of calcium. ZO-1 localization in both cell types at 48 h is indistinguishable from untreated, steady-state cells (data not shown). Both parental and MDCK/NZO-3 cells displayed no detectable differences in expression levels of ZO-1 (or any other junctional protein examined; see Fig. 9 immunoblot) at 6, 12, 24, and 48 h. The delayed targeting of the TJ protein ZO-1 in MDCK/NZO-3 cells correlates with the delay in TER recovery after calcium switch (Fig. 3 A). The localization of the TJ proteins ZO-2, endogenous ZO-3, and the integral membrane protein occludin was identical to that of ZO-1 in the MDCK/NZO-3 cells (data not shown).


Exogenous expression of the amino-terminal half of the tight junction protein ZO-3 perturbs junctional complex assembly.

Wittchen ES, Haskins J, Stevenson BR - J. Cell Biol. (2000)

NZO-3 expression alters the distribution of ZO-1 during the early stages of TJ assembly. (A) Parental MDCK (P) and MDCK/NZO-3 (NZO-3) cells were subjected to a calcium switch, and the localization of ZO-1 at 6, 12, 24, and 48 h after the re-addition of calcium was determined by immunofluorescence with anti–ZO-1 antibody. In parental cells, ZO-1 is completely localized to cell borders after 6 h in calcium-containing media. Over 48 h the cells become more tightly packed and uniform in size. In NZO-3 expressing cells, only scattered islands of cells showed ZO-1 localization at cell borders by 6 h. The number of cells with ZO-1 localized to cell–cell contact sites increases over time and is complete by 48 h after re-addition of calcium. (B) No MDCK/NZO-3 cells show ZO-1 localization around their peripheries at t = 0 in the calcium switch. Phase-contrast image shows that cells form a confluent monolayer at t = 0 of calcium switch. Anti–ZO-1 immunofluorescence shows ZO-1 staining in large intracellular aggregates and in thick bars at sites where two cells make contact. Bars: (A and B) 30 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169439&req=5

Figure 4: NZO-3 expression alters the distribution of ZO-1 during the early stages of TJ assembly. (A) Parental MDCK (P) and MDCK/NZO-3 (NZO-3) cells were subjected to a calcium switch, and the localization of ZO-1 at 6, 12, 24, and 48 h after the re-addition of calcium was determined by immunofluorescence with anti–ZO-1 antibody. In parental cells, ZO-1 is completely localized to cell borders after 6 h in calcium-containing media. Over 48 h the cells become more tightly packed and uniform in size. In NZO-3 expressing cells, only scattered islands of cells showed ZO-1 localization at cell borders by 6 h. The number of cells with ZO-1 localized to cell–cell contact sites increases over time and is complete by 48 h after re-addition of calcium. (B) No MDCK/NZO-3 cells show ZO-1 localization around their peripheries at t = 0 in the calcium switch. Phase-contrast image shows that cells form a confluent monolayer at t = 0 of calcium switch. Anti–ZO-1 immunofluorescence shows ZO-1 staining in large intracellular aggregates and in thick bars at sites where two cells make contact. Bars: (A and B) 30 μm.
Mentions: The physiological data suggesting impaired TJ-forming ability in NZO-3–expressing cells prompted us to assess the localization of proteins associated with both TJs and AJs in the calcium switch experimental system. Parental and MDCK/NZO-3 cells grown on filters were subjected to a calcium switch and protein localization was determined 6, 12, 24, and 48 h after re-addition of calcium. As shown in Fig. 4 A, ZO-1 is localized at cell–cell contacts within 6 h after re-addition of calcium. In the 12–48-h time period, the parental cells develop the cobblestone uniformity of shape and size that is typical of an MDCK epithelial cell monolayer at steady-state. The appearance of ZO-1 at cellular contacts 6 h after re-addition of calcium correlates well with the rapid increase in TER observed in the parental cells (Fig. 3 A). In contrast, cells expressing NZO-3 showed a significant delay in ZO-1 recruitment to cell borders after re-addition of calcium (Fig. 4 A). At 6 h, only small, isolated clusters of cells showed ZO-1 staining at cell borders. Over time the areas of cell monolayer that show ZO-1 localization at cell–cell contacts increase in size. At 6, 12, and 24 h when ZO-1 is not yet fully localized to cell borders, ZO-1 also localizes to large intracellular aggregates and linear planes of contact between neighboring cells. Complete localization of ZO-1 at the junctional membrane in NZO-3–expressing cells did not occur until 48 h after re-addition of calcium. ZO-1 localization in both cell types at 48 h is indistinguishable from untreated, steady-state cells (data not shown). Both parental and MDCK/NZO-3 cells displayed no detectable differences in expression levels of ZO-1 (or any other junctional protein examined; see Fig. 9 immunoblot) at 6, 12, 24, and 48 h. The delayed targeting of the TJ protein ZO-1 in MDCK/NZO-3 cells correlates with the delay in TER recovery after calcium switch (Fig. 3 A). The localization of the TJ proteins ZO-2, endogenous ZO-3, and the integral membrane protein occludin was identical to that of ZO-1 in the MDCK/NZO-3 cells (data not shown).

Bottom Line: Similarly, the adherens junction proteins E-cadherin and beta-catenin were also delayed in their recruitment to the cell membrane, and NZO-3 expression had striking effects on actin cytoskeleton dynamics.NZO-3 expression did not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100-soluble, signaling-active beta-catenin was increased in NZO-3-expressing cells during junction assembly.We hypothesize that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or beta-catenin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

ABSTRACT
The functional characteristics of the tight junction protein ZO-3 were explored through exogenous expression of mutant protein constructs in MDCK cells. Expression of the amino-terminal, PSD95/dlg/ZO-1 domain-containing half of the molecule (NZO-3) delayed the assembly of both tight and adherens junctions induced by calcium switch treatment or brief exposure to the actin-disrupting drug cytochalasin D. Junction formation was monitored by transepithelial resistance measurements and localization of junction-specific proteins by immunofluorescence. The tight junction components ZO-1, ZO-2, endogenous ZO-3, and occludin were mislocalized during the early stages of tight junction assembly. Similarly, the adherens junction proteins E-cadherin and beta-catenin were also delayed in their recruitment to the cell membrane, and NZO-3 expression had striking effects on actin cytoskeleton dynamics. NZO-3 expression did not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100-soluble, signaling-active beta-catenin was increased in NZO-3-expressing cells during junction assembly. In vitro binding experiments showed that ZO-1 and actin preferentially bind to NZO-3, whereas both NZO-3 and the carboxy-terminal half of the molecule (CZO-3) contain binding sites for occludin and cingulin. We hypothesize that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or beta-catenin.

Show MeSH
Related in: MedlinePlus