Differential roles for actin polymerization and a myosin II motor in assembly of the epithelial apical junctional complex - PubMed (original) (raw)

Differential roles for actin polymerization and a myosin II motor in assembly of the epithelial apical junctional complex

Andrei I Ivanov et al. Mol Biol Cell. 2005 Jun.

Abstract

Differentiation and polarization of epithelial cells depends on the formation of the apical junctional complex (AJC), which is composed of the tight junction (TJ) and the adherens junction (AJ). In this study, we investigated mechanisms of actin reorganization that drive the establishment of AJC. Using a calcium switch model, we observed that formation of the AJC in T84 intestinal epithelial cells began with the assembly of adherens-like junctions followed by the formation of TJs. Early adherens-like junctions and TJs readily incorporated exogenous G-actin and were disassembled by latrunculin B, thus indicating dependence on continuous actin polymerization. Both adherens-like junctions and TJs were enriched in actin-related protein 3 and neuronal Wiskott-Aldrich syndrome protein (N-WASP), and their assembly was prevented by the N-WASP inhibitor wiskostatin. In contrast, the formation of TJs, but not adherens-like junctions, was accompanied by recruitment of myosin II and was blocked by inhibition of myosin II with blebbistatin. In addition, blebbistatin inhibited the ability of epithelial cells to establish a columnar phenotype with proper apico-basal polarity. These findings suggest that actin polymerization directly mediates recruitment and maintenance of AJ/TJ proteins at intercellular contacts, whereas myosin II regulates cell polarization and correct positioning of the AJC within the plasma membrane.

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Figures

Figure 1.

Figure 1.

Dynamics of calcium-induced reassembly of apical junctions and F-actin reorganization in intestinal epithelial cells. In calcium-depleted T84 cells, an AJ protein E-cadherin and a TJ protein ZO-1 are localized in intracellular aggregates (A and B), whereas F-actin is primarily distributed at the cell cortex (C). Restoration of normal extracellular calcium level results in recruitment of AJ/TJ proteins to areas of cell-cell contacts to form a characteristic “chicken wire” staining pattern (G, H, J, K). Note the faster dynamics of formation of adherens junctions (D, G, J) compared with TJs (E, H, and K). The assembly of apical junctions is accompanied by dramatic reorganization of actin cytoskeleton manifested by early formation of radiating F-actin cables (F) that are later transformed into a perijunctional F-actin belt (I and H). Bar, 20 μm.

Figure 2.

Figure 2.

Nascent junctions and the mature AJC are composed of different sets of junctional proteins. Nascent junctions that are assembled after 0.5 h of calcium repletion, accumulate AJ proteins E-cadherin (A and B, red), p120 catenin (C, red), and β-catenin (D, green). Of all TJ proteins, only occludin (D, red) is present and colocalizes with β-catenin (D, arrows) in nascent junctions, whereas no significant accumulation JAM-A, ZO-1, and claudin-1 (A–C, green) is detected. In contrast, all of these TJ proteins colocalize with AJ components at AJC formed after 3 h of calcium repletion (E–H, arrows). Bar, 10 μm.

Figure 3.

Figure 3.

Formation of nascent junctions and TJs depends on the integrity of the actin cytoskeleton and is accompanied by global increase in intracellular F-actin. (A) In nascent junctions, E-cadherin (red) colocalizes with F-actin (green) cables that radiate between adjacent cells (arrows), whereas in TJs, occludin (red) colocalizes with a linear perijunctional F-actin belt (arrowheads). Bar, 10 μm. (B) Control T84 cells subjected to the calcium switch for either 1 or 3 h show typical adherens-like junctions and TJs at early and late stages of junctional assembly, whereas cells that were calcium-repleted for the same times in the presence of F-actin–disorganizing drug cytochalasin D (10 μM) develop neither nascent junctions nor TJs. Bar, 20 μm. (C) Representative Western blot and densitometric quantification show a dramatic decrease of G/F actin ratio starting from the early time point during calcium repletion. Data are presented as mean ± SE (n = 3); *p < 0.05 compared with the calcium-depleted group.

Figure 4.

Figure 4.

Formation of nascent junctions and TJs requires continuous polymerization of actin microfilaments, whereas ultimate maturation of the AJC decreases its dependence on actin polymerization. Calcium-depleted T84 cells were incubated in HCM for indicated times followed by additional 0.5-h incubation in HCM containing either G-actin–sequestering drug latrunculin B (1 μM) or vehicle. Latrunculin B treatment that prevents de novo actin polymerization causes rapid disassembly of preformed perijunctional actin bundles, and loss of junctional proteins from newly formed nascent junctions (A) and TJs (B), but it has no effect on the architecture of F-actin and occludin in mature AJC (C). Bar, 20 μm.

Figure 5.

Figure 5.

Nascent junctions and TJs represent the areas of active actin polymerization, and acceleration of F-actin polymerization promotes the assembly of TJs. (A) In calcium-repleted, saponin-permeabilized T84 cells, exogenous fluorescently labeled G-actin (green), is incorporated into nascent junctions and TJs where it colocalizes with, respectively, E-cadherin and occludin (arrows). Bar, 10 μm. (B) Jasplakinolide (1 μM) that stimulates F-actin polymerization accelerates recruitment of ZO-1 and occludin to the areas of cell-cell contact and promotes formation of circumferential TJs during calcium repletion. Data are presented as mean ± SE (n = 5); *p < 0.01 compared with the vehicle-treated group Bar, 20 μm.

Figure 6.

Figure 6.

N-WASP-Arp2/3–dependent actin nucleation is involved in formation of nascent junctions. (A) After 0.5 h of calcium repletion, nascent junctions are enriched in actin-nucleating proteins Arp3 and N-WASP (green) that colocalize with E-cadherin (arrows). Bar, 10 μm. (B) Control T84 cells calcium repleted for 1 h show typical assembly of F-actin bundles and accumulation of E-cadherin at nascent junctions, whereas cells that were calcium repleted in the presence of the N-WASP inhibitor wiskostatin (50 μM) demonstrate formation of neither F-actin–rich cell-cell contacts nor adherens-like junctions. Bar, 20 μm.

Figure 7.

Figure 7.

N-WASP-Arp2/3–dependent actin nucleation is critical for TJ assembly. (A) Arp3 and N-WASP (green) colocalize with occludin (arrows) in TJs formed after 3 h of calcium repletion. Bar, 10 μm. (B) Control T84 cells calcium repleted for 3 h demonstrate extensive assembly of both the perijunctional F-actin belt and TJs, whereas wiskostatin-treated cells form neither apical F-actin belt nor occludin-rich TJs. Bar, 20 μm.

Figure 8.

Figure 8.

Myosin II is not essential for assembly of nascent AJ-like junctions. (A) Representative Western blots and densitometric quantification show an increase in the amount of diphosphorylated (pp) but not total or monophosphorylated (p) RMLC in T84 cell lysates during calcium repletion. Data are presented as mean ± SE (n = 4); *p < 0.05 compared with the calcium-depleted group. (B) Neither MNMMIIA heavy chain nor pp-RMLC (green) accumulated at E-cadherin–based junctions (red) after 0.5 h of calcium repletion. Bar, 10 μm. (C) A selective inhibitor of MNMM motor activity, blebbistatin (50 μM), does not affect the assembly of F-actin–rich adherens-like junctions after 1 h of calcium repletion. Bar, 20 μm.

Figure 9.

Figure 9.

Myosin II is critical for assembly of apical TJs. (A) Both MNMMIIA heavy chain and pp-RMLC (green) colocalize with occludin (arrows) in TJs after 3 h of calcium repletion. Bar, 10 μm. (B) After 3 h of calcium repletion, the vehicle-treated T84 cells develop normal perijunctional F-actin belts and circumferential occludin strands, whereas the blebbistatin-treated cells demonstrate round F-actin–rich patches as well as small ring-like occludin staining (arrowheads). Bar, 20 μm.

Figure 10.

Figure 10.

Inhibition of myosin II activity prevents formation of the apico-basal cell polarity. (A) Early during calcium repletion (1 h), control T84 cells form ring-like structures containing the apical membrane domain marker syntaxin-3 (green) that are frequently located at lateral E-cadherin–rich contacts (arrows). Later (3 h) the rings disappear and syntaxin-3 becomes distributed evenly at the apical surface. In contrast, blebbistatin-treated cells retain undeveloped apical plasma membrane in a form of syntaxin-3–positive rings at areas of cell-cell contacts (arrowheads). Bar, 10 μm. (B) Transmission electron micrograph shows that after 3 h of calcium repletion, the vehicle-treated T84 monolayer is composed of columnar-shaped cells that possess typical apical surfaces with microvilli and electrondense apical junctions (arrows). In contrast, blebbistatin-treated monolayers contain rounded cells that do not have an extended apical surface with microvilli. Instead, these cells possess microvilli containing vacuoli (arrowhead) and lateral junctions at areas of cell-cell contacts (asterisks).

Figure 11.

Figure 11.

Three-step model of the formation of the apical junctional complex and apico-basal polarity in epithelial cells. Epithelial cells transiently lose columnar polarity and apical junctions after incubation at micromolar concentrations of extracellular calcium. Their TJ/AJ proteins are accumulated in the cytosol and F-actin is evenly distributed under the plasma membrane. Restoration of normal calcium level in cultural medium induces cell repolarization and reformation of the AJC in a process that can be divided into three major steps. The first step involves accumulation of F-actin bundles in areas of cell-cell contacts and the assembly of nascent adherens-like junctions. The second step results in the assembly of TJs encircling a primordial apical plasma membrane that is formed in a laterally located lumen with hepatic-type polarity. The final step is the transition from hepatic to apico-basal polarity resulting in formation of the apical plasma membrane domain and mature AJC.

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